CIRCADIAN REGULATION AND NATURAL VARIATION OF LOW TEMPERATURE SIGNALING IN ARABIDOPSIS By Malia Aratani Dong A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Plant Biology 2012 ABSTRACT CIRCADIAN REGULATION AND NATURAL VARIATION OF LOW TEMPERATURE SIGNALING IN ARABIDOPSIS By Malia Aratani Dong Many plants increase in freezing tolerance in response to low non-freezing temperature through a process known as cold-acclimation. In Arabidopsis, cold acclimation is associated with the induction or repression of over a thousand genes. The CBF cold response pathway has a central role in these changes. Within minutes of transfer to low temperature, genes encoding three closely related transcription factors, CBF1-3, are induced and alter expression of more than one hundred target genes, which go on to impart freezing tolerance. An abiotic stress such as freezing can limit the productivity and relative fitness of a plant. Consequently, there is considerable interest in finding upstream regulators of the CBF pathway in hopes of expanding the geographical range and yield of important food and biofuel crops. Previous studies have shown that CBF1-3 are subject to circadian regulation and that cold induction of CBF1-3 is gated by the circadian clock. We identify specific clock components involved in the circadian oscillations, and thus, upstream regulation of CBF genes. We also demonstrate the involvement of these identified clock components in gating coldregulated expression of CBF1-3 and CBF-target-genes. Furthermore, we show that these clock components affect plant freezing tolerance. Investigating natural variation of cold response pathways in ecotypes of Arabidopsis provides another means of distinguishing genes important for freezing tolerance. In previous studies, two Arabidopsis populations collected from Sweden (SW) and Italy (IT) were tested for fitness (survival and seed set) in reciprocal transplant experiments. Reciprocal transplant experiments revealed that home accessions at both sites had a strong advantage in terms of seed-set and survival. SW and IT recombinant inbred lines (RILs) were used to define fitness QTL in both locations. Since there is substantial variation in temperature across latitudes, genes associated with freezing tolerance may potentially underlie identified fitness QTL. This study shows that there are differences in the cold-acclimated freezing tolerance of SW and IT ecotypes under laboratory conditions. Through RNA-seq experiments, a set of genes that may contribute to differences in freezing tolerance between SW and IT is defined. RILs are used to map expression QTL (eQTL) for a subset of these low-temperature associated genes. Some of the eQTL mapped for these low-temperature associated genes overlap with previously identified fitness QTL and this study offers hypotheses as to the genes underlying these eQTL. This dissertation is dedicated to all of my family, but particularly to my parents, Jim and Gail, and my new parents John and Sara for their encouragement, to my undergraduate advisor J. Gary Tallman for his role in sparking my enthusiasm for scientific research, and especially to my husband Jackson Gehan for his support and the tent-cot. iv ACKNOWLEDGMENTS It is imperative that I thank Dr. Mike Thomashow for his support and direction throughout my graduate career, Dr. Sarah Gilmour for her role in reading and editing my writing as well as providing support and guidance, current and past members of the Thomashow lab for helpful conversations, friends for their support, especially Jackson, Merry, Mike, Molly, Kane, Starla, and Bonnie, and lastly, I need to acknowledge my committee members, Dr. David Arnosti, Dr. Robin Buell, Dr. Eva Farre, and Dr. Jonathan Walton for their guidance and strong encouragement to expand my skill-set. v PREFACE In Chapter Two, cca1-1, and the restored cca1-1 CCA1p:CCA1-GFP lines were procured by Eva Farre and generously donated by Jose Pruneda-Paz of the Kay Lab (San Diego, California). Other T-DNA mutant lines, used in Chapters Two and Four, were obtained from the Arabidopsis Biological Resource Center unless otherwise specified. In Chapter Three, the Schemske and Agren labs conducted reciprocal transplant experiments with the Swedish (SW) and Italian (IT) ecotypes and provided data for fitness QTL. Recombinant inbred lines (RILs) were generated and genotyped by members of the Schemske Lab. Avery Mendelsen and Nicolas Batora aided in sterilization, planting and transplanting of RILs used in eQTL analysis. SW and IT versions of CBF2 were sequenced by Chin-Mei Lee. Alignment and assembly of SW and IT RNA-seq data to the Columbia-0 (COL-0) genome was performed by Pingsha Hu. vi TABLE OF CONTENTS LIST OF TABLES……………………………………………………………………………….ix LIST OF FIGURES……………………………………………………………………………..xii KEY TO ABBREVIATIONS………………………………………………………………….xvii CHAPTER 1 LITERATURE REVIEW…………………………………………………………1 Cold-acclimation and freezing tolerance……………………………………………...2 Molecular changes associated with plant cold-acclimation…………………………3 CBF pathway of cold-acclimation………………………………...……………………7 CBF pathway in important plant species……………………………………………..8 Natural variation of CBF genes in Arabidopsis………………………………....……9 CBF-independent pathways of cold-acclimation in Arabidopsis……………….…11 Upstream regulation of CBF pathway in Arabidopsis……………………………...12 Upstream CBF regulation by the circadian clock…………………………………..15 Literature Cited…………………………………………………………………………19 CHAPTER 2 CCA1 and LHY regulate expression of the CBF cold response pathway and freezing tolerance in Arabidopsis……………………………………………………….31 Summary………………………………………………………………………………..32 Introduction……………………………………………………………………………..34 Results…………………………………………………………………………………..37 Discussion………………………………………………………………………………48 Materials and Methods………………………………………………………………..53 Appendix………………………………………………………………………………..56 Literature Cited………………………………………………………………………...63 CHAPTER 3 Natural variation in the low-temperature response of Arabidopsis ecotypes from Sweden and Italy………………………………………………………………………...69 Summary………………………………………………………………………………..70 Introduction……………………………………………………………………………..72 Results and Discussion: Freezing tolerance of SW and IT………………………..76 Results and Discussion: SW and IT RNA-seq experiments………..……………..80 Analysis of SW and IT basal transcriptomes.………………………………80 SW and IT cold-induced genes………………………………………………89 SW and IT transcriptomes at 1 week of cold-acclimation…......................98 Genes contributing to differences in freezing tolerance………………….132 Results and Discussion: SW and IT eQTL mapping……..………………………158 Conclusions…………………………………………………………………………...184 Materials and Methods………………………………………………………………187 Appendix………………………………………………………………………………194 Literature Cited……………………………………………………………………….312 vii CHAPTER 4 Interesting observations and future directions…………………………….320 Summary………………………………………………………………………………321 Results and Discussion……...………………………………………………………322 Materials and Methods………………………………………………………………351 Appendix………………………………………………………………………………353 Literature Cited……………………………………………………………………….357 viii LIST OF TABLES Table A2.1 Primer pairs used for qRT-PCR and ChIP experiments…………………..…61 Table 3.1. Locations of fitness QTL…………………………………………………………75 Table 3.2 GO categories significantly enriched in 372 genes with higher expression in SW under non-acclimated conditions………………………………………………………..87 Table 3.3. GO categories significantly enriched in 887 genes with higher expression in IT under non-acclimated conditions………………………………………………………….88 Table 3.4. GO categories significantly enriched in 608 genes only up-regulated in SW at 1 week of cold-acclimation…………………………………………………………………..106 Table 3.5. GO categories significantly enriched in 1012 genes only up-regulated in IT at 1 week of cold-acclimation…………………………………………………………………..108 Table 3.6. GO categories significantly enriched in 376 genes only down-regulated in SW at 1 week of cold-acclimation…………………………………………………………..112 Table 3.7. GO categories significantly enriched in 1319 genes only down-regulated in IT at 1 week of cold-acclimation……………………………………………………………….113 Table 3.8. GO categories significantly enriched in 77 genes with up-regulated induction pattern and higher expression level in SW at 1 week of cold-acclimation……………..120 Table 3.9. GO categories significantly enriched in 28 genes with up-regulated induction pattern and higher expression level in IT at 1 week of cold-acclimation……………….120 Table 3.10. GO categories significantly enriched in 69 genes with down-regulated induction pattern and higher expression level in IT at 1 week of cold-acclimation……124 Table 3.11. GO categories significantly enriched in 629 genes with up-regulated induction patterns and similar expression levels in SW and IT………………………….129 Table 3.12. GO categories significantly enriched in 841 genes with down-regulated induction patterns and similar expression levels in SW and IT………………………….131 Table 3.13. GO categories significantly enriched in 474 Category 1 genes with higher expression in SW at 2 weeks of cold-acclimation………………………………………...138 ix Table 3.14. GO categories significantly enriched in 1656 Category 1 genes with higher expression in IT at 2 weeks of cold-acclimation…………………………………………..139 Table 3.15. GO categories significantly enriched in 294 Category 2 genes with higher expression in SW at 2 weeks of cold-acclimation………………………………………...146 Table 3.16. GO categories significantly enriched in 504 Category 2 genes with higher expression in IT at 2 weeks of cold-acclimation…………………………………………..148 Table 3.17. 10 FTD candidate genes for eQTL analysis………………………………...171 Table 3.18. Locations of eQTL for Category 1 FTD candidate genes………………….174 Table 3.19. Locations of eQTL for Category 2 FTD candidate genes………………….175 Table 3.20. Expression of CBF genes…………………………………………………….182 Table A3.1. SW and IT markers used for mapping both fitness QTL and eQTL……..195 Table A3.2. GO categories of Hannah genes positively (277 genes) and negatively (466 genes) correlated with freezing tolerance………………………………………………….202 Table A3.3. 474 Category 1 FTD Candidate Genes with higher expression in SW at 2 weeks of cold-acclimation…………………………………………………………………...203 Table A3.4. 1656 Category 1 FTD Candidate Genes with higher expression in IT at 2 weeks of cold-acclimation…………………………………………………………………...220 Table A3.5. 294 Category 2 FTD Candidate Genes with higher expression in SW at 2 weeks of cold-acclimations………………………………………………………………….278 Table A3.6. 504 Category 2 FTD Candidate Genes with higher expression in IT at 2 weeks of cold-acclimations………………………………………………………………….289 Table A3.7. Primer pairs used for qRT-PCR and sequencing…………………………..307 Table A3.8. Number of reads passing the Illumina purity filter for each RNA-seq sample.………………………………………………………………………………………...308 Table A3.9. Summary of SW and IT RNA-seq samples…………………………………311 Table 4.1. Candidate cold-regulated transcription factors for SZF1 trans-QTL on chromosome 5 (between RIL markers 20.0 and 36.7)…………………………………...349 x Table A4.1. Primer pairs used for qRT-PCR……………………………………………...355 Table A4.2. Primer pairs used for ChIP qRT-PCR……………………………………….356 xi LIST OF FIGURES Figure 1.1. Regulation of central components of the Arabidopsis circadian clock based on previous literature…………………………………………………………………………..18 Figure 2.1. Effects of the cca1-11/lhy-21 double mutation on circadian regulation of CBF1, CBF2, CBF3 and CBF-targeted genes COR15A, COR47 and COR78…..……..40 Figure 2.2. Binding of CCA1 at the CBF1-3 locus……………………………………......41 Figure 2.3. Effect of the cca1-11/lhy-21 double mutation on plant freezing tolerance…44 Figure 2.4. Effects of the cca1-11/lhy-21double mutation on cold induction of CBF1, CBF2, CBF3 and CBF-targeted genes COR15A, COR47 and COR78………………….46 Figure 2.5. Model for circadian regulation and gated cold-induction of CBF1, CBF2 and CBF3…………………………………………………………………………………………….52 Figure A2.1. Circadian rhythms and gated expression of CBF genes in cca1-11 and lhy-21. …………………………………………………………………………………………..57 Figure A2.2. Robust circadian rhythms and gated expression of LHCB1.4 in cca1-11 and lhy-21 plants.………………………………………………………………………………58 Figure A2.3. Robust circadian rhythms and gated expression of LHCB1.4 in cca1-11/ lhy-21 plants..…………………………………………………………………………………..59 Figure A2.4. Mock experiments testing CCA1 binding at the CBF1-3 locus………...…69 Figure 3.1. Freezing tolerance of SW and IT ecotypes without cold-acclimation or with 1 week of cold-acclimation………………………………………………………………………78 Figure 3.2. Freezing tolerance of SW and IT ecotypes with 2 or 3 weeks of coldacclimation……………………………………………………………………………………...79 Figure 3.3 1259 genes differentially expressed between SW and IT under nonacclimated conditions………………………………………………………………………….85 Figure 3.4. Fold change of 40 Hannah genes positively correlated with freezing tolerance that have higher expression in IT. ………………………………………………86 Figure 3.5 Cold-responsive genes in SW…………………………………………………..94 Figure 3.6. Cold-responsive genes in IT…………………………………………….……...95 xii Figure 3.7. Overlap between the SW cold-responsive genes set and the Hannah genes set………………………………………………………………………………………………..96 Figure 3.8. Overlap between the IT cold-responsive genes set and the Hannah genes set………………………………………………………………………………………………..97 Figure 3.9. Overlap between SW and IT response at 1 week of cold-acclimation……103 Figure 3.10. Overlap between genes uniquely up-regulated in SW or IT at 1 week of cold-acclimation and the Hannah genes set………………………………………………104 Figure 3.11. Fold change of 55 Hannah genes that overlap with 608 genes only upregulated in SW at 1 week of cold-acclimation……………………………………………105 Figure 3.12. Overlap between genes uniquely down-regulated in SW or IT at 1 week of cold-acclimation and the Hannah genes set………………………………………………110 Figure 3.13. Fold change of 40 Hannah genes that overlap with 376 genes only downregulated in SW at 1 week of cold-acclimation……………………………………………111 Figure 3.14. Fold change of 32 Hannah genes that overlap with 1319 genes only down-regulated in IT at 1 week of cold-acclimation………………………………………111 Figure 3.15. 105 genes commonly up-regulated by cold but significantly different in expression level………………………………………………………………………………117 Figure 3.16. 105 genes commonly up-regulated by cold but significantly different in expression level and their overlap with the Hannah genes set………………………….118 Figure 3.17. Fold change of 13 Hannah genes that overlap with 77 genes with upregulated induction pattern, but higher expression level in SW…………………………119 Figure 3.18. 105 genes commonly down-regulated by cold but significantly different in expression level………………………………………………………………………………121 Figure 3.19. 105 genes commonly down-regulated by cold but significantly different in expression level and their overlap with the Hannah genes set………………………….122 Figure 3.20. Fold change of 6 Hannah genes that overlap with 69 genes with downregulated induction pattern, but higher expression level in IT…………………………...123 Figure 3.21. 629 genes with up-regulated induction patterns and similar expression levels and their overlap with the Hannah genes set……………………………………...128 xiii Figure 3.22. 841 genes with down-regulated induction patterns and similar expression levels and their overlap with the Hannah genes set……………………………………...130 Figure 3.23. 2130 Category 1 FTD candidate genes and overlap with the Hannah gene set………………………………………………………………………………………………136 Figure 3.24. Fold change of 15 Hannah genes that overlap with 474 Category 1 FTD genes with higher expression in SW……………………………………………………….137 Figure 3.25. Fold change of 20 Hannah genes that overlap with 474 Category 1 FTD genes with higher expression in SW……………………………………………………….137 Figure 3.26. 798 Category 2 FTD candidate genes and their overlap with the Hannah gene set……………………………………………………………………………………….144 Figure 3.27. Fold change of 32 Hannah genes that overlap with 294 Category 2 FTD genes with higher expression in SW……………………………………………………….145 Figure 3.28. Fold change of 12 Hannah genes that overlap with 504 Category 2 FTD genes with higher expression in IT…………………………………………………………147 Figure 3.29. Fold change of 25 Hannah genes that overlap with 504 Category 2 FTD genes with higher expression in IT…………………………………………………………147 Figure 3.30. Fold change of cold-regulated genes in SW and IT (7863 genes)………153 Figure 3.31. Fold change of CBF regulon genes in SW and IT………………………...154 Figure 3.32. Fold change of Hannah genes in SW and IT………………………………155 Figure 3.33. Fold change of differentially expressed biotic stress genes in SW and IT (348 genes)……………………………………………………………………………………156 Figure 3.34. Fold change of differentially expressed photosynthesis associated genes in SW and IT (53 genes)…………………………………………………………………….157 Figure 3.35. Category 1 FTD candidate genes for eQTL analysis…….……………….172 Figure 3.36. Category 2 FTD candidate genes for eQTL analysis……………………..173 Figure 3.37. eQTL for Category 2 FTD gene COR78……………………………………176 Figure 3.38. eQTL for Category 1 FTD gene COR15A………………………………….177 xiv Figure 3.39. eQTL for Category 2 FTD gene COR47……………………………………178 Figure 3.40. eQTL for Category 1 FTD gene COR314………………………………….179 Figure 3.41. eQTL for Category 2 FTD gene ERD10……………………………………180 Figure 3.42. eQTL for Category 1 FTD gene ZAT12…………………………………….181 Figure 3.43. CBF gene expression………………………………………………………...182 Figure 3.44. Protein prediction for CBF2 in SW, IT and COL-0………………………..183 Figure 4.1. Effects of the cca1-11/lhy-21double mutation on circadian and gated coldinduction of COR27…………………………………………………………………………..326 Figure 4.2. Binding of CCA1 on COR27…………………………………………………..327 Figure 4.3. Effects of the cca1-11/lhy-21double mutation on temperature-entrained circadian regulation of CBF1, CBF2, CBF3 and COR27.………………………………..333 Figure 4.4. Effects of the cca1-11/lhy-21double mutation on temperature-entrained circadian regulation of CBF target genes, COR15A, COR47 and COR78, and cycling control genes LHCB1.4, CAB2 and CCR2………………………………………………..334 Figure 4.5. Effects of the cca1-11/lhy-21double mutation on temperature-entrained circadian regulation of clock genes TOC1, PRR3, PRR5, PRR7 and PRR9.…………………………………………………………………………………………335 Figure 4.6. Effects of the cca1-11/lhy-21double mutation on temperature-entrained cold-induction of CBF1, CBF2, CBF3 and COR27……………………………………….336 Figure 4.7. Effects of the cca1-11/lhy-21double mutation on temperature-entrained cold-induction of CBF target genes, COR15A, COR47 and COR78, and cycling control genes LHCB1.4, CAB2 and CCR2…………………………………………………………337 Figure 4.8. Effects of the cca1-11/lhy-21double mutation on temperature-entrained cold-induction of PRR clock genes TOC1, PRR3, PRR5, PRR7 and PRR9…………..338 Figure 4.9. Cycling of CBF2 expression in plants grown under light-entrained conditions then sampled in constant-cold conditions………………………………………………….343 Figure 4.10. Cycling of CCA1 under warm, temperature cycling or constant-cold conditions……………………………………………………………………………………...344 xv Figure 4.11. SZF1-regulation candidate gene expression…………………………......350 Figure A4.1. Effects of the cca1-11 and lhy-21 single mutants on circadian and gated cold-induction of COR27…………………………………………………………………….354 xvi KEY TO ABBREVIATIONS ABA ACT AP2 BOA BP CAMTA CBF CBS CCA1 CHE CHIP COR COS CRT CVI DAVID DGDG DRE DREB DUF EE ELF EQTL FDR FTD GI GO ICE1 ICER IT LEA LER LHCB LHY LOD LUX MGDG NLS ABSCISIC ACID ACTIN APETALA2 BROTHER OF LUX ARRHYTHMO BASE PAIR CALMODULIN-BINDING TRANSCRIPTIONAL ACTIVATOR C-REPEAT BINDING FACTOR CCA1 BINDING SITE CIRCADIAN CLOCK ASSOCIATED 1 CCA1 HIKING EXPEDITION CHROMATIN IMMUNOPRECIPATION COLD-RESPONSIVE GENE COLD-STANDARD GENE C-REPEAT CAPE VERDE ISLANDS DATABASE FOR ANNOTATION VISUALIZATION AND INTEGRATED DISCOVERY DI-GALACTOSYLDIACYLGLYCEROL DROUGHT RESPONSE ELEMENT DEHYDRATION RESPONSIVE ELEMENT BINDING FACTOR DOMAIN OF UNKNOWN FUNCTION EVENING ELEMENT EARLY FLOWERING EXPRESSION QUANTITATIVE TRAIT LOCI FALSE DISCOVERY RATE FREEZING TOLERANCE DIFFERENCE GIGANTEA GENE ONTOLOGY INDUCER OF CBF EXPRESSION1 INDUCER OF CBF EXPRESSION REGION ITALIAN ECOTYPE LATE EMBRYOGENESIS ABUNDANT LANSBERG ERRECTA LIGH HARVESTING COMPLEX LATE ELONGATED HYPOCOTYL LOGARITHM OF ODDS LUX ARRHYTHMO MONO-GALACTOSYLDIACYLGLYCEROL NUCLEAR LOCALIZATION SIGNAL xvii P5CS PAG PBS PR PRR QTL RILS RVE SFR2 SUMO SW T1ME TBS TF TOC1 UBQ ZT Δ-PYRROLINE-5-CARBOXYLATE SYNTHASE PHOTOSYNTHESIS ASSOCIATED GENE PUTATIVE BOA BINDING SITE PATHOGENESIS-RELATED PSEUDO-RESPONSE REGULATOR QUANTITATIVE TRAIT LOCI RECOMBINANT INBRED LINES REVEILLE SENSITIVE TO FREEZING 2 SMALL UBIQUITIN-RELATED MODIFIER SWEDISH ECOTYPE TOC1 MORNING ELEMENT TCP BINDING SITE TRANSCRIPTION FACTOR TIMING OF CAB 1 UBIQUITIN ZEITGEBER TIME xviii CHAPTER ONE LITERATURE REVIEW     1 CHAPTER ONE LITERATURE REVIEW Cold-acclimation and freezing tolerance. The geographic range of plants is limited by a number of abiotic factors, including low temperature (1-3). Plants can vary greatly in their tolerance to temperature changes. In fact, in the deserts of Southern California plants thrive in an environment that goes from freezing to scorching in a single day, with temperature swings of approximately 50°C (4, 5). For plants growing in temperate and tropical climates the average difference between day and night temperatures is much narrower than these extreme desert conditions. However, temperate and tropical plants are still exposed to temperature fluctuations of approximately 10-15°C in a single day (5). In addition to constraining the geographical locations that a plant can grow, low temperatures can also decrease plant productivity and cause crop loss (6). Most temperate plants, such as winter wheat, barley and Arabidopsis, can increase their tolerance to freezing temperature by prior exposure to low non-freezing temperatures, a process known as cold-acclimation (6-8). Many economically important crops such as commercial citrus trees (9), maize (10), tomato (11) and soybean (12) are chillingsensitive and cannot cold-acclimate. In fact, in 2007, after 4 days of freezing temperatures, the California citrus industry suffered $480 million dollars of damage (13).     Chilling-sensitive crops cannot cold acclimate but can reduce damage by cold through prior exposure to low temperatures (8). Consequently, a better understanding of the mechanisms of cold-acclimation may also lead to enhancements in chilling tolerance (8,     2 14, 15). Freezing tolerance is an agronomically important trait and considerable effort has gone into breeding plants with greater freezing tolerance (6, 16-19). Closely related species of plants can differ significantly in their ability to cold-acclimate (15). For example, the common cultivated potato, Solanum tuberosum, and the wild potato Solanum commersonii, are closely related evolutionarily (15). Both species are chilling tolerant and have similar basal freezing tolerance, but Solanum commersonii can coldacclimate while Solanum tuberosum cannot (15). Even ecotypes within a species can vary greatly in their basal and cold-acclimated freezing tolerance (2, 3, 20-24). Traditional breeding methods, thus far, have only achieved limited success in increasing plant freezing tolerance (19). In comparison to traditional breeding methods, transgenic plants have been much more successful in increasing freezing tolerance of important crop species (25-30). Therefore there is considerable interest in continuing to elucidate the molecular mechanisms underlying the process of cold-acclimation. Molecular changes associated with plant cold-acclimation. At freezing temperatures ice forms on the outside of leaf surfaces and subsequently propagates to the intercellular space of plant tissue (31-33). This intercellular ice can cause physical damage to plant cells and tissue (31-33). However, the dehydration that accompanies freezing is the primary source of damage (31-33). The severe cellular dehydration that accompanies freezing, is the result of the chemical potential of the cell moving unfrozen water from the intracellular (cytoplasm) space to the intercellular space where the water     3 is frozen (31-33). Under non-acclimated conditions (no prior exposure to low temperatures), dehydration as a result of freezing can also instigate two forms of membrane damage. First, is endocytotic vesiculation, which alone is not damaging (31-33). However, the surface area reduction is not reversible, and cell lysis occurs when cells regain their volume during thawing (31-33). Secondly, during severe dehydration hexagonal II phase regions can form between the plasma membrane and endomembranes, such as the chloroplast envelope (31-33). These hexagonal II phase regions can result in a loss of osmotic responsiveness (31-33). Therefore, the plant cell can never recover from freeze-induced dehydration. Cold-acclimated (prior exposure to low, non-freezing temperatures) plant cells respond differently to cellular dehydration as a result of freezing (31-33). Rather than endocytotic vesiculation, exocytotic vesicles continuous with the plasma membrane are formed (31-33). Therefore cells are not disrupted when they regain their volume during thawing. Hexagonal II phase regions in the plasma membrane also do not form with cold-acclimation (31-33). Consequently, assessment of membrane damage after freeze/thaw is a means of quantitatively measuring plant freezing tolerance (electrolyte leakage assay) and distinguishing plants that can and cannot cold-acclimate. It has long been observed that there are significant changes in membrane lipid composition during cold-acclimation (31, 32, 34, 35). These changes include an increase in fatty acid unsaturation and phospholipid content as well as a drastic decrease in chloroplast-specific mono-galactosyldiacylglycerol (MGDG; (35)). A recent     4 study by Moellering et al. 2010 found that the gene SENSITIVE TO FREEZING 2 (SFR2) encodes a galactolipid remodeling enzyme vital to this chloroplast-specific decrease in MGDG (35). MGDG is prone to the formation of hexagonal II phase lipid structures (35). As previously mentioned, these hexagonal II phase regions can result in a loss of osmotic responsiveness during freezing (31-33). SFR2 is thus hypothesized to increase plant freezing tolerance by decreasing the formation of hexagonal II phase lipids by converting MGDG to di-galactosyldiacylglycerol (DGDG) and oligogalactolipids, which are not prone to hexagonal II phase formation in vitro (35). Along with changes in lipid composition, Guy et al. 1985, showed through in vitro translation of mRNA in non-acclimated and cold-acclimated samples, that there are many gene expression changes during the process of cold-acclimation (36). Since then, many studies have detailed the changes in gene expression that occur during the process of cold acclimation (37-45). These studies found that many of the coldresponsive genes encode hydrophilic proteins. These hydrophilic COLD-RESPONSIVE (COR) polypeptides have simple, repetitive, amino-acid composition (37-45). COR6.6, also known as KIN2, is almost identical to KIN1, a small hydrophilic protein that is hypothesized to act as an antifreeze protein due to sequence similarity with arctic fish antifreeze proteins (37, 39, 46-48). COR6.6 is targeted to the cytoplasm and has been shown to have cryoprotective properties by reducing the amount of freeze-induced membrane fusions in vitro (46-48). COR15A is a cold-regulated gene specifically localized to the stroma of chloroplasts (38). Current data support the hypothesis that COR15A increases freezing tolerance by preventing the formation of     5 hexagonal II phase regions, which normally form between the plasma membrane and chloroplasts (41, 46). Fittingly, overexpression of COR15A leads to an increase in nonacclimated freezing tolerance of chloroplasts in vitro and an increase in the acclimated and non-acclimated freezing tolerance of protoplasts (41). COR47 has sequence homology with LATE EMBRYOGENESIS ABUNDANT (LEA) proteins, which respond to ABSCISIC ACID (ABA), water stress, and drought (39). Like COR47, COR78 is another hydrophilic protein that resembles LEA proteins, which responds to cold, ABA and drought (49, 50). Mutation or overexpression of COR78 has little impact on plant freezing tolerance (51). Accordingly, the function of COR78 and many of these COR proteins in freezing tolerance have yet to be entirely elucidated (51). Transcriptional changes at low temperature are also accompanied by extensive changes in metabolic profile, which include the accumulation of certain amino acids and sugars, such as proline and sucrose (44, 52, 53). Free proline has been shown to have cryoprotective properties in plants (52) and the low-temperature induced increase in free 1 proline has been linked to the increase in transcripts for Δ -pyrroline-5-carboxylate synthase (P5CS), a key enzyme in the proline biosynthetic pathway (54). Fructose, glucose, and sucrose are sugars up-regulated by low-temperature and the synthesis of these sugars is associated with the raffinose pathway (44, 52, 53). These solutes accumulate in the cytoplasm, decreasing the chemical potential driving dehydration (31, 34, 53). The increase in these solutes with cold-acclimation also possibly reduces hexagonal II phase formation by increasing the distance between membranes (34). The up-regulation of the raffinose pathway by cold has been specifically linked to the gene     6 GALACTINOL SYNTHASE3 (GOLS3), a key enzyme in the raffinose biosynthetic pathway (55). GOLS1 and GOLS2 are regulated by other stresses (55). While these are two examples of genes driving metabolic changes during cold-acclimation, the genes responsible for many of the other metabolic changes remain unidentified (44). PATHOGENESIS-RELATED (PR) proteins accumulate in the intercellular fluid (apoplasts) of plants at low-temperature and have been shown to have antifreeze activity (56-58). High concentrations of these PR antifreeze proteins in the apoplast can decrease the freezing temperature of the intercellular fluid, thus preventing ice formation (56). At lower concentrations, the growth of ice in the intercellular space is also limited by PR antifreeze proteins, which adhere onto ice and prevent large ice crystals from forming (56). This decrease in intercellular ice formation by PR proteins thus, decreases the amount of membrane damage by intercellular ice and suggests an interesting relationship between cold and biotic stresses (56). CBF pathway of cold-acclimation. Promoter analysis of the COR genes identified the C-REPEAT/DEHYDRATION RESPONSE ELEMENT (CRT/DRE) as a novel and significantly overrepresented cis-element (40, 50). This element was found to be responsive to cold and drought but not ABA (40, 50). A transcriptional activator, CREPEAT BINDING FACTOR1 (CBF1), was identified as binding to the CRT/DRE through a yeast-1-hybrid screen (59). Two closely related genes CBF2 and CBF3, located in tandem to CBF1 on chromosome 4, were subsequently identified and shown to bind to the CRT/DRE (42). CBF1-3 all contain a NUCLEAR LOCALIZATION SIGNAL     7 (NLS), an APETALA2 (AP2) DNA binding domain, followed by an acidic activation domain (59). Within minutes of exposure to cold, CBF1-3 gene expression is rapidly induced in Arabidopsis (42). CBF transcription factors bind to the CRT/DRE element in the promoters of COR genes, which leads to activation of COR gene expression (42). Constitutive over-expression of CBFs in Arabidopsis results in expression of COR genes under warm temperatures and constitutive freezing tolerance without prior cold acclimation (45, 54, 60-62). Phenotypically, CBF over-expressing plants are dwarf compared to wild-type plants. CBF1-3 also regulate many of the metabolic changes associated with cold acclimation (44, 62). In fact, both P5CS and GOLS3 are CBF target genes (44, 54, 55). Genomic analysis also revealed the existence of a closely related AP2 transcription factor on chromosome 5 named CBF4 (63). Although closely related in terms of transcript sequence, the regulation of CBF4 seems to have diverged significantly from CBF1-3, since CBF4 appears to be specifically up-regulated by drought but not low-temperature (63). Overexpression of CBF4 leads to the activation of genes containing CRT/DRE elements (63). Consequently, both drought and freezing tolerance are increased in CBF4 overexpressing plants (63). CBF pathway in important plant species. Although the CBF pathway was identified in the model plant, Arabidopsis, it appears to be conserved in many economically important plant species that can cold-acclimate, such as wheat, rye,     8 poplar and barley (64-66). Overexpression of the CBF genes in these plants also leads to an increase in freezing tolerance (64-66). The CBF genes are also present in plants that do not cold-acclimate such as rice, and tomato (67, 68). Interestingly, overexpression of CBF genes in rice and tomato does not lead to an increase in freezing tolerance, but the CBF regulons of these two species are significantly smaller than other species that do cold-acclimate (67, 68). Solanum commersonii and Solanum tuberosum, two evolutionarily close potato species, differ in their ability to cold acclimate and also have significant differences in the genes that comprise their CBF regulons, although the number of CBF regulated genes is similar (15). Chapter Three of this study will detail variations in the low-temperature response of two Arabidopsis ectoypes with differing freezing tolerance. Natural variation of CBF genes in Arabidopsis. Previous studies on the natural variation of freezing tolerance in Arabidopsis have implicated the CBFs as a source of significant variation. Alonso-Blanco et al. 2005 mapped differences in freezing tolerance using recombinant inbred lines (RILs) from Arabidopsis accessions LANDSBERG ERECTA (LER) and CAPE VERDE ISLANDS (CVI) (20). Seven different quantitative trait loci (QTL) on chromosomes 1,4, and 5 were found, but the single QTL on chromosome 4 explained the most variance and also contained the CBF locus (20). CVI freezing tolerance was significantly reduced in comparison to LER, as was CBF2 expression. Introduction of the Ler-CBF2 transgene into CVI significantly reduced differences in freezing tolerance between LER and CVI (20).     9 CBF expression in the Versailles collection of Arabidopsis was also found to vary significantly (24). Although freezing tolerance did not simply correlate with CBF expression in the 8 accessions tested, the most freezing-sensitive Versailles accessions were also the accessions with the lowest cold-induced CBF expression levels (24). The most freezing tolerant Versailles accessions varied significantly in their CBF expression, suggesting the importance of CBF-independent pathways of freezing tolerance for some accessions (24). Freezing tolerance QTL mapped for two Versailles RIL populations were not completely overlapping (24). However, both populations had a common QTL on chromosome 4 that overlapped with the CBF locus and explained the most variance (24). Further analysis of CBF gene sequence in the 48 Versailles accessions showed a number of polymorphisms in both the promoter and coding sequences of CBF1-3 (22). A study by Zhen et al. 2008 further analyzed CBF sequence in 24 accession of Arabidopsis and found a large bias toward nonsynonymous changes in the activation domain compared to the DNA binding domain of CBF genes (23). Zhen et al. 2008 also showed that the CBF genes undergo relaxed purifying selection in Arabidopsis accession native to southern latitudes in comparison to accessions native to northern latitudes (23). The 10 southern latitude accessions tested had 1.5 to 4.6-fold more polymorphisms in the individual CBF genes in comparison to the 14 northern latitude accessions tested (23). The four accessions (CO-1, CVI-0, CVI-1, and ITA-0) with the lowest CBF expression originated from southern latitudes and had the lowest survival rates in freezing tolerance assays (23). CO-1, CVI-1, ITA-0 and two additional     10 southern accessions, Can-0 and L1-0, also had reduced expression of three CBF target genes (COR6.6, COR15A, COR78) in comparison to northern accessions (23). CBF-independent pathways of cold-acclimation in Arabidopsis. Microarray analysis of CBF overexpressing plants revealed that only 12% of the cold-responsive transcriptome is part of the CBF regulon (69). Furthermore, 28% of cold-responsive genes are not affected by expression of the CBF genes (69). This indicates that there are CBF-independent pathways regulating expression of cold-induced genes. Through an EMS mutant screen, ESKIMO1 (ESK1) was identified as a plant with constitutive freezing tolerance without prior cold acclimation (70). The esk1 mutant has constitutively high levels of proline (70), similar to CBF overexpressing plants (62). However, ESK1-regulated genes do not overlap significantly with CBF regulated genes, suggesting that ESK1 is part of a CBF-independent pathway (70, 71). ESK1 encodes DOMAIN OF UNKNOWN FUNCTION 231 (DUF231) protein and it was recently shown that ESK1 is important for production of functional xylem and consequently water transport (71, 72). Hos9 and hos10 mutant plants were identified in a mutant screen using plants expressing the COR78 promoter driving the expression of a luciferase reporter gene (73-75). The hos9 and hos10 mutants both have reduced freezing tolerance compared to wild-type plants in whole plant freeze tests (73, 74). Though the gene underlying the hos10 mutation has yet to be found (75), the gene underlying the hos9 mutation is a constitutively active homeodomain transcription factor (73). Microarray analysis of the     11 hos9 mutant reveals that genes regulated by HOS9 are significantly different from those regulated by the CBFs (73). Upstream regulation of CBF pathway in Arabidopsis. CBF genes significantly impact freezing tolerance in economically important species, and appear to be under substantial selective pressure (22-24). Consequently, there is considerable interest in finding upstream regulators of the CBF genes. In 2004, Novillo et al. isolated a cbf2 mutant that had increased freezing tolerance and higher expression of CBF1 and CBF3 (76). This therefore suggests that CBF2 negatively regulates CBF1 and CBF3. However, this hypothesis was not substantiated by microarray studies (45, 61). Plants overexpressing CBF2 did not have significantly reduced CBF1 or CBF3 expression (45, 61). In 2003, Zarka et al. used 5’ and 3’ deletions of the CBF2 promoter to identify a ~155 base pair (bp) fragment required for cold induction (77). Interestingly, the Arabidopsis accession, CVI, which was found to have reduced CBF2 expression in comparison to LER, has a 1630 bp deletion in the CBF2 promoter that intrudes on this important ~155 bp region (20). Zarka et al. 2003 also identified two short elements, INDUCTION OF CBF EXPRESSION REGION 1 (ICER1) and ICER2, within this ~155 bp region important for cold-regulated expression (77). Subsequent mutations performed within this ~155 bp region further identified sequences important for coldinduced CBF expression (78). One element found through this mutational analysis was identified as a binding site for the CALMODULIN BINDING TRANSCRIPTION     12 ACTIVATOR (CAMTA) family of calmodulin binding transcription factors (78). CAMTA3 was found to positively regulate CBF1 and CBF2, but not CBF3, which does not contain a CAMTA binding site (78). Interestingly, exposure to cold temperature results in a rapid spike of cytosolic calcium (79). Although the connection between calcium and cold is still not well understood, these cold-induced increases in cytosolic calcium are also required for normal activation of CBF regulated genes (79). In plants, calcium can be perceived by calmodulin (78). Therefore, as a calmodulin binding transcription factor, CAMTA3, is a putative connection between calcium, cold and the CBF genes (80). In 2003, INDUCER OF CBF EXPRESSION1 (ICE1) was identified through a mutant screen using plants expressing a luciferase reporter gene under the control of the CBF3 promoter (81). ICE1, is a constitutively expressed, nuclear localized, MYC family transcription factor that positively regulates CBF3, but has little effect on CBF1 or CBF2 (81). Upon exposure to cold, ICE1 transcripts are only slightly up-regulated (81), but ICE1 is post-transcriptionally regulated through sumoylation by SIZ1, a SMALL UBIQUITIN-RELATED MODIFIER (SUMO) E3 ligase (82). Sumoylation is a reversible post-transcriptional modification that is most notably known to affect subnuclear targeting, transcriptional regulation and ubiquitin mediated-protein degradation (82). Miura et al. 2007 demonstrated that cold-induced sumoylation of ICE1 leads to enhanced protein stability by reducing polyubiquitination, which normally leads to degradation (82).     13 MYB15 was identified through microarray experiments as a cold-regulated gene with higher expression in ice1 mutants (83). This suggests that ICE1 normally represses expression of MYB15. Consistent with this idea, ICE1 sumoylation by SIZ1 also represses expression of MYB15 (82). Subsequent yeast-2-hybrid and pull-down assays also suggested interaction of MYB15 with ICE1 (83). Overexpression of MYB15 reduces expression of CBFs and MYB15 RNAi lines have been shown to increase CBF expression (83). CBF1-3 promoters contain the known binding site for MYB15 and in vitro binding assays suggest that MYB15 negatively regulates CBF genes directly (83). But CBF target genes are not affected by overexpression or reduction of MYB15 expression (83). Agarwal et al. 2006 explain this confounding result by suggesting that MYB15 negatively regulates CBF genes, but induces expression of other cold-regulated transcription factors, which up-regulate COR genes (83). This is a reasonable hypothesis, however, in vivo binding assays are needed to confirm the MYB15-CBF interaction. ZAT12 is a transcription factor that was found to have a similar cold induction pattern to the CBF genes (45, 69). ZAT12 overexpressing plants had significantly reduced CBF expression but only slightly reduced CBF target gene expression (45). ZAT12 is thus another potential negative regulator of the CBF genes. ZAT12 overexpressing plants are dwarf as are CBF overexpressing plants, but are only slightly more freezing tolerant than wild-type plants (45). Microarray analysis of ZAT12 overexpressing plants further supports the role of ZAT12 in cold signaling (45).     14 However, further evidence is needed to show that ZAT12 is directly involved in the negative regulation of CBF genes. Upstream CBF regulation by the circadian clock. Circadian clocks give an internal estimate of time, allowing organisms to predict daily changes in their environment such as the onset of day or night, or changes between day and night temperature (84, 85). Not surprisingly, the ability to anticipate and respond to environmental changes provides an adaptive advantage for plants (84, 86, 87). The Arabidopsis circadian clock is composed of several interlocking feedback loops and additional components of the Arabidopsis clock continue to be found each year (88, 89). Morning-expressed MYB transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) directly repress the expression of TIMING OF CAB1 (TOC1) by binding to an EVENING ELEMENT (EE; AAAATATCT) in the TOC1 promoter (90). EARLY FLOWERING 3 (ELF3), ELF4, LUX ARRHYTHMO (LUX), and GIGANTEA (GI) promote expression of CCA1 and LHY, but there is no evidence that these transcription factors directly regulate CCA1 or LHY (9092). Recently, BROTHER OF LUX ARRHYTHMO (BOA) was identified as a clock component that positively regulates CCA1 expression by binding to PUTATIVE BOA BINDING SITE 3 (PBS3) and PBS4 elements in the CCA1 promoter (89). TOC1, up until very recently (93), was also widely hypothesized to promote expression of CCA1 and LHY since toc1 mutants have reduced expression of CCA1 and LHY (90). However, this hypothesis was confounded for many years by the result     15 that TOC1 overexpressing plants also have low expression of CCA1 and LHY (94). In 2012, Gendron et al. showed that TOC1 generally acts as a repressor, which can bind to a new cis-element TOC1 MORNING ELEMENT (T1ME) through its CCT domain (93). Although, Gendron et al. 2012 also demonstrated that TOC1 represses expression of CCA1 and LHY, they do not negate genetic data (90) that suggests that TOC1 is somehow necessary for activation of CCA1 and LHY (93). Although the role of TOC1 in the circadian clock still seems unsettled there is evidence that suggests that the increase in CCA1 and LHY could be partially explained by the repression of CCA1 HIKING EXPEDITION (CHE) by TOC1 (93, 95). Pruneda-Paz et al. 2009 demonstrated that CHE represses CCA1 through the TCP-BINDING SITE (TBS; GGTCCCAC) in the CCA1 promoter (95). CCA1 and LHY also repress CHE through the CCA1-BINDING SITE (CBS; AAAAATCT) in the CHE promoter (95). The TOC1 protein binds directly to the N-terminal domain of CHE, which binds directly to TBS, and chromatin immunoprecipitation (ChIP) experiments have shown enrichment of TOC1 at TBS elements in the CCA1 promoter (95). TOC1 is thus hypothesized to promote expression of CCA1 by antagonizing the CCA1 inhibitor, CHE (95). PRR1 was found to be identical to TOC1 (96) and PRR7 and 9, were subsequently identified as components of the circadian clock that form a ‘morning expressed’ feedback loop with CCA1 and LHY (97-102). CCA1 and LHY positively regulate PRR7, and 9, which then directly repress CCA1 and LHY expression (97-102). In 2000, Harmer et al. demonstrated that components of key transcriptional pathways such as photosynthesis, phenylpropanoid biosynthesis, cell elongation, and     16 starch mobilization, are all under concerted regulation by the circadian clock (84). Harmer et al. 2000 also showed that CBF3 expression is rhythmic under warm conditions (84). This result led Fowler et al. 2005 to examine the expression of CBF genes under circadian conditions (103). In 2005, Fowler et al. used northern blot analysis to demonstrate that the amplitude of CBF1-3 cold-induction is dependent on the time of day that the plants are exposed to cold (103). This gating of cold-induced gene expression was observed under both diurnal and light-entrained circadian conditions (103). Peak CBF expression was at ZEITGEBER TIME 4 (4 hours after dawn; ZT4) and ZT28, and troughs of expression were at ZT16 and ZT40 (103). Fowler et al. 2005 used CCA1 overexpressing plants to show that gated coldinduced expression of CBF genes is dependent on the circadian clock (103), since overexpression of central clock component CCA1 disrupts normal clock function (90, 104). However, the specific clock components regulating CBF expression are still unclear. A study by Nakamichi et al. 2009 showed that the prr9/prr7/prr5 triple mutant disrupted gated cold-induced expression of CBF genes. The triple mutant had constitutively high CBF expression (105). Therefore, PRR9, PRR7 and PRR5, potentially act as negative regulators of CBF expression, though no evidence of in vivo interaction with the CBF genes was provided (105). In Chapter Two of this study an alternate mechanism is suggested for both the circadian regulation and gated coldinduction of CBF genes.     17 PRR5,7,9 PRR7 PRR9 LHY LHY CCA1 CCA1 CHE CHE TOC1 TOC1 BOA LUX ELF3 ELF4 GI Figure 1.1. Regulation of central components of the Arabidopsis circadian clock based on previous literature. Ovals represent proteins, and rectangles represent a gene and promoter region. Solid lines ending in arrows (that are not attached to genes) represent positive regulation, while solid lines ending in a perpendicular line represent negative regulation. Dotted lines represent transcription and translation of a gene to a protein. For details on the regulation of central clock components please refer ‘upstream CBF regulation by the circadian clock’ section of Chapter One, which begins on page 15.     18 LITERATURE CITED     19 LITERATURE CITED 1. Zhen Y & Ungerer MC (2008) Clinal Variation In Freezing Tolerance Among Natural Accessions Of Arabidopsis Thaliana. New Phytologist 177(2):419-427. 2. Lefebvre V, Kiani SP, & Durand-Tardif M (2009) A Focus On Natural Variation For Abiotic Constraints Response In The Model Species Arabidopsis Thaliana. 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Plant and Cell Physiology 50(3):447-462.     30 CHAPTER TWO CIRCADIAN CLOCK-ASSOCIATED 1 and LATE ELONGATED HYPOCOTYL regulate expression of the CBF cold response pathway and freezing tolerance in Arabidopsis The following chapter was published in Proceedings of the National Academy of Sciences of the United States of America. Dong MA, Farre EM, & Thomashow MF (2011) CIRCADIAN CLOCK-ASSOCIATED 1 and LATE ELONGATED HYPOCOTYL regulate expression of the C-REPEAT BINDING FACTOR (CBF) pathway in Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 108:7241-7246   31   CHAPTER TWO CIRCADIAN CLOCK-ASSOCIATED 1 and LATE ELONGATED HYPOCOTYL regulate expression of the CBF cold response pathway and freezing tolerance in Arabidopsis SUMMARY The C-REPEAT BINDING FACTOR (CBF) cold response pathway has a prominent role in cold acclimation, the process whereby certain plants increase in freezing tolerance in response to low non-freezing temperatures. In Arabidopsis, the CBF pathway is characterized by rapid induction of the C-REPEAT BINDING FACTOR 1 (CBF1), CBF2 and CBF3 genes, which encode transcriptional activators, followed by induction of the CBF-targeted genes known as the CBF regulon. Expression of the CBF regulon results in an increase in freezing tolerance. Previous studies established that CBF1, CBF2 and CBF3 are subject to circadian regulation and that their cold induction is gated by the circadian clock. Here we present the results of genetic analysis and chromatinimmunoprecipitation experiments indicating that both of these forms of regulation involve direct positive action of two transcription factors that are core components of the clock, i.e., CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). In plants carrying the cca1-11/lhy-21 double mutation, cold induction of CBF1, CBF2 and CBF3 was greatly impaired and circadian regulation of CBF1 and CBF3 was essentially eliminated; circadian regulation of CBF2 continued, though with significantly reduced amplitude. Circadian regulation and cold induction of the CBF regulon genes COLD-REGULATED GENE15A (COR15A), COR47 and   32   COR78 were also greatly diminished in plants carrying the cca1-11/lhy-21 double mutation. Furthermore, the cca1-11/lhy-21 double mutation resulted in impaired freezing tolerance in both non-acclimated and cold-acclimated plants. These results indicate that CCA1/LHY-mediated output from the circadian clock contributes to plant cold tolerance through regulation of the CBF cold response pathway.   33   INTRODUCTION A general feature of plants from temperate environments is that they increase in freezing tolerance in response to low non-freezing temperatures, a process called cold acclimation (1, 2). It is now well established that cold acclimation involves extensive changes in gene expression (3-6). The best understood cold regulatory pathway is the CBF pathway. This pathway, which is widely conserved in plants (7), is best characterized in Arabidopsis (8, 9). When Arabidopsis plants are transferred from warm to cold temperature, C-REPEAT BINDING FACTOR 1 (CBF1), CBF2 and CBF3–also known as DROUGHT REPONSE ELEMENT BINDING FACTOR1B (DREB1B), DREB1C and DREB1A, respectively—are rapidly induced. These genes, which are physically linked in tandem array, encode transcription factors that are members of the AP2/ERF family of DNA binding proteins (10). The CBF proteins bind to the CRT/DRE regulatory element present in the promoters of about 100 cold-regulated (COR) genes, known as the CBF regulon, and induce their expression (4, 6, 11). Constitutive overexpression of CBF1, CBF2 and CBF3 at warm temperature results in constitutive expression of the CBF regulon and an increase in freezing tolerance (12-14). The mechanisms whereby expression of the CBF regulon promotes freezing tolerance are not completely understood, but involve the synthesis of low molecular weight cryoprotectants such as sucrose and raffinose and proteins that have cryoprotective properties (1, 2). Given their importance in cold acclimation, efforts have been directed at understanding the mechanisms involved in cold-induction of CBF1, CBF2 and CBF3. To date, two positive regulators have been identified: INDUCER OF CBF EXPRESSION   34   1 (ICE1), a MYC family transcription factor that positively regulates CBF3 (15), and CALMODULIN BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3), a CAMTA family transcription factor that positively regulates CBF1 and CBF2 (16). The ICE1 and CAMTA3 genes are transcribed at warm temperature, indicating that their activities involve posttranscriptional regulatory mechanisms that are responsive to low temperature (16-18). Another factor that affects the expression of CBF1, CBF2 and CBF3 is the circadian clock (19-22). At warm temperature, the transcript levels for CBF1, CBF2 and CBF3 oscillate with a peak at about 8 h after dawn (ZEITGEBER TIME 8; ZT8), and a trough at about ZT20. Moreover, cold-induction of CBF1, CBF2 and CBF3 is “gated” by the clock (22); if plants are exposed to low temperature at ZT4, the increase in CBF1, CBF2 and CBF3 transcript levels is much greater than if plants are exposed to low temperature at ZT16. These results indicate that cold induction of CBF1, CBF2 and CBF3 involves the integration of low temperature and clock regulatory pathways. The circadian clock of Arabidopsis consists of multiple interlocking regulatory feedback loops (23, 24). Key components of the core feedback loop are CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), MYB transcription factors that have partially overlapping functions (25-28), and TIMING OF CAB 1 (TOC1), a PSEUDO RESPONSE REGULATOR (PRR) protein (29). Expression of CCA1 and LHY peaks just after dawn, while the expression of TOC1 peaks in the early evening. CCA1 and LHY bind to the Evening Element (EE) (19) present in the promoter of TOC1 and repress its transcription (30). TOC1 is necessary for the   35   induction of both CCA1 and LHY (28). TOC1 is known to inhibit the repression of CCA1 by the TCP transcription factor CCA1 HIKING EXPEDITION 1 (CHE1) (31), but the means by which LHY expression is activated by TOC1 remains unknown. CCA1 and LHY also regulate expression of PSEUDO RESPONSE REGULATOR 7 (PRR7) and PRR9 (32, 33), two components of the morning regulatory loop. CCA1 and LHY bind to the promoters of these two genes to induce their expression and the PRR7 and PRR9 proteins then negatively regulate CCA1 and LHY (32, 33). It was recently reported that Arabidopsis plants carrying the prr5/prr7/prr9 triple mutation constitutively express CBF1, CBF2 and CBF3 at high levels and display constitutively high levels of freezing tolerance (34). Thus, it was proposed that PRR5, PRR7 and PRR9 might act as direct negative regulators of CBF1, CBF2 and CBF3 (34). Here we present results indicating that the clock also provides positive regulation of the CBF cold response pathway and enhances freezing tolerance through action of the core clock components CCA1 and LHY.   36   RESULTS CCA1 and LHY have a direct role in circadian regulation of CBF1, CBF2 and CBF3. Consistent with previous reports (19-21), we found that CBF1, CBF2 and CBF3 are subject to circadian regulation (Fig. 2.1). Transcript levels for CBF1, CBF2 and CBF3 oscillated with a peak occurring at about ZT8 followed by a second peak about 24 h later. The oscillation patterns for the three CBF genes were similar, though we observed one consistent difference; whereas the ZT8 peak for CBF1 was lower than the second peak, the ZT8 peaks for CBF2 and CBF3 were greater than the second peak. Thus, the transition from dark to light may have a specific effect on the regulation of CBF1. Three lines of evidence led us to think that CCA1 and LHY might drive circadian regulation of CBF1, CBF2 and CBF3. First, the protein levels for CCA1 and LHY peak in the early morning (ZT1-3) (26, 35, 36), just prior to when the transcript levels of CBF1, CBF2, and CBF3 begin to increase. Second, the transcript levels for PRR7 (32, 36) and LIGHT HARVESTING COMPLEX B (LHCB) (24, 35, 37), both of which are induced by CCA1 and LHY, peak similarly to the CBF genes. Lastly, the promoter regions of CBF1, CBF2 and CBF3 have several EE (AAAATATCT) (19) and CCA1Binding Sites (CBS; AATCT) (35) (Fig. 2.2), which mediate binding of CCA1 and LHY (30, 35, 38) to target promoters. To determine whether CCA1 and LHY were involved in circadian regulation of CBF1, CBF2 and CBF3, we asked whether their expression was affected in plants carrying either the single cca1-11 or lhy-21 null mutations or the cca111/lhy-21 double mutation. We found that the single mutations had differing effects on   37   the three CBF genes; whereas cycling of CBF1 was severely disrupted, CBF2 and CBF3 transcript levels cycled with approximately the same amplitudes as in the wildtype plants, though the peaks were about 2 h earlier than in the wild-type plants (Fig. A2.1). Period shortening of output genes such as LHCB has been previously observed in cca1 and lhy mutant plants (39, 40), which we also observed in our experiments (Fig. A2.2). The cca1-11/lhy-21 double mutations also had differing effects on the circadian regulation of the three CBF genes (Fig. 2.1); whereas circadian regulation of CBF1 and CBF3 was essentially eliminated in the double mutant plants, CBF2 transcript levels clearly continued to cycle, though the amplitude was diminished and the period was greatly shortened. Period shortening of output genes such as LHCB has also been previously observed in cca1-11/lhy-21 double mutant plants (40, 41) and in our experiments (Fig. A2.3). From these results we concluded that circadian regulation of CBF1 and CBF3 is dependent on the action of either CCA1 or LHY and that circadian regulation of CBF2 involves action of CCA1 and LHY, but can be driven to a considerable degree by other unknown factors. CCA1 and LHY may impart circadian regulation of CBF1, CBF2 and CBF3 by binding to the EE and CBS motifs present in the CBF promoters and act as positive regulators stimulating transcription. To test this hypothesis, we conducted chromatin immunoprecipitation (ChIP) experiments to determine whether CCA1 binds directly to the promoter regions of CBF1, CBF2 and CBF3. This was assessed by comparing ChIP results obtained with plants carrying the cca1-1 mutation and cca1-1 plants that had been restored with a construct encoding the CCA1 protein tagged with green   38   fluorescent protein (GFP) under the endogenous CCA1 promoter (31). Chromatin was isolated from plants harvested at ZT4, the point when CBF transcript levels begin to rise. In mock experiments where rabbit immunoglobulin was used for precipitation, no specific binding was detected for any of the sub-regions (A to O) of the CBF1-3 locus tested or for TOC1, ACTIN7 or UBIQUITIN10 (Fig. A2.4). In contrast, test experiments indicated that specific binding of the CCA1-GFP protein occurred throughout most of the CBF1-3 locus and as well as within the promoter region of TOC1, a positive control, but not in the promoters of ACTIN7 or UBIQUITIN10, two negative controls (31) (Fig. 2.2). Significant CCA1-GFP binding occurred in the promoter regions of CBF1 (C), CBF2 (L), and CBF3 (G and I). We also observed CCA1 associated with the coding region of CBF1 (E). This could be due to the tandem connection of the CBF genes and consequently, close proximity to several CCA1 binding sites located in the adjacent CBF3 promoter (Fig. 2.2). Thus, although it is possible that there is CCA1 binding within the CBF1 transcript region, the actual binding site could be downstream in the CBF3 promoter. In sum, the results of our genetic and ChIP experiments supported the model that circadian regulation of CBF1, CBF2 and CBF3 involves action of CCA1 and LHY binding to the promoters of these genes and up-regulating their transcription during the morning hours.   39   RELATIVE EXPRESSION CBF1 CBF2 WS cca1-11/lhy-21 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# 0.00# 4# 12# 20# 28# 36# 44# 4# 12# 20# 28# 36# 44# CBF3 5.00# 4.00# 3.00# 2.00# 1.00# 0.00# 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# 0.00# COR15A 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# 0.00# 4# 12# 20# 28# 36# 44# COR47 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# 0.00# 4# 12# 20# 28# 36# 44# 4# 12# 20# 28# 36# 44# COR78 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# 0.00# 4# 12# 20# 28# 36# 44# TIME (ZT) Figure 2.1. Effects of the cca1-11/lhy-21 double mutation on circadian regulation of CBF1, CBF2, CBF3 and CBF-targeted genes COR15A, COR47 and COR78. Wild-type Ws-2 (WS) and cca111/lhy-21 double mutant plants were grown at 22⁰C under a 12 h photoperiod to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were harvested every 2 h and the transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from three independent biological experiments (n=3). Error bars indicate ±SEM.   40   Fig. 2 1 KB CBF Locus RELATIVE IP (NORMALIZED TO INPUT) A B C 20# 18# 16# 14# 12# 10# 8# 6# 4# 2# 0# D CBF1 E F G H I CBF3 J K L M N *# CBF2 O *# *# *# *# *# Figure 2.2. Binding of CCA1 at the CBF1-3 locus. cca1-1 and cca1-1 CCA1p:CCA1GFP plants were grown at 22⁰C under a 12 h photoperiod to the four leaf stage. Tissue was fixed at ZT4 and ChIP was performed using anti-GFP antibody. Immunoprecipitated DNA was quantified by qRT-PCR using primers specific to regions within the CBF1-3 locus (boxes A through O). The levels of immunoprecipitated DNA were normalized to the respective input DNA. Immunoprecipitation in cca1-1 CCA1p:CCA1-GFP plants (black bars) is relative to cca1-1 plants (gray bars) set to a value of 1. Primer pairs directed to the 3’-untranslated region of TOC1 (TOC1-3’UTR), ACTIN7 (ACTIN) and UBIQUITIN10 (UBQ10) were used as negative controls (31). Primers near the EE element in the TOC1 promoter (TOC1-EE) were used as a positive control. Values represent the average of five independent biological experiments (n=5). Error bars indicate ± SEM. *=p<0.05 using a paired, one-tailed t-test. In the CBF locus diagram, the transcribed regions are indicted with white boxes and the approximate positions of CBS (AATCT), and EE (AAAATATCT) motifs are indicated by gray circles, and white squares respectively.   41   Rhythmic expression of CBF regulon COR genes and freezing tolerance is impaired in plants carrying the cca1-11/lhy-21 double mutation. Harmer et al. (19) suggested that the circadian regulation of CBF1, CBF2 and CBF3 could potentially result in rhythmic expression of CBF regulon COR genes. To test this, we examined the transcript levels for three CBF-inducible genes—COR15A, COR47 and COR78—in wild-type plants and in plants carrying the cca1-11/lhy-21 double mutation. The results indicated that the transcript levels for all three COR genes oscillated with a period of about 24 h in wild-type plants, though the amplitude of the second peak was much less than that of the first for COR15A and COR78 (Fig. 2.1). For all three genes, the first peak was at about ZT10, which was consistent with the transcript levels of CBF1, CBF2 and CBF3 peaking just before this at about ZT8 (19, 20) (Fig. 2.1). Moreover, the oscillation in COR transcript levels was largely reduced in the cca1-11/lhy-21 double mutant plants (Fig. 2.1). These results were consistent with the model that circadianregulated expression of CBF1, CBF2 and CBF3 imparts rhythmic expression of CBFtargeted COR genes at “basal” non-acclimating temperatures. The decrease in expression of the CBF-targeted COR genes in the cca1-11/lhy21 double mutant plants could potentially result in a decrease in basal freezing tolerance. We tested this using the electrolyte leakage assay to compare the freezing tolerance of wild-type and cca1-11/lhy-21 plants. The results indicated that the cca111/lhy-21 double mutation reduced freezing tolerance by about 50%; whereas the EL50 (temperature at which cell damage results in release of 50% of total electrolytes) of wild-   42   type plants was about -4°C, it was about -2°C in the cca1-11/lhy-21 mutant plants (Fig. 2.3). Thus, the circadian clock is required for maximum basal freezing tolerance.   43   PERCENT ELECTROLYTE LEAKAGE NON-ACCLIMATED 120 100 80 60 40 20 0 WS cca1-11/lhy-21 0 -2 -3 -4 -5 -6 -7 -8 -9 -10 -11 -12 COLD-ACCLIMATED 120 100 80 60 40 20 0 0 -2 -3 -4 -5 -6 -7 -8 -9-10 -12 -14 -11 -13 -15 TEMPERATURE (°C) Figure 2.3. Effect of the cca1-11/lhy-21 double mutation on plant freezing tolerance. cca1-11/lhy-21 double mutant and wild-type Ws-2 (WS) plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days and either tested directly for freezing tolerance (non-acclimated plants; top graph) or were transferred at ZT4 to 4⁰C for 7 days under a 12 h photoperiod and then tested for freezing tolerance (coldacclimated plants; bottom graph). Freezing tolerance was tested using the electrolyte leakage test. The results presented are average values from three independent experiments (n=3). Error bars indicate ±SEM.   44   CCA1 and LHY regulate cold induction of CBF1, CBF2 and CBF3. Fowler et al. (22) reported that cold induction of CBF1, CBF2, and CBF3 is gated by the circadian clock. Given our results indicating a role for CCA1 and LHY in the circadian regulation of CBF1, CBF2 and CBF3, we asked whether these transcription factors also had a role in the gating phenomenon. As previously reported (22, 34), cold-induction of CBF1, CBF2, and CBF3 was much greater in the subjective day than in the subjective evening (Fig. 2.4). This cold induction was little affected by the single cca1-11 and lhy-21 mutations (Fig. A2.1), but was greatly reduced in the cca1-11/lhy-21 double mutants and the period of cycling was shortened (Fig. 2.4) as was the period of cycling for LHCB (Fig. A2.3). Thus, CCA1 and LHY have a major role in the induction of CBF1, CBF2 and CBF3 expression in response to low temperature.   45   CBF2 WS 1.80# cca1-11/lhy-21 1.60# 0.80# 1.40# 1.20# 0.60# 1.00# 0.80# 0.40# 0.60# 0.40# 0.20# 0.20# 0.00# 0.00# 4# 12# 20# 28# 36# 44# 4# 12# 20# 28# 36# 44# COR15A COR47 1.20# 1.20# 1.00# 1.00# 0.80# 0.80# 0.60# 0.60# 0.40# 0.40# 0.20# 0.20# 0.00# 0.00# 4# 12# 20# 28# 36# 44# 4# 12# 20# 28# 36# 44# RELATIVE EXPRESSION CBF1 1.00# CBF3 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# 0.00# 4# 12# 20# 28# 36# 44# COR78 1.20# 1.00# 0.80# 0.60# 0.40# 0.20# 0.00# 4# 12# 20# 28# 36# 44# TIME TRANSFERRED TO COLD (ZT) Figure 2.4. Effects of the cca1-11/lhy-21double mutation on cold induction of CBF1, CBF2, CBF3 and CBF-targeted genes COR15A, COR47 and COR78. Wild-type Ws-2 (WS) and cca1-11/lhy-21 double mutant plants were grown at 22⁰C under a 12 h photoperiod to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were transferred to cold temperature (4⁰C) for 2 h, every 2 h (CBF genes) or for 4 h, every 4 h (COR genes) at the start of constant light conditions. Transcript levels for the indicated genes were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were transferred to cold temperature. Gene expression was normalized to UBQ10 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from three independent biological experiments (n=3). Error bars indicate ±SEM. See Table A2.1 for primer pair sequences.   46   Cold induction of CBF-targeted COR genes and freezing tolerance is impaired in plants carrying the cca1-11/lhy-21 double mutation. The finding that cold induction of CBF1, CBF2 and CBF3 was impaired in plants carrying the cca111/lhy-21 double mutation prompted us to determine whether the double mutation also impaired cold induction of CBF-targeted COR genes and the freezing tolerance of coldacclimated plants. In wild-type plants, the degree to which COR15A, COR47 and COR78 were induced by low temperature cycled with peaks in the late day and troughs in the late subjective evening (Fig. 2.4), times that were consistent with the cycling of CBF1, CBF2 and CBF3 cold induction (Fig. 2.4). In plants carrying the cca1-11/lhy-21 double mutation, the peak following the night to day transition was little affected, but the subsequent peaks were greatly diminished and the period of cycling was shortened (Fig. 2.4). In addition, the freezing tolerance of cold-acclimated plants carrying the cca1-11/lhy-21 double mutation was about 1°C less than that of cold-acclimated wildtype plants (Fig. 2.3). These results indicate that CCA1 and LHY are required for Arabidopsis plants to attain maximum levels of COR gene induction and freezing tolerance in response to low temperature.   47   DISCUSSION The CBF cold response pathway is highly conserved among plants and has a major role in plant freezing tolerance (1-3, 9). Accordingly, there is considerable interest in understanding the mechanisms that control expression of this stress response pathway. Here we establish that the CBF pathway is subject to positive regulation by the circadian clock components CCA1 and LHY. We show that these factors have roles in both circadian regulation and cold induction of the pathway and that they are required for plants to attain maximum freezing tolerance at both basal and cold acclimating temperatures. At basal growth temperature, the transcript levels for CBF1, CBF2, and CBF3 oscillate with peaks and troughs occurring at about ZT8 and ZT20, respectively (19-21) (Fig. 2.1). Our genetic and ChIP analyses indicate that this circadian regulation is due to direct action of CCA1 and LHY binding at the CBF1-3 locus—presumably at the EE, CBS, and related motifs—and inducing transcription of the CBF genes. In the morning hours when CCA1 and LHY protein levels peak (25, 35, 36), the transcript levels of CBF1, CBF2, and CBF3 peak; and in the evening hours, when CCA1 and LHY protein levels are low, the transcript levels for CBF1, CBF2, and CBF3 are low (Fig. 2.5). The finding that circadian regulation of CBF1 and CBF3 is nearly eliminated in plants carrying the cca1-11/lhy-21 double mutation indicates that no other regulatory proteins are sufficient to impart positive circadian regulation of these genes. In contrast, circadian regulation of CBF2 continues in plants carrying the cca1-11/lhy-21 double mutation, albeit with reduced amplitude and shortened periodicity. Thus, at least one   48   additional regulatory protein appears to drive positive circadian regulation of CBF2. Prime candidates for this residual regulation are the four REVEILLE (RVE) proteins RVE1, RVE3, RVE4 and RVE8 (42). These MYB-like transcription factors fall into the CCA1 subfamily, bind to the EE motif, are circadian-regulated, and like CCA1 and LHY, have peak transcript levels at dawn (42). Kidokoro et al. (21) reported that circadian regulation of CBF1, CBF2, and CBF3 also involves negative regulation. These investigators found that PIF7 binds to a G-box element in the promoter of CBF2 and that this element was required for down-regulation of the CBF2 promoter during the subjective evening. In addition, it was found that PIF7 physically interacts with TOC1 (21). Thus, circadian-controlled down-regulation of the CBF genes appears to involve action of a PIF7-TOC1 protein complex binding to G-box elements in their promoters (Fig. 2.5). In addition to establishing a role for CCA1 and LHY in circadian regulation of CBF1, CBF2 and CBF3, our results indicate that CCA1 and LHY also act as positive regulators of CBF cold induction. This is evidenced by the finding that cold induction of CBF1, CBF2 and CBF3 is greatly impaired in plants carrying the cca1-11/lhy-21 double mutation (Fig. 2.4). We propose that the gating of CBF1, CBF2 and CBF3 cold induction results, in part, from positive synergistic interaction between cold-signaling and clock output pathways, the former mediated by ICE1 and CAMTA3, and the latter by CCA1 and LHY (Fig. 2.5). If the temperature drops in the morning, CCA1 and LHY are present at the CBF locus and can act with ICE1 and CAMTA3 to induce high level expression of CBF1, CBF2 and CBF3. In contrast, if the temperature drops in the   49   evening, CCA1 and LHY are at low levels and consequently, there is little synergy between the low temperature and clock pathways and the induction of CBF1, CBF2 and CBF3 is low, approximating the peak levels obtained with circadian regulation (Fig. 2.5). Cold induction of CBF1, CBF2 and CBF3 during the evening hours may also involve negative regulation. Such regulation would not appear to involve PIF7, as Kidokoro et al. (21) showed that the gating of CBF1, CBF2, and CBF3 expression is not impaired in plants carrying the pif7-2 mutation (21). However, Nakamichi et al. (34) found that circadian regulation of CBF1, CBF2 and CBF3, and the gating of their cold induction, does not occur in plants carrying the prr9-11/prr7-10/prr5-10 triple mutation. When plants were grown at basal temperature, the transcript levels for CBF1, CBF2 and CBF3 remained high throughout the day in the triple mutant plants (34). Similarly, the cold induction of CBF1, CBF2 and CBF3 in the triple mutant plants remained at about the peak levels observed in wild-type plants regardless of the time of day at which the mutant plants were exposed to low temperature (34). Nakamichi et al. (34) concluded that PRR9, PRR7 and PRR5 are negative regulators of CBF1, CBF2 and CBF3 and proposed two possible mechanisms. One was that PRR9, PRR7 and PRR5 directly repress expression of the CBF genes. Alternatively, they suggested that aberrant expression of the CBF genes might result from the “circadian disorder” caused by the prr9-11/prr7-10/prr5-10 triple mutation. Our results provide no direct evidence in favor of, or against, the first model. However, the consistently high CBF expression may be partially explained by the constitutively elevated expression of CCA1 and LHY in the triple mutant plants (34, 43).   50   A final point regards the role of the clock in freezing tolerance. Our results indicate that CCA1 and LHY are required for Arabidopsis to attain maximum levels of freezing tolerance at both non-acclimating and cold-acclimating temperatures (Fig. 2.3). Recently, Espinoza et al. (44) independently reached the same conclusion as they also found that the cca1-11/lhy-21 double mutation resulted in impaired freezing tolerance. Our results also indicate a mechanism whereby cold-signaling and clock regulatory pathways are integrated to condition freezing tolerance; the positive regulation of the CBF cold response pathway mediated through CCA1 and LHY binding at the CBF1-3 locus and inducing expression of CBF1, CBF2 and CBF3 (Fig. 2.5). Taken together, our results suggest that the integration of cold signaling pathways with the circadian clock may have been an important evolutionary event that has contributed to plant adaptation to cold environments.   51   DAY RELATIVE TRANSCRIPT LEVELS WARM CBF CBF CBF 1 3 2 CCA1# LHY# CBF1 CCA1# LHY# CBF3 CCA1# LHY# 40 CBF2 70 30 COLD CM3# CCA1# CCA1# CCA1# ICE1# CM3# 900 3000 2000 LHY# LHY# LHY# CBF1 CBF3 CBF2 NIGHT PRR# 5,7,9# WARM PIF7# PIF7# CBF1 PIF7# CBF3 1 1 1 40 20 100 CBF2 PRR# 5,7,9# COLD CM3# CBF1 ICE1# CM3# CBF3 CBF2 Figure 2.5. Model for circadian regulation and gated cold-induction of CBF1, CBF2 and CBF3. During the day, CCA1 and LHY bind throughout the CBF locus and promote CBF transcription. In the evening, CCA1 and LHY are at low levels and have little effect on CBF expression. Oscillations in CCA1 and LHY binding at the CBF locus largely account for the circadian regulation of the CBF genes. Circadian regulation also involves repression in the evening hours mediated by PIF7 (21) and PRR5, PRR7 and PR9 (34). Transfer of plants to low temperature in the day or evening results in activation of ICE1 (15) and CAMTA3 (CM3) (16), and possibly other transcription factors, that stimulate transcription of the CBF genes. If the temperature drops during the day, the clock and cold-signaling pathways act synergistically to induce CBF expression to high levels. If plants are exposed to cold temperatures in the evening, there is no positive synergy between the two pathways and there is repression by PRR5, PRR7 and PR9 (34) leading to CBF induction at moderate levels. Relative transcript levels were calculated using peak and trough values obtained for the CBF genes in the experiments presented in Fig. 2.2.1 and Fig. 4 (the values obtained for plants grown at warm temperature and harvested in the evening were set to 1). See Discussion for details.   52   MATERIALS AND METHODS Plant Material and Growth Conditions. Arabidopsis thaliana ecotype WS-2 and mutants in this background were grown as described previously (16). Homozygous T-DNA mutant lines were obtained from the Arabidopsis Biological Resource Center (45). Null mutations were checked by qRT-PCR. These lines were cca1-11(CS9378), lhy-21 (CS9379) and cca1-11/lhy-21(CS9380). Restored cca1-1 line, CCA1p:CCA1GFP under the CCA1 endogenous promoter and cca1-1 (31), used in ChIP experiments, were generously donated by the Kay Lab (University of California, San Diego). All seeds were stratified for 3 to 5 days in the dark at 4⁰C. Except for freezing tolerance tests, plants were grown at 22⁰C under sterile conditions on Gamborg’s B5 -2 -1 medium (Caisson Laboratories) without sucrose at ~100 µmol m s in a 12 h photoperiod. For circadian experiments, plants were sampled at 22⁰C in 100 µmol m 2 -1 s -2 -1 constant light or at 4⁰C in 35 µmol m s - constant light. For electrolyte leakage -2 -1 experiments plants were grown as described (16) at ~100 µmol m s under a 12 h photoperiod. Cold temperature treatment for plants grown on soil was at 4⁰C in light at -2 -1 35 µmol m s under a 12 h photoperiod. RNA Analysis. RNA extraction was performed as described (16). For qRT-PCR (Applied Biosystems 7500 FAST Real-Time PCR System in FAST mode), cDNA was made as described in (16) except that total RNA of either 0.2 or 0.025 µg was used for a   53   40 µL reverse-transcription reaction. In the 10 µL PCR reactions, 2 µL of diluted cDNA was used. UBQ10 or IPP2 were used as reference genes. All primer sets can be found in Table A2.1. Chromatin Immunoprecipatation. ChIP experiments were carried out as described by Pruneda-Paz et al. (31) with a few modifications. CCA1p:CCA1-GFP and cca1-1 lines were sampled at ZT4 instead of ZT3. DNA was also purified by PCR Clean-Up Kit (Qiagen) instead of phenol-chloroform extraction. Immunoprecipitated DNA was analyzed with Applied Biosystems FAST real-time PCR in FAST mode (using presets). For each biological replicate immunoprecipitated DNA was normalized to the input DNA as in (31) and each of these values were expressed relative to the cca1-1 line set to a value of 1. A one-tailed paired t-test was performed to assess the statistical significance of enrichment in the CCA1p:CCA1-GFP line compared to cca1-1 plants for each primer pair used across biological replicates. Primers pairs used in ChIP experiments can be found in Table A2.1. Freezing Tolerance Tests. Electrolyte leakage assays were performed as described in (16). For cold acclimation, plants were transferred to 4⁰C at ZT4 for 7 days under a 12 h photoperiod. Assays for acclimated and non-acclimated plants started at ~ZT2 in all biological replicates.   54   ACKNOWLEDGMENTS We thank Steve Kay for providing us with the Arabidopsis lines carrying the cca1-1 mutation and the restored line expressing the CCA1-GFP fusion and are grateful to Sarah Gilmour for her help in preparing the manuscript. This research was funded by grants from the NSF Plant Genome Project (DBI 0701709); the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy (DE-FG02-91ER20021); and the Michigan Agricultural Experiment Station.   55   APPENDIX   56   WS cca1-11 lhy-21 B. CBF2 1.50 C. CBF3 1.50 4.00 1.00 1.00 2.00 RELATIVE EXPRESSION A. CBF1 6.00 0.50 0.50 0.00 0.00 0.00 4 12 20 28 36 44 4 12 20 28 36 44 4 12 20 28 36 44 TIME (ZT) D. CBF1 2.00 E. CBF2 F. CBF3 3.00 2.00 1.50 1.50 2.00 1.00 1.00 1.00 0.50 0.00 0.50 0.00 2 10 18 26 34 42 0.00 2 10 18 26 34 42 2 10 18 26 34 42 TIME TRANSFERRED TO COLD (ZT) Figure A2.1. Circadian rhythms and gated expression of CBF genes in cca1-11 and lhy-21. qRT-PCR analysis for CBF1, CBF2 and CBF3 in cca1-11(white boxes), lhy21(grey triangles) and wild-type (WS) (black diamonds). Expression for each gene was relative to one wild-type sample (WS) set to a value of 1 in each biological replicate. Values are averages of three independent experiments (n=3) for WS, error bars indicate ± SEM. Values are average of two independent experiments (n=2) for cca1-11 and lhy21, error bars indicate ± range. (A-C) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were harvested every 2 h and the transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. (D-F) Plants were grown with a 12 h-photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were dropped to cold temperature (4⁰C) for 2 h, every 2 h, for 48 h at the start of constant light conditions and the transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to UBQ10 for each sample. See Table A2.1 for primer pair sequences.   57   WS( cca1$11% lhy$21% A.#LHCB1.4# 3.00( RELATIVE(EXPRESSION( 2.00( 1.00( 0.00( 4( 12( 20( 28( 36( 44( TIME((ZT)( B.#LHCB1.4# 2.00 1.50 1.00 0.50 0.00 2( 10( 18( 26( 34( 42( TIME(TRANSFERRED(TO(COLD((ZT)( Figure A2.2. Robust circadian rhythms and gated expression of LHCB1.4 in cca1-11 and lhy-21 plants. qRT-PCR analysis of LHCB1.4 in cca1-11 (white boxes) , lhy-21 (grey triangles) and wild-type (WS) (black diamonds). Expression for LHCB1.4 was relative to one wild-type sample (WS) set to a value of 1 in each biological replicate. Values are averages from three independent experiments (n=3) for WS, error bars indicate ± SEM. Values are averages from two independent experiments (n=2) for cca111 and lhy-21, error bars indicate ± range. (A) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were harvested every 2 h and the transcript levels for LHCB1.4 were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. (B) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were dropped to cold temperature (4⁰C) for 2 h, every 2 h, for 48 h at the start of constant light conditions and the transcript levels for LHCB1.4 were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were dropped to cold temperature. Gene expression was normalized to UBQ10 for each sample. See Table A2.1 for primer pair sequences   58   RELATIVE(EXPRESSION( A.#LHCB1.4# 1.20( 1.00( 0.80( 0.60( 0.40( 0.20( 0.00( WS( cca1$11/lhy$21% 4( 12( 20( 28( 36( 44( B.#LHCB1.4# 1.20( 1.00( 0.80( 0.60( 0.40( 0.20( 0.00( TIME((ZT)( C.#LHCB1.4# 1.20( 1.00( 0.80( 0.60( 0.40( 0.20( 0.00( 2( 10( 18( 26( 34( 42( 4( 12( 20( 28( 36( 44( TIME(TRANSFERRED(TO(COLD((ZT)( Figure A2.3. Robust circadian rhythms and gated expression of LHCB1.4 in cca1-11/ lhy-21 plants. qRT-PCR analysis LHCB1.4 in cca1-11/lhy-21 (white boxes), and wildtype (WS) (black diamonds). Expression for LHCB1.4 was relative to one wild-type sample (WS) set to a value of 1 in each biological replicate. Values are averages of three independent experiments (n=3). Error bars indicate ± SEM. (A) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were harvested every 2 h and the transcript levels for LHCB1.4 were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. (B) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were dropped to cold temperature (4⁰C) for 2 h, every 2 h, for 48 h at the start of constant light conditions and the transcript levels for the indicated genes were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were dropped to cold temperature. Gene expression was normalized to UBQ10 for each sample. (C) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were dropped to cold temperature for 4 h, every 4 h, for 48 h at the start of constant light conditions and the transcript levels for LHCB1.4 were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were dropped to cold temperature. Gene expression was normalized to UBQ10 for each sample. See Table A2.1 for primer pair sequences.   59   RELATIVE(IP((NORMALIZED(TO(INPUT)( CBF Locus 20( 18( 16( 14( 12( 10( 8( 6( 4( 2( 0( A( B( C( CBF1( D( E( CBF3( F( G( H( I( J( K( 1 KB CBF2( L( M( N( O( Figure A2.4. Mock experiments testing CCA1 binding at the CBF1-3 locus. cca1-1 and cca1-1 CCA1p:CCA1-GFP plants were grown at 22⁰C under a 12 h photoperiod to the four leaf stage. Tissue was fixed at ZT4 and ChIP was performed using rabbit-IgG (Jackson ImmunoResearch) antibody. Immunoprecipitated DNA was quantified by qRT-PCR using primers specific to regions within the CBF1-3 locus (boxes A through O). The levels of immunoprecipitated DNA were normalized to the respective input DNA. Immunoprecipitation in cca1-1 CCA1p:CCA1-GFP plants (black bars) is relative to cca1-1 plants (gray bars) set to a value of 1. Primer pairs directed to the 3’untranslated region of TOC1 (TOC1-3’UTR), ACTIN7 (ACT) and UBBIQUITIN10 (UBQ10) were used as negative controls (32). Primers near the EE element in the TOC1 promoter (TOC1-EE) were used as a positive control. Values represent the average of five independent biological experiments (n=5). Error bars indicate ± SEM. *=p<0.05 using a paired, one-tailed t-test. See Table A2.1 for primer pair sequences. In the CBF locus diagram, the transcribed regions are indicted with white boxes and the approximate positions of CBS (AATCT), and EE (AAAATATCT) motifs are indicated by gray circles, and white squares.   60   Table A2.1. Primer pairs used for qRT-PCR and ChIP experiments. Primers used in this study were designed with Primer Express 3.0, using presets optimized for qRT-PCR. For locations of primer pairs on CBF1-3 please see Fig. 2.2 or Fig. A2.4. qRT-­‐PCR   Primers                   Name   Forward  (FW)   FW     Reverse  (RV)   RV     ATG   CBF1   CBF2   CBF3   COR15A   COR47   COR78   IPP2   UBQ10   LHCB1.4   GGAGACAATGTTTGGGATGC   CGACGGATGCTCATGGTCTT   TTCCGTCCGTACAGTGGAAT   GAAAAAAACAGTGAAACCGCAGAT   CGGTACCAGTGTCGGAGAGT   GAAAGGAGGAGGAGGAATGG   ATTTGCCCATCGTCCTCTGT   GGCCTTGTATAATCCCTGATGAATAAG   GCCTTCGCTACCAACTTCGTC   +671   +560   +694   +704   +748   +2251   +115   +1833   +829   CGACTATCGAATATTAGTAACTCC   TCTTCATCCATATAAAACGCATCTTG   AACTCCATAACGATACGTCGTC   CCACATACGCCGCAGCTT   ACAGCTGGTGAATCCTCTGC   AACCAGCCAGATGATTTTGG   GAGAAAGCACGAAAATTCGGTAA   AAAGAGATAACAGGAACGGAAACATAGT   AACCGGATACACACAACTCGATC   +793   +628   +741   +750   +855   +2351   +155   +1866   +873   AT4G25490     AT4G25470   AT4G25480   AT2G42540   AT1G20440   AT5G52310   AT3G02780   AT4G05320       AT2G34430     ChIP   Primers                                 Name   Forward  (FW)   FW     Reverse  (RV)   RV     ATG   ACTIN   UBQ10   TOC1   TOC1   3'UTR   CBF1-­‐A   CBF1-­‐B         CGTTTCGCTTTCCTTAGTGTTA   TCCAGGACAAGGAGGTATTCCTCCG   TTTTATGGCCTGCACTTTTTATTG   +54   AGCGAACGGATCTAGAGACTC   +1616   CCACCAAAGTTTTACATGAAACGAA   -­‐186   GGTGGGACTTGGGATATTTTAGG   GCTACAGCCAAAAAAACATCGA   +3212   GAGCCGCAAGAGCCAACAT   AGAACCACGACGATATAGAGAGTGAA   -­‐1997   CGCCTGCCAAAATCATTCTAC   TGCTTTCAAGGCCGAATGAT   -­‐1312   CGTTCTCATTCCACGTGTGATG   61 +167   AT5G09810   +1796   AT4G05320       -­‐144   AT5G61380     +3256   AT5G61380     -­‐1951   AT4G25490     -­‐1274   AT4G25490     Table A2.1. (cont’d) CBF1-­‐C   CBF1-­‐D   CBF1-­‐E   CBF3-­‐F   CBF3-­‐G   CBF3-­‐H   CBF3-­‐I   CBF3-­‐J   CBF2-­‐K   CBF2-­‐L   CBF2-­‐M   CBF2-­‐N   CBF2-­‐O   TTACCACTCTTTTTTTCCCTCTTTG   TCTTTACAAGGGTCAAAGGACACA   GGAGACAATGTTTGGGATGC   AGTTCTATCGGACTAATTCTTGGCTTA   TGACTAAGGACGTGGTGGTTGA   TGTTACATTTGATCATTCACCCAAA   CGTGGCATTACCAGAGACACA   TTCCGTCCGTACAGTGGAAT   CAAGAGAGCACTGTCCGTAGCTT   TTTGCCGGAAAACTCAACTCA   GAGAGATGCTGGAAATTGTGATCA   GGGATCGCTTAGCTGTTTCTTA   CGACGGATGCTCATGGTCTT   -­‐845   -­‐186   +671   -­‐1859   -­‐1235   -­‐604   -­‐124   +694   -­‐1851   -­‐1147   -­‐943   -­‐142   +560   CTCGCTCTCACGTTATTGACATTT   GCGAAGCAATCCCACGAT   CGACTATCGAATATTAGTAACTCC   GATGATCAAGCGTAATTGCTTTGT   AGCGCACTTCCTTCTCACTCA   CGTATATAAGCACGTAAGTCACCAAGT   GCGGAAGATATTTTAGAGGCAAAA   AACTCCATAACGATACGTCGTC   TGGTTACAAGAGGAGCCACGTA   CCTTCTTTTTGGTCTGAAA   AAATATGGTAAGTGGTTAGGCGAAA   TCGAACGCGGAGTTTCTGT   TCTTCATCCATATAAAACGCATCTTG       62 -­‐801   -­‐142   +793   -­‐1752   -­‐1178   -­‐550   -­‐83   +741   -­‐1811   -­‐1108   -­‐897   -­‐100   +628   AT4G25490     AT4G25490     AT4G25490     AT4G25480   AT4G25480   AT4G25480   AT4G25480   AT4G25480   AT4G25470   AT4G25470   AT4G25470   AT4G25470   AT4G25470   LITERATURE CITED   63 LITERATURE CITED 1. 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Science 301:653-657.     68 CHAPTER THREE Natural variation in the low temperature response of Arabidopsis ecotypes from Sweden and Italy   69 CHAPTER THREE Natural variation in the low temperature response of Arabidopsis ecotypes from Sweden and Italy SUMMARY In previous studies, three accessions from two Arabidopsis ecotypes collected from Sweden (SW) and Italy (IT) were tested for fitness (survival and fruit-number) in reciprocal transplant experiments. There is substantial variation in temperature across latitudes and minimum winter soil temperature was found to correlated with fitness in these experiments. Consequently, genes associated with freezing tolerance may underlie identified fitness QTLs. This project assessed differences in freezing tolerance between SW and IT accessions using electrolyte leakage assays under laboratory conditions. The SW accession was able to maintain greater freezing tolerance, compared to IT, after 2 weeks of cold-acclimation. RNA-seq experiments on non-acclimated, 1 week cold-acclimated, and 2 week cold-acclimated SW and IT accessions were analyzed to find genes that may contribute to differences in freezing tolerance. This transcriptomic data was also used to determine general differences between SW and IT and assess if SW and IT reach similar levels of freezing tolerance with similar transcriptional changes. SW and IT recombinant inbred lines (RILs) were used in expression QTL (eQTL) analysis on genes potentially contributing to differences in freezing tolerance. CBF regulon genes were overrepresented in the list of genes that may potentially contribute to differences in freezing tolerance. Variation in CBF regulon genes, which were   70 selected for eQTL analysis, indeed mapped to the CBF locus. Interestingly, the location of the CBF locus also overlaps with a fitness QTL. Based on gene expression levels, CBF2 regulatory sequences may have diverged between SW and IT. However, there is also a 13 base pair deletion in the coding region of IT-CBF2 that results in a predicted protein with a DNA binding domain but without an activation domain. This truncated protein could potentially act as a dominant negative version of CBF2 in IT, able to bind to downstream target genes but incapable of activating transcription. Consistent with this hypothesis, expression of upregulated CBF target genes is generally lower in IT compared to SW. This would potentially be the first example of a naturally occurring dominant negative version of a CBF. Further work is necessary to confirm this hypothesis. Altogether, results from this study suggest that CBF2 is an excellent candidate gene for differences in freezing tolerance and possibly differences in fitness between SW and IT.   71 INTRODUCTION Adaptation of plants and other organisms to their local environment has been frequently observed and is driven by random mutation and natural selection (1-5). With rapid change in global environmental conditions, understanding the molecular mechanisms underlying local adaptation are of particular interest (1-5). Arabidopsis is a model plant native to a wide geographical range (Europe to Central Asia) and thus wide range of climatic conditions (6, 7). Two naturally occurring populations of Arabidopsis from Rodasen, Sweden (SW) and Castelnuovo di Porto, Italy (IT) were recently collected (8). In reciprocal transplant experiments with three accessions from each SW and IT population, accessions originating from the experimental sites had a strong advantage in terms of fruit-number and survival throughout five years of experimentation (8). Barrett et al. 2011 defined an adaptive allele as one that functionally affects a phenotypic trait to increase fitness (5). Consequently, it can be very difficult to demonstrate that specific alleles are adaptive (1-5). Reciprocal transplant experiments with recombinant inbred lines (RILs) allow fitness to be measured then mapped to specific genetic regions (quantitative trait loci; QTL) and for segregating traits to be identified (5, 9). This method thus connects specific genetic regions to a phenotypic trait that impacts fitness, though these regions can still often span several hundred genes (5). The Schemske lab sequenced SW and IT accessions and 141,437 single nucleotide polymorphisms (SNPs) were found (Schemske, unpublished). A population of RILs was created by crossing a randomly selected individual from the SW ecotype   72 (male parent) to a randomly selected individual from the IT ecotype (female parent), seeds from the F1 generation were used to establish a large number of lines that were selfed by single-seed descent for nine generations (Schemske and Agren, unpublished). SW and IT RILs used in this study were from the F10 generation. 384 of the 141,437 SNPs with an average spacing of ~1.1 cm across the Arabidopsis genome were selected to genotype the RIL population (Schemske, unpublished). 400 genotyped RILs were then grown at both the Swedish and Italian sites and QTL for fitness were mapped by measuring both fruit-number and survival over these 384 markers (Table 3.1; Schemske and Agren, unpublished). Both minimum winter soil temperature and flowering-time were found to correlate with fitness of the parent lines during previous reciprocal transplant experiments (8). Other recent studies on local adaptation in various Arabidopsis ecotypes have also shown temperature variation to be particularly linked to identified ‘local alleles’ (1, 2). Sweden has significantly lower average air and soil temperatures in comparison to Italy, especially during winter months (8). Therefore, freezing tolerance is a potential trait underlying fitness QTL mapped in the RILs. Ideally, the SW and IT RILs would be quantitatively (or even semi-quantitatively) tested for differences in freezing tolerance (10) in order to map freezing tolerance QTL and determine if they overlap with fitness QTL. The Schemske lab is currently optimizing freezing tolerance protocols for mass screening of RILs. Keeping this in mind, a reductionist approach has been taken to determine if freezing tolerance related genes underlie previously identified fitness QTL (Table 3.1; Schemske and Agren, unpublished).   73 Results and analyses are broken into three sections. First, significant differences in SW and IT freezing tolerance are shown under controlled laboratory conditions. Second, RNA-seq experiments are used to define a set of genes possibly contributing to differences in freezing tolerance. This is the first look at the transcriptomes of these two Arabidopsis accessions. Therefore in this second section, basal differences between SW and IT transcriptomes are observed. Further analysis of RNA-seq data in this section also suggests that the cold-response of the SW and IT accessions is significantly different. Third, a subset of the genes that possibly contribute to differences in freezing tolerance are used to map QTL for differences in gene expression (expression QTL; eQTL; (11-14)). Some of the eQTL were found to overlap with previously defined fitness QTL (Table 3.1). Consequently, the genes underlying eQTL mapped for genes possibly contributing to differences in freezing tolerance might also underlie fitness QTL. This study therefore provides evidence in support of freezing tolerance as a trait underlying SW and IT fitness QTL (Table 3.1) and suggests candidate genes contributing to these QTL.       74 Table 3.1. Locations of fitness QTL. 400 RILs were grown at both Swedish and Italian field sites in 2009. Fruit-number and survival-until-fruit-set were fitness traits measured (Data courtesy of the Schemske Lab, unpublished). RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/) for each fitness trait at each location. To detect QTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LOD) values were calculated for each trait using 1000 permutations in RQTL. To estimate the location of each QTL a Bayes credible interval (99%) was calculated in RQTL. No QTL are reported for survival-until-fruit-set at the Italian field site because no QTL were detected above the calculated significance threshold of 3.42 (α=0.01) or even a less stringent LOD threshold of 2.54 (α=0.05). For locations of markers based on the Col-0 genome please see Table A3.1. ‘MB’ indicates the megabases contained within the QTL. ‘# GENES’ indicates the number of genes contained within the QTL based on the Col-0 genome. %VAR indicates the estimated proportion of phenotypic variance explained by the QTL. FRUIT-­‐NUMBER  ITALY  2009  (LOD  THRESHOLD  3.17)       CHR   LEFT  (CM)   PEAK  (CM)   RIGHT  (CM)   MB     1   57.35   58.00   62.48   2.00   2   22.00   26.84   46.00   6.59   4   44.31   57.69   60.00   6.91   5   0.00   1.40   4.59   1.16   5   68.69   75.80   77.42   3.19   FRUIT-­‐NUMBER  SWEDEN  2009  (LOD  THRESHOLD  3.08)   CHR   LEFT  (CM)   PEAK  (CM)   RIGHT  (CM)   MB     2   50.00   55.27   58.59   4.32   SURVIVAL  SWEDEN  2009  (LOD  THRESHOLD  2.99)   CHR   LEFT  (CM)   PEAK  (CM)   RIGHT  (CM)     MB     1   76.9   78.6   83   2.31   5   64   71.3   78.2   4.89           75         #  GENES   LOD   584   18.35   1803   8.04   2065   14.37   365   9.18   988   18.14           #  GENES   LOD   1427   12.26       %VAR   11.66   4.81   8.91   5.52   11.52       %VAR   11.68    #  GENES     LOD   695   6.26   1505   4.63     %VAR   7.27   4.8   RESULTS AND DISCUSSION: FREEZING TOLERANCE OF SW AND IT Summary: Freezing tolerance of SW and IT. Minimum winter soil temperature was correlated with the relative fitness of the parent lines during previous reciprocal transplant experiments (8). If freezing tolerance is a trait underlying fitness QTL mapped for the RILS, then SW and IT accessions should differ in freezing tolerance. SW and IT have similar freezing tolerance without cold-acclimation and with 1 week of cold-acclimation (Fig 3.1). But SW was significantly more freezing tolerant than IT with both 2 and 3 weeks of cold-acclimation (Fig. 3.2). Consequently, genes contributing to differences in freezing tolerance between SW and IT may also be candidates for genes underlying fitness QTL. Freezing tolerance of SW and IT. If freezing tolerance is a trait underlying fitness QTL found using the SW and IT RILs, then a significant difference in the freezing tolerance of the SW and IT accessions might be expected. Electrolyte leakage assays were used to assess the freezing tolerance of both SW and IT accessions (16). The EL50 (temperature at which cell damage results in release of 50% of total electrolytes) of SW and IT accessions was the same under non-acclimated conditions (EL50=-2.5; Fig. 3.1). Hannah et al. 2006 found that nine accessions of Arabidopsis generally had similar non-acclimated freezing tolerance despite differing levels of freezing tolerance at 2 weeks of cold-acclimation (6). Accordingly, differences in the cold-acclimated freezing tolerance of the SW and IT accessions were determined. After 1 week of cold-acclimation at 4°C, both SW and IT can increase their freezing tolerance to a similar degree (EL50=-5.5; Fig. 3.1). However after 2 weeks of   76 cold-acclimation at 4°C, IT is unable to maintain freezing tolerance similar to 1 week of cold-acclimation (Fig 3.1) and decreases in freezing tolerance (EL50=-4.5; Fig. 3.2). On the other hand, the SW accession increases in freezing tolerance after 2 weeks of coldacclimation (EL50=-6.5; Fig. 3.2) in comparison to 1 week of cold-acclimation (Fig 3.1). After 3 weeks of cold-acclimation at 4°C there is still a substantial difference in freezing tolerance between SW (EL50=-5.5) and IT (EL50=-4.5) though the SW line has decreased its freezing tolerance in comparison to 2 weeks of cold-acclimation (Fig 3.2). The study by Hannah et al. 2006 also found that the cold-acclimated freezing tolerance of nine accessions of Arabidopsis correlated considerably with the accession’s average home temperature in the coldest month of the growing season (6). A separate study using 71 accessions of Arabidopsis found a similar correlation with the latitude of origin (17). Data from this study corroborates both of these previous studies. SW, the accession with the colder home winter temperature and higher latitude (8), has greater freezing tolerance than IT after 2 weeks of cold-acclimation (Fig. 3.2). The SW accession was significantly more freezing tolerant than the IT accession at both 2 (p≤0.026 at EL50 calculated by Student’s t test) and 3 (p≤0.015 at EL50 calculated by Student’s t test) weeks of cold-acclimation (Fig 3.2). Therefore, freezing tolerance is a potential trait contributing to local adaptation (1-5). Consequently, genes contributing to differences in freezing tolerance between SW and IT may also be candidates for genes underlying fitness QTL.   77 PERCENT"ELECTROLYTE"LEAKAGE" NON#ACCLIMATED, 120" 110" 100" 90" 80" 70" 60" 50" 40" 30" 20" 10" 0" IT" SW" 0" ,2" ,3" ,4" ,5" ,6" ,7" ,8" ,9" ,10" ,11" ,12" 1,WEEK,COLD#ACCLIMATED, 110" 100" 90" 80" 70" 60" 50" 40" 30" 20" 10" 0" IT" SW" 0" ,2" ,3" ,4" ,5" ,6" ,7" ,8" ,9" ,10" ,11" ,12" TEMPERATURE"(°C)" Figure 3.1. Freezing tolerance of SW and IT accessions without cold-acclimation or with 1 week of cold-acclimation. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days and either tested directly for freezing tolerance (non-acclimated plants; top graph) or were transferred at ZT4 to 4⁰C for 7 days under a 12 h photoperiod and then tested for freezing tolerance (cold-acclimated plants; bottom graph). Freezing tolerance was tested using the electrolyte leakage test. For non-acclimated samples the results presented are average values from six independent experiments (n=6). For 1 week cold-acclimated samples the results presented are average values from four independent experiments (n=4). Error bars indicate ±SEM.   78 PERCENT"ELECTROLYTE"LEAKAGE" 2,WEEKS,COLD#ACCLIMATED, 110" 100" 90" 80" 70" 60" 50" 40" 30" 20" 10" 0" IT" SW" 0" ,2" ,3" ,4" ,5" ,6" ,7" ,8" ,9" ,10" ,11" ,12" 3 WEEKS COLD-ACCLIMATED 110" 100" 90" 80" 70" 60" 50" 40" 30" 20" 10" 0" IT SW 0" ,2" ,3" ,4" ,5" ,6" ,7" ,8" ,9" ,10" ,11" ,12" TEMPERATURE"(°C)" Figure 3.2. Freezing tolerance of SW and IT accessions with 2 or 3 weeks of coldacclimation. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days then transferred at ZT4 to 4⁰C for 2 weeks (2 week cold-acclimated plants; top graph) or 3 weeks (3 week cold-acclimated plants; bottom graph) under a 12 h photoperiod and then tested for freezing tolerance. Freezing tolerance was tested using the electrolyte leakage test. For 2 week cold –acclimated samples the results presented are average values from five independent experiments (n=5). For 3 week coldacclimated samples the results presented are average values from four independent experiments (n=4). Error bars indicate ±SEM.   79 RESULTS AND DISCUSSION: SW AND IT RNA-SEQ EXPERIMENTS This second section of experiments analyzes RNA-seq data from nonacclimated, 1 week cold-acclimated and 2 week cold-acclimated SW and IT samples. The ultimate goal of these experiments is to determine the genes that potentially contribute to differences in freezing tolerance seen after 2 weeks of cold-acclimation in SW and IT. However, the transcriptomes of these accessions have never been examined and transcriptional changes prior to 2 weeks of cold-acclimation may contribute to differences in freezing tolerance. Furthermore, the freezing tolerance of SW and IT are the same at 1 week of cold-acclimation, which allows the opportunity to determine if SW and IT attain similar levels of freezing tolerance with similar transcriptional changes. Therefore analysis of RNA-seq data is partitioned into four subsections. First, basal differences between SW and IT transcriptomes are examined. Second, sets of cold-regulated genes for SW and IT are defined. Third, SW and IT coldregulated transcripts at 1 week of cold-acclimation are compared. Fourth, two sets of genes that may contribute to differences in freezing tolerance are defined. ANALYSIS OF SW AND IT BASAL TRANSCRIPTOMES Summary: Analysis of SW and IT basal (non-acclimated) transcriptomes. In the first section of results it is demonstrated that SW has significantly (p≤0.026) higher freezing tolerance in comparison to IT after 2 weeks of cold-acclimation (Fig 3.2). Although SW and IT have similar levels of freezing tolerance under non-acclimated conditions, basal (non-acclimated) transcriptome differences were first evaluated to examine general variance and to determine if genes previously associated with cold-   80 acclimation are enriched in differentially expressed genes. There are a total of 1259 genes differentially expressed between SW and IT under non-acclimated conditions. Analysis of these differentially expressed genes does not overtly suggest that there will be greater freezing tolerance in SW after 2 weeks of cold-acclimation but does indicate that there might be differences in known pathways of cold-acclimation between SW and IT. Analysis of SW and IT basal transcriptomes. SW has significantly (p≤0.026) higher freezing tolerance in comparison to IT after 2 weeks of cold-acclimation (Fig 3.2). RNA-seq experiments were conducted on rosette tissue collected before the start of electrolyte leakage assays (Fig 3.1 and 3.2). For RNA-seq experiments average 2 correlation between biological replicates was R =0.90, suggesting that RNA-seq data in this study is repeatable (Table A3.9). Altogether, SW and IT transcriptomes under nonacclimated, 1 week cold-acclimated, and 2 week cold-acclimated conditions, represent a total of 25,678 genes (76.4%) from the Arabidopsis Col-0 genome. On average, there were 23,264 genes (69.2%) represented in each sample (Table A3.9). Pairwise comparisons between non-acclimated and cold-acclimated samples or between SW and IT samples were calculated using a two-tailed Student’s t test in the cuffdiff program within Cufflinks (18). Genes were considered differentially expressed between nonacclimated and cold-acclimated samples or between SW and IT samples if the Benjamini-Hochberg (false discovery rate; FDR) corrected p-value was ≤0.050. While analyzing differentially expressed genes a ≥3 fragments per kilobase of exon per million   81 fragments mapped (FPKM) threshold (i.e. one sample in the pairwise comparison had to be ≥3FPKM) was used (19, 20). Genes differentially expressed between SW and IT without treatment (nonacclimated conditions) were first evaluated to examine general variance and to determine if genes previously associated with cold-acclimation are enriched in differentially expressed genes. Altogether, 1259 genes were differentially expressed between SW and IT under non-acclimated conditions that met the ≥3FPKM criteria (19, 20) in either SW or IT (Fig. 3.3). Of these differentially expressed genes, 887 genes have higher expression in IT and 372 are higher in SW (Fig. 3.3). Although these basal differences in gene expression do not manifest into differences in non-acclimated freezing tolerance, it is of interest to determine if the 1259 genes differentially expressed between SW and IT have previously been correlated with increases in freezing tolerance. In 2006, Hannah et al. analyzed the freezing tolerance and transcriptomes of nine accessions of Arabidopsis after two weeks of cold acclimation to define a set of genes (henceforth referred to as Hannah genes) positively and negatively correlated with increases in freezing tolerance associated with coldacclimation (6). The Hannah gene set is significantly enriched in genes from the CBF regulon (6). The CBFs are a fundamental component of the most well described pathway of cold-acclimation. Overexpression of CBF genes leads to constitutive activation of CBF target genes and a plant with much higher freezing tolerance (21-25). The Hannah gene set is also enriched in genes from the ZAT12 regulon, and overexpression of ZAT12 leads to a slight increase in plant freezing tolerance (25).   82 There significant overlap (40 genes; p=1.5E-08; calculated using a hypergeometric distribution in R) between the 887 genes with higher expression in IT and Hannah genes positively correlated with freezing tolerance (Fig. 3.3). A significant number of these (36 of the 40) overlapping genes have greater than 2-fold higher expression in IT compared to SW (Fig 3.4). Since the CBF regulon is enriched in the Hannah gene set, it was determined if the genes with higher basal expression in IT are also enriched in CBF regulated genes (25). Indeed, the genes with higher basal expression in IT are enriched (26 genes; p=0.00056) in genes up-regulated by CBFs (25). The CBF pathway of cold-acclimation appears to be differentially regulated between SW and IT. Since there is no difference in freezing tolerance between SW and IT under non-acclimated conditions (Fig 3.1) significant overlap between genes with higher in expression in SW, Hannah genes positively correlated with freezing tolerance, and CBF regulon genes, might also be expected. This was not the case. There is not significant overlap between Hannah genes positively or negatively correlated with freezing tolerance and the 372 genes with higher expression in SW (Fig 3.3). These 372 genes also are not enriched in CBF regulon genes. Consequently, it was of interest to determine what types of genes are enriched. Gene ontology (GO) enrichment analysis showed that no cold related GO categories were specifically enriched in the 372 genes with higher expression in SW (Table 3.2). ‘Response to abiotic stimulus’ and ‘response to water deprivation’ were enriched GO categories in the 887 genes with higher expression in IT (Table 3.3). Defense response related GO categories were significantly enriched in both sets of   83 genes (Table 3.2 and 3.3). Cold-related GO categories were not specifically enriched in SW or IT, but previous research by Hon et al. 1995 showed that defense response related genes, PR2, PR3 and PR5 encode proteins with antifreeze properties (26). Interestingly, PR3 expression is significantly higher (17.5 fold; p≤0.05) in SW compared to IT at non-acclimated conditions. In summary, SW has higher basal expression of 372 genes, which includes a pathogenesis related gene with antifreeze properties. IT has higher basal expression of 887 genes, which are significantly enriched in genes previously positively correlated with cold-acclimation. Given this enrichment of genes positively correlated with freezing tolerance, is it interesting that IT is not more freezing tolerant than SW under nonacclimated conditions. Ultimately, comparison of SW and IT basal transcriptomes does not reveal any obvious suggestion for the higher level of freezing tolerance in SW seen later at 2 weeks of cold-acclimation, but does suggest that genes associated with coldacclimation may be differentially regulated between SW and IT.   84 1259" differenJally" expressed" 372/1259"" higher"in"SW" SWEDEN" HIGH" 5" NON,ACC" 367" 887/1259"" higher"in"IT" HANNAH" POSITIVE" COLD" 272" ITALY" HANNAH" HIGH" POSITIVE" NON,ACC" 40" COLD" 847" 237" p=0.76" p=1.5"x"10,08" SWEDEN" HANNAH" HIGH" NEGATIVE" 13" NON,ACC" COLD" 359" 453" ITALY" HANNAH" HIGH" NEGATIVE" 15" NON,ACC" COLD" 872" 451" p=0.27" p=0.99" Figure 3.3 1259 genes differentially expressed between SW and IT under nonacclimated conditions. (Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression. P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes; 277 Hannah genes positively correlated with freezing tolerance and 466 Hannah genes negatively correlated with freezing tolerance; (6)).   85 ITALY" HANNAH" HIGH" POSITIVE" 40" NON,ACC" COLD" 847" 237" p=1.5"x"10,08" 8"are">4"fold" higher"in"in"IT" 28"are">2"fold" higher"in"IT" 4"are"<2"fold" higher"in"in"IT" p=0.99" p=1.1"x"10,06" p=1.5"x"10,08" Figure 3.4. Fold change of 40 Hannah genes positively correlated with freezing tolerance that have higher expression in IT. P-values represent the significance of overlap between ‘IT High’ and Hannah genes if 4- fold, 2-fold, or no fold change cut-offs were applied (from left to right).   86 Table 3.2 GO categories significantly enriched in 372 genes with higher expression in SW under non-acclimated conditions. GO enrichment was determined using the database for annotation visualization and integrated discovery (DAVID; david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in the 372 genes with higher expression in SW. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric pvalue was ≤0.050. GO  TERM   #   #  GENES   CATEGORY   HIGH  IN  SW  (372  GENES)   HITS   IN  GO   BP   Apoptosis   15   172   BP   Programmed  Cell  Death   16   223   BP   Defense  Response   36   1030   BP   Cell  Death   16   254   MF   Amine  Biosynthetic  Process   13   204   BP   Carbohydrate  Binding   14   215   BP   Nitrogen  Biosynthetic  Process   19   506   CC   Oxidation  Reduction   32   1186   BP   Biogenic  Amine  Biosynthetic  Process   6   40   CC   External  Encapsulating  Structure   22   620   BP   Cell  Wall   22   611   MF   Immune  Response   13   293   BP   Amino  Acid  Biosynthetic  Process   10   184   MF   Amino  Acid  Derivative  Biosynthetic  Process   10   194   BP   Innate  Immune  Response   12   275   MF   Sugar  Binding   9   124   BP   Biogenic  Amine  Metabolic  Process   6   59       87 FDR     P-­‐VALUE   3.45E-­‐05   4.76E-­‐05   5.30E-­‐05   1.87E-­‐04   1.79E-­‐03   2.62E-­‐03   1.16E-­‐02   1.23E-­‐02   1.25E-­‐02   1.26E-­‐02   2.05E-­‐02   3.00E-­‐02   3.86E-­‐02   4.31E-­‐02   4.59E-­‐02   4.74E-­‐02   4.85E-­‐02   Table 3.3. GO categories significantly enriched in 887 genes with higher expression in IT under non-acclimated conditions. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in the 887 genes with higher expression in IT. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric pvalue was ≤0.050. GO  TERM   CATEGORY   HIGH  IN  IT  (887  GENES)   BP   Secondary  Metabolic  Process   BP   Defense  Response   BP   Apoptosis   BP   Phenylpropanoid  Metabolic  Process   BP   Programmed  Cell  Death   BP   Flavonoid  Biosynthetic  Process   BP   Cell  Death   BP   Flavonoid  Metabolic  Process   BP   Phenylpropanoid  Biosynthetic  Process   BP   Immune  Response   BP   Amino  Acid  Derivative  Metabolic  Process   BP   Response  To  Water  Deprivation   BP   Response  To  Abiotic  Stimulus   BP   Oxidation  Reduction   BP   Response  To  Water   BP   Amino  Acid  Derivative  Biosynthetic  Process   BP   Innate  Immune  Response   BP   Response  To  Salicylic  Acid  Stimulus   BP   Aromatic  Compound  Biosynthetic  Process   BP   Response  To  Organic  Substance     88 #   HITS   37   65   21   20   23   11   24   11   16   25   24   18   64   63   18   18   22   15   18   59   #  GENES   IN  GO   423   1030   172   166   223   53   254   57   128   293   280   176   1197   1186   185   194   275   150   215   1176   FDR     P-­‐VALUE   1.24E-­‐04   1.62E-­‐04   2.49E-­‐04   4.47E-­‐04   6.89E-­‐04   1.03E-­‐03   1.25E-­‐03   1.53E-­‐03   1.80E-­‐03   2.99E-­‐03   3.77E-­‐03   4.74E-­‐03   6.22E-­‐03   7.34E-­‐03   7.55E-­‐03   1.17E-­‐02   1.43E-­‐02   1.97E-­‐02   3.23E-­‐02   4.06E-­‐02   SW AND IT COLD-INDUCED GENES Summary: SW and IT cold-induced genes. In this second subsection of RNAseq analysis cold-regulated genes for SW and IT are defined. SW and IT cold-regulated genes are also compared in greater detail in the third and fourth RNA-seq experiment subsections. However general analysis reveals that SW cold-regulated genes are more enriched in genes previously associated with increases in freezing tolerance than IT cold-regulated genes. The quantity of cold-regulated genes in SW and IT also suggests that there could be differences their capacity to cold-acclimate. SW and IT cold-induced genes. To find genes contributing to differences in freezing tolerance seen after 2 weeks of cold-acclimation (Fig 3.2) sets of coldresponsive genes were first defined for both SW and IT. RNA-seq data generated from SW and IT rosette tissue under non-acclimated, 1week cold-acclimated and 2 week cold acclimated conditions, was collected before electrolyte leakage experiments. Fold change cut-offs were not used while defining sets of cold-responsive genes. A fold change threshold is an arbitrary value and not necessarily biologically significant especially since transcription factors can have compounding downstream effects (28). Furthermore, if fold change cut-offs were applied when defining cold-induced genes important similarities between SW and IT cold-responsive genes might later have been missed. For example, a gene up-regulated 2-fold by cold in SW and 1.9-fold in IT would have been removed from the list of IT cold-responsive genes if a 2-fold threshold had been applied. Instead, genes were considered differentially expressed in response to cold if the FDR corrected p-value comparing non-acclimated to cold samples was   89 ≤0.050 (as determined by cuffdiff program within Cufflinks). Although a fold-change threshold was not used, a ≥3FPKM cut-off for differential expression was used while defining cold-responsive genes (19, 20). This means that the sample with higher expression (FPKM) was greater or equal to ≥3FPKM, otherwise interesting genes may have been removed. For example, a gene down-regulated from 15,000FPKM to 1FPKM, after 1 week of cold-acclimation, would have been filtered out if both nonacclimated and cold samples were required to have ≥3FPKM. While a threshold of ≥3FPKM (19, 20) for differentially expressed genes is still an arbitrary cut-off, it allows small but significant (≤0.050) fold-change differences to be included in analysis. Based on the methods above, there are a total of 2307 genes up-regulated by cold in SW and 2310 genes up-regulated by cold in IT (Fig. 3.5 and 3.6). There are also a total of 3252 genes down-regulated by cold in SW and 2658 genes down-regulated by cold in IT (Fig. 3.5 and 3.6). Both SW and IT increase their freezing tolerance after 1 and 2 weeks of cold-acclimation in comparison to basal freezing tolerance (Fig. 3.1 and 3.2). The defined cold-regulated genes are presumed to instigate these increases in freezing tolerance associated with cold-acclimation. A previous study by Hannah et al. 2006 defined a set of genes positively and negatively correlated with increases in freezing tolerance associated with cold acclimation (this set of genes is detailed in earlier analysis of basal transcriptomes; (6)). Therefore it was determined if SW and IT cold-regulated genes are enriched in genes previously associated with cold-acclimation. Fittingly, genes up-regulated by cold in SW and IT are enriched for Hannah genes positively correlated but not negatively correlated with increases in freezing tolerance   90 (Fig 3.7 and 3.8). Interestingly, the number of Hannah genes positively correlated with freezing tolerance, which overlap with SW genes up-regulated by cold, is more significant than with the genes up-regulated by cold in IT (Fig. 3.7 and 3.8). In SW, genes down-regulated by cold are significantly enriched in Hannah genes negatively correlated with freezing tolerance (Fig. 3.6). However, this is not also the case for the 2658 IT genes down-regulated by cold (Fig. 3.8). This suggests that there may be significant differences in the genes that comprise SW and IT cold-regulated transcriptomes, which will be further analyzed in the following subsections. SW and IT have similar freezing tolerance at 1 week of cold-acclimation and differing freezing tolerance at 2 weeks of cold-acclimation. Therefore it was also of interest to determine if there is significant overlap between cold-regulated genes at 1 and 2 weeks of cold-acclimation, or if discrete sets of genes are cold-regulated with 1 and 2 weeks of cold-acclimation (Fig 3.5 and 3.6). There is significant overlap in genes up-regulated by cold at 1 week and 2 weeks of cold-acclimation in both SW (1013 genes; Fig 3.5) and IT (1007; Fig. 3.6). There is also significant overlap in genes downregulated by cold at 1 and 2 weeks of cold acclimation in both SW (1275 genes; Fig 3.5) and IT (1376 genes; Fig 3.6). Hannah et al. 2006 found that the cold-acclimation capacity of nine accessions of Arabidopsis was positively correlated with the number of genes that significantly change in expression in response to cold (6). Consequently, it was of interest to determine if this correlation was also corroborated with sets of SW and IT cold-regulated genes. At 2 weeks of cold-acclimation there are more cold-regulated genes in SW (1978 genes   91 up-regulated by cold; 3205 genes down-regulated by cold; Fig 3.5) than in IT (1552 genes up-regulated by cold; 1796 genes down-regulated by cold; Fig 3.6). Based on this result Hannah et al. 2006 would hypothesize that SW has a greater capacity to cold acclimate than IT. This hypothesis is substantiated in electrolyte leakage assays performed in this study. SW has greater freezing tolerance than IT with 2 weeks of cold-acclimation (Fig. 3.2). The number of gene expression changes from 1 to 2 weeks of cold-acclimation are also consistent with the ‘gene number to cold-acclimation correlation’ hypothesis by Hannah et al. 2006. Electrolyte leakage assays show that SW increases in freezing tolerance from 1 to 2 weeks of cold-acclimation (Fig 3.1 and 3.2), and the number of gene expression changes in SW also increases from 2664 cold-regulated genes at 1 week of cold-acclimation to 5183 cold-regulated genes at 2 weeks of cold-acclimation (Fig 3.5). Furthermore, IT decreases in freezing tolerance from 1 to 2 weeks of coldacclimation and the number of gene expression changes also decreases from 4011 cold-regulated genes at 1 week of cold-acclimation to 3348 cold-regulated genes at 2 weeks of cold-acclimation. Unfortunately, the ‘gene number to cold-acclimation correlation’ hypothesis by Hannah et al. 2006 is not infallible and is inconsistent with gene expression changes at 1 week of cold-acclimation. At 1 week of cold-acclimation there are 2664 cold-regulated genes in SW (1342 genes up-regulated by cold; 1322 genes down-regulated by cold; Fig 3.5), and 4011 cold-regulated genes in IT (1746 genes up-regulated by cold; 2265 genes down-regulated by cold; Fig 3.6). According to Hannah et al. 2006, this would   92 suggest that IT has a greater capacity to cold-acclimate than SW. But electrolyte leakage assays determined that SW and IT have similar freezing tolerance with 1 week of cold-acclimation (Fig 3.1). Altogether, SW cold-regulated genes appear to be more enriched in genes previously associated with increases in freezing tolerance than IT cold-regulated genes. The quantity of cold-regulated genes in SW and IT, also suggests that there could be differences their capacity to cold-acclimate. Therefore in the next subsections of this study SW and IT cold-regulated genes will be compared in more detail. Since both SW and IT have similar freezing tolerance after 1 week of cold-acclimation, a more detailed comparison of transcriptomes at this time-point may reveal if SW and IT achieve this level of freezing tolerance by similar or divergent mechanisms.   93 SWEDEN" UP,COLD"" 1013" 1WK" 329" SWEDEN"" UP,COLD" 2WK" 965" p<2.2"x"10,16" SWEDEN" SWEDEN"" DOWN,COLD"" 1275" DOWN,COLD" 1WK" 2WK" 47" 1930" p<2.2"x"10,16" Figure 3.5 Cold-responsive genes in SW. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 or 2 weeks under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values represent overlap between differentially expressed cold-responsive genes at 1 and 2 weeks of coldacclimation (calculated using a hypergeometric distribution in R).   94 ITALY" UP,COLD"" 1007" 1WK" 740" ITALY" UP,COLD" 2WK" 563" p<2.2"x"10,16" ITALY" ITALY" DOWN,COLD"" DOWN,COLD" 1376" 1WK" 2WK" 889" 393" p<2.2"x"10,16" Figure 3.6. Cold-responsive genes in IT. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 or 2 weeks under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values represent overlap between differentially expressed cold-responsive genes at 1 and 2 weeks of coldacclimation (calculated using a hypergeometric distribution in R).   95 SWEDEN" HANNAH" UP,COLD" POSITIVE" 101" ALL" COLD" 2206" 176" SWEDEN" HANNAH" DOWN, POSITIVE" COLD" 24" COLD" ALL" 253" 3228" p<2.2"x"10,16" p=0.99" SWEDEN" HANNAH" UP,COLD" NEGATIVE" 46" ALL" COLD" 2261" 420" SWEDEN" HANNAH" DOWN, NEGATIVE" COLD" 101" COLD" ALL" 176" 2206" p=0.75" p=9.4"x"10,05" Figure 3.7. Overlap between the SW cold-responsive genes set and the Hannah genes set (6). Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 Hannah genes positively correlated with freezing tolerance and 466 Hannah genes negatively correlated with freezing tolerance; (6)).   96 ITALY" DOWN, COLD" ALL" 2618" ITALY" HANNAH" UP,COLD" POSITIVE" 47" ALL" COLD" 2263" 230" p=0.0011" HANNAH" POSITIVE" 40" COLD" 237" p=0.17" ITALY" DOWN, COLD" ALL" 2595" ITALY" HANNAH" UP,COLD" NEGATIVE" 33" ALL" COLD" 2277" 433" p=0.99" HANNAH" NEGATIVE" 63" COLD" 403" p=0.24" Figure 3.8. Overlap between the IT cold-responsive genes set and the Hannah genes set (6). Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 Hannah genes positively correlated with freezing tolerance and 466 Hannah genes negatively correlated with freezing tolerance; (6)).   97 SW AND IT TRANSCRIPTOMES AT 1 WEEK OF COLD-ACCLIMATION. Summary: SW and IT transcriptomes at 1 week of cold-acclimation. SW and IT have similar freezing tolerance after 1 week of cold-acclimation. Therefore, the SW and IT cold-response at 1 week of cold-acclimation was examined to determine if this level of freezing tolerance is attained by similar transcriptional changes. Although there are a significant number of genes that respond similarly to cold in SW and IT, the majority of cold-responsive genes are differentially regulated, either in terms of induction pattern (up-regulated by cold / down-regulated by cold / not regulated by cold) or in terms of expression level (FPKM). Furthermore, these differentially expressed coldregulated genes are enriched in genes that have previously been correlated with coldacclimation. Analysis of RNA-seq data also suggests that there may be differences in the regulation of the CBF pathway of cold-acclimation between SW and IT. Overall, data suggests that SW and IT may reach similar levels of freezing tolerance through significantly different transcriptional changes at 1 week of cold-acclimation. SW and IT transcriptomes at 1 week of cold-acclimation: Analysis of nonoverlapping cold-regulated genes. SW and IT have similar freezing tolerance after 1 week of cold-acclimation. Therefore, the SW and IT cold-response at 1 week of coldacclimation was examined to determine if this level of freezing tolerance is attained by similar transcriptional mechanisms. First, for a gene to ‘respond similarly’ to cold it must have a similar induction pattern (increase in response to cold or decrease in response to cold). Even if a gene ultimately has a similar level of expression at 1 week of cold-   98 acclimation despite dissimilar induction patterns (i.e. the gene is constitutively on in one ecotype) the change in induction would indicate altered regulation of that gene. There are a significant number of genes commonly up-regulated (734 genes) and down-regulated (946 genes) by cold between SW and IT with 1 week of coldacclimation (Fig. 3.9). But there are also 608 genes only up-regulated by cold in SW and 1012 genes only up-regulated by cold in IT at 1 week of cold-acclimation (Fig. 3.9). There are also 376 genes only down-regulated by cold in SW and 1319 genes only down-regulated by cold in IT at 1 week of cold-acclimation (Fig. 3.9). To determine if SW and IT use similar transcriptional mechanisms to attain similar levels of freezing tolerance, the cold-genes, which are uniquely expressed in SW or IT, were first examined. The genes with overlapping induction patterns in SW and IT were next examined to determine the genes that have significantly different levels of expression. Lastly, genes that do appear to respond similarly to cold in SW and IT, in terms of induction pattern and expression level, were further analyzed. It is possible that cold-responsive genes that are differentially regulated between SW and IT do not play a role in increases in freezing tolerance associated with coldacclimation. To appraise if non-overlapping cold-responsive genes have been previously associated with cold-acclimation enrichment of Hannah genes (6), which have been previously positively and negatively correlated with increases in freezing tolerance associate with cold-acclimation, was tested (Fig. 3.10). The 608 genes uniquely up-regulated by cold in SW at 1 week of cold-acclimation are enriched (55 genes overlapping) in Hannah genes that are positively correlated with freezing   99 tolerance (Fig. 3.10). Of the 55 overlapping Hannah genes, 20 are greater than 4-fold induced by cold in SW, 33 are greater than 2-fold induced and only 2 are less than 2fold induced (Fig. 3.11). This suggests that genes uniquely up-regulated by cold in SW may contribute to cold-acclimation in some way. The 608 genes only up-regulated in SW are also enriched (56 genes; p< 2.2E-16) in genes up-regulated by the CBFs (CBF regulon; (25)). The CBFs are central components of the most well described pathway of cold-acclimation. Since these CBF regulon genes are only expressed in SW it suggests that there might be some difference in the CBF pathway of cold-acclimation between SW and IT. ‘Response to cold’ and other related abiotic stress GO categories are also enriched in the 608 genes only up-regulated in SW (Table 3.4). Interestingly, the 1012 genes that are exclusively up-regulated by cold in IT at 1 week of cold-acclimation are not enriched in Hannah genes (Fig. 3.10) or in CBF regulon genes. ‘Response to cold’ or other related abiotic stress categories are also markedly absent from the GO terms enriched in the 1012 genes only up-regulated by cold in IT (Table 3.5). The 376 genes that are uniquely down-regulated by cold in SW are enriched (40 genes) in Hannah genes that negatively correlate with freezing tolerance (Fig 3.12). Of the 40 overlapping Hannah genes, 5 are greater than 4-fold reduced by cold, 30 of these genes are greater than 2-fold reduced and 5 are less than 2-fold reduced (Fig 3.13). For these 376 genes there are no biological process GO categories significantly enriched above the threshold (FDR p-value ≤0.050) but molecular function GO categories enriched include ‘water transport’ (Table 3.6). The 1319 genes only downregulated by cold in IT are not significantly enriched (5 genes) in Hannah genes   100 negatively correlated with freezing tolerance (Fig. 3.12). Remarkably, the 1319 genes only down-regulated in IT are enriched (32 genes) in Hannah genes that are positively correlated with increases in freezing tolerance (Fig. 3.12). Of the 32 overlapping Hannah genes, 6 are greater than 4-fold reduced by cold, 20 are greater than 2-fold reduced 6 are less than 2-fold reduced (Fig. 3.14). Furthermore, these 1319 gene that are only down-regulated by cold in IT are enriched (23 genes; p=0.021) in genes upregulated by the CBFs (25). This indicates that CBF regulon genes may be uniquely repressed in IT and again suggests that the CBF pathway of cold-acclimation may be regulated differently between SW and IT. For the 1319 genes only down-regulated in IT, ‘defense response’ related GO categories such as ‘innate immune response’ and ‘response to biotic stimulus’ are significantly enriched (Table 3.7). This is interesting considering that ‘defense response’ was also enriched in genes with higher expression in IT under basal conditions (Table 3.3). Altogether, analysis of RNA-seq data at 1 week of cold-acclimation indicates that genes uniquely cold-regulated in SW are enriched in genes previously associated with cold acclimation. This enrichment of genes correlated with cold-acclimation in coldregulated genes uniquely expressed SW is curious considering that there is no significant difference in freezing tolerance between SW and IT at 1 week of cold acclimation (Fig. 3.1). Therefore cold-regulated genes uniquely expressed in IT at 1 week of cold-acclimation may potentially be part of pathways of cold-acclimation currently not well described allowing IT to achieve a similar level of freezing tolerance to SW at 1 week of cold-acclimation (Fig 3.1). Differences in gene expression at 1 week of   101 cold-acclimation may also take time to manifest into differences in freezing tolerance (Fig 3.2). Consequently genes differentially regulated between SW and IT at 1 week of cold-acclimation, which remain differentially regulated at 2 weeks of cold-acclimation, are included in the list of genes that potentially contribute to differences in freezing tolerance. Genes that potentially contribute to differences in freezing tolerance will be described in more detail in the next subsection of RNA-seq analysis but first genes with similar cold induction patterns but significantly different levels of expression will be examined.   102 SWEDEN" ITALY" UP,COLD"" UP,COLD" 734" 1WK" 1WK" 608" 1012" p<2.2"x"10,16" SWEDEN" DOWN, COLD"" 946" 1WK" 376" ITALY" DOWN, COLD" 1WK" 1319" p<2.2"x"10,16" Figure 3.9. Overlap between SW and IT response at 1 week of cold-acclimation. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05.≥3FPKM was used as the cut-off for differential expression (19, 20). Pvalues represent overlap between SW and IT cold-responsive genes (calculated using a hypergeometric distribution in R).   103 SWEDEN" ITALY" UP,COLD"" UP,COLD" 734" 1WK" 1WK" 608" 1012" p<2.2"x"10,16" ITALY" UP,COLD" 1WK" 1012" SWEDEN" HANNAH" UP,COLD"" POSITIVE" 55" 1WK" COLD" 553" 222" 0" HANNAH" POSITIVE" COLD"" 277" p<2.2"x"10,16" p=NOT"SIGNIFICANT" SWEDEN" HANNAH" UP,COLD"" NEGATIVE" 0" 1WK" COLD" 608" 466" ITALY" HANNAH" UP,COLD" NEGATIVE" 24" 1WK" COLD" 988" "442" p=NOT"SIGNIFICANT" p=0.28" Figure 3.10. Overlap between genes uniquely up-regulated in SW or IT at 1 week of cold-acclimation and the Hannah genes set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 genes positively correlated with freezing tolerance and 466 genes negatively correlated with freezing tolerance; (6)).   104 SWEDEN" HANNAH" UP,COLD"" POSITIVE" 55" 1WK" COLD" 553" 222" p<2.2"x"10,16" 20"are">4" fold"induced" 33"are">2" fold"induced" 2"are"<2"fold" induced" p=0.00013" p<2.2"x"10,16" p<2.2"x"10,16" Figure 3.11. Fold change of 55 Hannah genes, which overlap with 608 genes only upregulated in SW at 1 week of cold-acclimation. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-offs were applied (from left to right).   105 Table 3.4. GO categories significantly enriched in 608 genes only up-regulated in SW at 1 week of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in the 608 genes only up-regulated in SW at 1 week of cold-acclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   SW  ONLY  UP-­‐COLD  (608  GENES)   CATEGORY   BP   Response  To  Organic  Substance   BP   Response  To  Endogenous  Stimulus   BP   Response  To  Ethylene  Stimulus   BP   Secondary  Metabolic  Process   BP   Phenylpropanoid  Biosynthetic  Process   BP   Ethylene  Mediated  Signaling  Pathway   BP   Response  To  Abiotic  Stimulus   BP   Response  To  Jasmonic  Acid  Stimulus   BP   Response  To  Abscisic  Acid  Stimulus   BP   Response  To  Water   BP   Phenylpropanoid  Metabolic  Process   BP   Response  To  Cold   BP   Response  To  Temperature  Stimulus   BP   Response  To  Water  Deprivation   MF   UDP-­‐Glucosyltransferase  Activity   MF   Glutathione  Transferase  Activity   BP   Response  To  Oxidative  Stress   BP   Two-­‐Component  Signal  Transduction   BP   Amino  Acid  Derivative  Biosynthetic  Process   BP   Toxin  Metabolic  Process   MF   Quercetin  3-­‐O-­‐Glucosyltransferase  Activity   MF   Transcription  Regulator  Activity   BP   Hormone-­‐Mediated  Signaling   BP   Cellular  Response  To  Hormone  Stimulus   BP   Carbohydrate  Transport   MF   Glucosyltransferase  Activity   MF   Transcription  Factor  Activity   BP   Response  To  Wounding   BP   Response  To  Chitin   MF   Solute:  Cation  Symporter  Activity   BP   Response  To  Carbohydrate  Stimulus     106 #   #  GENES   HITS   IN  GO   66   1176   59   975   22   256   29   423   15   128   17   170   56   1197   16   156   22   285   17   185   16   166   19   233   24   354   16   176   13   119   9   49   20   287   17   218   16   194   8   46   7   23   72   1913   25   432   25   432   10   83   13   137   64   1678   13   147   12   127   12   126   15   199   FDR  P-­‐ VALUE   1.70E-­‐07   2.24E-­‐07   2.96E-­‐04   3.36E-­‐04   4.21E-­‐04   4.63E-­‐04   5.27E-­‐04   6.27E-­‐04   6.41E-­‐04   9.88E-­‐04   9.97E-­‐04   1.16E-­‐03   1.33E-­‐03   1.59E-­‐03   3.36E-­‐03   3.57E-­‐03   4.32E-­‐03   4.42E-­‐03   4.55E-­‐03   4.92E-­‐03   4.94E-­‐03   5.96E-­‐03   7.29E-­‐03   7.29E-­‐03   7.33E-­‐03   8.06E-­‐03   8.12E-­‐03   9.93E-­‐03   1.01E-­‐02   1.15E-­‐02   1.31E-­‐02       Table 3.4 (cont’d)   BP   BP   BP   CC   MF   BP   MF   CC   BP     Response  To  Inorganic  Substance   Regulation  Of  Transcription   Aromatic  Compound  Biosynthetic  Process   Vacuole   Sugar:  Hydrogen  Symporter  Activity   Negative  Regulation  Of  ABA  Signaling   Symporter  Activity   Plasma  Membrane   Flavonoid  Biosynthetic  Process   107 28   73   15   30   10   5   12   76   7   540   2024   215   643   99   19   146   2228   53   1.32E-­‐02   2.14E-­‐02   2.58E-­‐02   2.80E-­‐02   2.81E-­‐02   2.83E-­‐02   3.14E-­‐02   3.85E-­‐02   4.65E-­‐02   Table 3.5. GO categories significantly enriched in 1012 genes only up-regulated in IT at 1 week of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in the 1012 genes only up-regulated in IT at 1 week of cold-acclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   CATEGORY   MF   CC   CC   CC   CC   CC   CC   BP   CC   CC   BP   BP   MF   CC   MF   MF   MF   CC   CC   CC   MF   CC   BP   BP   MF   BP   CC   BP   BP   BP   MF     IT  ONLY  UP-­‐COLD  (1012  GENES)   Structural  Constituent  Of  Ribosome   Non-­‐Membrane-­‐Bounded  Organelle   Cytosolic  Part   Ribosomal  Subunit   Nucleolus   Cytosol   Membrane-­‐Enclosed  Lumen   Ribosome  Biogenesis   Nuclear  Lumen   Intracellular  Organelle  Lumen   Translation   RNA  Processing   RNA  Binding   Mitochondrion   Purine  NTP-­‐Dependent  Helicase  Activity   ATP-­‐Dependent  Helicase  Activity   Translation  Factor  Activity   Plastid   Chloroplast   Cell  Wall   ATPase  Activity   External  Encapsulating  Structure   Embryonic  Development,  Seed  Dormancy   RNA  Modification   Macromolecule  Transmembrane  Transporter     Chromatin  Assembly   Plasma  Membrane   Nucleosome  Assembly   Seed  Development   Mitochondrion  Organization   RNA  Methyltransferase  Activity   108 #   #  GENES   HITS   IN  GO   147   394   232   1144   126   271   128   294   103   332   145   708   134   667   84   242   113   472   132   659   173   1231   72   436   128   1351   114   1349   26   113   26   113   27   149   205   3259   197   3192   52   611   44   467   52   620   37   372   20   133   12   51   14   71   135   2228   13   69   38   432   9   32   8   26   FDR     P-­‐VALUE   1.30E-­‐97   6.95E-­‐96   1.09E-­‐95   1.94E-­‐93   3.13E-­‐57   1.01E-­‐56   4.44E-­‐51   1.21E-­‐50   1.54E-­‐50   3.72E-­‐50   3.19E-­‐46   4.78E-­‐21   5.49E-­‐16   1.58E-­‐10   8.97E-­‐10   8.97E-­‐10   7.50E-­‐08   1.76E-­‐07   1.67E-­‐06   1.06E-­‐04   1.22E-­‐04   1.51E-­‐04   3.10E-­‐04   3.47E-­‐04   3.83E-­‐04   6.54E-­‐04   7.25E-­‐04   1.94E-­‐03   2.29E-­‐03   2.48E-­‐03   3.05E-­‐03   Table 3.5 (cont’d) CC   BP   BP   MF   BP   BP   BP   BP   MF   CC   MF   BP   MF   CC   MF   BP   MF   BP   BP   MF   BP   BP   MF   MF   BP   CC   BP   MF   BP   MF     Small  Nucleolar  Ribonucleoprotein  Complex   Cellular  Macromolecular  Complex  Assembly   Nucleotide  Biosynthetic  Process   Pseudouridine  Synthase  Activity   Fruit  Development   Nitrogen  Compound  Biosynthetic  Process   Pseudouridine  Synthesis   Response  To  Cadmium  Ion   DNA-­‐Directed  RNA  Polymerase  Activity   Vacuole   Exonuclease  Activity   Regulation  Of  Macromolecule  Biosynthetic  Process   RNA  Polymerase  Activity   CUL4  RING  Ubiquitin  Ligase  Complex   Ribonuclease  Activity   Purine  Nucleotide  Metabolic  Process   GTPase  Activity   Protein  Folding   Translational  Initiation   Nuclease  Activity   Protein  Localization  In  Organelle   Negative  Regulation  Of  DNA  Replication   3'-­‐5'-­‐Exoribonuclease  Activity   Intramolecular  Transferase  Activity   Response  To  Metal  Ion   Nucleosome   Pyrimidine  Nucleotide  Metabolic  Process   GTP  Binding   Regulation  Of  DNA  Replication   Exoribonuclease  Activity   109 14   23   20   7   38   41   7   29   11   46   8   11   11   14   12   16   13   23   11   19   11   4   7   9   30   9   6   23   5   7   100   208   170   20   452   506   21   327   63   643   34   69   69   122   84   140   97   251   74   188   76   6   30   53   384   63   22   263   14   33   3.83E-­‐03   3.93E-­‐03   4.66E-­‐03   4.81E-­‐03   4.87E-­‐03   4.87E-­‐03   6.82E-­‐03   1.01E-­‐02   1.02E-­‐02   1.22E-­‐02   1.52E-­‐02   1.68E-­‐02   1.91E-­‐02   1.98E-­‐02   2.26E-­‐02   2.28E-­‐02   2.36E-­‐02   2.52E-­‐02   2.60E-­‐02   2.80E-­‐02   3.11E-­‐02   3.19E-­‐02   3.29E-­‐02   3.70E-­‐02   4.14E-­‐02   4.20E-­‐02   4.23E-­‐02   4.29E-­‐02   4.61E-­‐02   4.84E-­‐02   SWEDEN" ITALY" DOWN, DOWN, COLD"" 946" COLD" 1WK" 1WK" 376" 1319" p<2.2"x"10,16" ITALY" DOWN, COLD" 1WK" 1314" SWEDEN" HANNAH" DOWN, NEGATIVE" COLD" 40" COLD" 1WK" 426" 336" p=NOT"SIGNIFCANT" p<2.2"x"10,16" SWEDEN" DOWN, COLD" 1WK" 375" 1" HANNAH" NEGATIVE" 5" COLD"" 461" ITALY" DOWN, COLD" 1WK" 1287" HANNAH" POSITIVE" COLD" 276" HANNAH" POSTIVE" 32" COLD" "245" p=0.00043" p=0.99" Figure 3.12. Overlap between genes uniquely down-regulated in SW or IT at 1 week of cold-acclimation and the Hannah genes set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 genes positively correlated with freezing tolerance and 466 genes negatively correlated with freezing tolerance; (6)).   110 SWEDEN" HANNAH" DOWN, NEGATIVE" COLD" 40" COLD" 1WK" 426" 336" p<2.2"x"10,16" 5"are">4"fold" repressed" 30"are">2"fold" repressed" 5"are"<2"fold" repressed" p=0.91" p=4.6"x"10,13" p<2.2"x"10,16" Figure 3.13. Fold change of 40 Hannah genes that overlap with 376 genes only downregulated in SW at 1 week of cold-acclimation. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-offs were applied (from left to right). ITALY" DOWN, COLD" 1WK" 1287" HANNAH" POSTIVE" 32" COLD" "245" p=0.00043" 6"are">4"fold" repressed" 20"are">2"fold" repressed" 6"are"<2"fold" repressed" p=0.99" p=0.020" p=0.00043" Figure 3.14. Fold change of 32 Hannah genes that overlap with 1319 genes only down-regulated in IT at 1 week of cold-acclimation. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-offs were applied (from left to right).   111 Table 3.6. GO categories significantly enriched in 376 genes only down-regulated in SW at 1 week of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in the 376 genes only down-regulated in SW at 1 week of cold-acclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   SW  ONLY  DOWN-­‐COLD  (376  GENES)   CATEGORY   CC   Anchored  To  Plasma  Membrane   CC   Anchored  To  Membrane   CC   External  Encapsulating  Structure   CC   Cell  Wall   MF   Channel  Activity   MF   Passive  Transmembrane  Transporter  Activity   CC   Apoplast   CC   Intrinsic  To  Plasma  Membrane   MF   Substrate  Specific  Channel  Activity   CC   Intrinsic  To  Membrane   MF   Water  Transporter  Activity   MF   Water  Channel  Activity   CC   Plasma  Membrane     112 #   HITS   9   18   26   25   11   11   18   9   11   66   6   6   56   #  GENES   IN  GO   66   320   620   611   126   126   382   103   125   2658   37   37   2228   FDR     P-­‐VALUE   2.08E-­‐03   2.44E-­‐03   2.70E-­‐03   4.29E-­‐03   4.76E-­‐03   4.76E-­‐03   8.16E-­‐03   8.33E-­‐03   8.87E-­‐03   2.21E-­‐02   2.89E-­‐02   2.89E-­‐02   4.16E-­‐02   Table 3.7. GO categories significantly enriched in 1319 genes only down-regulated in IT at 1 week of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 1319 genes only down-regulated in IT at 1 week of cold-acclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   CATEGORY   BP   BP   BP   BP   BP   BP   BP   CC   BP   CC   BP   BP   BP     IT  ONLY  DOWN-­‐COLD  (1319  GENES)   Immune  Response   Cell  Death   Response  To  Organic  Substance   Apoptosis   Response  To  Chitin   Defense  Response   Response  To  Carbohydrate  Stimulus   Intrinsic  To  Membrane   Response  To  Wounding   CCAAT-­‐Binding  Factor  Complex   Plant-­‐Type  Hypersensitive  Response   Amine  Transport   Protein  Ubiquitination   113 #   HITS   50   38   102   28   21   84   26   169   20   6   10   12   17   #  GENES   IN  GO   293   254   1176   172   127   1030   199   2658   147   13   45   67   125   FDR  P-­‐ VALUE   1.06E-­‐10   2.26E-­‐06   1.37E-­‐05   2.85E-­‐05   9.75E-­‐04   1.82E-­‐03   3.29E-­‐03   1.79E-­‐02   1.89E-­‐02   2.50E-­‐02   3.97E-­‐02   4.65E-­‐02   4.83E-­‐02   SW and IT transcriptomes at 1 week of cold-acclimation: Analysis of coldinduced genes with overlapping induction patterns but different levels of expression. Though there are 1620 genes exclusively up-regulated by cold in either SW or IT there are also a significant number of genes commonly up-regulated by cold (734 genes) between SW and IT (Fig. 3.9). There are also a significant number of genes commonly down-regulated by cold (946 genes) in addition to the 1695 genes exclusively down-regulated in either SW or IT (Fig. 3.9). The 734 commonly upregulated and 946 commonly down-regulated genes have similar cold-induction patterns and thus met the first requirement of ‘responding similarly’ (Fig. 3.9). The second measure for ‘responding similarly’ was that the gene must have a similar level of expression between SW and IT. Genes must have similar levels of expression in order to ‘respond similarly’ because statistically significant variances would again indicate that the gene has altered regulation between SW and IT. Statistically significant differences in expression level (FPKM) were measured by a FDR corrected p-value of ≤0.050. A significant number of genes (105 genes out of 734) commonly up-regulated by cold in SW and IT have different induction levels (Fig. 3.15). There are 77 differentially expressed up-regulated genes with higher expression in SW and 28 with higher expression in IT (Fig 3.15). Again, these differentially expressed genes may not be associated with cold-acclimation. Therefore it was determined if these differentially expressed genes were enriched in Hannah genes that have been previously positively and negatively correlated with increases in freezing tolerance associated with coldacclimation (6). The 77 genes with higher expression in SW are significantly enriched   114 (13 genes) in Hannah genes positively correlated with increases in freezing tolerance (Fig. 3.16). Of the 13 overlapping Hannah genes, 7 have greater than 4-fold higher expression in SW compared to IT, 5 are greater than 2-fold higher and 1 is less than 2fold higher (Fig. 3.17). The 77 genes up-regulated by cold with higher expression in SW are enriched in cold-related GO terms such as ‘response to cold’, ‘response to abiotic stimulus’ and ‘cold-acclimation’ (Table 3.8). The 77 genes with higher expression in SW are also significantly enriched (14 genes; p=1.3E-13) in genes up-regulated by the CBFs (25). The 28 genes up-regulated by cold with higher expression in IT do not include any genes regulated by the CBFs. This suggests that SW and IT may differ in the CBF pathway of cold-acclimation, which is similar what was observed in genes that were uniquely cold regulated in either SW or IT. Furthermore, there is also no overlap between the 28 up-regulated genes with higher expression in IT and Hannah genes positively correlated with freezing tolerance (Fig. 3.16). These 28 genes are also not enriched in GO terms related to stress (Table 3.9). A significant number of genes (105 out of 946 genes) commonly down-regulated by cold in SW and IT have significant differences in expression level (Fig. 3.18). There are 36 differentially expressed genes with higher expression in SW (more repressed in IT) and 69 with higher expression in IT (more repressed in SW; Fig 3.18). The 36 genes that are more repressed by cold in IT (higher expression in SW) do not significantly overlap with Hannah genes negatively correlated with increases in freezing tolerance or with CBF regulon genes (Fig. 3.19). These 36 genes do not have any statistically overrepresented GO terms. There is significant overlap (6 genes) between the 63   115 genes more repressed by cold in SW (higher in IT) and Hannah genes negatively correlated with freezing tolerance (Fig. 3.19). Of the 6 overlapping Hannah genes, 2 are greater than 4-fold repressed in SW compared to IT and 4 are greater than 2-fold repressed (Fig. 3.20). These 69 genes are not enriched in GO terms specifically related to cold but are enriched in GO terms such as ‘response to endogenous stimulus’ (Table 3.10). The 69 genes down-regulated by cold that are more repressed in SW (higher in IT) are also significantly enriched (4 genes; p=0.0072) in genes down-regulated by CBFs, again suggesting that SW and IT have differences in the CBF pathway of coldacclimation. Analysis of both non-overlapping cold genes and genes with common induction patterns but significantly different levels of expression suggests that the differentially expressed genes in SW are more enriched in genes previously correlated (Hannah genes) and associated (CBF regulon) with cold-acclimation. There is no difference in freezing tolerance between SW and IT at 1 week of cold-acclimation (Fig. 3.1). This suggests that IT may reach a similar level of freezing tolerance to SW through pathways of cold-acclimation not yet well defined. However, there are a significant number of genes with similar induction patterns and no significant difference in expression level (629 genes commonly up-regulated, and 841 genes commonly down-regulated). Next, these cold-regulated genes that respond similarly in SW and IT will be further analyzed to determine if they have been previously associated with cold-acclimation.   116 SWEDEN" ITALY" UP,COLD"" UP,COLD" 734" 1WK" 1WK" 608" 1012" p<2.2"x"10,16" 629"GENES"WITH" SIMILAR"LEVELS" OF"EXPRESSION" 105"GENES" DIFFERENTIALLY" EXPRESSED" p=5.9"x"10,09" 77"GENES"ARE" HIGHER"IN"SW" 28"GENES"ARE" HIGHER"IN"IT" Figure 3.15. 105 genes commonly up-regulated by cold but significantly different in expression level. 77 genes are higher in expression in SW and 28 genes are higher in IT. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. Genes were considered differentially expressed between SW and IT if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values represent overlap between SW and IT cold-responsive genes (calculated using a hypergeometric distribution in R).   117 105"SW+IT"UP, REG"GENES" DIFFERENTIALLY" EXPRESSED" 77"GENES"ARE" HIGHER"IN"SW" 28"GENES"ARE" HIGHER"IN"IT" ITALY" HIGHER" UP,COLD" 1WK" 28" SWEDEN" HANNAH" HIGHER" POSITIVE" UP,COLD" 13" COLD" 1WK" 264" 64" p=NOT"SIGNIFICANT" p=1.77x"10,11" SWEDEN" HIGHER" UP,COLD" 1WK" 76" 0" " HANNAH" POSITIVE" COLD"" 277" ITALY" HIGHER" UP,COLD" 1WK" 28" HANNAH" NEGATIVE" 1" COLD" 465" p=0.81" HANNAH" NEGATIVE" 0" COLD" "466" p=NOT"SIGNIFICANT" Figure 3.16. 105 genes commonly up-regulated by cold but significantly different in expression level and their overlap with the Hannah genes set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. Genes were considered differentially expressed between SW and IT if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 genes positively correlated with freezing tolerance and 466 genes negatively correlated with freezing tolerance; (6)).   118 SWEDEN" HANNAH" HIGHER" POSITIVE" UP,COLD" 13" COLD" 1WK" 264" 64" p=1.77x"10,11" 7"are">4"fold" higher"in"SW" 5"are">2"fold" higher"in"SW" 1"are"<2"fold" higher"in"SW" p=6.0"x"10,05" p=1.8"x"10,11" p=1.8"x"10,11" Figure 3.17. Fold change of 13 Hannah genes that overlap with 77 genes with upregulated induction pattern, but higher expression level in SW. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4fold, 2-fold, or no fold change cut-offs were applied (from left to right).   119 Table 3.8. GO categories significantly enriched in 77 genes with up-regulated induction pattern and higher expression level in SW at 1 week of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 77 genes with up-regulated induction pattern and higher expression level in SW at 1 week of cold-acclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   SW  HIGHER  UP-­‐COLD  (77  GENES)   CATEGORY   BP   Response  To  Temperature  Stimulus   BP   Response  To  Cold   BP   Response  To  Abiotic  Stimulus   BP   Response  To  Water   BP   Response  To  Abscisic  Acid  Stimulus   BP   Response  To  Water  Deprivation   BP   Oxidation  Reduction   BP   Secondary  Metabolic  Process   BP   Aromatic  Compound  Biosynthetic  Process   BP   Heat  Acclimation   BP   Response  To  Desiccation   BP   Cold  Acclimation   #  HITS   11   9   17   8   9   7   14   8   6   3   3   3   #  GENES   IN  GO   354   233   1197   185   285   176   1186   423   215   16   18   20   FDR  P-­‐ VALUE   1.92E-­‐04   3.22E-­‐04   3.93E-­‐04   5.03E-­‐04   5.66E-­‐04   2.17E-­‐03   1.46E-­‐02   3.21E-­‐02   3.50E-­‐02   3.81E-­‐02   4.38E-­‐02   4.95E-­‐02   Table 3.9. GO categories significantly enriched in 28 genes with up-regulated induction pattern and higher expression level in IT at 1 week of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 28 genes with up-regulated induction pattern and higher expression level in IT at 1 week of cold-acclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   IT  HIGHER  UP-­‐COLD  (28  GENES)   CATEGORY   BP   Ribonucleoprotein  Complex  Biogenesis     120 #   HITS   5   #  GENES   IN  GO   249   FDR  P-­‐ VALUE   1.02E-­‐02   SWEDEN" DOWN, COLD"" 946" 1WK" 376" ITALY" DOWN, COLD" 1WK" 1319" p<2.2"x"10,16" 841"GENES"WITH" SIMILAR"LEVELS" OF"EXPRESSION" 105"GENES" DIFFERENTIALLY" EXPRESSED" p=0.00054" 36"GENES"ARE" HIGHER"IN"SW" 69"GENES"ARE" HIGHER"IN"IT" Figure 3.18. 105 genes commonly down-regulated by cold but significantly different in expression level. 36 genes more repressed in IT (higher in expression in SW) and 69 genes more repressed in SW (higher expression in IT). Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. Genes were considered differentially expressed between SW and IT if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). Pvalues represent overlap between SW and IT cold-responsive genes (calculated using a hypergeometric distribution in R).   121 105"SW+IT" DOWN,REG" DIFFERENTIALLY" EXPRESSED" 36"GENES"ARE" HIGHER"IN"SW" 69"GENES"ARE" HIGHER"IN"IT" SWEDEN" HANNAH" HIGHER" NEGATIVE" DOWN, 0" COLD" COLD"1WK" 466" 36" ITALY" HANNAH" HIGHER" NEGATIVE" DOWN, 6" COLD"" COLD"1WK" " 460" 63" p=NOT"SIGNIFICANT" p=0.0038" SWEDEN" HIGHER" 0" DOWN, COLD"1WK" 36" ITALY" HIGHER" 1" DOWN, COLD"1WK" 68" HANNAH" POSITIVE" COLD" 277" p=NOT"SIGNIFICANT" HANNAH" POSITIVE" COLD" "276" p=0.59" Figure 3.19. 105 genes commonly down-regulated by cold but significantly different in expression level and their overlap with the Hannah genes set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. Genes were considered differentially expressed between SW and IT if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 genes positively correlated with freezing tolerance and 466 genes negatively correlated with freezing tolerance; (6)).   122 ITALY" HANNAH" HIGHER" NEGATIVE" DOWN, 6" COLD"" COLD"1WK" " 460" 63" p=0.0038" 2"are">4"fold" higher"in"IT" 4"are">2"fold" higher"in"IT" p=0.44" p=0.0038" 0"are"<2"fold" higher"in"IT" Figure 3.20. Fold change of 6 Hannah genes that overlap with 69 genes with downregulated induction pattern, but higher expression level in IT. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4fold, 2-fold, or no fold change cut-offs were applied (from left to right).   123 Table 3.10. GO categories significantly enriched in 69 genes with down-regulated induction pattern and higher expression level in IT at 1 week of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 69 genes with down-regulated induction pattern and higher expression level in IT at 1 week of cold-acclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   IT  HIGHER  UP-­‐COLD  (69  GENES)   CATEGORY   BP   Response  To  Organic  Substance   BP   Response  To  Endogenous  Stimulus   BP   Response  To  Hormone  Stimulus     124 #   HITS   19   13   12   #  GENES   IN  GO   1176   975   909   FDR     P-­‐VALUE   1.81E-­‐06   5.03E-­‐03   8.78E-­‐03   SW and IT transcriptomes at 1 week of cold-acclimation: Analysis of genes that respond similarly to cold in SW and IT. Analysis of cold-responsive genes, which are differentially regulated between SW and IT, suggests that SW and IT may differ in their transcriptional mechanisms of cold-acclimation. However, there are a significant number of genes that are regulated similarly between SW and IT. There are 629 genes up-regulated by cold in both SW and IT with similar expression level. However, these genes may not be associated with increasing freezing tolerance. Therefore it was subsequently determined if genes commonly up-regulated by cold are enriched in genes previously associated with cold-acclimation. Hannah genes have been positively and negatively correlated with increases in freezing tolerance associated with cold-acclimation (6). The 629 genes commonly up-regulated by cold in SW and IT are not significantly enriched in Hannah genes positively or negatively correlated with increases in freezing tolerance (Fig. 3.21). However, these 629 up-regulated genes are enriched (15 genes; p=0.0045) in genes up-regulated by the CBFs. Abiotic stress GO terms, such as ‘response to cold’, ‘response to temperature stimulus’, ‘cold-acclimation’, ‘response to salt stress’ and ‘response to osmotic stress’ are significantly enriched in these 629 genes (Table 3.11). Similar GO terms are also enriched in the 277 Hannah genes positively correlated with increases in freezing tolerance (Table A3.2). The 841 genes commonly down-regulated by cold with similar expression levels in SW and IT are not significantly enriched in Hannah genes (Fig. 3.22) or in CBF regulon genes (11 genes; p=0.33). Noteworthy overrepresented GO terms for these 841 genes include biotic stress related categories such as ‘immune response’, ‘defense   125 response to bacterium’ and ‘apoptosis’ (Table 3.12). Photosynthesis and light related GO categories are also enriched (Table 3.12). Defense response and photosynthesis related GO terms are also overrepresented in the 466 Hannah genes negatively correlated with increases in freezing tolerance (Table A3.2). In summary, 1725 out of 2354 genes up-regulated by 1 week of cold are exclusively expressed in either SW or IT (Fig. 3.9) or have significantly different levels of expression between SW and IT (Fig. 3.15). 1800 out of 2641 genes down-regulated by 1 week of cold are exclusively expressed in either SW or IT (Fig 3.9) or have significantly different levels of expression between SW and IT (Fig 3.18). Therefore, the majority of cold-responsive genes are not regulated similarly between SW and IT. Although all of these differentially regulated genes may not play a role in coldacclimation, comparisons of differentially-regulated genes to the Hannah gene set (6) and to the CBF regulon (25) suggests enrichment of genes correlated with coldacclimation. There are a significant number of genes regulated similarly by cold in SW and IT. Though genes commonly up-regulated by cold are enriched in CBF regulon genes, common cold-regulated genes between SW and IT are not as enriched in genes previously associated with cold-acclimation in comparison to differentially regulated genes (Fig 3.10, 3.12, 3.16, 3.19, 3.21, 3.22). Overall, analysis of RNA-seq data suggests that SW and IT reach similar levels of freezing tolerance at 1 week of coldacclimation (Fig. 3.2) through different transcriptional changes. Furthermore, IT appears to be consistently less enriched in Hannah genes and CBF regulated genes than SW, suggesting that IT possibly reaches a similar level of freezing tolerance   126 through CBF-independent pathways, which are much less well described. These differences in cold-response will be taken into account while defining sets of candidate genes that may contribute to differences in freezing tolerance (FTD genes) in the next section of this study.   127 SWEDEN" ITALY" UP,COLD"" UP,COLD" 734" 1WK" 1WK" 608" 1012" p<2.2"x"10,16" 629"GENES"WITH" SIMILAR"LEVELS"OF" EXPRESSION" SW+IT" HANNAH" UP, POSITIVE" COMMON" 13" COLD" 1WK" 264" 616" SW+IT" HANNAH" UP, NEGATIVE" COMMON" 4" COLD" 1WK" 426" 625" 105"GENES" DIFFERENTIALLY" EXPRESSED" p=0.064" p=NOT"SIGNIFICANT" Figure 3.21. 629 genes with up-regulated induction patterns and similar expression levels and their overlap with the Hannah genes set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. Genes were not differentially expressed between SW and IT if the FDR corrected p-value was ≥0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 genes positively correlated with freezing tolerance and 466 genes negatively correlated with freezing tolerance; (6)).   128 Table 3.11 GO categories significantly enriched in 629 genes with up-regulated induction patterns and similar expression levels in SW and IT. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 629 genes with up-regulated induction patterns and similar expression levels in SW and IT. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   SW+IT  COMMON  UP-­‐COLD  (629  GENES)   CATEGORY   CC   Cytosolic  Ribosome   CC   Ribosome   MF   Structural  Constituent  Of  Ribosome   CC   Ribonucleoprotein  Complex   MF   Structural  Molecule  Activity   CC   Non-­‐Membrane-­‐Bounded  Organelle   CC   Cytosol   CC   Membrane-­‐Enclosed  Lumen   CC   Nucleolus   CC   Nuclear  Lumen   CC   Organelle  Lumen   BP   Ribosome  Biogenesis   BP   Circadian  Rhythm   BP   Translation   BP   Response  To  Abiotic  Stimulus   BP   Response  To  Osmotic  Stress   BP   Response  To  Temperature  Stimulus   BP   Response  To  Cold   CC   Plastid   CC   External  Encapsulating  Structure   CC   Chloroplast   BP   Response  To  Salt  Stress   CC   Plant-­‐Type  Cell  Wall   BP   Response  To  Cadmium  Ion   CC   Plasma  Membrane   BP   Cold  Acclimation   CC   Mitochondrion   BP   Response  To  Metal  Ion   BP   Response  To  Inorganic  Substance   BP   Protein  Folding     129 #  HITS   62   73   65   81   71   103   75   63   44   51   60   31   12   69   65   31   29   23   128   36   125   26   20   23   89   6   58   24   30   18   #  GENES   IN  GO   317   470   394   671   538   1144   708   667   332   472   659   242   39   1231   1197   399   354   233   3259   620   3192   369   270   327   2228   20   1349   384   540   251   FDR     P-­‐VALUE   2.73E-­‐31   9.99E-­‐31   9.01E-­‐29   7.15E-­‐27   7.25E-­‐26   1.55E-­‐24   1.99E-­‐21   1.78E-­‐15   1.89E-­‐15   1.92E-­‐14   8.27E-­‐14   2.45E-­‐09   1.11E-­‐06   1.77E-­‐06   1.17E-­‐05   3.87E-­‐05   3.89E-­‐05   4.19E-­‐05   1.05E-­‐03   1.27E-­‐03   1.37E-­‐03   1.96E-­‐03   3.06E-­‐03   6.06E-­‐03   8.74E-­‐03   1.03E-­‐02   1.69E-­‐02   1.96E-­‐02   2.02E-­‐02   2.57E-­‐02   SWEDEN" ITALY" DOWN, DOWN, COLD"" 946" COLD" 1WK" 1WK" 376" 1319" p<2.2"x"10,16" 841"GENES"WITH" SIMILAR"LEVELS"OF" EXPRESSION" 105"GENES" DIFFERENTIALLY" EXPRESSED" SW+IT" HANNAH" DOWN" NEGATIVE" COMMON" 22" COLD" 1WK" 444" 819" SW+IT" DOWN, COMMON" 4" 1WK" 837" HANNAH" POSITIVE" COLD" 273" p=0.21" p=NOT"SIGNIFICANT" Figure 3.22. 841 genes with down-regulated induction patterns and similar expression levels and their overlap with the Hannah genes set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. Genes were not differentially expressed between SW and IT if the FDR corrected p-value was ≥0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 genes positively correlated with freezing tolerance and 466 genes negatively correlated with freezing tolerance; (6)).   130 Table 3.12. GO categories significantly enriched in 841 genes with down-regulated induction patterns and similar expression levels in SW and IT. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 841 genes with down-regulated induction patterns and similar expression levels in SW and IT. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   CATEGORY   BP   CC   CC   BP   BP   BP   BP   CC   BP   CC   BP   BP   BP   BP   CC   MF   BP   MF   BP   BP   MF   MF   MF   BP   BP   CC   MF   BP   CC   CC     SW+IT  COMMON  DOWN-­‐COLD  841  GENES)   Response  To  Abiotic  Stimulus   Organelle  Subcompartment   Thylakoid  Membrane   Response  To  Light  Stimulus   Programmed  Cell  Death   Response  To  Radiation   Response  To  Bacterium   Photosynthetic  Membrane   Cell  Death   Chloroplast   Response  To  Blue  Light   Response  To  Far  Red  Light   Protein  Amino  Acid  Phosphorylation   Immune  Response   Plastid   Protein  Serine/Threonine  Kinase  Activity   Response  To  Red  Light   Protein  Kinase  Activity   Apoptosis   Photosynthesis   Nucleoside  Binding   Adenyl  Ribonucleotide  Binding   Adenyl  Nucleotide  Binding   Phosphorylation   Response  To  Organic  Substance   Vacuole   ATP  Binding   Phosphate  Metabolic  Process   Cell  Wall   Interchromatin  Granule   131 #   HITS   75   30   27   39   25   39   25   27   27   145   11   10   65   27   146   58   10   66   18   18   123   115   123   66   66   38   112   69   35   3   #  GENES   IN  GO   1197   349   305   466   223   482   244   330   254   3192   55   44   1065   293   3259   975   56   1110   172   175   2536   2368   2528   1178   1176   643   2340   1273   611   3   FDR     P-­‐VALUE   5.31E-­‐04   5.70E-­‐04   6.39E-­‐04   6.43E-­‐04   7.54E-­‐04   8.59E-­‐04   1.21E-­‐03   1.37E-­‐03   1.45E-­‐03   1.71E-­‐03   2.34E-­‐03   2.52E-­‐03   2.68E-­‐03   2.76E-­‐03   2.87E-­‐03   9.41E-­‐03   1.05E-­‐02   1.10E-­‐02   1.11E-­‐02   1.18E-­‐02   1.20E-­‐02   1.29E-­‐02   1.54E-­‐02   1.57E-­‐02   1.60E-­‐02   2.02E-­‐02   2.38E-­‐02   2.68E-­‐02   4.49E-­‐02   4.79E-­‐02   GENES CONTRIBUTING TO DIFFERENCES IN FREEZING TOLERANCE Summary: Genes contributing to differences in freezing tolerance. In the next section of RNA-seq analysis two sets of genes that may possibly contribute to differences in freezing tolerance between SW and IT at 2 weeks of cold-acclimation are defined (Fig. 3.2). Category 1 FREEZING TOLERANCE DIFFERENCE (FTD) candidate genes are cold-regulated genes, which are differentially expressed between SW and IT at only 2 weeks of cold-acclimation, the point at which freezing tolerance differs. Analysis of SW and IT transcriptomes with 1 week of cold- suggests that SW and IT may potentially differ in their transcriptional mechanisms of cold-acclimation. Therefore, Category 2 FTD candidate genes are differentially expressed between SW and IT at both 1 week and 2 weeks of cold-acclimation. Analysis of Category 1 and Category 2 FTD genes suggests that the CBF pathway of cold-acclimation may be differentially regulated between SW and IT. Genes contributing to differences in freezing tolerance: Category 1 FREEZING TOLERANCE DIFFERENCE (FTD) candidate genes. SW and IT have no difference in freezing tolerance with 1 week of cold-acclimation or under non-acclimated conditions (Fig. 3.1). But after 2 weeks of cold-acclimation there is a significant difference in freezing tolerance between SW and IT (Fig 3.2; p ≤0.026). To determine if genes associated with differences in freezing tolerance possibly underlie fitness QTL, candidate genes contributing to differences in freezing tolerance were first defined. It is possible that differentially expressed cold genes between SW and IT are involved in differences between SW and IT not related to freezing tolerance. However, even if   132 these differentially expressed genes were not related to freezing tolerance, these genes would still represent differences between SW and IT that could potentially contribute to differences in fitness between SW and IT. Examination of SW and IT transcriptomes at 1 week of cold suggests that SW and IT may differ in their mechanisms of coldacclimation, since the majority of cold-regulated genes are differentially regulated between SW and IT. Although is possible that genes expressed similarly between SW and IT are responsible for cold-acclimation (Fig. 3.21-3.22), it seems likely that at least a portion of differentially regulated cold genes between SW and IT contribute coldacclimation in some way since differentially regulated cold genes at 1 week of coldacclimation are significantly enriched in genes regulated by the CBFs, master regulators of the most well defined pathway of cold-acclimation (21, 24, 29). Consequently, differences in gene expression at 1 week of cold-acclimation may contribute to the differences in freezing tolerance seen later at 2 weeks of cold-acclimation. These genes were defined as Category 2 FTD genes and will be described later. However, genes that are differentially expressed only at 2 weeks of cold-acclimation also seem like good candidates for genes contributing to differences in freezing tolerance. Therefore, Category 1 FTD candidate genes are cold-regulated genes (in either SW or IT) that have no difference in expression level between SW and IT at 1 week of coldacclimation and non-acclimated conditions, but different levels of expression at 2 weeks of cold-acclimation. Again, differential expression was determined by a FDR corrected p-value ≤0.050 (30) and a ≥3FPKM threshold for either sample in the pairwise comparison (19, 20).   133 There are 2130 Category 1 FTD candidate genes (Fig. 3.23; Table A3.3 and A3.4). Of these 2130 Category 1 FTD genes, 474 have higher expression in SW at 2 weeks of cold-acclimation (Fig. 3.23, Table A3.3). Again, all these differentially expressed genes may not necessarily contribute to cold-acclimation. Consequently, enrichment of genes that have been previously associated with cold-acclimation was tested. The CBFs are central components of the most well described pathway of coldacclimation. The 474 Category 1 FTD candidate genes with higher expression in SW are significantly enriched (33 genes; p=3.2E-17; Table A3.3) in genes up-regulated by the CBFs. This suggests that the CBF pathway of cold-acclimation differs between SW and IT, which is similar to what was seen at 1 week of cold-acclimation. These 474 genes are also significantly enriched in Hannah genes positively and negatively correlated with freezing tolerance (Fig 3.23). There are 15 Hannah genes positively correlated with freezing tolerance that overlap with the Category 1 genes with higher expression in SW. This overlap is statistically significant however the majority of these genes have less than 2-fold higher expression in SW compared to IT (Fig 3.24). There are 20 Hannah genes negatively correlated with freezing tolerance that overlap with the Category 1 genes with higher expression in SW. This overlap is again statistically significant but all of these genes are less than 2-fold higher in SW compared to IT (Fig. 3.25). Interesting GO categories overrepresented in these 474 genes include ‘response to temperature’, ‘response to cold’ and ‘photosynthesis’ (Table 3.13). Out of 2130 Category 1 FTD genes, 1656 Category 1 FTD candidate genes have higher expression in IT at 2 weeks of cold-acclimation (Fig 2.23; Table A.3.4). These   134 1656 genes are not significantly enriched in Hannah genes positively or negatively correlated with freezing tolerance (Fig. 3.23) and are also not enriched in genes upregulated (6 genes; p=0.99) or down-regulated (21 genes; p=0.29) by the CBFs. However, this genes set is significantly enriched in a number of notable GO terms, most of which related to defense response and biotic stress (Table 3.14). Temperaturerelated GO terms are not enriched in these 1656 Category 1 genes, but other abiotic stress categories such as ‘response to salt stress’ and ‘response to osmotic stress’ are enriched. Altogether, Category 1 FTD candidate genes with higher expression in SW appear to be more enriched in genes that have been previously associated with coldacclimation than IT. This difference is consistent with analysis of SW and IT transcriptomes at 1 week of cold-acclimation and again suggests that IT may coldacclimate using pathways not yet well defined.   135 2130"CATEGORY"1" CANDIDATE"GENES" 474"GENES"ARE" HIGHER"IN"SW"AT" 2WKS" CAT."1"" SW, HIGHER" 459" 1656"GENES"ARE" HIGHER"IN"IT"AT" 2WKS" HANNAH" CAT."1"" POSITIVE" IT,HIGHER" 17" COLD"" 1639" " 260" HANNAH" POSITIVE" 15" COLD" 262" p=0.00091" CAT."1" SW, HIGHER" 454" p=0.82" HANNAH" NEGATIVE" 20" COLD" 446" HANNAH" CAT."1"" NEGATIVE" IT,HIGHER" 27" COLD" 1629" "439" p=0.0025" p=0.93" Figure 3.23. 2130 Category 1 FTD candidate genes and overlap with the Hannah gene set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 Hannah genes positively correlated with freezing tolerance and 466 Hannah genes negatively correlated with freezing tolerance; (6)).   136 CAT."1"" SW, HIGHER" 459" HANNAH" POSITIVE" 15" COLD"" " 262" p=0.00091" 0"are">4"fold" higher"in"SW" 3"are">2"fold" higher"in"SW" 12"are"<2"fold" higher"in"SW" p=0.94" p=0.00091" Figure 3.24. Fold change of 15 Hannah genes that overlap with 474 Category 1 FTD genes with higher expression in SW. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-offs were applied (from left to right). CAT."1"" SW, HIGHER" 454" HANNAH" NEGATIVE" 20" COLD"" " 446" p=0.0025" 0"are">4"fold" higher"in"SW" "0"are">2"fold" higher"in"SW" 20"are"<2"fold" higher"in"SW" p=0.0025" Figure 3.25. Fold change of 20 Hannah genes that overlap with 474 Category 1 FTD genes with higher expression in SW. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-offs were applied (from left to right).   137 Table 3.13. GO categories significantly enriched in 474 Category 1 genes with higher expression in SW at 2 weeks of cold-acclimation. ‘GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 474 Category 1 genes with higher expression in SW at 2 weeks of coldacclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   #   #  GENES   SW  HIGH  CATEGORY  1  (474  GENES)   CATEGORY   HITS   IN  GO   CC   Chloroplast   155   3192   CC   Plastid   156   3259   CC   Organelle  Subcompartment   32   349   CC   Thylakoid  Membrane   29   305   CC   Photosynthetic  Membrane   29   330   CC   Organelle  Membrane   45   849   BP   Starch  Metabolic  Process   10   34   BP   Photosynthesis   19   175   CC   Plastid  Envelope   28   461   CC   Chloroplast  Envelope   26   440   CC   Photosystem   9   72   BP   Response  To  Abiotic  Stimulus   45   1197   BP   Carbohydrate  Biosynthetic  Process   15   189   BP   Cellular  Glucan  Metabolic  Process   12   124   BP   Response  To  Temperature  Stimulus   20   354   BP   Response  To  Cold   16   233   BP   Oligosaccharide  Metabolic  Process   8   51   BP   Starch  Catabolic  Process   5   13   BP   Carbohydrate  Biosynthetic  Process   16   254   BP   Cellular  Polysaccharide  Catabolic  Process   5   15   BP   Glucan  Metabolic  Process   12   151   BP   Cellular  Polysaccharide  Metabolic  Process   12   154   BP   Photosynthesis,  Light  Reaction   9   85   BP   Starch  Biosynthetic  Process   5   17   BP   Generation  Of  Precursor  Metabolites  And  Energy   19   404   CC   Photosystem  II   6   52   CC   Plastid  Outer  Membrane   5   34   CC   Chloroplast  Photosystem  II   4   18   CC   Oxygen  Evolving  Complex   4   18   BP   Glycoside  Metabolic  Process   8   84     138 FDR  P-­‐ VALUE   3.54E-­‐24   5.20E-­‐24   6.21E-­‐10   2.44E-­‐09   1.24E-­‐08   9.29E-­‐07   3.53E-­‐06   4.41E-­‐06   3.35E-­‐05   1.23E-­‐04   1.43E-­‐03   2.26E-­‐03   2.75E-­‐03   2.89E-­‐03   3.83E-­‐03   4.10E-­‐03   4.22E-­‐03   5.73E-­‐03   6.92E-­‐03   8.16E-­‐03   8.73E-­‐03   8.90E-­‐03   9.97E-­‐03   1.12E-­‐02   3.06E-­‐02   3.53E-­‐02   3.86E-­‐02   3.94E-­‐02   3.94E-­‐02   4.23E-­‐02   Table 3.14. GO categories significantly enriched in 1656 Category 1 genes with higher expression in IT at 2 weeks of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 1656 Category 1 genes with higher expression in IT at 2 weeks of coldacclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   IT  HIGH  CATEGORY  1  GENES  (1656  GENES)   CATEGORY   CC   Plasma  Membrane   BP   Response  To  Chitin   MF   Nucleoside  Binding   BP   Response  To  Carbohydrate  Stimulus   BP   Immune  Response   BP   Protein  Amino  Acid  Phosphorylation   BP   Response  To  Organic  Substance   MF   Protein  Kinase  Activity   MF   Purine  Nucleotide  Binding   MF   Adenyl  Ribonucleotide  Binding   BP   Innate  Immune  Response   BP   Response  To  Wounding   CC   Endoplasmic  Reticulum   MF   ATP  Binding   BP   Phosphate  Metabolic  Process   BP   Response  To  Bacterium   CC   Intrinsic  To  Membrane   BP   Phosphorylation   BP   Cell  Death   BP   Defense  Response  To  Bacterium   BP   Ribonucleotide  Binding   BP   Nucleotide  Binding   BP   Programmed  Cell  Death   BP   Defense  Response   BP   Callose  Deposition  During  Defense  Response   BP   Plant-­‐Type  Hypersensitive  Response   BP   Polysaccharide  Localization   CC   External  Encapsulating  Structure   CC   Integral  To  Membrane   CC   Cell  Wall   BP   Amino  Acid  Derivative  Metabolic  Process     139 #   #  GENES   HITS   IN  GO   277   2228   43   127   288   2536   50   199   63   293   146   1065   157   1176   150   1110   300   2785   262   2368   58   275   40   147   74   446   257   2340   159   1273   52   244   262   2658   148   1178   52   254   42   184   274   2621   332   3333   45   223   127   1030   12   18   17   45   12   21   78   620   207   2193   76   611   44   280   FDR  P-­‐ VALUE   9.35E-­‐25   1.38E-­‐14   2.55E-­‐13   6.49E-­‐12   6.57E-­‐12   6.95E-­‐12   7.34E-­‐12   2.27E-­‐11   2.77E-­‐11   6.58E-­‐11   6.76E-­‐11   1.11E-­‐10   1.12E-­‐10   2.42E-­‐10   6.15E-­‐10   6.63E-­‐10   9.77E-­‐10   2.08E-­‐09   2.15E-­‐09   6.69E-­‐09   8.30E-­‐09   1.08E-­‐08   8.10E-­‐08   1.94E-­‐07   4.77E-­‐07   3.31E-­‐06   3.45E-­‐06   6.92E-­‐06   7.12E-­‐06   1.06E-­‐05   1.14E-­‐04   Table 3.14 (cont’d) CC   CC   BP   BP   BP   BP   BP   BP   MF   BP   BP   BP   BP   BP   MF   MF   BP   BP   BP   BP   MF   BP   BP   BP   BP   BP   BP   MF   BP   BP   BP   MF   BP   BP   MF   BP   MF   MF   BP   BP     Endoplasmic  Reticulum  Part   20   Vacuole   72   Phospholipid  Transport   8   Indole  Derivative  Metabolic  Process   13   Aromatic  Compound  Biosynthetic  Process   34   Response  To  Inorganic  Substance   66   Secondary  Metabolic  Process   55   Apoptosis   29   Calcium  Ion  Binding   54   Response  To  Abiotic  Stimulus   123   Response  To  Endogenous  Stimulus   104   Defense  Response  To  Fungus   10   Cell  Wall  Modification   24   Response  To  Jasmonic  Acid  Stimulus   26   Phospholipid-­‐Translocating  ATPase  Activity   7   Aminophospholipid  Transporter  Activity   7   Response  To  Salt  Stress   47   Indole  Derivative  Biosynthetic  Process   11   Sulfur  Metabolic  Process   27   Response  To  Endoplasmic  Reticulum  Stress   7   FAD  Binding   27   Response  To  Osmotic  Stress   49   Response  To  Oxidative  Stress   38   Positive  Regulation  Of  Response  To  Stimulus   13   Regulation  Of  Innate  Immune  Response   10   Response  To  Light  Stimulus   54   Salicylic  Acid  Mediated  Signaling  Pathway   9   Calmodulin  Binding   27   S-­‐Glycoside  Metabolic  Process   11   Glucosinolate  Metabolic  Process   11   Positive  Regulation  Of  Defense  Response   9   Protein  Tyrosine  Kinase  Activity   40   Response  To  Radiation   54   Response  To  Ozone   8   Acid-­‐Amino  Acid  Ligase  Activity   38   Amino  Acid  Derivative  Biosynthetic  Process   27   Ligase  Activity   43   Cofactor  Binding   59   Intracellular  Signaling  Cascade   80   Response  To  Hormone  Stimulus   90   140 92   643   14   42   215   540   423   172   405   1197   975   26   135   156   12   12   369   37   171   14   164   399   287   55   35   466   29   175   44   44   31   312   482   25   294   194   349   525   790   909   2.89E-­‐04   6.41E-­‐04   1.16E-­‐03   1.42E-­‐03   1.50E-­‐03   1.52E-­‐03   1.52E-­‐03   1.88E-­‐03   1.95E-­‐03   2.22E-­‐03   2.24E-­‐03   2.28E-­‐03   3.87E-­‐03   5.30E-­‐03   6.64E-­‐03   6.64E-­‐03   7.50E-­‐03   7.62E-­‐03   8.46E-­‐03   8.55E-­‐03   9.39E-­‐03   1.16E-­‐02   1.41E-­‐02   1.45E-­‐02   2.02E-­‐02   2.06E-­‐02   2.27E-­‐02   2.46E-­‐02   2.71E-­‐02   2.71E-­‐02   3.50E-­‐02   3.85E-­‐02   4.00E-­‐02   4.01E-­‐02   4.27E-­‐02   4.35E-­‐02   4.62E-­‐02   4.63E-­‐02   4.81E-­‐02   4.87E-­‐02   Genes contributing to differences in freezing tolerance: Category 2 FTD candidate genes. Though there is no difference in freezing tolerance between SW and IT with 1 week of cold-acclimation, analysis of SW and IT transcriptomes suggests that they reach this similar level of freezing tolerance by different mechanisms (Fig 3.9). It is possible that transcriptional differences at 1 week of cold-acclimation do not result in differences in freezing tolerance until 2 weeks of cold-acclimation. It is also possible that differences in cold-regulated gene expression at 1 week and 2 weeks of coldacclimation couple to result in differences in freezing tolerance at 2 weeks of coldacclimation. Based on this, Category 2 FTD candidate genes are cold-induced genes (in either SW or IT) with significantly different levels of expression at both 1 and 2 weeks of cold-acclimation. There are 798 Category 2 FTD candidate genes (Fig 3.26). Out of 798 Category 2 genes, 294 have higher expression in SW compared to IT at 2 weeks of coldacclimation (Fig 3.26; Table A3.5). It was also determined if these genes have been previously associated with cold-acclimation. These 294 genes are significantly enriched (39 genes; p<2.2E-16) in genes up-regulated the CBFs (Table A3.5). These 294 genes are also significantly enriched in Hannah genes positively correlated with freezing tolerance (Fig 3.26). There are 32 Hannah genes positively correlated with freezing tolerance that overlap with the Category 2 genes with higher expression in SW. This overlap is statistically significant, and 8 genes have greater than 4-fold higher expression in SW compared to IT, 15 are greater than 2-fold higher and 9 are less than 2-fold higher (Fig 3.27). ‘Response to cold’ is the GO category most overrepresented in   141 these 294 genes, and other abiotic stress GO terms such as ‘response to water deprivation’ are also enriched (Table 3.15). There are 504 out of 798 Category 2 FTD candidate genes with higher expression in IT compared to SW at 2 weeks of cold-acclimation (Fig. 3.26; Table A3.6). The 504 genes with higher expression in IT are not enriched in genes up-regulated (2 genes; p=0.97) or down-regulated (3 genes; p=0.92) by the CBFs (Table A3.6). However, these 504 genes are significantly enriched in Hannah genes both positively and negatively correlated with increases in freezing tolerance. There are 12 Hannah genes positively correlated with freezing tolerance that overlap with the Category 2 genes with higher expression in IT than SW. This overlap is statistically significant, but less significant than the overlap between Category 2 genes with higher expression in SW and Hannah genes positively correlated with freezing tolerance (Fig 3.27). Of the 12 genes high in IT that overlap with the Hannah gene set, 2 are greater than 4-fold higher in IT compared to SW, 7 are greater than 2 fold higher, and 3 are less than 2-fold higher (Fig. 3.28). There are 25 Hannah genes negatively correlated with freezing tolerance that overlap with the Category 2 genes with higher expression in IT compared to SW. These 25 genes negatively correlated with freezing tolerance thus, have lower expression in SW. Of these 25 genes, 4 have greater than 4-fold higher expression in IT, 10 are greater than 2-fold higher, and 11 are less than 2-fold higher (Fig 3.29). The 504 Category 2 FTD candidate genes are not enriched in temperature-related GO terms. But ‘defense response’ and ‘biotic interaction’ related GO terms are highly enriched (Table 3.16).   142 In Summary, Category 2 FTD candidate genes with higher expression in SW are more enriched in genes that have been previously associated with cold-acclimation than IT. This is consistent with analysis of Category 1 FTD genes and cold-regulated transcriptomes at 1 week of cold-acclimation. This again suggests that IT may coldacclimate using pathways not yet well defined. Since IT decreases in freezing tolerance from 1 to 2 weeks of cold-acclimation it also suggests that IT pathways of coldacclimation are not as efficient as pathways in SW.   143 798"CATEGORY"2" CANDIDATE"GENES" 294"GENES"ARE" HIGHER"IN"SW"AT" 2WKS" CAT."2"" SW, HIGHER" 262" HANNAH" POSITIVE" 32" COLD" 245" p<2.2"x"10,16" CAT."2" SW, HIGHER" 293" 504"GENES"ARE" HIGHER"IN"IT"AT" 2WKS" HANNAH" CAT."2"" POSITIVE" IT,HIGHER" 12" COLD"" 492" " 265" p=0.024" HANNAH" NEGATIVE" 1" COLD" 465" p=0.99" HANNAH" CAT."2"" NEGATIVE" IT,HIGHER" 25" COLD" 479" "441" p=6.6"x"10,05" Figure 3.26. 798 Category 2 FTD candidate genes and their overlap with the Hannah gene set. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 week under a 12 h photoperiod then sampled. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Genes were considered differentially expressed from non-acclimated conditions if the FDR corrected p-value was ≤0.05. ≥3FPKM was used as the cut-off for differential expression (19, 20). Genes were considered differentially expressed between SW and IT if the FDR corrected p-value was ≤0.05. P-values (calculated using a hypergeometric distribution in R) represent overlap between differentially expressed genes and Hannah genes (277 genes positively correlated with freezing tolerance and 466 genes negatively correlated with freezing tolerance; (6)).   144 CAT."2"" SW, HIGHER" 262" HANNAH" POSITIVE" 32" COLD"" " 245" p<2.2"x"10,16" 8"are">4"fold" higher"in"SW" "15"are">2"fold" higher"in"SW" 9"are"<2"fold" higher"in"SW" p=0.031" p=2.7"x"10,12" p<2.2"x"10,16" Figure 3.27. Fold change of 32 Hannah genes that overlap with 294 Category 2 FTD genes with higher expression in SW. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-off were applied (from left to right).   145 Table 3.15. GO categories significantly enriched in 294 Category 2 genes with higher expression in SW at 2 weeks of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 294 Category 2 genes with higher expression in SW at 2 weeks of coldacclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   #   SW  HIGH  CATEGORY  2  (294  GENES)   CATEGORY   HITS   BP   Response  To  Cold   22   BP   Response  To  Temperature  Stimulus   25   BP   Response  To  Water   17   BP   Response  To  Water  Deprivation   16   BP   Response  To  Abiotic  Stimulus   40   BP   Cold  Acclimation   6   BP   Response  To  Abscisic  Acid  Stimulus   13   BP   Programmed  Cell  Death   11   BP   Response  To  Organic  Substance   30     146 #  GENES   IN  GO   233   354   185   176   1197   20   285   223   1176   FDR   P-­‐VALUE   1.81E-­‐09   1.04E-­‐08   4.77E-­‐07   1.41E-­‐06   8.06E-­‐06   4.48E-­‐04   2.96E-­‐02   4.33E-­‐02   4.46E-­‐02   HANNAH" CAT."2"" POSITIVE" IT,HIGHER" 12" COLD"" 492" " 265" p=0.024" 2"are">4"fold" higher"in"IT" "7"are">2"fold" higher"in"IT" 3"are"<2"fold" higher"in"IT" p=0.99" p=0.18" p<=0.024" Figure 3.28. Fold change of 12 Hannah genes that overlap with 504 Category 2 FTD genes with higher expression in IT. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-off were applied (from left to right). HANNAH" CAT."2"" NEGATIVE" IT,HIGHER" 25" COLD"" 479" " 441" p=6.6"x"10,05" 4"are">4"fold" higher"in"IT" "10"are">2"fold" higher"in"IT" 11"are"<2"fold" higher"in"IT" p=0.99" p=0.17" p=6.6"x"10,05" Figure 3.29. Fold change of 25 Hannah genes that overlap with 504 Category 2 FTD genes with higher expression in IT. P-values represent the significance of overlap between gene only up-regulated in SW and Hannah genes if 4-fold, 2-fold, or no fold change cut-off were applied (from left to right).   147 Table 3.16. GO categories significantly enriched in 504 Category 2 genes with higher expression in IT at 2 weeks of cold-acclimation. GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in 504 Category 2 genes with higher expression in IT at 2 weeks of coldacclimation. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   CATEGORY   BP   BP   BP   BP   BP   BP   BP   CC   BP   BP   CC   CC   BP   CC   BP   BP   CC   BP   CC   CC     IT  HIGH  CATEGORY  2  (504  GENES)   Innate  Immune  Response   Defense  Response   Response  To  Bacterium   Response  To  Organic  Substance   Response  To  Endogenous  Stimulus   Secondary  Metabolic  Process   Response  To  Salicylic  Acid  Stimulus   Vacuole   Response  To  Hormone  Stimulus   Oxidation  Reduction   Extracellular  Region   Apoplast   Toxin  Metabolic  Process   Intrinsic  To  Plasma  Membrane   Cell  Death   Systemic  Acquired  Resistance   External  Encapsulating  Structure   Response  To  Jasmonic  Acid  Stimulus   Anchored  To  Plasma  Membrane   Cell  Wall   148 #   HITS   26   56   21   54   44   25   14   31   39   47   44   20   7   9   16   6   26   12   7   25   #  GENES   IN  GO   275   1030   244   1176   975   423   150   643   909   1186   1225   382   46   103   254   32   620   156   66   611   FDR     P-­‐VALUE   1.55E-­‐06   1.74E-­‐06   1.51E-­‐04   1.77E-­‐04   2.46E-­‐03   3.26E-­‐03   3.59E-­‐03   4.25E-­‐03   1.60E-­‐02   1.70E-­‐02   2.13E-­‐02   2.82E-­‐02   3.46E-­‐02   3.75E-­‐02   3.82E-­‐02   3.96E-­‐02   4.25E-­‐02   4.29E-­‐02   4.76E-­‐02   4.91E-­‐02   Summary of SW and IT transcriptomes. Through analysis of basal, as well as 1 and 2 week cold-acclimated transcriptomes two sets of FTD candidate genes have been defined. Furthermore, several interesting trends regarding differences between SW and IT seem to have emerged through this investigation. Analysis of RNA-seq data suggests that SW and IT cold-acclimate through significantly different transcriptional changes. Comparisons of SW and IT cold-regulated transcriptomes to CBF regulon genes (25) and Hannah genes (6), which are enriched in CBF regulon genes, seem to indicate that SW is more enriched in genes that have been previously associated with cold-acclimation. This suggests that SW may use previously defined pathways of coldacclimation, such as the CBF pathway, while IT may use pathways of cold-acclimation not yet well defined. However, differences between SW and IT were determined without fold-change thresholds. Therefore, the fold-change of expression data was visually examined using heatmaps to ensure that these trends are consistent. Visual analysis of cold-regulated genes suggests that most genes have similar rather than opposite cold-induction patterns in SW and IT, however most cold-regulated genes differ between SW and IT in terms of expression level (Fig 3.9, 3.23, 3.26). This seems consistent with previous analysis using FDR corrected p-value thresholds. Under non-acclimated conditions, genes up-regulated by the CBFs (25) appear to have higher expression in IT, which is consistent with previous analysis of basal transcriptomes using FDR corrected p-value thresholds. The difference in basal expression of CBF regulon genes again suggests that the CBF pathway of coldacclimation is regulated differently between SW and IT. The CBFs are cold-responsive   149 genes (29). With the addition of cold, genes up-regulated by the CBFs generally have higher expression in SW than IT, and genes down-regulated by the CBFs are more repressed in SW (Fig 3.31). There also appears to be more CBF regulon genes responsive to cold in SW in comparison to IT (Fig. 3.3.1). Altogether, analysis of RNAseq data using fold-change values still indicates that the CBF pathway of coldacclimation may be differentially regulated between SW and IT. Fold-change analysis of Hannah genes (6), shows that SW generally has higher expression of genes previously positively correlated with freezing tolerance, and has lower expression of genes previously negatively correlated with freezing tolerance in comparison to IT (Fig. 3.32). Altogether, analysis of RNA-seq data by fold-change still suggests that SW is more enriched in genes that have been previously associated with cold-acclimation than IT (Fig. 3.31 and 3.32). GO enrichment analysis also revealed several trends in SW and IT transcriptomes. Under all conditions, IT seems to be enriched in genes related to biotic stress. Therefore, biotic stress related genes were pulled from TAIR (www.arabidopsis.org), to determine if this is generally the case. Differentially expressed biotic genes between SW and IT were defined by a FDR corrected p-value ≤0.050 (384 genes differentially expressed out of 925). A heatmap was generated for these biotic stress genes to visually compare fold-change differences between SW and IT expression. IT does appear to have higher expression of biotic stress related genes under both warm and cold conditions in comparison to SW (Fig 3.33), but this difference is most pronounced at 2 weeks of cold-acclimation. Also, more biotic stress related   150 genes appear to be up-regulated by cold in IT at 2 weeks of cold-acclimation in comparison to SW (Fig. 3.33). Examination of IT transcripts suggests that IT is less enriched in pathways of cold-acclimation that have been previously defined. Consequently, some of these biotic stress related genes may function in coldacclimation, especially since pathogens related proteins have previously been shown to have antifreeze properties (26). Furthermore, resistance to biotic stress may be an interesting trait segregating between the SW and IT ecotypes and should be tested in the SW and IT RILs in the future. Photosynthesis was another interesting reoccurring GO term for SW and IT. It is well known that many transcriptional as well a metabolic changes occur during coldacclimation (23, 31-34). Photosynthesis provides the energy for these necessary changes to occur (35). Increased photosynthetic capacity is consequently corrleated with cold-acclimation (24, 36-39). Therefore, differentially expressed photosynthesis associated genes (list of genes provided by Hu and Thomashow, unpublished) between SW and IT were determined by a FDR corrected p-value ≤0.050 (52 genes differentially expressed out of 244). A heatmap was generated for these photosynthesis genes to visually compare the fold-change differences between SW and IT expression (Fig 3.34). Although most photosynthesis related genes appear to be down-regulated by cold in both SW and IT, SW appears to have higher expression of photosynthesis related genes under both warm and cold conditions in comparison to IT (Fig 3.34). This may indicate that there are differences in the photosynthetic capacity of SW and IT. Differences in photosynthetic capacity would correlate with differences in freezing   151 tolerance between SW and IT at 2 weeks of cold-acclimation. Nevertheless, differences in photosynthetic capacity would need to be measured experimentally. Variations in photosynthetic capacity would also be an interesting trait to screen and map in the SW and IT RILs. In the next section of this study, expression of Category 1 and Category 2 FTD genes are examined in the SW and IT RILs, which were constructed by the Schemske Lab. This eQTL analysis allows expression differences to be mapped to specific genetic locations, and genes potentially important for differences in freezing tolerance as well as fitness QTL can also be determined (Table 3.1). FTD candidate genes that are also CBF regulon genes will be targeted for this study since CBF regulon genes appear to be enriched in differentially expressed genes. Furthermore, FTD candidate genes annotated as ‘photosynthesis related’ and ‘biotic stress related’ will be included in eQTL analysis since those GO terms appear to be overrepresented.   152 SW" SW" IT" IT" 1WK" 2WK" 1WK" 2WK" NON" 1WK" 2WK" ITALY" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " SWEDEN" LOG(2)" LOG(2)" UP,REG" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " DOWN,REG" Figure 3.30. Fold change of cold regulated genes in SW and IT (7863 genes). Gene expression was measured by RNA-seq. The results are averages from three independent experiments (n=3). ≥3FPKM was used as the cut-off for differential expression (19, 20). Genes were considered differentially regulated between nonacclimated and cold conditions if the FDR corrected p-value was ≤0.050. Left panel values are the logarithm (base=2) of the fold-change between non-acclimated and cold conditions; negative values denote genes down-regulated by cold (green), positive values denote genes up-regulated by cold (red). Right panel values are the logarithm (base=2) of the fold-change between SW and IT; negative values denote higher fold expression in SW (green) and positive values denote higher fold expression IT (red). Hierarchical clustering of genes was performed with the program, Cluster (40), and heatmap was generated using MapleTree (40). For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation.   153 SW" SW" IT" IT" 1WK" 2WK" 1WK" 2WK" NON" 1WK" 2WK" ITALY" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " SWEDEN" LOG(2)" LOG(2)" UP,REG" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " DOWN,REG" Figure 3.31. Fold change of CBF regulon genes in SW and IT. Top panels: 159 genes up-regulated by CBFs; Bottom panels: 55 genes down-regulated by CBFs (25). Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). ≥3FPKM was used as the cut-off for differential expression (19, 20). Left panels are the logarithm (base=2) of the fold-change between non-acclimated and cold conditions; negative values denote genes down-regulated by cold (green), positive values denote genes up-regulated by cold (red). Right panels are the logarithm (base=2) of the fold-change between SW and IT; negative values denote higher fold expression in SW (green) and positive values denote higher fold expression IT (red). Hierarchical clustering of genes was performed with the program, Cluster (40), and heatmap was generated using MapleTree (40).   154 SW" SW" IT" IT" 1WK" 2WK" 1WK" 2WK" NON" 1WK" 2WK" ITALY" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " SWEDEN" LOG(2)" LOG(2)" UP,REG" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " DOWN,REG" Figure 3.32. Fold change of Hannah genes in SW and IT. Top panels: 253 Hannah genes positively correlated with freezing tolerance; Bottom panels: 461 Hannah genes negatively correlated with freezing tolerance; (6). Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). ≥3FPKM was used as the cut-off for differential expression (19, 20). Left panels are the logarithm (base=2) of the fold-change between non-acclimated and cold conditions; negative values denote genes down-regulated by cold (green), positive values denote genes up-regulated by cold (red). Right panels are the logarithm (base=2) of the fold-change between SW and IT; negative values denote higher fold expression in SW (green) and positive values denote higher fold expression IT (red). Hierarchical clustering of genes was performed with the program, Cluster (40), and heatmap was generated using MapleTree (40).   155 SW" SW" IT" IT" 1WK" 2WK" 1WK" 2WK" NON" 1WK" 2WK" ITALY" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " SWEDEN" LOG(2)" LOG(2)" UP,REG" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " DOWN,REG" Figure 3.33. Fold change of differentially expressed biotic stress genes in SW and IT (348 genes). Gene expression was measured by RNA-seq. The results are averages from three independent experiments (n=3). ≥3FPKM was used as the cut-off for differential expression (19, 20). Genes were included if they were differentially expressed (FDR corrected p-value<0.05) between SW and IT in any sample (nonacclimated, 1 week of cold-acclimation, or 2 weeks of cold-acclimation). Left panels are the logarithm (base=2) of the fold-change between non-acclimated and cold conditions; negative values denote genes down-regulated by cold (green), positive values denote genes up-regulated by cold (red). Right panels are the logarithm (base=2) of the foldchange between SW and IT; negative values denote higher fold expression in SW (green) and positive values denote higher fold expression IT (red). Hierarchical clustering of genes was performed with the program, Cluster (40), and heatmap was generated using MapleTree (40).   156 SW" SW" IT" IT" 1WK" 2WK" 1WK" 2WK" NON" 1WK" 2WK" ITALY" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " SWEDEN" LOG(2)" LOG(2)" UP,REG" 3.00" 2.00" 1.00" 0.00" ,1.00" ,2.00" ,3.00" " DOWN,REG" Figure 3.34. Fold change of differentially expressed photosynthesis associated genes in SW and IT (53 genes). Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). ≥3FPKM was used as the cut-off for differential expression (19, 20). Genes were included in the heatmap if they were differentially expressed (FDR corrected p-value<0.05) between SW and IT in any sample (non-acclimated, 1 week of cold-acclimation, or 2 weeks of cold-acclimation). Left panels are the logarithm (base=2) of the fold-change between non-acclimated and cold conditions; negative values denote genes down-regulated by cold (green), positive values denote genes up-regulated by cold (red). Right panels are the logarithm (base=2) of the fold-change between SW and IT; negative values denote higher fold expression in SW (green) and positive values denote higher fold expression IT (red). Hierarchical clustering of genes was performed with the program, Cluster (40), and heatmap was generated using MapleTree (40).   157 RESULTS AND DISCUSSION: SW AND IT EQTL MAPPING Summary: SW and IT eQTL mapping: In the second section of this study Category 1 and Category 2 FTD genes, which possibly contribute to differences in freezing tolerance between SW and IT accessions at 2 weeks of cold-acclimation, were determined. FTD genes are enriched in CBF regulon genes and QTL mapped for differences in expression (eQTL) for several CBF-regulated FTD candidate genes mapped to the CBF locus on chromosome 4. This eQTL on chromosome 4 also overlaps with a fitness QTL mapped in the SW and IT RILs (Table 3.1). Consequently, the CBFs are candidates for regulating differences in FTD gene expression and fitness between SW and IT. Analysis of IT-CBF2 coding sequence reveals a deletion in the activation domain. Based on previous studies conducted in the Thomashow lab, it is hypothesized that IT-CBF2 functions as a dominant negative version of CBF2. RNAseq data is generally consistent with this hypothesis. SW and IT eQTL mapping: The ultimate goal of this project is to determine the genes underlying the fitness QTL mapped using the SW and IT RILs (Table 3.1). In general, QTL peaks can span a large genomic region encompassing numerous genes (12, 13, 41). The fitness QTL found for the SW and IT RILs are certainly no exception (Table 3.1). For example, the fruit-set fitness QTL on chromosome 4 spans a region containing 2065 genes (Table 3.1). Many of these genes might contain polymorphisms, which may or may not contribute to fitness (12, 13). Moreover, 24% of genes in the Arabidopsis (Col-0) genome are included within SW and IT fitness QTL (Table 3.1).   158 Consequently, determining the specific genes responsible for a QTL can be extremely challenging (5, 12, 13, 41). Minimum winter temperature was strongly correlated with fitness of the SW and IT parent lines in reciprocal transplant experiments (8), and freezing tolerance was found to be significantly different (p≤0.026 at 2 weeks of cold-acclimation) between SW and IT in laboratory experiments (Fig. 3.2). From analysis of RNA-seq data, genes that may contribute to differences in freezing tolerance between SW and IT have been defined (Table A3.3-A3.6). Similar to traditional QTL, an eQTL marks a position in the genome where a polymorphism is associated with differences in the relative abundance of a specific gene (12). Although correlative, eQTL mapped for FTD candidate genes, which also overlap with specific fitness QTL, can be determined. This will help to prioritize the tedious construction of near-isogenic lines (9) or complementation lines (41), which are needed to confirm if a gene affects freezing tolerance as well as plant fitness. An eQTL is normally classified as being cis- or trans- to the gene used for mapping. Trans-eQTL are not physically linked to the gene of interest, and thus, are indicative of a polymorphism that alters the regulation of the gene of interest (12). Likely candidates for trans-eQTL are transcription factor polymorphisms. A cis-eQTL is located in or near the gene of interest. This can result when there is a deletion of an entire gene or a polymorphism in the promoter or coding region that affects regulation or function of that gene (12).   159 10 FTD candidate genes (Table 3.17; Fig. 3.35 and 3.36) were selected for eQTL analysis with the SW and IT RIL population generated by the Schemske Lab (Schemske and Agren, unpublished). From analysis of SW and IT transcriptomes in the second section of this study, CBF regulon genes are consistently differentially regulated between SW and IT. Therefore, 1-Category 1 and 3-Category 2 FTD genes that are also regulated by the CBFs, were selected for eQTL analysis (Table 3.17). Even if eQTL mapped for these CBF regulated genes do not overlap with fitness QTL, it will determine if differences in CBF regulated genes map cis- to the gene itself, or trans- to novel loci or the CBF locus. COR314, SZF1, and DI21 were chosen for eQTL analysis because they are FTD candidate genes not included in the CBF regulon, and thus represent genes that are potentially regulated by CBF-independent (42) pathways of cold-acclimation (Table 3.17). ZAT12, a Category 1 FTD gene was selected for eQTL analysis because ZAT12 regulon genes are enriched in the Hannah gene set (6), which was found to have differing enrichment in SW and IT in the second section of this study. Biotic stress was a GO category overrepresented in Category 2 FTD genes (Table 3.16) and a number of biotic stress genes appear to be differentially regulated by cold between SW and IT (Fig 3.33). Therefore, PR5, a Category 2 FTD gene that encodes a protein, which has been previously been shown to have antifreeze properties (26), was selected for eQTL analysis (Table 3.17). Photosynthesis was a GO category overrepresented in Category 1 FTD candidate genes (Table 3.13), and a number of photosynthesis related genes are differentially regulated between SW and IT (Fig. 3.34). Therefore ZAT10, a Category 1 FTD gene and a photosynthesis-related transcription   160 factor, was selected for eQTL analysis (Table 3.17). Altogether, 4 of these FTD genes have higher expression in IT and 6 have higher expression in SW at 2 weeks of coldacclimation (Fig 3.35 and 3.36; Table A3.3-3.6). SW and IT accessions, sampled at the same time as RNA-seq experiments, were also analyzed by qRT-PCR (Fig. 3.35 and 3.36). This ensured that expression differences measured by qRT-PCR were similar to RNA-seq experiments (Fig. 3.35 and 3.36). 544 genotyped RILs were grown similarly to plants used in electrolyte leakage experiments and sampled at 2 weeks of cold-acclimation, which is the acclimation period with the greatest difference in freezing tolerance (Fig. 3.2). For the RILs, expression of the 10 FTD genes was measured using qRT-PCR and differences were mapped using RQTL (15). RQTL was also used to calculate genome wide logarithm of odds (LOD) threshold values as well as the percent of phenotypic variance explained by each QTL (Table 3.18 and 3.19). Distinct eQTL above LOD thresholds were found for all genes except ZAT10 (Table 3.18 and 3.19). Quantitative traits like differences in gene expression are defined by small and intermediate effect QTL (43). The average percent of variance explained by each eQTL in this study (Table 3.18 and 3.19) is lower (5.32%) than previous genome wide eQTL studies in Arabidopsis, which see percent variance explained by eQTL averaging from 10-30% (13, 44). However, the ‘Beavis effect’ indicates that percent variance of a QTL is greatly overestimated if only 100 lines are tested, and are only slightly overestimated with 500 lines tested (43). This study uses 544 RILs, more than double the number of RILs used in eQTL studies by West et al. 2007 (211 lines used) and Keurentjes et al.   161 2007 (160 lines used) (13, 44). Consequently, the lower percent variance explained by eQTL in this study may be due to the Beavis effect (43). When 160 SW and IT RILs are randomly selected for COR15A eQTL analysis, the same QTL on chromosome 4 is found (Table 3.18). However, the LOD score for the COR15A eQTL decreases from 10.09 to 6.19 with 160 samples, while the percent variance explained by the eQTL increases from 8.39% to 16.33% with 160 samples suggesting that the lower percent variance explained by eQTL with 544 RILs may be due to the Beavis effect (Table 3.18). The eQTL, which were found to overlap with fitness QTL (Table 3.1), will be analyzed next and candidates for the genes underlying them will be provided. Genes with eQTL that do not overlap with fitness QTL will be discussed in more detail in Chapter Four of this study. Expression differences in CBF regulon gene, COR78, map to two eQTL (Fig 3.37; Table 3.1 and 3.18). The first eQTL maps to a region on chromosome 5, which overlaps with a previously identified fitness QTL (Fig 3.37; Table 3.1 and 3.18). This may be a cis-eQTL since the COR78 gene is located (between markers 60.60 and 61.28) within the eQTL peak on chromosome 5. COR78 is a Hannah gene positively correlated with increases in freezing tolerance and is regularly used as a marker gene for both cold and salt stress (6, 16, 45, 46). Although the coding sequence of COR78 is conserved between SW, IT and Col-0, it is known that the response of COR78 to cold and salt stress is driven through its promoter (47). In fact, the COR78 promoter is often employed as a stress-inducible promoter for other genes (48-52). Therefore, polymorphisms in this region are likely to influence expression. According to the effect   162 plot, the SW genotype at this eQTL is associated with higher expression of COR78 (Fig. 3.35). Currently, there is little evidence to indicate that COR78 alone can affect freezing tolerance (53). However, these results advocate sequencing of the COR78 promoter, construction of near-isogenic lines (9) for the region containing COR78 and suggest that complementation lines (41) should be made. There is an alternate hypothesis for the COR78 eQTL on chromosome 5. COR78 is known to be regulated by cold-induced transcription factors CBF1-3 (24), which are located on chromosome 4. But CBF4, a closely related transcription factor, is located within the peak of the chromosome 5 eQTL (Fig 3.37; Table 3.19), and moreover between the same SW and IT RIL markers as COR78 (markers 60.60 and 61.28). CBF4 has also been shown to regulate COR78, and overexpression of CBF4 results in constitutive freezing and drought tolerance (54). CBF4 expression is not known to be cold-regulated but it is regulated by drought (54). The RNA-seq data is consistent with this result and CBF4 is not significantly (p>0.050) induced by cold (Fig. 3.43; Table 3.20). There are also no differences in CBF4 expression level between SW and IT (Fig. 3.43; Table 3.20). However, CBF4 may be regulated by cold posttranscriptionally and this regulation may differ between SW and IT. Therefore, CBF4 coding and promoter regions in SW and IT should be sequenced and complementation lines (41) for CBF4 should be made. COR78, also has a trans-eQTL on chromosome 4, which explains more of the expression variance than the trans/cis-eQTL on chromosome 5 (Fig 3.37; Table 3.18). Interestingly, this trans-eQTL on chromosome 4 overlaps with a previously defined   163 fitness QTL (Table 3.1 and 3.20). 5 other FTD genes used for eQTL analysis, COR15A, COR47, COR314, EARLY RESPONSIVE TO DEHYDRATION10 (ERD10) and ZAT12, also have an eQTL that overlaps with this fitness QTL on chromosome 4 (Fig. 3.373.42; Table 3.1, 3.18 and 3.19). CBF1-3 are located in tandem array on chromosome 4 between SW and IT RIL markers 50.20 and 50.60. The CBF locus is thus contained within the chromosome 4 eQTL of all 6 FTD genes (Fig. 3.37-3.42; Table 3.1, 3.18 and 3.19). Expression of COR15A, COR47, COR78, and ERD10 was mapped knowing that they were previously found to be regulated by the CBF genes (Table 3.17; (25)). Based on the CBF regulon defined by Vogel et al. 2005, expression differences mapping to the CBF locus for COR314 and ZAT12 were not expected (25). However, in a separate analysis of CBF overexpression lines by Maruyama et al. 2004, COR314 was found to be regulated by CBF genes (55). Furthermore, though ZAT12 is not part of the CBF regulon, it does have a regulatory relationship with the transcription factors (25). The 6 effect plots for the chromosome 4 eQTL also all indicate that the SW genotype is associated with higher expression (Fig. 3.37-3.42). This is interesting considering that ZAT12 has an opposite induction pattern compared to the other genes used for mapping (Fig. 3.35 and 3.36). However, the details of the CBF-ZAT12 relationship have yet to be elucidated. ZAT12 is a cold-regulated transcription factor known to induce and repress cold-responsive genes (25, 42). CBF and ZAT12 regulons overlap and both regulons are overrepresented in the Hannah gene set (6, 25). Overexpression of ZAT12 down-regulates CBF expression and these plants are slightly more freezing tolerant than wild-type plants, but not nearly as freezing tolerant as CBF   164 overexpressing plants (25). CBF and ZAT12 overexpressing plants both have a dwarf phenotype. However plants overexpressing both ZAT12 and CBF2 are similar in size to wild-type plants (Doherty, unpublished). Consequently, it may be possible that the CBFs regulate ZAT12 in some way, but further work is necessary to elucidate the relationship of the CBFs to ZAT12. Previous natural variation studies on freezing tolerance have also implicated the CBFs as a source of variation. Alonso-Blanco et al. 2005 used RILs constructed from Arabidopsis accessions LER and CVI to map differences in freezing tolerance (10). Seven different QTL on chromosomes 1,4, and 5 were found, but the single QTL on chromosome 4, which contained the CBF locus, explained the most variance (10). CVI freezing tolerance was significantly reduced in comparison to LER, as was CBF2 expression. The CVI-CBF2 promoter was found to have a 1630 bp deletion (starting at 160), reducing the promoter region to 378 bp (10). Introduction of the Ler-CBF2 transgene into CVI significantly reduced differences in freezing tolerance between LER and CVI (10). CBF expression in the Versailles core collection of Arabidopsis (48 accessions tested) was also found to vary significantly (56). However, freezing tolerance did not correlate simply with CBF expression level in the 8 accessions tested (56). The most freezing-sensitive Versailles accessions were also the accessions with the lowest maximal cold-induced CBF expression levels. But the most freezing tolerant Versailles accessions varied significantly in their CBF expression, suggesting the importance of CBF-independent pathways of freezing tolerance for some accessions (56). It should   165 be pointed out, however, that in this Versailles study, CBF expression measured after hours in cold was correlated with freezing tolerance assays conducted after 2 weeks of cold-acclimation (56). Therefore in the Versailles study, CBF expression and freezing tolerance assays were conducted at very different time-points. In future studies, CBF expression should be measured at several time-points, but especially at time-points just as to 2 week cold-acclimated freezing tolerance assays are conducted to determine if CBF expression and CBF regulon gene expression correlates with differences in freezing tolerance between accessions. Freezing tolerance QTL differed between two Versailles RIL populations (56). However, in both populations a common QTL on chromosome 4 explained the most variance in freezing tolerance and overlapped with the CBF locus (56). Further analysis of CBF gene sequence in the 48 Versailles accessions showed a number of polymorphisms in both the promoter and coding sequences of CBF genes (57). The most notable of these accessions were Bl-1 with a 10 amino acid deletion at the end of the activation domain in CBF1, Condara with a premature stop codon in the activation domain of CBF3, and Gre-0 with a glycine to arginine change in the activation domain of CBF2 (57). Unfortunately, these 3 particular accessions were not chosen for freezing tolerance assays or tested for CBF target gene expression (57). A recent study by Zhen et al. 2008 further showed that CBF genes undergo relaxed selection in Arabidopsis accessions native to southern latitudes(58). Analysis of CBF sequence in 24 accession of Arabidopsis (9 accessions overlap with 48 Versailles accessions previously mentioned (56, 57)) found a large bias toward nonsynonymous   166 changes in the activation domain compared to DNA binding domain of CBF genes (58). Southern latitude accession had 1.3 to 4.6-fold more polymorphisms in the individual CBF genes and also had reduced expression of CBF target genes (58). CBF1 and CBF3 coding sequences were found to be conserved between SW and IT. Transcripts for CBF1 and CBF3 are also not significantly (p>0.050) differentially regulated between SW and IT at 2 weeks of cold-acclimation in RNA-seq experiments (Fig. 3.43; Table 3.20). This suggests that any polymorphisms in CBF1 and CBF3 promoters have little effect on gene expression. On the other hand, CBF2 transcripts are differentially regulated between SW and IT at 2 weeks of cold-acclimation (Fig. 3.43; Table 3.20). CBF2 expression is 1.5-fold higher in SW compared to IT at 2 weeks of cold-acclimation (Table 3.20). CBF1, 2 and 3 are known to regulate over a hundred target genes (25, 59, 60). Therefore, polymorphisms in the CBF2 promoter could contribute to differences in CBF2 expression and have compounding effects on target gene regulation. The coding sequence of CBF2 is conserved between SW and Col-0 (Fig. 3.44). However, there is a 13 basepair (bp) deletion (+384 to +397) in the activation domain (61) of IT-CBF2, though surrounding coding sequences are conserved between SW, IT, and Col-0 (Fig 3.44). This leads to a predicted protein (Fig. 3.42) with a nuclear localization signal (NLS) and DNA binding domain, but missing activation domain (61). This predicted protein would be similar to the Versailles accession, Condara, which has a premature stop codon in the activation domain of CBF3 (57). Freezing tolerance and CBF target gene expression was not tested in   167 Condara (56, 57). However, Condara freezing tolerance and CBF target gene expression may be predicted to be diminished in comparison to SW, similar to IT. A similar truncated version of CBF2 (CBF2ΔC), predicted to have a missing activation domain, was previously identified through an EMS screen of CBF2 overexpressing plants (Gilmour and Thomashow, unpublished). At 2 weeks of coldacclimation, the combined expression level of IT-CBF1 (3.02 FPKM) and IT-CBF3 (35.85 FPKM) is similar to IT-CBF2 (33.23 FPKM). Interestingly, CBF2ΔC was shown to function as a dominant negative protein in plants with equal expression of CBF2 and CBF2ΔC (Doherty, unpublished). CBF2ΔC overexpressing plants are also significantly impaired in cold-acclimation (Doherty, unpublished) and IT is less freezing tolerant than SW at 2 weeks of cold-acclimation. Transcriptional analysis of CBF2ΔC overexpressing plants revealed that 40% of cold-induced genes were affected (Doherty, unpublished). CBF regulon genes are overrepresented in genes differentially expressed between SW and IT; Category 1 FTD genes include 61 CBF regulon genes (p=4.3E-13; Table A3.33.4) and Category 2 FTD genes include 51 gene CBF regulon genes (p<2.2E-16; Table A3.5-A3.6). In general, cold-regulated gene expression is consistent with IT-CBF2 acting as a dominant negative version of CBF2; genes up-regulated by CBFs have lower expression in IT than SW and gene down-regulated by CBFs have higher expression in in IT than SW (Fig 3.31). Furthermore, there are fewer CBF regulon genes up-regulated by cold in IT in comparison to SW (Fig 3.31). Based on these results, I postulate that IT-CBF2 may function as a natural version of CBF2ΔC, in addition to effects of possible promoter polymorphisms. More experiments are necessary to test   168 this hypothesis, which would include determining CBF1-3 protein levels in IT and SW, as well as transforming the IT-CBF2 and SW-CBF2 into SW and IT, respectively. It is interesting that differences in freezing tolerance are not seen at both 1 and 2 weeks of cold-acclimation since IT-CBF2 would presumably act as a dominant negative protein at both times. Category 2 FTD candidate genes are enriched in CBF regulon genes and were defined as cold-regulated genes that are differentially expressed between SW and IT at both 1 and 2 weeks of cold acclimation. Therefore, IT-CBF2 does appear to be acting as a dominant negative version of CBF2 at both 1 and 2 weeks of cold-acclimation. However, there are also known to be CBF-independent pathways of cold acclimation (42), which are of varying importance to Arabidopsis accessions (56). Therefore, there may be CBF-independent pathways of coldacclimation, which compensate for the dominant negative IT-CBF2 at 1 week of cold but not 2 weeks. The analysis of IT transcriptomes at 1 and 2 weeks of cold-acclimation did reveal numerous genes, which are exclusively cold-regulated at either 1 or 2 weeks of cold-acclimation (Fig. 3.6). It is also of note that at 2 weeks of cold-acclimation SW freezing tolerance increases in addition to IT freezing tolerance decreasing. SW-CBF2 expression is significantly higher in comparison to IT-CBF2 at only 2 weeks of cold-acclimation. Therefore, in addition to IT-CBF2 possibly acting as a dominant negative protein, reducing freezing tolerance through interruption of the CBF pathway of cold-acclimation, polymorphisms in the SW-CBF2 promoter could be contributing to higher CBF2 expression and subsequent increase in freezing tolerance at 2 weeks of cold-   169 acclimation. Overall, CBF2 appears to be an excellent candidate gene to explain differences in cold-regulated gene expression and differences in freezing tolerance and fitness between SW and IT. Sequencing the CBF2 promoter in SW and IT, nearisogenic lines (9) for the region containing CBF2, and complementation lines (41) should be made a top priority in order to test how great a role CBF2 plays in differential expression of FTD candidate genes, freezing tolerance, and fitness.   170     Table 3.17. 10 FTD candidate genes for eQTL analysis. Genes are ordered by their AGI numbers. Genes were considered significantly different between SW and IT under non-acclimated conditions if they had a Benjamini-Hochberg corrected p-value (FDR P-VALUE) of <0.05 and a minimum of ≥3FPKM in either SW or IT. The differential expression of each gene is also represented by the logarithm (base=2) of the fold change (LOG2.FC). This table also denotes if a gene was previously described as positively or negatively correlating with freezing tolerance (HANNAH;(6)), a transcription factor (TF), a gene previously described as up- or down-regulated by 1 week of cold (COS, (25)), identified as an up- or down-regulated component of the CBF regulon (CBF REGULON; (25)) or identified as a photosynthesis associated genes (PAG; Hu and Thomashow, unpublished). Shaded genes have higher expression in SW and non-shaded genes have higher expression in IT. CATEGORY  1  FTD  CANDIDATES  FOR  eQTL  ANALYSIS       SW.IT.2WK     SW.IT.2WK           CBF     AGI   NAME   SHORT  DESCRIPTION   LOG2FC   FDR  P-­‐VALUE   HANNAH?   TF?   COS?   REG?   PAG?   AT1G27730   ZAT10   Salt  Tolerance  Zinc  Finger   2.11   0.00E+00   NO   YES   UP   NO   YES   AT1G29390   COR314   Cold  Regulated  314     -­‐0.97   1.74E-­‐08   NO   NO   NO   NO   NO   AT2G42540   COR15A   Cold-­‐Regulated  15a   -­‐1.88   2.90E-­‐08   NO   NO   UP   UP   NO   AT3G55980   SZF1   Salt-­‐Inducible  Zinc  Finger  1   1.63   0.00E+00   NO   YES   DOWN   NO   NO   AT5G59820   ZAT12   C2H2-­‐Type  Zinc  Finger  Family     1.75   1.85E-­‐14   NO   YES   NO   NO   NO     CATEGORY  2  FTD  CANDIDATES  FOR  eQTL  ANALYSIS      SW.IT.2WK   SW.IT.2WK.           CBF     AGI   NAME   SHORT  DESCRIPTION   LOG2FC   FDR  P-­‐VALUE   HANNAH?   TF?   COS?   REG?   PAGs   AT1G20440   COR47   Cold-­‐Regulated  47   -­‐2.51   0.00E+00   NO   NO   UP   UP   NO   AT1G20450   ERD10   Dehydrin  Family  Protein   -­‐1.96   0.00E+00   NO   NO   UP   UP   NO   AT1G75040   PR5   Pathogenesis-­‐Related  5   4.50   0.00E+00   NO   NO   UP   NO   NO   AT4G15910   DI21   Drought-­‐Induced  21   -­‐2.58   0.00E+00   NO   NO   NO   NO   NO   AT5G52310   COR78   Cold-­‐Regulated  78   -­‐2.83   0.00E+00   POSITIVE   NO   UP   UP   NO     171     ZAT10,RNA#SEQ, 50" 0" 0" NON" NON" FPKM, 20000" 0" 50" 0" 2WK" 1WK" 2WK" COR15A,RNA#SEQ, NON" 1WK" SFZ1,RNA#SEQ, NON" 1WK" 2WK" 20" RELATIVE,EXPRESSION, 0" 0" 1WK" COR314,RNA#SEQ, 500" 50" 5" SW" IT" 1WK" IT" COR314,QRT#PCR, SW" IT" 5" 0" 5" 5" 0" 2WK" SW" COR15A,QRT#PCR, 0" 2WK" ZAT12,RNA#SEQ, NON" 0" ZAT10,QRT#PCR, SW" IT" SW" IT" SFZ1,QRT#PCR, ZAT12,QRT#PCR, SW" IT" Figure 3.35. Category 1 FTD candidate genes for eQTL analysis. IT samples are shown in red, and SW in blue. The results presented are average values from three independent experiments (n=3). Error bars indicate ±SEM. For RNA-seq experiments, plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 or 2 weeks under a 12 h photoperiod then sampled. For qRT-PCR experiments, plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was transferred to 4⁰C for 2 weeks under a 12 h photoperiod then sampled. IPP2 was used as a housekeeping gene. Primer sequences can be found in Table A3.6.   172     COR47,RNA#SEQ, 2000" SW" IT" 0" 0" NON" SW" IT" SW" IT" ERD10,QRT#PCR, 0" 1WK" PR5,RNA#SEQ, NON" 1WK" DI21,RNA#SEQ, NON" 1WK" COR78,RNA#SEQ, 2WK" 2WK" RELATIVE,EXPRESSION, FPKM, NON" 10000" 2000" 2WK" COR47,QRT#PCR, 5" 0" 0" 1WK" ERD10,RNA#SEQ, 1000" 500" 5" 0" 10" 0" 50" 0" 2WK" 10" 0" 0" PR5,QRT#PCR, SW" IT" SW" IT" DI21,QRT#PCR, COR78,QRT#PCR, SW" IT" NON" 1WK" 2WK" Figure 3.36. Category 2 FTD candidate genes for eQTL analysis. IT samples are shown in red, and SW in blue. The results presented are average values from three independent experiments (n=3). Error bars indicate ±SEM. For RNA-seq experiments, plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 or 2 weeks under a 12 h photoperiod then sampled. For qRT-PCR experiments, plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was transferred to 4⁰C for 2 weeks under a 12 h photoperiod then sampled. IPP2 was used as a housekeeping gene. Primer sequences can be found in Table A3.6.   173     Table 3.18. Locations of eQTL for Category 1 FTD candidate genes. 544 RILs were used for eQTL mapping. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/). To detect eQTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LOD) values were calculated for each trait using 1000 permutations in RQTL. To estimate the location of each eQTL a Bayes credible interval (99%) was calculated in RQTL. For locations of markers based on the Col-0 genome please see Table A3.1. QTL are categorized as cis- or trans- (CIS/TRANS?). Shaded QTL overlap with previously identified fitness QTL (Schemske and Agren, unpublished). ‘MB’ indicates the number of megabases contained within the eQTL. ‘# GENES’ indicates the number of genes contained within the eQTL based on the Col-0 genome. %VAR indicates the estimated proportion of phenotypic variance explained by the QTL. AT1G27730-­‐ZAT10  (LOD  THRESHOLD=3.04)   CHR   LEFT  (CM)           PEAK  (CM)   NO  QTL         RIGHT  (CM)             AT1G29390-­‐COR314  (LOD    THRESHOLD=3.33)   CHR   LEFT  (CM)   4   50.00   PEAK  (CM)   55.52   RIGHT  (CM)   58.00           AT2G42540-­‐COR15A  (LOD  THRESHOLD=3.13)   CHR   LEFT  (CM)   4   50.000   PEAK  (CM)   50.004   PEAK  (CM)   33.42   22.00     PEAK  (CM)   11.18   50.60   48.00         MB   4.94           RIGHT  (CM)   36.00   36.74           AT5G59820-­‐ZAT12  (LOD  THRESHOLD=3.15)   CHR   LEFT  (CM)   3   9.64   4   50.00   5   10.00   MB           CIS/   #  GENES   TRAN?                 CIS/   #  GENES   TRAN?   1575   TRANS                         LOD               %VAR       LOD   6.35             %VAR   5.35       CIS/   RIGHT  (CM)   MB   #  GENES   TRAN?   LOD   %VAR   55.520   3.790   1153   TRANS   10.09   8.390           AT3G55980-­‐STZ1  (LOD  THRESHOLD=2.81)   CHR   LEFT  (CM)   4   32.00   5   20.00       MB   1.73   3.50               CIS/   #  GENES   TRAN?   477   TRANS   805   TRANS           CIS/   RIGHT  (CM)   MB   #  GENES   TRAN?   22.00   3.20   951   TRANS   56.60   4.66   1410   TRANS   56.60   15.66   4071   TRANS   174     LOD   5.46   3.87       LOD   5.25   4.46   6.54       %VAR   3.85   2.40       %VAR   4.10   4.37   4.91       Table 3.19. Locations of eQTL for Category 2 FTD candidate genes. 544 RILs were used for eQTL mapping. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/). To detect eQTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LOD) values were calculated for each trait using 1000 permutations in RQTL. To estimate the location of each eQTL a Bayes credible interval (99%) was calculated in RQTL. For locations of markers based on the Col-0 genome please see Table A3.1. QTL are categorized as cis- or trans- (CIS/TRANS?). Shaded QTL overlap with previously identified fitness QTL (Schemske and Agren, unpublished). ‘MB’ indicates the number of megabases contained within the eQTL. ‘# GENES’ indicates the number of genes contained within the eQTL based on the Col-0 genome. %VAR indicates the estimated proportion of phenotypic variance explained by the QTL. AT1G20440-­‐COR47  (LOD  THRESHOLD=2.83)                   CIS/   CHR   LEFT  (CM)   PEAK  (CM)   RIGHT  (CM)   MB   #  GENES   TRAN?   LOD   1   10   14.05   18.12   3.19   967   CIS   7.59   4   46.00   50.20   58.00   5.77   1977   TRANS   6.76                                   AT1G20450-­‐ERD10  (LOD  THRESHOLD=3.25)                   CIS/   CHR   LEFT  (CM)   PEAK  (CM)   RIGHT  (CM)   MB   #  GENES   TRAN?   LOD   4   50.00   55.52   56.30   4.36   1322   TRANS   12.79   CHR   2   3   5   MB   #  GENES   1.73   450   1.16   356   4.68   1697   CIS/   TRAN?   TRANS   TRANS   TRANS           AT4G15910-­‐DI21  (LOD  THRESHOLD=2.85)           CHR   4   CIS/   MB   #  GENES   TRAN?   LOD   %VAR   0.64   179   CIS   28.96   11.92   LEFT  (CM)   34.00   PEAK  (CM)   RIGHT  (CM)   34.49   35.77               AT5G52310-­‐COR78  (LOD  THRESHOLD=3.00)   CHR   4   5         CIS/   MB   #  GENES   TRAN?   6.35   1910   TRANS   7.83   2342   CIS   LEFT  (CM)   48.00   54.83   PEAK  (CM)   RIGHT  (CM)   55.52   60.44   64.62   78.17   175             %VAR   10.50       PEAK  (CM)   RIGHT  (CM)   46.95   48.04   11.18   12.64   54.27   56.60       %VAR   6.89   6.35                   AT1G75040-­‐PR5  (LOD  THRESHOLD=3.05)   LEFT  (CM)   40.25   9.64   42.00           LOD   6.07   7.63   5.87           LOD   6.39   4.20       %VAR   3.87   4.25   4.63           %VAR   5.18   3.50       AT5G52310#COR78, 6 6" 5 5" lod LOD" 4 4" 3 3" 2 2" 1 1" 0 0" 1" 2" 3" 4" 5" CHROMOSOME" EFFECT"PLOT,CH5@64.6" EFFECT"PLOT,CH4@55.5" 1 2 3 4 5 Chromosome Effect plot for *L4_38 13" 14" 13 14 12" 12 12" 11" 12 COR78 11 COR78 COR78"EXPRESSION" Effect plot for *L5_69 10" 10 9" 8" 10 8 7" 10" 9 7 8" IT" AA 8 SW" IT" AA BB *L5_69 SW" BB *L4_38 Figure 3.37. eQTL for Category 2 FTD gene COR78. 544 RILs were used for eQTL mapping by qRT-PCR. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/) for each fitness trait at each location. To detect QTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LODs) values were calculated for each trait using 1000 permutations in RQTL (shown in blue). Effects plots represent expression averages for the genotype groups (IT or SW) at eQTL on chromosome 4 and 5. Effect plots were constructed using ‘effectplot’ function in R/QTL(15).   176     AT2G42540#COR15A,, 10 10" 6 6" lod LOD" 8 8" 4 4" 2 2" 0 0" 1" 1 2" 3" 4" CHROMOSOME" 2 3 4 5" 5 Chromosome EFFECT"PLOT,CH4@50.0" 110" 110 100" 100 COR15A COR15A"EXPRESSION" Effect plot for *L4_37 90" 90 80" 80 70" 70 IT" AA SW" BB *L4_37 Figure 3.38. eQTL for Category 1 FTD gene COR15A. 544 RILs were used for eQTL mapping by qRT-PCR. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/) for each fitness trait at each location. To detect QTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LODs) values were calculated for each trait using 1000 permutations in RQTL (shown in blue). Effect plot represents expression averages for the genotype groups (IT or SW) at eQTL on chromosome 4. Effect plots were constructed using ‘effectplot’ function in R/QTL(15).   177     AT1G20440#COR47,, lod LOD" 6 6" 4 4" 2 2" 0 0" 1" 2" 3" 4" 5" CHROMOSOME" EFFECT"PLOT,CH4@50.2" EFFECT"PLOT,CH1@14.1" 1 2 3 4 5 Chromosome Effect plot for *L4_38 30" 30" 30 25" 25 25" COR47 25 COR47 COR47"EXPRESSION" Effect plot for *L1_23 30 20" 20 20" 20 15" 15 15" 15 IT" AA SW" IT" BB *L1_23 AA SW" BB *L4_38 Figure 3.39. eQTL for Category 2 FTD gene COR47. 544 RILs were used for eQTL mapping by qRT-PCR. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/) for each fitness trait at each location. To detect QTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LODs) values were calculated for each trait using 1000 permutations in RQTL (shown in blue). Effect plots represent expression averages for the genotype groups (IT or SW) at eQTL on chromosome 1 and 4. Effect plots were constructed using ‘effectplot’ function in R/QTL(15).   178     AT1G29390#COR314, 6 6" 5 5" lod LOD" 4 4" 3 3" 2 2" 1 1" 0 0" 1" 1 2" 3" 4" CHROMOSOME" 2 3 4 Chromosome 5" 5 EFFECT"PLOT,CH4@55.5" 10" COR314 COR314"EXPRESSION" Effect plot for *L4_50 10 9" 9 8" 8 7" 7 IT" AA SW" BB *L4_50 Figure 3.40. eQTL for Category 1 FTD gene COR314. 544 RILs were used for eQTL mapping by qRT-PCR. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/) for each fitness trait at each location. To detect QTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LODs) values were calculated for each trait using 1000 permutations in RQTL (shown in blue). Effect plot represents expression averages for the genotype groups (IT or SW) at eQTL on chromosome 4. Effect plots were constructed using ‘effectplot’ function in R/QTL(15).   179     AT1G20450#ERD10, 12 12" 10 10" lod LOD" 8 8" 6 6" 4 4" 2 2" 0 0" 1" 1 2" 3" 4" CHROMOSOME" 2 3 4 Chromosome 5" 5 EFFECT"PLOT,CH4@55.5" ERD10 ERD10"EXPRESSION" Effect plot for *L4_37 9" 9 8" 8 7" 7 6" 6 5" 5 IT" AA SW" BB *L4_37 Figure 3.41. eQTL for Category 2 FTD gene ERD10. 544 RILs were used for eQTL mapping by qRT-PCR. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/) for each fitness trait at each location. To detect QTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LODs) values were calculated for each trait using 1000 permutations in RQTL (shown in blue). Effect plot represents expression averages for the genotype groups (IT or SW) at eQTL on chromosome 4. Effect plots were constructed using ‘effectplot’ function in R/QTL(15).   180     AT5G59820#ZAT12, 6 6" lod LOD" 5 5" 4 4" 3 3" 2 2" 1 1" 0 0" 1" 2" 3" 4" CHROMOSOME" 1 2 3 5" 4 5 Chromosome EFFECT"PLOT,CH3@11.2" EFFECT"PLOT,CH4@50.6" EFFECT"PLOT,CH5@48.0" Effect plot for *L4_41 0.55" 0.50" Effect plot for *L5_47 0.55" 0.50" 0.45" 0.40" 0.35" 0.30" 0.55" 0.50" 0.55 0.55 0.55 0.50 0.50 0.45" 0.45" 0.40" 0.40" 0.35" AT5G59820 0.50 AT5G59820 AT5G59820 ZAT12"EXPRESSION" Effect plot for *L3_19 0.35" 0.45 0.40 0.45 0.40 0.40 0.35 0.35 0.35 IT" AA SW" BB *L3_19 0.45 0.30 AA IT" BB *L4_41 SW" AA IT" BB *L5_47 SW" Figure 3.42. eQTL for Category 1 FTD gene ZAT12. 544 RILs were used for eQTL mapping by qRT-PCR. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/) for each fitness trait at each location. To detect QTL, estimated genome-wide significance thresholds (α=0.01) for the logarithm of odds (LODs) values were calculated for each trait using 1000 permutations in RQTL (shown in blue). Effect plots represent expression averages for the genotype groups (IT or SW) at eQTL on chromosomes 1, 4 and 5. Effect plots were constructed using ‘effectplot’ function in R/QTL(15).   181 Table 3.20. Expression of CBF genes. Gene expression was measured by RNA-seq. The results presented are average values from three independent experiments (n=3). Comparisons of SW and IT under non-acclimated conditions, 1 week of cold-acclimation, and 2 weeks of cold-acclimation and the corrected p-value (FDR P-VALUE) for that comparison. The comparison of each gene is also represented by the logarithm (base=2) of the fold change (LOG2FC). This table also denotes if a gene was previously described as positively or negatively correlating with freezing tolerance (HANNAH;(6)), a transcription factor (TF), a gene previously described as up- or down-regulated by 1 week of cold (COS, (24)), identified as an up- or down-regulated component of the CBF regulon (CBF REGULON; (24)). Shaded genes have significantly different expression between SW and IT at 2 weeks of cold-acclimation. 20" 0" 1WK" 2WK" 2WK.FDR. P-­‐VALUE   2.03E-­‐01   8.06E-­‐03   6.84E-­‐02   6.76E-­‐01   HANNAH?   POSITIVE   NO   NO   NO   CBF3#RNA#seq, 50" 1" 0" 0" NON" 1WK" 2WK" TF?   COS?   YES   NO   YES   UP   YES   UP   YES   NO   CBF   REG?   NO   UP   NO   NO   CBF4#RNA#seq, 100" 40" 2" SW.IT. 2WK   -­‐0.73   -­‐0.58   -­‐0.40   0.89   CBF2#RNA#seq, 60" SW" IT" NON" 1WK.FDR. P-­‐VALUE   8.52E-­‐01   5.24E-­‐01   4.65E-­‐01   3.85E-­‐01   FPKM, 4" CBF1#RNA#seq, SW.IT. 1WK   -­‐0.33   -­‐0.40   -­‐0.44   -­‐1.96   FPKM, FPKM, 6" NON.FDR. P-­‐VALUE   5.32E-­‐01   7.41E-­‐01   2.01E-­‐02   1.00E+00   FPKM, AGI   NAME   AT4G25490   CBF1   AT4G25470   CBF2   AT4G25480   CBF3   AT5G51990   CBF4   SW.IT. NON   1.66   0.55   1.76   0.00   0.5" 0" NON" 1WK" 2WK" NON" 1WK" 2WK" Figure 3.43. CBF gene expression. IT samples are shown in red, and SW in blue. Gene expression was measured by RNA-seq (FPKM). The results presented are average values from three independent experiments (n=3). Error bars indicate ±SEM. Plants were grown at 22⁰C under a 12 h photoperiod for approximately 18 days before rosette tissue was sampled directly or transferred to 4⁰C for 1 or 2 weeks under a 12 h photoperiod then sampled.   182 MAJORITY, SW, IT, COL#0, MAJORITY, SW, IT, COL#0, MAJORITY, SW, , COL#0, Figure 3.44. Protein prediction for CBF2 in SW, IT and COL-0.     183 CONCLUSIONS SW and IT ecotypes are adapted to their respective environments and both minimum winter temperature and flowering time were correlated with relative fitness of the SW and IT accessions in reciprocal transplant experiments (8). Temperature has previously been shown to be a strong selective pressure and freezing tolerance correlates with both latitude and minimum average temperature (6, 8, 17, 62). This study shows that SW and IT have significant differences in freezing tolerance at 2 weeks of cold acclimation, but not at 1 week of cold-acclimation or under nonacclimated conditions. This difference in freezing tolerance at 2 weeks of cold acclimation is due to both an increase in SW freezing tolerance as well as a reduction in IT freezing tolerance. eQTL mapping revealed CBF2 as an excellent candidate gene to explain differences in cold-regulated gene expression and consequently freezing tolerance. Analysis of SW and IT CBF2 coding sequence exposed a 13 bp deletion in IT-CBF2 resulting in a predicted premature stop in the activation domain. Based on transcriptome and freezing tolerance analysis of a similar truncated version of CBF2, ITCBF2 is predicted to function as a dominant negative version of CBF2. Transcriptional analyses are generally consistent with IT-CBF2 acting as a dominant negative version of CBF2, though additional experiments are needed to confirm. This is by no means the first example of CBF genes significantly contributing to differences in freezing tolerance between different accessions of Arabidopsis (10, 56-58). A similar premature stop in the activation domain of CBF3 was previously identified in the Condara accession, but   184 the effect of this truncation was not tested on CBF target genes or freezing tolerance (57). Therefore, this could possibly be the first natural example of a dominant negative version of CBF2. The higher level of freezing tolerance in SW at 2 weeks of cold-acclimation could also be explained through CBF2 expression, which is significantly higher than in IT at only this time-point. Therefore, the CBF2 promoter should be sequenced in SW and IT in the near future. SW photosynthetic capacity may also be greater than that of IT based on analysis of differentially regulated photosynthesis associate genes. However, photosynthetic capacity would need to be measured experimentally. Increased photosynthetic capacity is associated with higher levels of freezing tolerance, so differences in photosynthesis may also be a segregating trait between SW and IT. Transcriptional analysis suggests that SW and IT reach similar levels of freezing tolerance at 1 week of cold-acclimation through significantly divergent transcriptional mechanisms. Both transcriptomic analysis and CBF2 sequence analysis suggest that IT may use CBF-independent mechanisms to cold-acclimate. These CBF-independent mechanisms might include genes annotated as biotic stress genes, since cold-regulated genes only found in IT were enriched in biotic stress GO terms. Biotic stress genes were also more highly expressed in IT compared to SW, regardless of cold-regulation. The mechanisms by which IT cold-acclimates do not seem to be as efficient as SW since there is a decrease in freezing tolerance at 2 weeks of cold-acclimation. I speculate that lower photosynthetic capacity in IT could be associated with the reduced number of cold-regulated genes from 1 to 2 weeks of cold-acclimation in IT, which may   185 synergistically combine with the dominant negative effect of IT-CBF2 to result in reduced freezing tolerance at 2 weeks of cold-acclimation. Altogether, progress has been made towards discerning genetic differences between SW and IT that potentially contribute to differences in freezing tolerance and fitness. Other potentially segregating traits such as pathogen resistance, and photosynthetic capacity, that may contribute to fitness have also been identified. Genomic sequence of SW and IT parent lines should soon be available. This will make the RNA-seq data generated in this study more powerful, since locations of sequence differences across the SW and IT genomes will allow FTD candidate genes to be further prioritized for eQTL screening. In the near future, the Schemske lab plans to map differences in freezing tolerance in the SW and IT RILs. Based on results from this study overlap between freezing tolerance QTL and fitness QTL is expected (Table 3.1). A freezing tolerance QTL overlapping with the CBF locus is also expected. It will be interesting to see how much overlap there is between fitness and freezing tolerance QTL.   186 MATERIALS AND METHODS Plant Material and Growth Conditions. Swedish (SW) and Italian (IT) accessions used in this study were collected from their native habitats by members of Schemske and Agren labs (Schemske and Agren, unpublished). Recombinant inbred lines (RILs) used in this study were constructed by the Schemske lab by crossing a randomly selected individual from the SW ecotype (male parent) to a randomly selected individual from the IT ecotype (female parent), seeds from the F1 generation were used to establish a large number of lines that were selfed by single-seed descent for nine generations (Schemske and Agren, unpublished). SW and IT RILs used in this study were from the F10 generation. All seeds were stratified for 5 days in the dark at 4⁰C. For electrolyte leakage experiments, plants were grown as described by Dong et al. 2011(16), with slight modification. Plant were germinated on plates at 22⁰C under sterile conditions on -2 -1 Gamborg’s B5 medium (Caisson Laboratories) with 1% sucrose at ~100 µmol m s in a 12 h photoperiod for 7 days before transfer to soil for an additional 11 days, plants were then assayed for freezing tolerance directly (non-acclimated conditions) or -2 -1 transferred cold-acclimated conditions for 1, 2 or 3 weeks at ~35 µmol m s in a 12 h photoperiod. For eQTL experiments RILs were grown as described by Dong et al. 2011(16), with slight modification. RILs were germinated on plates at 22⁰C under sterile conditions on Gamborg’s B5 medium (Caisson Laboratories) with 1% sucrose at ~100   187 -2 -1 µmol m s in a 12 h photoperiod for 7 days then transfer to soil for an additional 11 -2 -1 days before 2 week cold treatment at ~35 µmol m s in a 12 h photoperiod. Freezing Tolerance Tests. Electrolyte leakage assays were performed as described in Dong et al. 2011(16). For cold acclimation, plants were transferred to 4⁰C -2 -1 at ZT4 for 1 week, 2 weeks, or 3 weeks at ~35 µmol m s under a 12 h photoperiod. Electrolyte leakage assays for acclimated and non-acclimated plants were started at ~ZT4 in for all biological replicates. Tissue for RNA-seq experiments described below was collected before the start of each electrolyte leakage assay. RNA-seq Experiments. Randomly selected rosette tissue (three technical replicates for each biological replication) from each electrolyte leakage experiment (nonacclimated, 1 week acclimated, 2 week acclimated, 3 week acclimated) was collected for RNA-seq analysis. RNA from 3 out of the 6 biological replication sets (SW, IT, at non-acclimated, 1 week acclimated, and 2 week acclimated) was chosen at random for further analysis. RNA was isolated as described in Dong et al. 2011 (16) from 2 technical replicates from selected biological replications; one technical replication was used for qRT-PCR analysis experiments and the other was submitted for RNA-seq analysis at MSU’s Research Technology Support Facility (RTSF). For qRT-PCR analysis, cDNA was made as described in Dong et al. 2011 (16). Expression of known cold-induced genes (CBF1, CBF2, CBF3, COR15A, COR47,   188 COR78) was tested using qRT-PCR (Applied Biosystems 7500 FAST Real-Time PCR System in FAST mode) as a means of ensuring that submitted RNA-seq samples were responding to cold. IPP2 (AT3G02780) was used as a reference gene. For RNA-seq samples RNA quality was determined by an Agilent 2100 Bioanalyzer using manufacturer protocols ((Agilent RNA 6000 Nano Kit, Cat. 50671511, Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed on an Illumina Genome Analyzer II (GA II). Like-treated samples (i.e. 3 biological replicates of SW and IT non-acclimated samples) were multiplexed in two lanes resulting in singleend reads ~75 bp in length with an average of 45,257,092  reads passing the Illumina purity filters for each sample (please see Table A3.8 for details). Sample preparation, including mRNA purification, cDNA preparation, end repair of cDNA, adaptor ligation, and cDNA amplification, was performed by MSU RTSF and conducted according to the manufacturer protocols (mRNA-Seq Sample Preparation Kit, Cat. RS-930-1001, Illumina Inc., San Diego, CA, USA). RNA-seq Analysis. SW and IT RNA-seq data was aligned to TAIR10 sequence using Tophat (18). For Tophat, minimum intron length was set to 7 bp and maximum intron length was set to 12000 bp, all other parameters were set to defaults (18). Cufflinks was used to estimate transcript abundance based on unique reads in order to address the challenges associated with ‘multi-mapping reads’ (63), rRNA was also masked to improve robustness of transcript abundance estimates, all other parameters were set to default values (18). When comparing treatments for differential expression,   189 the cuffdiff program within Cufflinks was used for pairwise comparisons with default settings (18). Cuffdiff assigns p-values based a two-tailed Student’s t-test; this value is then adjusted using a Benjamini-Hochberg correction for ‘multiple-testing’(18). While analyzing differentially expressed genes in RNA-seq data, a cut-off of ≥3FPKM was used for differentially expressed genes (19, 20). This means that, when treatments were compared, one value in the comparison had to be ≥3FPKM. When testing for significant overlap between two sets of genes, a hypergeometric probability was calculated using the ‘stats’ package in R (64). Gene ontology (GO) enrichment was determined using the database for annotation visualization and integrated discovery tool (DAVID; david.abcc.ncifcrf.gov; (27)). The default DAVID gene ontology settings ‘GOTERM_BP_FAT’, ‘GOTERM_MF_FAT’, and ‘GOTERM_CC_FAT’ were used for analysis (27). GO category was considered significantly enriched if its BenjaminiHochberg corrected (FDR corrected) hypergeometric p-value was <0.050. eQTL Experiments. RIL seed stratification, germination, and movement to low temperature conditions was staggered to allow all RILs to be grown then cold acclimated in the same set of growth chambers and to ensure that a manageable number of RILs, at the same age, could be samples within an hour. Plant tissue was collected and quickly frozen in liquid nitrogen between ZT4-ZT5 after 2 weeks of cold acclimation. RNA extraction and cDNA synthesis for RILs was performed as described by Dong et al. 2011(16). These samples were tested for expression of 10 Freezing Tolerance Difference (FTD) candidate genes (see Table 3.17) by qRT-PCR (Applied   190 Biosystems 7500 FAST Real-Time PCR System in FAST mode) with IPP2 (AT3G02780) as a housekeeping gene. All primer sets can be found in Table A3.7. eQTL Mapping and Analysis. SW and IT parent lines were sequenced by the Schemske lab using the Illumina GAII platform. Paired end reads were mapped to the COL-0 reference sequence (TAIRv9) and approximately 141,437 single nucleotide polymorphisms (SNPs) were found (Schemske, unpublished). 384 of these SNPs with an average spacing of ~1.1 cm across the Arabidopsis genome were selected to genotype the RIL population (Schemske, unpublished). Expression data for the10 FTD candidate genes generated during the eQTL experiments described above was used to map eQTL over these 384 markers. RQTL (15) was used to perform interval mapping using the multiple imputation method as described by the reference manual (http://www.rqtl.org/). To detect QTL, estimated genome-wide significance thresholds (α=0.01 and α=0.05) for the logarithm of odds (LOD) values were calculated for each FTD gene of interest using 1000 permutations in RQTL. To estimate the location of each QTL both a Bayes credible interval (99%) and LOD support interval were calculated in RQTL. The SeqViewer tool on TAIR (www.arabidopsis.org) was used to identify all genes within the QTL interval and this list was compared to SW and IT RNAseq data described above to find candidate transcription factors. CBF sequencing. Genomic DNA was extracted from SW, IT and COL-0 using a QIAGEN DNeasy Plant Mini Kit using manufacturer protocols (Cat. 69104, Qiagen,   191 Valencia, CA). CBF1-3, COR78, COR15A and COR47 coding sequences were amplified by PCR using TaKaRa LA Taq Hot-Start DNA Polymerase (Cat. RR042A, Mountain View, CA). Primers used to amplify CBF1, 2 and 3 coding sequence in SW, IT and Col-0, can be found in Table A3.8. PCR products were cloned into PCR2.1TOPO, transformed into E. coli competent cells, and screened using manufacturer protocols (Cat. K4500-02, Invitrogen, Carlsbad, CA). Selected clones were sequenced by MSU RTSF. Sequence alignment was performed using the Lasergene Suite of tools (www.dnastar.com).   192 ACKNOWLEDGEMENTS I am especially grateful to Pingsha Hu who performed alignment and assembly of the RNA-seq data generated in this study. Chin-Mei Lee helped with the cloning and sequencing of SW and IT cold genes. Thanks to the Schemske lab for providing the Arabidopsis ecotypes from Sweden and Italy as well as the recombinant inbred lines. I am also grateful to Nicholas Bartoa and Avery Mendelson for their help with plant growth and care.   193 APPENDIX   194 Table A3.1. SW and IT markers used for mapping both fitness QTL and eQTL. Marker locations in centimorgans (CM), and marker locations in megabases (MB) based on the Col-0 genome. CHROMOSOME 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1   MARKER (CM) 0 1.422 2.075 3.013 3.384 3.943 4.408 5.25 5.343 6.112 6.206 8.36 8.842 9.507 9.916 10.312 11.03 11.501 11.97 12.636 13.483 14.053 14.525 16.069 18.117 18.877 19.638 20.792 21.941 22.704 23.652 24.623 25.681 26.545 28.103 29.502 30.882 32.413 33.796 35.512 37.885 38.268 38.363 38.459 38.459 38.554 39.911 40.59 195 MARKER LOCATIONS (MB) 0.371484 0.658928 0.95079 1.247666 1.545309 1.831853 2.112359 2.402241 2.689578 2.97831 3.270056 4.139064 4.434414 4.716592 5.008647 5.305697 5.588245 5.876257 6.486531 6.750734 7.043318 7.332032 7.621705 7.906548 8.199225 8.511642 8.846085 9.081675 9.389545 9.64586 9.948571 10.232305 10.517662 10.817536 11.101365 11.402119 11.697338 11.966262 12.344267 12.843656 13.156629 13.451072 13.753592 14.004113 15.522314 15.889605 16.136207 16.445556     Table A3.1 (cont’d) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2   42.502 43.493 46.721 47.977 48.931 49.788 50.352 51.496 52.543 53.291 54.325 54.603 55.197 55.601 55.904 56.275 57.347 58.632 58.823 60.236 60.984 61.076 61.824 62.477 63.146 64.88 66.846 67.637 68.419 70.447 71.997 74.435 75.399 76.971 77.163 77.86 78.551 79.56 80.38 80.886 82.142 83.088 83.845 0 4.606 6.33 8.55 10.17 11.4 12.822 14.736 15.107 16.923537 17.203021 17.775164 18.059668 18.352147 18.65907 18.926903 19.240146 19.529287 19.800653 20.108938 20.38011 20.668556 20.964571 21.251788 21.551115 22.152521 22.412767 22.723886 22.988965 23.292064 23.580047 23.857523 24.155803 24.435268 24.765127 25.032697 25.324254 25.609182 25.889912 26.181454 26.760155 27.057077 27.352821 27.626189 27.917735 28.220678 28.509198 28.806377 29.077284 29.384089 29.659444 30.245282 0.365473 0.661681 0.954162 1.258538 1.532339 1.871886 2.135186 2.433194 4.621443 196     Table A3.1 (cont’d) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3   15.2 15.48 15.761 17.687 22.613 24.312 25.601 26.841 27.893 29.449 30.714 31.706 31.992 32.589 34.308 35.592 37.365 38.247 39.837 40.254 41.761 42.345 43.114 44.28 46.948 48.042 48.734 49.409 49.516 49.704 50.366 50.646 51.02 52.453 53.036 53.415 54.399 54.885 55.269 56.022 56.402 56.779 56.874 58.025 58.313 58.594 59.444 60.487 60.86 0 0.094 1.032 5.049931 5.295348 5.587569 5.95692 6.764214 7.047117 7.340228 7.626054 7.920385 8.197294 8.502011 8.779839 9.065286 9.36707 9.647248 9.940989 10.225771 10.536215 10.837886 11.106932 11.38934 11.679435 11.966391 12.267422 12.547427 12.838094 13.135793 13.420085 13.716369 13.995766 14.319012 14.575482 14.868193 15.16817 15.447154 15.747054 16.06694 16.319037 16.60741 16.898396 17.18517 17.476046 17.779 18.063832 18.358707 18.639532 18.932927 19.233555 19.529865 0.370926 0.656579 0.961179 197     Table A3.1 (cont’d) 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3   1.688 2.155 2.533 2.82 3.487 3.96 4.523 5.092 6.349 7.208 7.313 8.176 9.637 10.681 11.184 11.853 12.641 13.533 14.728 15.618 16.51 18.033 19.705 22.343 23.319 25.381 28.021 29.512 31.527 32.697 37.858 39.55 40.641 41.121 41.309 41.402 42.553 43.813 44.974 45.949 49.119 50.183 51.952 53.323 54.095 54.672 55.367 57.064 58.618 59.188 60.383 61.53 1.270326 1.525377 1.825965 2.112696 2.398036 2.692472 2.975352 3.269092 3.565378 4.141096 4.427835 4.718296 5.295658 5.619526 5.875889 6.175656 6.457498 6.74919 7.051377 7.329844 7.628106 7.914025 8.200995 8.497891 8.776721 9.13927 9.654017 9.940341 10.234401 10.587664 11.114247 11.399488 11.747164 12.041893 12.56772 15.210147 15.514979 15.759118 16.058567 16.36229 16.648133 16.899341 17.225206 17.486181 17.781386 18.077874 18.350962 18.651512 18.955604 19.220884 19.514107 19.799867 198     Table A3.1 (cont’d) 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4   63.124 63.604 64.18 64.928 65.487 65.858 66.606 66.791 67.54 68.386 69.618 70.466 0 2.329 6.129 7.648 8.975 9.255 12.891 14.399 15.265 15.674 15.767 18.335 19.776 20.73 24.028 25.332 26.733 27.984 29.948 31.047 32.149 33.418 34.491 35.766 37.38 39.077 41.05 42.292 43.507 44.312 46.649 48.883 50.004 50.195 50.574 51.142 51.331 51.993 52.369 52.847 20.092191 20.401811 20.665136 20.960795 21.25848 21.558962 21.840004 22.14465 22.441322 22.70487 22.986945 23.275556 0.384888 0.680041 0.982791 1.238231 1.535171 2.717267 2.396469 2.133635 1.828832 3.900172 5.080132 5.587712 5.924099 6.171429 6.498644 6.754691 7.042854 7.346579 7.616384 7.918274 8.211444 8.498174 8.815569 9.140013 9.650672 9.942702 10.236673 10.522219 10.831675 11.15739 11.714851 12.292822 12.5471 12.843072 13.151997 13.421012 13.720933 14.008832 14.292115 14.584632 199     Table A3.1 (cont’d) 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5   53.543 53.935 54.131 54.523 55.222 55.523 55.916 56.299 56.593 57.696 59.526 60.435 60.721 0 1.428 2.63 3.939 4.594 4.913 5.695 6.678 7.459 7.652 8.525 9.384 10.048 10.434 10.717 10.811 11.39 11.972 13.126 13.602 14.471 14.668 14.963 15.919 16.811 18.508 20.419 21.967 27.139 28.533 29.833 32.507 34.144 36.082 36.645 36.738 37.016 38.26 40.107 14.869919 15.162178 15.450267 15.735751 16.026122 16.337059 16.606958 16.90214 17.202249 17.486207 17.796648 18.063371 18.347312 0.367158 0.655985 0.949897 1.245151 1.528949 1.839919 2.111365 2.408921 2.685965 2.985496 3.270887 3.57043 3.852246 4.13647 4.426649 4.726262 5.013068 5.336868 5.656316 5.889105 6.473301 6.752582 7.071294 7.334179 7.618492 7.914975 8.232952 8.569949 9.0801 9.376062 9.6691 9.969215 10.233797 10.639041 10.929784 11.415254 13.106228 13.707451 14.015641 200     Table A3.1 (cont’d) 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5   41.4 43.043 44.949 46.891 48.063 49.947 50.244 50.244 50.244 50.45 50.962 51.576 52.441 53.407 54.267 54.836 56.611 57.476 57.664 58.578 59.597 60.101 60.596 61.278 62.603 64.099 64.62 66.055 66.456 67.732 68.693 69.359 70.123 70.596 70.983 71.373 72.063 72.931 73.61 74.474 75.802 77.422 78.174 14.545953 14.876427 15.185072 15.464485 15.768401 16.043932 16.326685 16.611838 16.897426 17.196046 17.503065 17.787766 18.072654 18.382907 18.640297 18.926019 19.227434 19.550188 19.797944 20.09524 20.390151 20.67101 20.961565 21.249178 21.548963 21.864028 22.116026 22.406706 22.711593 23.006666 23.277096 23.580206 23.87063 24.172305 24.444383 24.725353 25.017566 25.350635 25.599724 25.889576 26.180944 26.468215 26.757861 201   Table A3.2. GO categories of Hannah genes positively (277 genes) and negatively (466 genes) correlated with freezing tolerance (6). GO enrichment was determined using DAVID (david.abcc.ncifcrf.gov; (27)). ‘GO TERM CATEGORY’ denotes if the GO term was categorized as a biological process (BP), cellular component (CC), or molecular function (MF) in the DAVID database. ‘# HITS’ denotes the number of genes with that GO category in Hannah genes positively (277 genes) and negatively (466 genes) correlated with freezing tolerance. ‘# GENES IN GO’ denotes the total number of genes in that GO category included in the DAVID database. GO category was considered significantly enriched if the FDR corrected hypergeometric p-value was ≤0.050. GO  TERM   CATEGORY   BP   BP   BP   BP   BP   BP   HANNAH  POSITIVE  (277GENES)   Response  To  Water  Deprivation   Response  To  Abiotic  Stimulus   Response  To  Cold   Response  To  Oxidative  Stress   Carbohydrate  Biosynthetic  Process   Response  To  Osmotic  Stress   #   HITS   16   34   11   12   11   14   #  GENES   IN  GO   176   1197   233   287   254   399   GO  TERM   #   HANNAH  NEGATIVE  (466  GENES)   CATEGORY   HITS   CC   Chloroplast   225   CC   Thylakoid   80   CC   Photosynthetic  Membrane   56   MF   rRNA  Binding   21   CC   Organelle  Membrane   65   MF   Structural  Constituent  Of  Ribosome   32   MF   Structural  Molecule  Activity   37   CC   Ribosome   38   BP   Photosynthesis   18   BP   Chlorophyll  Biosynthetic  Process   8   BP   Pigment  Metabolic  Process   11   BP   Nitrogen  Compound  Biosynthetic  Process   26   BP   Tetrapyrrole  Biosynthetic  Process   8   CC   Anchored  To  Plasma  Membrane   8   CC   NAD(P)H  Dehydrogenase  Complex     4   BP   Porphyrin  Metabolic  Process   8   BP   Response  To  Cold   15   BP   Heterocycle  Biosynthetic  Process   11   BP   Ribosome  Biogenesis   15   CC   Non-­‐Membrane-­‐Bounded  Organelle   46     202 #  GENES   IN  GO   3192   491   330   76   849   394   538   470   175   34   101   506   50   66   10   61   233   132   242   1144   FDR     P-­‐VALUE   2.68E-­‐07   1.22E-­‐04   3.66E-­‐02   3.82E-­‐02   4.13E-­‐02   4.43E-­‐02   FDR     P-­‐VALUE   3.34E-­‐51   1.27E-­‐37   7.22E-­‐27   4.55E-­‐15   8.10E-­‐13   6.55E-­‐09   1.04E-­‐08   5.16E-­‐08   7.19E-­‐05   1.69E-­‐03   4.09E-­‐03   4.52E-­‐03   5.27E-­‐03   1.11E-­‐02   1.14E-­‐02   1.71E-­‐02   1.81E-­‐02   2.06E-­‐02   2.27E-­‐02   4.37E-­‐02   Table A3.3. 474 Category 1 FTD Candidate Genes with higher expression in SW at 2 weeks of cold-acclimation. Genes are ordered by their AGI numbers. FPKM values are the average of three biological replicates (n=3). Standard error (SE) values are also given for each FPKM value. Genes were considered significantly different between SW and IT under nonacclimated conditions if they had a Benjamini-Hochberg corrected p-value (FDR P-VALUE) of <0.05 and a minimum of ≥3FPKM in IT. The differential expression of each gene is also represented by the logarithm (base=2) of the fold change (LOG2.FC). This table also denotes if a gene was previously described as positively or negatively correlating with freezing tolerance (HANNAH;(6)), a transcription factor (TF), a gene previously described as up- or down-regulated by 1 week of cold (COS, (25)), identified as an up- or down-regulated component of the CBF regulon (CBF REGULON; (25)). AGI   AT1G01210   AT1G01640   AT1G02300   AT1G02460   AT1G03310   AT1G04240   AT1G04350   AT1G04570   AT1G04620   AT1G06360   AT1G06700   AT1G06980   AT1G07200   AT1G07280   AT1G08500   AT1G08570   AT1G09750   AT1G10522     SW.2WK   SW.2WK   FPKM   SE   25.47   0.77   10.27   0.35   30.44   0.44   17.43   0.27   36.19   1.34   61.42   1.64   20.72   0.41   132.07   1.41   75.99   0.87   67.20   1.07   59.72   0.38   16.78   0.31   12.97   0.13   131.04   0.92   10.71   0.34   53.28   0.33   80.53   1.54   64.52   1.31   IT.2WK   FPKM   14.97   6.01   20.14   10.21   25.42   35.21   14.58   40.21   52.13   44.29   40.10   6.63   9.36   96.96   6.35   39.07   53.50   47.36   IT.2WK   SE   0.99   0.37   0.38   0.22   0.39   0.91   0.65   0.74   0.61   2.60   1.11   0.48   0.30   0.44   0.26   0.74   1.12   0.80   SW.IT.2WK   SW.IT.2WK   FDR  P-­‐ LOG2FC   VALUE   HANNAH?   -­‐0.77   1.60E-­‐02   NO   -­‐0.77   4.09E-­‐02   NO   -­‐0.60   6.08E-­‐03   NO   -­‐0.77   3.71E-­‐04   NO   -­‐0.51   1.53E-­‐03   NO   -­‐0.80   2.00E-­‐05   NO   -­‐0.51   3.30E-­‐02   NO   -­‐1.72   0.00E+00   NO   -­‐0.54   3.74E-­‐03   NO   -­‐0.60   1.52E-­‐03   NO   -­‐0.57   4.17E-­‐04   NO   -­‐1.34   6.39E-­‐05   NO   -­‐0.47   1.02E-­‐02   NO   -­‐0.43   5.00E-­‐03   NO   -­‐0.75   4.21E-­‐02   NO   -­‐0.45   1.10E-­‐02   NO   -­‐0.59   1.43E-­‐03   NO   -­‐0.45   2.86E-­‐02   NO   203 TF?   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   COS?   NO   NO   NO   UP   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   CBF   REG?   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO     Table A3.3 (cont’d) AT1G10760   109.27   AT1G11700   23.34   AT1G12090   1320.94   AT1G12250   70.97   AT1G12710   47.22   AT1G13560   56.05   AT1G14150   156.49   AT1G14200   20.79   AT1G15405   155.32   AT1G15470   25.73   AT1G16410   152.60   AT1G18360   31.45   AT1G19570   236.89   AT1G19960   407.82   AT1G20010   306.54   AT1G20610   9.37   AT1G21500   93.57   AT1G21790   21.97   AT1G22540   5.53   AT1G22700   210.12   AT1G22740   21.91   AT1G22940   37.65   AT1G23080   30.07   AT1G25422   3.05   AT1G26230   25.78   AT1G26761   42.88   AT1G27050   18.95   AT1G27385   39.61   AT1G27910   10.36     1.78   0.66   3.95   0.66   1.05   1.41   3.14   0.48   3.67   0.62   2.38   0.78   2.40   4.55   1.71   0.37   1.25   0.81   0.05   1.87   0.48   0.29   0.51   0.18   0.28   0.49   0.27   1.11   0.36   75.43   15.93   788.36   52.09   27.27   44.22   87.89   13.80   75.96   18.39   114.96   17.57   180.50   204.76   203.60   6.25   66.71   13.68   2.57   155.66   15.01   22.38   23.66   1.05   16.38   29.43   11.42   28.36   7.23   0.21   0.52   2.37   0.62   0.94   0.22   0.69   0.25   1.76   0.19   3.13   0.35   2.14   3.28   3.80   0.12   1.82   0.45   0.22   1.31   0.47   0.42   1.11   0.17   0.28   0.43   0.75   0.48   0.19   -­‐0.53   -­‐0.55   -­‐0.74   -­‐0.45   -­‐0.79   -­‐0.34   -­‐0.83   -­‐0.59   -­‐1.03   -­‐0.48   -­‐0.41   -­‐0.84   -­‐0.39   -­‐0.99   -­‐0.59   -­‐0.58   -­‐0.49   -­‐0.68   -­‐1.10   -­‐0.43   -­‐0.55   -­‐0.75   -­‐0.35   -­‐1.54   -­‐0.65   -­‐0.54   -­‐0.73   -­‐0.48   -­‐0.52   204 1.53E-­‐02   4.48E-­‐02   6.44E-­‐04   1.99E-­‐02   2.17E-­‐05   3.15E-­‐02   7.20E-­‐10   4.86E-­‐02   2.04E-­‐04   3.06E-­‐02   6.93E-­‐03   1.06E-­‐05   1.65E-­‐02   3.13E-­‐08   3.35E-­‐03   3.38E-­‐02   2.30E-­‐02   2.96E-­‐03   6.12E-­‐04   1.49E-­‐02   4.26E-­‐02   3.87E-­‐05   4.99E-­‐02   2.17E-­‐02   3.47E-­‐04   7.04E-­‐03   1.56E-­‐02   3.05E-­‐02   2.46E-­‐02   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP     Table A3.3 (cont’d) AT1G28070   23.20   AT1G28410   16.64   AT1G29390   119.79   AT1G30250   32.50   AT1G30500   6.98   AT1G30510   16.04   AT1G30910   21.79   AT1G31230   37.24   AT1G31460   6.20   AT1G31850   37.44   AT1G32070   67.46   AT1G32090   10.05   AT1G32860   47.13   AT1G33230   47.77   AT1G33265   96.99   AT1G35420   106.21   AT1G35720   1410.82   AT1G44800   32.53   AT1G45191   6.22   AT1G46480   9.94   AT1G47580   26.14   AT1G47710   97.26   AT1G48600   27.78   AT1G49160   10.37   AT1G49560   32.95   AT1G50840   16.86   AT1G51400   461.22   AT1G51610   78.50   AT1G51650   424.94     1.28   0.43   1.15   3.31   0.10   0.32   0.33   0.75   0.22   0.79   0.29   0.13   0.47   0.56   2.21   0.47   2.15   1.45   0.41   0.05   0.50   1.00   1.09   0.47   0.48   0.55   4.33   0.56   1.52   10.83   10.44   61.03   16.63   2.87   11.62   14.17   23.59   3.68   21.90   41.51   5.76   20.18   25.24   60.14   65.65   881.87   24.01   3.89   5.86   16.71   62.81   13.49   5.44   14.60   12.58   326.84   52.66   305.12   0.12   0.42   0.97   1.77   0.13   0.57   0.25   0.43   0.23   0.66   0.68   0.30   0.94   0.51   0.97   0.21   13.58   0.58   0.17   0.26   0.87   1.00   2.54   0.16   0.47   0.17   8.80   0.20   5.02   -­‐1.10   -­‐0.67   -­‐0.97   -­‐0.97   -­‐1.28   -­‐0.47   -­‐0.62   -­‐0.66   -­‐0.75   -­‐0.77   -­‐0.70   -­‐0.80   -­‐1.22   -­‐0.92   -­‐0.69   -­‐0.69   -­‐0.68   -­‐0.44   -­‐0.68   -­‐0.76   -­‐0.65   -­‐0.63   -­‐1.04   -­‐0.93   -­‐1.17   -­‐0.42   -­‐0.50   -­‐0.58   -­‐0.48   205 1.48E-­‐04   5.00E-­‐03   1.74E-­‐08   2.57E-­‐03   5.45E-­‐03   3.60E-­‐02   1.12E-­‐02   2.71E-­‐04   1.98E-­‐02   1.75E-­‐07   3.57E-­‐06   2.72E-­‐04   7.28E-­‐12   2.64E-­‐07   1.94E-­‐04   7.39E-­‐05   1.10E-­‐02   4.12E-­‐02   3.94E-­‐02   3.02E-­‐02   1.52E-­‐02   6.18E-­‐04   3.27E-­‐11   2.19E-­‐05   9.27E-­‐10   4.15E-­‐02   8.19E-­‐03   2.07E-­‐03   1.31E-­‐02   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   POSITIVE   POSITIVE   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   UP   DOWN   NO   NO   NO   DOWN   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT1G52220   644.30   AT1G53035   118.69   AT1G53160   28.70   AT1G53520   30.41   AT1G53590   25.97   AT1G54000   6.48   AT1G54410   5206.40   AT1G55690   30.71   AT1G56300   139.89   AT1G58235   19.26   AT1G60590   10.21   AT1G60680   5.12   AT1G61520   3383.70   AT1G61740   29.12   AT1G62290   4.52   AT1G62960   21.40   AT1G63240   13.97   AT1G64770   162.85   AT1G65590   8.75   AT1G65960   428.15   AT1G66430   84.72   AT1G67080   26.70   AT1G67105   10.03   AT1G69523   18.79   AT1G69830   74.14   AT1G69910   7.96   AT1G70560   9.96   AT1G70640   130.29   AT1G71000   5.59     1.76   0.58   0.45   0.64   0.66   0.16   93.83   0.68   1.20   0.37   0.50   0.26   29.25   0.79   0.25   0.28   0.17   1.36   0.17   4.52   0.21   0.67   0.25   0.25   1.14   0.14   0.47   1.29   0.75   383.59   81.77   11.38   17.49   19.04   2.46   3059.92   18.98   57.61   12.75   6.22   2.45   2573.50   18.64   1.02   15.66   6.57   105.16   4.97   315.59   48.52   18.08   4.38   12.89   49.51   4.99   4.21   29.31   0.45   3.87   0.85   0.44   1.51   0.24   0.35   56.05   0.47   0.35   0.23   0.31   0.14   20.66   0.19   0.40   0.53   0.19   0.89   0.51   1.53   1.26   1.23   0.75   0.25   0.25   0.11   0.28   1.21   0.04   -­‐0.75   -­‐0.54   -­‐1.33   -­‐0.80   -­‐0.45   -­‐1.40   -­‐0.77   -­‐0.69   -­‐1.28   -­‐0.60   -­‐0.71   -­‐1.06   -­‐0.39   -­‐0.64   -­‐2.15   -­‐0.45   -­‐1.09   -­‐0.63   -­‐0.82   -­‐0.44   -­‐0.80   -­‐0.56   -­‐1.20   -­‐0.54   -­‐0.58   -­‐0.67   -­‐1.24   -­‐2.15   -­‐3.64   206 2.38E-­‐05   4.63E-­‐03   3.57E-­‐08   3.78E-­‐04   2.64E-­‐02   7.26E-­‐05   1.53E-­‐02   1.03E-­‐07   1.91E-­‐14   3.05E-­‐02   4.05E-­‐03   1.63E-­‐02   4.52E-­‐02   1.12E-­‐03   1.62E-­‐07   4.27E-­‐02   7.02E-­‐07   5.19E-­‐06   2.04E-­‐03   1.43E-­‐03   5.16E-­‐06   1.87E-­‐02   6.29E-­‐03   2.88E-­‐02   2.02E-­‐03   1.21E-­‐02   3.36E-­‐05   0.00E+00   6.49E-­‐04   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT1G71710   36.99   AT1G71720   61.67   AT1G73655   305.45   AT1G74070   33.66   AT1G74520   88.76   AT1G74560   103.91   AT1G74680   17.75   AT1G74840   79.97   AT1G75750   829.59   AT1G75780   8.56   AT1G76020   34.81   AT1G76790   39.90   AT1G77490   80.16   AT1G77540   80.90   AT1G77580   18.10   AT1G78610   21.05   AT1G78820   36.71   AT1G78995   92.65   AT1G79790   44.79   AT1G80940   28.45   AT2G01275   3.28   AT2G02080   48.63   AT2G02100   1972.26   AT2G02510   218.11   AT2G02740   60.75   AT2G12400   6.92   AT2G13550   12.11   AT2G14045   36.12   AT2G14170   42.86     0.91   1.19   3.02   0.36   0.82   1.15   0.34   0.42   17.38   0.28   0.39   0.40   0.27   0.53   0.58   0.41   0.99   1.12   0.57   0.61   0.06   7.69   17.76   1.02   1.14   0.26   0.16   0.61   0.45   23.05   42.54   187.61   20.20   46.38   63.58   12.35   48.23   397.91   5.13   18.91   22.05   43.63   55.05   9.44   13.62   23.28   63.84   30.37   15.51   1.00   33.63   1127.29   144.88   45.57   2.77   6.50   25.84   33.09   0.25   0.72   1.76   0.58   1.35   0.98   0.22   0.36   13.34   0.16   0.21   0.50   0.63   1.53   0.32   0.52   0.47   0.17   0.34   0.42   0.02   6.37   16.33   2.60   0.56   0.06   0.29   0.58   0.31   -­‐0.68   -­‐0.54   -­‐0.70   -­‐0.74   -­‐0.94   -­‐0.71   -­‐0.52   -­‐0.73   -­‐1.06   -­‐0.74   -­‐0.88   -­‐0.86   -­‐0.88   -­‐0.56   -­‐0.94   -­‐0.63   -­‐0.66   -­‐0.54   -­‐0.56   -­‐0.88   -­‐1.71   -­‐0.53   -­‐0.81   -­‐0.59   -­‐0.41   -­‐1.32   -­‐0.90   -­‐0.48   -­‐0.37   207 1.14E-­‐04   6.14E-­‐03   6.71E-­‐05   2.03E-­‐04   3.37E-­‐07   1.62E-­‐05   2.03E-­‐02   4.45E-­‐05   0.00E+00   1.31E-­‐02   1.38E-­‐04   9.83E-­‐06   3.79E-­‐07   1.86E-­‐02   5.71E-­‐08   9.33E-­‐04   6.50E-­‐04   5.56E-­‐03   5.87E-­‐03   2.64E-­‐06   3.80E-­‐03   2.69E-­‐03   6.08E-­‐05   2.54E-­‐03   4.83E-­‐02   7.71E-­‐06   1.66E-­‐03   3.52E-­‐02   4.08E-­‐02   NO   NO   NEGATIVE   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT2G16600   3296.96   AT2G18328   279.10   AT2G18890   18.94   AT2G19170   8.96   AT2G19860   38.60   AT2G20270   116.11   AT2G21130   94.42   AT2G21250   186.46   AT2G21320   143.22   AT2G21340   26.91   AT2G21370   38.41   AT2G21380   19.13   AT2G21590   21.42   AT2G22080   166.15   AT2G22122   38.51   AT2G22190   50.97   AT2G22240   28.57   AT2G22260   3.76   AT2G22540   84.61   AT2G23340   20.71   AT2G23670   257.19   AT2G23910   13.22   AT2G24120   35.58   AT2G24150   25.03   AT2G24270   308.66   AT2G27530   746.39   AT2G28840   243.86   AT2G29170   43.52   AT2G29320   43.57     32.69   1.40   0.81   0.40   0.69   2.13   0.81   1.25   1.58   0.52   0.32   0.26   0.90   1.73   0.36   1.12   0.59   0.09   0.49   0.63   2.55   0.17   1.32   0.54   1.27   6.61   5.26   4.05   0.77   2148.89   156.19   12.95   6.41   25.07   90.45   69.47   124.95   107.75   14.72   24.58   11.92   7.47   118.32   20.34   24.29   19.55   0.97   61.71   11.81   192.22   8.49   26.22   17.12   221.62   599.41   150.48   22.93   25.32   37.50   0.16   0.52   0.32   0.73   1.59   1.15   2.36   0.65   0.19   0.05   0.29   0.30   1.75   0.34   0.55   1.04   0.17   0.44   0.61   6.07   0.20   0.27   0.86   3.10   9.34   0.45   2.63   0.77   -­‐0.62   -­‐0.84   -­‐0.55   -­‐0.48   -­‐0.62   -­‐0.36   -­‐0.44   -­‐0.58   -­‐0.41   -­‐0.87   -­‐0.64   -­‐0.68   -­‐1.52   -­‐0.49   -­‐0.92   -­‐1.07   -­‐0.55   -­‐1.96   -­‐0.46   -­‐0.81   -­‐0.42   -­‐0.64   -­‐0.44   -­‐0.55   -­‐0.48   -­‐0.32   -­‐0.70   -­‐0.92   -­‐0.78   208 3.34E-­‐02   1.94E-­‐06   1.88E-­‐02   4.55E-­‐02   8.34E-­‐04   3.40E-­‐02   3.53E-­‐02   1.36E-­‐03   3.95E-­‐02   2.93E-­‐06   5.96E-­‐05   1.98E-­‐04   0.00E+00   9.38E-­‐03   1.15E-­‐03   7.57E-­‐09   2.72E-­‐03   1.19E-­‐03   1.74E-­‐02   3.15E-­‐03   3.45E-­‐02   2.32E-­‐02   2.04E-­‐02   1.59E-­‐02   3.31E-­‐04   2.99E-­‐02   7.13E-­‐08   4.64E-­‐02   6.28E-­‐05   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   UP   NO   NO   NO   NO   UP   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT2G29630   895.67   AT2G30170   37.95   AT2G31230   35.16   AT2G31360   401.48   AT2G31840   70.59   AT2G33860   28.37   AT2G34650   9.26   AT2G34720   77.83   AT2G34850   5.32   AT2G35040   118.97   AT2G35700   21.89   AT2G35840   152.42   AT2G35960   85.68   AT2G36145   303.95   AT2G36290   26.32   AT2G36390   100.24   AT2G36460   213.52   AT2G36470   24.80   AT2G37020   34.09   AT2G37600   127.87   AT2G37990   91.18   AT2G39470   170.35   AT2G40350   4.18   AT2G40840   109.73   AT2G41870   25.76   AT2G42130   144.72   AT2G42530   3092.92   AT2G42540   12067.70   AT2G43550   107.72     12.20   0.32   0.60   3.45   1.42   0.72   0.46   0.49   0.17   2.09   0.61   1.79   1.59   3.11   0.45   1.80   2.93   0.29   0.78   1.66   1.43   0.85   0.43   1.47   0.64   1.35   21.48   189.81   0.78   601.79   25.86   22.22   243.17   50.54   14.92   5.41   48.17   1.80   52.20   6.63   113.57   60.07   193.43   18.51   50.23   137.55   14.94   25.16   82.01   67.54   112.01   0.57   49.12   15.18   87.94   1922.89   3281.28   60.57   3.34   0.53   0.41   1.55   0.54   0.56   0.14   0.37   0.11   1.24   0.41   0.20   1.26   0.86   0.14   0.53   2.00   0.34   0.53   1.93   1.43   0.33   0.15   0.61   0.25   1.79   30.40   31.62   2.05   -­‐0.57   -­‐0.55   -­‐0.66   -­‐0.72   -­‐0.48   -­‐0.93   -­‐0.78   -­‐0.69   -­‐1.56   -­‐1.19   -­‐1.72   -­‐0.42   -­‐0.51   -­‐0.65   -­‐0.51   -­‐1.00   -­‐0.63   -­‐0.73   -­‐0.44   -­‐0.64   -­‐0.43   -­‐0.60   -­‐2.87   -­‐1.16   -­‐0.76   -­‐0.72   -­‐0.69   -­‐1.88   -­‐0.83   209 2.82E-­‐02   2.65E-­‐03   4.59E-­‐03   1.83E-­‐04   1.61E-­‐02   1.91E-­‐07   4.67E-­‐03   2.51E-­‐04   1.63E-­‐03   0.00E+00   8.95E-­‐06   6.53E-­‐03   9.64E-­‐03   2.30E-­‐04   2.14E-­‐02   9.34E-­‐09   2.49E-­‐04   3.06E-­‐04   1.77E-­‐02   2.04E-­‐04   3.15E-­‐02   1.10E-­‐04   1.15E-­‐02   3.96E-­‐11   1.82E-­‐04   2.64E-­‐10   8.67E-­‐03   2.90E-­‐08   1.59E-­‐05   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   YES   NO   NO   YES   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   UP   UP   NO     Table A3.3 (cont’d) AT2G44040   AT2G45560   AT2G46660   AT2G46680   AT2G47260   AT2G47420   AT2G47840   AT3G02020   AT3G02630   AT3G02730   AT3G03770   AT3G04860   AT3G05640   AT3G05730   AT3G06035   AT3G06040   AT3G06510   AT3G08940   AT3G09540   AT3G09910   AT3G10920   AT3G11260   AT3G11410   AT3G11670   AT3G11720   AT3G12120   AT3G12490   AT3G12670   AT3G12990     37.40   29.52   8.99   81.38   5.95   48.41   75.32   76.86   38.21   303.82   30.27   7.36   5.18   822.32   82.79   120.35   103.37   829.78   18.48   9.47   493.76   4.94   58.31   44.57   18.99   314.36   638.39   52.00   19.86   0.68   0.25   0.69   2.39   0.44   0.75   1.30   1.20   0.17   0.78   1.04   0.22   0.34   4.21   1.81   2.40   0.41   18.07   0.87   0.14   4.17   0.67   1.19   0.27   0.67   0.78   4.59   0.38   0.36   26.32   13.52   4.76   44.11   3.32   31.99   54.88   55.74   27.17   224.60   21.27   4.14   2.96   458.72   59.35   94.29   78.63   577.56   8.35   5.33   346.63   1.95   31.89   27.41   13.54   248.61   403.22   35.81   12.46   0.42   0.61   0.04   1.95   0.12   0.74   1.18   1.34   0.54   2.28   0.56   0.28   0.21   6.35   1.30   2.11   0.56   7.35   0.52   0.34   2.61   0.18   1.05   0.42   0.27   2.27   5.61   0.78   0.47   -­‐0.51   -­‐1.13   -­‐0.92   -­‐0.88   -­‐0.84   -­‐0.60   -­‐0.46   -­‐0.46   -­‐0.49   -­‐0.44   -­‐0.51   -­‐0.83   -­‐0.81   -­‐0.84   -­‐0.48   -­‐0.35   -­‐0.39   -­‐0.52   -­‐1.15   -­‐0.83   -­‐0.51   -­‐1.34   -­‐0.87   -­‐0.70   -­‐0.49   -­‐0.34   -­‐0.66   -­‐0.54   -­‐0.67   210 1.42E-­‐02   5.15E-­‐11   4.05E-­‐04   2.87E-­‐07   9.12E-­‐03   2.48E-­‐03   2.99E-­‐02   2.32E-­‐02   1.53E-­‐02   2.46E-­‐02   3.92E-­‐04   1.50E-­‐02   3.24E-­‐02   5.89E-­‐06   2.29E-­‐02   3.35E-­‐02   3.14E-­‐03   2.85E-­‐02   2.17E-­‐07   2.26E-­‐02   1.22E-­‐02   3.93E-­‐02   1.07E-­‐06   6.02E-­‐05   6.04E-­‐03   2.71E-­‐02   4.40E-­‐05   4.39E-­‐03   2.00E-­‐03   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT3G13350   AT3G14200   AT3G14210   AT3G15140   AT3G15270   AT3G15357   AT3G15460   AT3G16140   AT3G16670   AT3G17120   AT3G17998   AT3G18090   AT3G18420   AT3G18490   AT3G18500   AT3G19800   AT3G19820   AT3G20300   AT3G20470   AT3G21870   AT3G22150   AT3G22200   AT3G23570   AT3G23580   AT3G23640   AT3G23940   AT3G24420   AT3G24590   AT3G25770     32.03   50.43   121.91   13.31   5.97   1.59   19.88   858.71   151.47   17.75   67.66   1.72   97.34   204.13   16.81   60.07   162.19   13.84   12.86   47.52   14.86   86.31   35.98   31.55   24.94   76.05   20.37   56.38   495.10   0.63   0.68   3.44   0.97   0.11   0.04   0.71   6.55   3.78   0.13   0.67   0.02   3.08   1.77   0.35   0.55   1.79   0.93   0.97   0.39   0.43   0.81   0.54   0.31   0.34   1.43   2.34   0.64   1.10   19.79   37.16   75.14   7.44   2.12   0.21   14.01   562.03   90.51   11.82   33.05   0.62   46.79   146.43   12.18   36.03   117.02   9.15   6.78   31.29   9.59   65.14   26.35   19.44   17.78   61.22   11.95   37.88   352.86   0.30   0.72   2.50   0.21   0.43   0.03   0.43   5.84   3.67   0.62   2.28   0.06   1.01   1.98   0.30   0.96   2.96   0.27   0.20   0.51   0.27   0.82   0.56   0.82   0.42   0.32   0.81   0.64   7.04   -­‐0.69   -­‐0.44   -­‐0.70   -­‐0.84   -­‐1.49   -­‐2.89   -­‐0.50   -­‐0.61   -­‐0.74   -­‐0.59   -­‐1.03   -­‐1.47   -­‐1.06   -­‐0.48   -­‐0.47   -­‐0.74   -­‐0.47   -­‐0.60   -­‐0.92   -­‐0.60   -­‐0.63   -­‐0.41   -­‐0.45   -­‐0.70   -­‐0.49   -­‐0.31   -­‐0.77   -­‐0.57   -­‐0.49   211 5.90E-­‐04   3.15E-­‐02   1.17E-­‐04   2.06E-­‐03   5.11E-­‐03   1.68E-­‐02   4.51E-­‐02   2.52E-­‐03   7.43E-­‐05   2.60E-­‐03   1.13E-­‐09   8.10E-­‐04   1.59E-­‐09   2.08E-­‐02   2.93E-­‐02   6.52E-­‐06   7.85E-­‐05   1.36E-­‐02   1.44E-­‐02   8.81E-­‐03   1.55E-­‐03   5.30E-­‐03   4.87E-­‐02   3.93E-­‐04   9.54E-­‐04   3.96E-­‐02   2.11E-­‐03   4.77E-­‐03   1.75E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT3G26290   35.84   AT3G26300   58.94   AT3G26450   49.29   AT3G26900   54.10   AT3G27180   49.64   AT3G27690   622.51   AT3G29320   107.63   AT3G29390   9.87   AT3G46780   320.82   AT3G46970   413.39   AT3G47160   49.82   AT3G47860   255.85   AT3G48460   23.89   AT3G48610   23.50   AT3G48700   3.15   AT3G49560   171.74   AT3G49910   1235.69   AT3G50500   31.10   AT3G50740   19.33   AT3G52070   26.66   AT3G52390   34.31   AT3G53000   18.41   AT3G53530   17.90   AT3G54050   1147.94   AT3G55120   95.30   AT3G55580   87.91   AT3G55610   306.79   AT3G56490   131.05   AT3G57030   97.85     0.13   0.52   1.06   0.68   1.28   11.88   1.08   0.21   3.82   5.52   0.74   0.78   0.69   0.46   0.48   0.51   9.19   0.08   1.25   2.64   0.74   0.22   0.42   11.42   1.72   1.88   5.19   0.44   0.36   20.30   41.05   24.14   39.69   35.96   312.38   61.50   5.84   219.87   211.20   36.99   136.79   15.27   15.44   0.82   123.01   887.91   20.57   13.74   15.97   20.61   12.50   11.74   836.04   64.13   40.95   173.37   96.96   63.76   0.34   0.14   0.64   0.63   0.63   7.42   0.61   0.04   1.36   0.97   0.96   1.99   1.51   0.65   0.13   2.72   12.03   0.31   0.42   1.15   0.11   0.29   0.93   5.08   1.81   0.93   2.72   1.71   1.80   -­‐0.82   -­‐0.52   -­‐1.03   -­‐0.45   -­‐0.47   -­‐0.99   -­‐0.81   -­‐0.76   -­‐0.55   -­‐0.97   -­‐0.43   -­‐0.90   -­‐0.65   -­‐0.61   -­‐1.93   -­‐0.48   -­‐0.48   -­‐0.60   -­‐0.49   -­‐0.74   -­‐0.74   -­‐0.56   -­‐0.61   -­‐0.46   -­‐0.57   -­‐1.10   -­‐0.82   -­‐0.43   -­‐0.62   212 1.07E-­‐05   7.50E-­‐03   5.97E-­‐07   1.59E-­‐02   1.92E-­‐02   1.37E-­‐07   1.24E-­‐05   2.19E-­‐03   9.05E-­‐03   1.20E-­‐05   4.32E-­‐02   1.35E-­‐07   3.40E-­‐03   2.93E-­‐03   1.83E-­‐03   1.18E-­‐02   4.98E-­‐02   2.41E-­‐03   3.10E-­‐02   3.34E-­‐02   5.88E-­‐05   2.17E-­‐02   2.38E-­‐02   3.00E-­‐02   4.25E-­‐03   1.72E-­‐10   3.00E-­‐07   3.12E-­‐02   7.39E-­‐04   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT3G57062   51.13   AT3G58010   59.01   AT3G58070   21.20   AT3G59980   126.01   AT3G60530   31.80   AT3G61080   41.04   AT3G62750   9.31   AT3G63160   4669.45   AT4G00310   11.29   AT4G00670   0.32   AT4G00810   390.27   AT4G01130   18.25   AT4G01460   12.04   AT4G01670   17.36   AT4G01940   336.24   AT4G02920   396.75   AT4G04330   118.04   AT4G04470   53.27   AT4G04630   9.18   AT4G04840   14.78   AT4G09970   28.64   AT4G10380   13.18   AT4G12000   19.93   AT4G12470   1853.39   AT4G12600   273.29   AT4G12900   33.53   AT4G13150   33.31   AT4G13220   126.85   AT4G13493   338.15     1.30   0.74   0.51   1.31   0.22   0.22   0.16   13.44   0.23   0.07   2.31   0.13   0.29   0.08   0.38   3.16   0.56   0.63   0.03   0.57   1.28   0.61   0.49   67.10   5.11   0.88   0.05   1.96   0.56   35.45   40.30   13.84   91.51   20.07   20.25   6.14   2926.92   6.19   0.05   290.01   6.22   7.81   9.26   190.23   265.80   74.91   39.34   4.29   8.56   20.16   8.80   11.64   1159.34   190.84   19.01   20.37   94.97   222.03   1.02   0.56   0.64   1.92   1.03   0.31   0.08   14.12   0.41   0.03   4.20   0.28   1.05   0.26   2.93   1.68   0.38   0.30   0.71   0.80   0.93   0.54   0.56   45.80   3.16   0.19   0.19   0.55   1.90   -­‐0.53   -­‐0.55   -­‐0.61   -­‐0.46   -­‐0.66   -­‐1.02   -­‐0.60   -­‐0.67   -­‐0.87   -­‐2.60   -­‐0.43   -­‐1.55   -­‐0.63   -­‐0.91   -­‐0.82   -­‐0.58   -­‐0.66   -­‐0.44   -­‐1.10   -­‐0.79   -­‐0.51   -­‐0.58   -­‐0.78   -­‐0.68   -­‐0.52   -­‐0.82   -­‐0.71   -­‐0.42   -­‐0.61   213 3.99E-­‐02   5.71E-­‐03   1.55E-­‐02   1.76E-­‐02   2.12E-­‐03   1.23E-­‐07   4.31E-­‐02   8.93E-­‐03   1.60E-­‐03   1.47E-­‐02   2.63E-­‐02   1.97E-­‐09   2.20E-­‐02   3.65E-­‐04   2.64E-­‐06   2.87E-­‐04   3.64E-­‐04   4.54E-­‐02   1.98E-­‐03   1.81E-­‐02   3.23E-­‐02   2.69E-­‐02   6.39E-­‐04   8.50E-­‐03   6.55E-­‐03   1.09E-­‐03   5.84E-­‐04   4.24E-­‐02   4.49E-­‐02   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT4G13840   59.95   AT4G14020   167.14   AT4G14105   1.06   AT4G14605   45.34   AT4G15510   91.04   AT4G16060   37.62   AT4G17090   1829.44   AT4G17550   60.35   AT4G17730   90.08   AT4G17770   34.77   AT4G18670   13.97   AT4G18700   105.13   AT4G19120   123.77   AT4G19410   85.26   AT4G20170   33.27   AT4G21215   50.47   AT4G22890   240.03   AT4G23630   353.03   AT4G24010   4.40   AT4G24930   124.10   AT4G24960   78.24   AT4G25470   45.16   AT4G25910   83.37   AT4G26530   147.61   AT4G26555   35.33   AT4G26670   80.33   AT4G26950   19.08   AT4G27440   1757.23     0.59   2.61   0.84   1.17   1.31   0.67   6.20   1.96   0.57   1.66   0.48   1.34   0.93   1.17   0.09   0.34   1.93   2.56   0.07   1.51   1.44   0.68   0.41   1.37   0.47   1.74   0.69   14.33   30.00   70.37   0.55   27.90   69.27   20.73   912.16   34.18   68.08   16.44   10.34   69.84   85.47   56.20   18.15   20.32   182.99   246.82   2.06   88.81   51.39   30.27   58.94   98.04   22.77   51.52   11.05   765.72   0.47   1.95   0.38   0.20   0.47   0.53   11.92   0.87   1.10   0.76   0.25   0.88   1.30   0.46   0.18   0.52   2.69   2.95   0.16   0.88   1.56   0.13   1.60   1.27   0.79   1.11   0.85   10.88   -­‐1.00   -­‐1.25   -­‐0.94   -­‐0.70   -­‐0.39   -­‐0.86   -­‐1.00   -­‐0.82   -­‐0.40   -­‐1.08   -­‐0.44   -­‐0.59   -­‐0.53   -­‐0.60   -­‐0.87   -­‐1.31   -­‐0.39   -­‐0.52   -­‐1.10   -­‐0.48   -­‐0.61   -­‐0.58   -­‐0.50   -­‐0.59   -­‐0.63   -­‐0.64   -­‐0.79   -­‐1.20   214 7.31E-­‐09   1.79E-­‐11   4.13E-­‐06   1.68E-­‐04   3.95E-­‐02   2.11E-­‐05   1.04E-­‐03   2.90E-­‐06   4.27E-­‐02   2.66E-­‐10   4.68E-­‐02   1.67E-­‐03   6.11E-­‐04   6.08E-­‐04   1.52E-­‐06   1.53E-­‐13   1.18E-­‐03   1.87E-­‐02   8.23E-­‐04   1.01E-­‐02   1.76E-­‐03   8.06E-­‐03   1.19E-­‐02   7.07E-­‐04   6.71E-­‐03   5.13E-­‐04   2.74E-­‐02   2.50E-­‐06   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   DOW N   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT4G27520   AT4G27560   AT4G27570   AT4G27800   AT4G28706   AT4G28750   AT4G29490   AT4G30710   AT4G31780   AT4G31810   AT4G32520   AT4G32590   AT4G32950   AT4G33480   AT4G33490   AT4G33520   AT4G33905   AT4G33980   AT4G34560   AT4G34950   AT4G34990   AT4G35450   AT4G36240   AT4G36570   AT4G37320   AT4G37330   AT4G37470   AT4G37660   AT4G38050     417.18   26.69   6.79   135.76   30.01   902.84   22.58   7.60   86.15   29.04   82.38   123.17   2.98   67.68   22.09   65.85   5.26   90.40   10.01   270.64   17.70   404.87   12.02   24.70   25.27   14.23   112.63   46.33   4.99   7.47   0.49   0.09   1.46   0.17   5.77   0.42   0.21   1.80   0.38   1.40   1.51   0.00   1.35   0.29   0.59   0.37   1.76   0.46   0.74   0.31   3.60   0.14   1.42   0.44   0.11   1.04   0.80   0.10   256.97   19.70   3.52   103.14   16.80   658.46   16.51   4.02   61.32   21.09   58.89   98.43   0.79   47.50   15.51   33.09   1.70   67.35   5.55   165.98   7.96   335.41   3.79   12.03   8.77   9.79   72.40   31.63   3.05   2.89   0.70   0.19   0.39   0.42   5.73   0.26   0.10   0.70   0.55   0.48   1.10   0.22   0.31   0.47   0.57   0.34   1.26   0.50   1.53   0.27   4.30   0.68   0.97   0.19   0.34   1.12   0.98   0.07   -­‐0.70   -­‐0.44   -­‐0.95   -­‐0.40   -­‐0.84   -­‐0.46   -­‐0.45   -­‐0.92   -­‐0.49   -­‐0.46   -­‐0.48   -­‐0.32   -­‐1.91   -­‐0.51   -­‐0.51   -­‐0.99   -­‐1.63   -­‐0.42   -­‐0.85   -­‐0.71   -­‐1.15   -­‐0.27   -­‐1.67   -­‐1.04   -­‐1.53   -­‐0.54   -­‐0.64   -­‐0.55   -­‐0.71   215 3.76E-­‐04   4.33E-­‐02   4.41E-­‐03   4.10E-­‐02   1.79E-­‐06   3.40E-­‐02   4.23E-­‐02   4.71E-­‐05   1.25E-­‐02   3.12E-­‐02   1.07E-­‐03   2.59E-­‐02   7.88E-­‐03   8.13E-­‐03   6.67E-­‐03   1.69E-­‐13   1.64E-­‐03   2.81E-­‐02   1.22E-­‐02   3.14E-­‐04   4.92E-­‐05   8.71E-­‐03   1.23E-­‐05   3.28E-­‐02   1.85E-­‐14   2.89E-­‐02   3.71E-­‐04   1.57E-­‐02   2.60E-­‐02   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   UP   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   UP   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT4G39260   253.08   AT4G39510   16.21   AT4G39540   31.11   AT4G40065   63.05   AT5G01520   15.75   AT5G02190   11.45   AT5G02240   661.26   AT5G02790   206.73   AT5G03230   20.02   AT5G03670   6.61   AT5G06530   66.07   AT5G07580   53.96   AT5G07690   57.12   AT5G08030   3.70   AT5G08100   35.93   AT5G08260   137.93   AT5G08410   380.19   AT5G09672   5.57   AT5G10170   4.62   AT5G11110   12.76   AT5G11310   7.07   AT5G11450   255.86   AT5G11790   53.17   AT5G11840   44.16   AT5G13930   643.47   AT5G14090   9.91   AT5G14545   3469.79   AT5G14565   53.30   AT5G14570   32.90     5.38   0.42   0.54   0.41   0.44   0.66   3.99   0.43   0.42   0.19   0.74   0.80   1.04   0.28   0.22   0.92   2.36   1.39   0.11   0.72   0.08   0.78   0.81   0.24   16.94   0.74   48.24   1.68   0.35   170.07   11.05   22.28   31.93   4.32   7.93   396.65   143.53   11.76   3.65   45.83   37.87   27.38   1.13   26.49   95.57   287.35   2.65   1.95   8.00   2.65   182.63   39.02   31.62   222.62   4.73   606.23   20.90   21.95   3.23   1.06   0.16   0.50   0.29   0.28   0.69   0.10   0.52   0.26   1.12   1.23   1.51   0.13   0.80   1.34   1.14   0.59   0.02   0.14   0.27   2.88   1.09   0.38   3.79   0.30   4.05   0.40   0.28   -­‐0.57   -­‐0.55   -­‐0.48   -­‐0.98   -­‐1.87   -­‐0.53   -­‐0.74   -­‐0.53   -­‐0.77   -­‐0.86   -­‐0.53   -­‐0.51   -­‐1.06   -­‐1.71   -­‐0.44   -­‐0.53   -­‐0.40   -­‐1.07   -­‐1.24   -­‐0.67   -­‐1.41   -­‐0.49   -­‐0.45   -­‐0.48   -­‐1.53   -­‐1.07   -­‐2.52   -­‐1.35   -­‐0.58   216 1.07E-­‐08   1.37E-­‐02   2.50E-­‐02   9.46E-­‐08   7.72E-­‐12   4.54E-­‐02   1.37E-­‐04   4.85E-­‐03   6.75E-­‐03   3.68E-­‐03   3.16E-­‐03   1.06E-­‐02   1.81E-­‐09   2.22E-­‐03   4.09E-­‐02   6.21E-­‐03   4.39E-­‐02   3.51E-­‐04   9.25E-­‐04   5.34E-­‐04   1.06E-­‐06   1.18E-­‐02   2.93E-­‐02   2.92E-­‐02   0.00E+00   6.12E-­‐04   5.50E-­‐07   0.00E+00   2.58E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP     Table A3.3 (cont’d) AT5G15160   AT5G15190   AT5G15360   AT5G15650   AT5G16010   AT5G16290   AT5G17840   AT5G18680   AT5G19090   AT5G19150   AT5G19460   AT5G20280   AT5G20700   AT5G21020   AT5G21100   AT5G22020   AT5G22070   AT5G22650   AT5G22880   AT5G24300   AT5G24490   AT5G25890   AT5G26570   AT5G28150   AT5G28300   AT5G37360   AT5G39240   AT5G39350   AT5G42420     9.81   19.10   80.07   517.49   154.66   43.91   65.80   18.33   38.88   31.04   32.24   129.33   901.96   196.73   6.66   27.41   32.55   254.73   253.00   157.63   429.59   22.52   173.21   28.30   53.64   52.59   274.06   4.44   49.40   0.13   1.07   1.93   9.35   2.62   0.94   0.82   0.14   0.34   0.05   0.65   2.10   9.56   1.04   0.06   0.63   0.61   2.29   1.73   1.66   1.89   0.63   2.26   0.82   1.63   0.34   2.87   0.23   0.24   4.03   9.97   50.32   340.35   111.70   30.25   40.85   12.50   23.06   23.37   22.62   86.86   620.43   127.91   4.05   20.13   21.75   187.60   161.08   108.50   247.88   14.95   84.75   14.78   29.25   31.46   158.24   2.50   37.08   0.23   0.18   0.93   2.74   1.10   0.44   0.20   0.13   1.18   0.27   0.40   0.54   2.19   1.72   0.53   0.34   0.32   3.36   3.85   0.63   0.76   0.10   0.16   0.37   1.23   0.51   5.08   0.14   0.65   -­‐1.28   -­‐0.94   -­‐0.67   -­‐0.60   -­‐0.47   -­‐0.54   -­‐0.69   -­‐0.55   -­‐0.75   -­‐0.41   -­‐0.51   -­‐0.57   -­‐0.54   -­‐0.62   -­‐0.72   -­‐0.45   -­‐0.58   -­‐0.44   -­‐0.65   -­‐0.54   -­‐0.79   -­‐0.59   -­‐1.03   -­‐0.94   -­‐0.87   -­‐0.74   -­‐0.79   -­‐0.83   -­‐0.41   217 1.85E-­‐02   2.84E-­‐03   7.02E-­‐04   6.11E-­‐03   1.81E-­‐02   3.87E-­‐03   7.49E-­‐04   2.10E-­‐02   1.30E-­‐08   3.20E-­‐02   1.44E-­‐02   6.47E-­‐03   2.36E-­‐02   5.07E-­‐04   1.39E-­‐02   4.64E-­‐02   2.63E-­‐03   2.09E-­‐02   4.14E-­‐04   7.17E-­‐03   1.80E-­‐05   3.32E-­‐02   8.40E-­‐08   2.14E-­‐06   4.43E-­‐07   9.76E-­‐05   1.25E-­‐04   2.14E-­‐02   4.47E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   UP   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT5G42530   3652.28   AT5G42900   200.37   AT5G43150   70.15   AT5G43400   1.17   AT5G43850   101.90   AT5G44005   34.19   AT5G44530   12.75   AT5G45680   279.32   AT5G45930   178.18   AT5G46160   84.25   AT5G46600   13.77   AT5G46710   30.15   AT5G47060   66.36   AT5G47540   28.64   AT5G47700   460.51   AT5G48630   37.21   AT5G48790   55.97   AT5G49190   3.15   AT5G50240   14.84   AT5G50250   179.70   AT5G50360   22.39   AT5G51820   150.21   AT5G54580   63.49   AT5G55280   177.75   AT5G57345   205.84   AT5G57760   35.34   AT5G58490   128.35   AT5G58700   14.42   AT5G59080   20.23     130.06   3.24   1.13   0.10   0.30   0.58   0.29   1.44   1.66   1.38   0.30   0.53   1.15   0.41   2.63   0.85   0.42   0.26   0.17   1.35   0.45   1.44   0.89   2.58   2.27   4.42   1.49   0.58   0.26   1728.50   157.06   33.67   0.43   70.42   13.55   8.85   142.13   93.94   52.30   7.64   15.99   29.78   18.57   317.94   26.28   28.74   1.55   8.63   130.69   11.93   89.79   46.48   125.73   147.91   14.51   71.78   5.98   9.75   9.29   0.76   0.91   0.05   1.53   0.71   0.16   1.01   1.35   1.64   0.29   0.46   0.89   0.61   9.62   0.41   0.45   0.03   0.44   2.00   0.22   0.62   1.70   1.13   0.85   2.76   0.87   0.36   0.59   -­‐1.08   -­‐0.35   -­‐1.06   -­‐1.44   -­‐0.53   -­‐1.34   -­‐0.53   -­‐0.97   -­‐0.92   -­‐0.69   -­‐0.85   -­‐0.91   -­‐1.16   -­‐0.63   -­‐0.53   -­‐0.50   -­‐0.96   -­‐1.02   -­‐0.78   -­‐0.46   -­‐0.91   -­‐0.74   -­‐0.45   -­‐0.50   -­‐0.48   -­‐1.28   -­‐0.84   -­‐1.27   -­‐1.05   218 2.54E-­‐06   5.63E-­‐03   1.21E-­‐06   3.44E-­‐02   6.66E-­‐03   7.59E-­‐07   1.40E-­‐02   7.27E-­‐09   6.20E-­‐08   7.77E-­‐05   1.42E-­‐04   1.74E-­‐05   3.09E-­‐10   1.93E-­‐03   3.40E-­‐03   3.38E-­‐02   1.83E-­‐07   7.50E-­‐03   8.58E-­‐03   1.88E-­‐02   7.07E-­‐05   4.52E-­‐05   3.67E-­‐02   9.98E-­‐03   1.26E-­‐02   2.01E-­‐05   1.27E-­‐06   2.18E-­‐09   2.01E-­‐04   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.3 (cont’d) AT5G59680   1.00   AT5G59700   2.08   AT5G59950   79.63   AT5G60540   94.80   AT5G61520   30.16   AT5G61660   145.45   AT5G61810   39.39   AT5G62360   149.35   AT5G62960   12.13   AT5G63810   44.33   AT5G64040   3250.16   AT5G64240   11.61   AT5G64350   330.08   AT5G64460   104.70   AT5G64550   19.16   AT5G65220   1230.84   AT5G65420   8.51   AT5G65480   95.46   AT5G66420   25.91   AT5G66530   271.43   AT5G66610   4.51   AT5G66720   118.40     0.16   0.08   0.18   0.50   0.18   1.28   0.22   2.66   1.00   0.39   81.42   0.28   0.58   0.94   0.19   17.13   0.21   1.35   0.49   2.45   0.15   0.94   0.28   0.97   53.71   51.24   20.95   106.65   26.32   97.20   4.77   31.87   1924.23   7.14   247.18   76.87   11.35   737.48   4.92   65.95   19.23   192.21   2.31   81.66   0.05   0.08   1.11   0.66   0.22   2.18   0.72   3.60   0.23   0.39   33.98   0.08   3.57   0.87   0.76   17.73   0.14   0.29   0.42   2.00   0.17   1.59   -­‐1.82   -­‐1.10   -­‐0.57   -­‐0.89   -­‐0.53   -­‐0.45   -­‐0.58   -­‐0.62   -­‐1.35   -­‐0.48   -­‐0.76   -­‐0.70   -­‐0.42   -­‐0.45   -­‐0.76   -­‐0.74   -­‐0.79   -­‐0.53   -­‐0.43   -­‐0.50   -­‐0.97   -­‐0.54   219 9.25E-­‐03   1.40E-­‐02   1.48E-­‐04   3.14E-­‐07   6.38E-­‐04   2.71E-­‐02   2.24E-­‐03   7.36E-­‐04   2.43E-­‐06   1.43E-­‐02   5.00E-­‐03   6.58E-­‐03   3.42E-­‐02   1.25E-­‐03   7.49E-­‐05   4.90E-­‐04   1.49E-­‐02   5.62E-­‐03   1.99E-­‐02   9.57E-­‐03   6.22E-­‐03   1.84E-­‐03   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4. 1656 Category 1 FTD Candidate Genes with higher expression in IT at 2 weeks of cold-acclimation. Genes are ordered by their AGI numbers. FPKM values are the average of three biological replicates (n=3). Standard error (SE) values are also given for each FPKM value. Genes were considered significantly different between SW and IT under nonacclimated conditions if they had a Benjamini-Hochberg corrected p-value (FDR P-VALUE) of <0.05 and a minimum of ≥3FPKM in IT. The differential expression of each gene is also represented by the logarithm (base=2) of the fold change (LOG2.FC). This table also denotes if a gene was previously described as positively or negatively correlating with freezing tolerance (HANNAH;(6)), a transcription factor (TF), a gene previously described as up- or down-regulated by 1 week of cold (COS, (25)), identified as an up- or down-regulated component of the CBF regulon (CBF REGULON; (25)). AGI   AT1G01180   AT1G01240   AT1G01340   AT1G01440   AT1G01490   AT1G01560   AT1G02335   AT1G02450   AT1G02500   AT1G02640   AT1G02920   AT1G02930   AT1G03060   AT1G03210   AT1G03290   AT1G03370   AT1G03400   AT1G03730     SW.2WK   SW.2WK   FPKM   SE   3.95   0.21   12.36   0.26   1.05   0.18   5.27   0.06   31.27   0.73   3.21   0.76   4.60   0.26   2.66   0.39   49.68   1.00   1.31   0.04   113.78   4.47   207.21   14.24   4.82   0.20   21.79   0.75   20.73   0.70   11.32   0.61   27.38   0.96   6.51   0.21   IT.2WK   FPKM   11.81   17.96   11.58   8.17   46.49   22.16   8.63   28.09   139.80   2.49   449.52   1012.24   7.04   31.44   45.39   19.25   43.81   10.90   IT.2WK   SE   0.30   0.17   0.46   0.14   0.39   0.42   0.13   1.90   2.80   0.25   0.57   4.71   0.26   0.35   0.30   0.41   0.16   0.38   SW.IT.2WK   LOG2FC   1.58   0.54   3.46   0.63   0.57   2.79   0.91   3.40   1.49   0.93   1.98   2.29   0.55   0.53   1.13   0.77   0.68   0.74   220 SW.IT.2WK   FDR  P-­‐ VALUE   HANNAH?   7.19E-­‐07   NO   2.13E-­‐02   NO   0.00E+00   NO   1.71E-­‐02   NO   1.57E-­‐03   NO   0.00E+00   NO   4.10E-­‐02   NO   7.37E-­‐13   NO   0.00E+00   NO   2.73E-­‐02   NEGATIVE   0.00E+00   NO   0.00E+00   NO   4.99E-­‐03   NO   3.63E-­‐02   NO   0.00E+00   POSITIVE   3.27E-­‐05   NO   6.71E-­‐04   NO   2.74E-­‐02   NO   TF?   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   COS?   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   CBF   REG?   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G04140   18.15   AT1G04425   30.21   AT1G04530   34.88   AT1G04770   8.61   AT1G04780   20.89   AT1G04980   8.28   AT1G05000   3.05   AT1G05010   96.25   AT1G05230   6.76   AT1G05570   12.02   AT1G05630   5.93   AT1G05880   0.33   AT1G05910   14.38   AT1G06410   26.90   AT1G06430   55.48   AT1G06620   1.91   AT1G06645   8.20   AT1G06650   52.94   AT1G07000   7.43   AT1G07128   2.12   AT1G07135   9.88   AT1G07590   141.59   AT1G07600   4144.39   AT1G07620   5.92   AT1G07640   5.78   AT1G07650   19.03   AT1G07900   3.75   AT1G08050   1.68   AT1G08060   3.04     0.43   0.54   0.58   0.44   0.32   0.38   0.34   0.92   0.24   0.43   0.24   0.14   0.27   0.39   0.90   0.33   0.40   0.09   1.16   0.11   1.10   1.64   75.33   0.72   0.28   0.32   0.97   0.17   0.08   23.81   63.27   77.34   21.16   39.76   32.65   6.33   193.56   9.59   20.02   10.10   5.50   20.19   43.52   86.22   5.83   15.11   75.50   22.05   5.22   24.64   208.87   6055.84   18.54   10.15   31.75   9.84   13.21   4.42   0.27   3.12   1.48   0.86   0.39   0.84   0.65   0.98   0.39   0.31   0.23   0.14   0.23   0.29   1.44   0.24   0.19   0.52   0.53   0.14   0.26   1.46   37.14   0.49   0.08   0.41   1.08   0.54   0.32   0.39   1.07   1.15   1.30   0.93   1.98   1.05   1.01   0.50   0.74   0.77   4.07   0.49   0.69   0.64   1.61   0.88   0.51   1.57   1.30   1.32   0.56   0.55   1.65   0.81   0.74   1.39   2.98   0.54   221 1.35E-­‐02   5.43E-­‐05   1.85E-­‐11   4.52E-­‐08   7.32E-­‐08   0.00E+00   7.42E-­‐03   3.09E-­‐09   1.06E-­‐02   2.55E-­‐05   1.44E-­‐05   0.00E+00   1.20E-­‐02   1.08E-­‐04   4.06E-­‐04   1.27E-­‐04   6.60E-­‐04   6.95E-­‐04   1.93E-­‐14   2.64E-­‐02   6.79E-­‐06   1.16E-­‐02   1.62E-­‐02   0.00E+00   2.26E-­‐03   1.02E-­‐08   1.11E-­‐02   0.00E+00   5.85E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G08450   AT1G08600   AT1G08620   AT1G08650   AT1G08830   AT1G08930   AT1G08940   AT1G09180   AT1G09210   AT1G09415   AT1G09560   AT1G09850   AT1G09970   AT1G10020   AT1G10050   AT1G10140   AT1G10155   AT1G10170   AT1G10550   AT1G10920   AT1G10960   AT1G11050   AT1G11190   AT1G11310   AT1G11410   AT1G11790   AT1G11905   AT1G12110   AT1G12140     41.91   5.08   5.50   31.91   215.63   70.02   2.45   1.80   159.65   24.92   62.06   28.52   28.73   7.35   3.83   6.55   29.68   8.59   5.43   4.66   121.53   3.59   0.93   35.64   2.23   15.75   27.86   25.71   18.41   1.71   0.18   0.17   0.91   0.76   0.64   0.33   0.12   2.84   0.72   1.61   0.84   0.71   0.02   0.30   0.30   0.41   0.17   0.78   0.19   1.38   0.37   0.22   0.34   0.12   0.69   0.87   0.17   0.08   157.81   7.18   7.58   43.35   418.66   95.18   15.47   9.33   360.11   40.42   221.01   43.26   89.69   13.02   5.97   14.11   59.89   12.58   14.71   7.46   167.31   11.14   3.11   66.00   3.74   21.96   41.86   35.47   28.13   2.45   0.23   0.19   0.75   6.35   0.93   0.03   0.99   5.38   1.17   1.82   0.56   2.41   0.70   0.40   0.39   0.46   0.29   0.53   0.37   2.05   0.56   0.63   0.67   0.23   0.05   1.08   0.43   0.67   1.91   0.50   0.46   0.44   0.96   0.44   2.66   2.38   1.17   0.70   1.83   0.60   1.64   0.83   0.64   1.11   1.01   0.55   1.44   0.68   0.46   1.63   1.74   0.89   0.75   0.48   0.59   0.46   0.61   222 0.00E+00   3.07E-­‐03   4.15E-­‐02   3.67E-­‐02   3.24E-­‐08   2.20E-­‐02   6.78E-­‐12   3.46E-­‐06   3.99E-­‐10   5.69E-­‐03   0.00E+00   1.87E-­‐03   0.00E+00   2.52E-­‐04   1.39E-­‐02   2.78E-­‐03   1.39E-­‐05   6.96E-­‐03   1.35E-­‐06   3.21E-­‐03   1.95E-­‐02   1.16E-­‐11   2.16E-­‐03   5.82E-­‐08   2.66E-­‐02   2.99E-­‐02   2.10E-­‐03   2.09E-­‐02   2.27E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G12240   AT1G12290   AT1G12360   AT1G12420   AT1G12440   AT1G12520   AT1G12990   AT1G13210   AT1G13245   AT1G13260   AT1G13950   AT1G14010   AT1G14330   AT1G14360   AT1G14370   AT1G14820   AT1G14870   AT1G14890   AT1G15020   AT1G15340   AT1G15430   AT1G15520   AT1G15670   AT1G15750   AT1G15860   AT1G15950   AT1G16090   AT1G16110   AT1G16260     20.47   2.66   24.86   8.25   144.56   37.47   13.24   16.94   47.19   61.71   2.37   33.14   4.07   18.38   7.13   40.45   1.17   4.94   8.43   52.61   5.88   2.98   1.05   32.47   44.88   54.65   3.95   3.08   6.19   0.28   0.17   0.12   0.23   1.06   0.20   0.33   0.87   1.42   1.05   0.25   0.33   0.19   0.48   0.35   0.40   0.34   0.11   0.12   0.62   0.48   1.38   0.22   0.41   0.67   0.85   0.15   0.31   0.12   30.92   5.24   39.12   17.87   209.85   60.71   24.86   44.47   107.41   92.94   6.15   64.06   9.15   50.23   17.52   54.23   8.73   9.45   11.66   82.58   9.48   76.00   4.62   43.96   65.95   101.87   11.99   4.95   9.59   0.62   0.29   0.14   0.30   1.45   0.78   0.25   0.86   2.25   1.26   0.28   0.45   0.38   1.00   0.60   0.57   0.38   0.16   0.16   1.26   0.40   2.25   0.57   0.27   0.99   0.98   0.69   0.24   0.33   0.59   0.98   0.65   1.12   0.54   0.70   0.91   1.39   1.19   0.59   1.38   0.95   1.17   1.45   1.30   0.42   2.91   0.94   0.47   0.65   0.69   4.67   2.14   0.44   0.56   0.90   1.60   0.69   0.63   223 1.59E-­‐03   1.73E-­‐04   3.36E-­‐04   1.14E-­‐07   3.32E-­‐03   3.88E-­‐05   1.82E-­‐06   0.00E+00   2.44E-­‐09   1.32E-­‐03   1.53E-­‐02   5.13E-­‐07   3.19E-­‐06   0.00E+00   2.38E-­‐09   3.67E-­‐02   5.19E-­‐07   1.56E-­‐02   4.70E-­‐02   3.16E-­‐04   4.31E-­‐02   0.00E+00   2.25E-­‐06   3.40E-­‐04   3.90E-­‐04   1.58E-­‐07   7.17E-­‐08   3.19E-­‐02   5.35E-­‐03   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G16270   AT1G16420   AT1G16670   AT1G17070   AT1G17330   AT1G17340   AT1G17360   AT1G17380   AT1G17430   AT1G17440   AT1G17550   AT1G17600   AT1G17620   AT1G17745   AT1G18210   AT1G18260   AT1G18300   AT1G18370   AT1G18390   AT1G18570   AT1G19000   AT1G19020   AT1G19180   AT1G19250   AT1G19300   AT1G19350   AT1G19360   AT1G19380   AT1G19490     4.23   0.97   7.14   4.09   11.06   3.04   11.15   1.70   5.31   20.43   16.07   2.33   13.67   80.82   116.67   15.39   2.23   6.90   6.18   5.38   40.63   5.24   34.47   0.17   2.84   59.81   22.54   1.98   6.86   0.10   0.49   0.48   0.12   0.33   0.15   0.32   0.37   0.35   0.13   0.24   0.15   0.24   1.50   1.37   0.12   0.25   0.14   0.48   0.46   0.56   1.34   0.30   0.13   0.24   1.67   0.18   0.39   0.10   6.10   15.44   18.33   6.35   25.29   6.18   19.29   8.78   8.83   30.36   25.23   6.35   27.82   152.73   156.06   26.32   8.91   9.69   9.98   22.46   55.29   73.67   78.94   13.41   8.02   73.70   33.82   17.52   11.08   0.12   0.82   0.03   0.32   0.11   0.08   0.47   0.49   0.33   0.70   0.71   0.33   0.84   3.89   1.16   0.77   0.64   0.45   0.89   1.50   1.61   1.47   1.22   0.91   0.41   1.89   0.35   1.11   0.25   0.53   3.99   1.36   0.63   1.19   1.02   0.79   2.37   0.73   0.57   0.65   1.45   1.03   0.92   0.42   0.77   2.00   0.49   0.69   2.06   0.44   3.81   1.20   6.32   1.50   0.30   0.59   3.14   0.69   224 1.20E-­‐02   0.00E+00   4.88E-­‐10   1.60E-­‐02   1.14E-­‐06   5.23E-­‐05   1.67E-­‐05   1.59E-­‐08   4.24E-­‐02   5.83E-­‐05   6.09E-­‐04   5.11E-­‐09   3.46E-­‐06   1.97E-­‐07   5.02E-­‐03   4.14E-­‐05   7.29E-­‐07   3.28E-­‐02   1.91E-­‐03   0.00E+00   8.55E-­‐03   0.00E+00   1.08E-­‐12   0.00E+00   4.47E-­‐06   2.43E-­‐02   2.66E-­‐03   2.59E-­‐11   7.79E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   YES   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G19660   AT1G19670   AT1G19835   AT1G20100   AT1G20330   AT1G20510   AT1G20630   AT1G20780   AT1G20920   AT1G21100   AT1G21240   AT1G21250   AT1G21310   AT1G21370   AT1G21450   AT1G21460   AT1G21520   AT1G21580   AT1G21900   AT1G21910   AT1G21920   AT1G22280   AT1G22330   AT1G22410   AT1G22500   AT1G22650   AT1G22750   AT1G22930   AT1G23000     27.48   49.97   15.87   47.59   40.01   38.11   32.92   11.23   8.87   1.06   3.98   56.92   88.27   7.89   10.27   53.05   15.09   8.55   82.80   0.78   15.55   28.43   1.28   42.80   2.04   3.06   56.76   33.99   0.81   0.52   0.99   0.60   0.42   0.51   0.92   0.69   0.37   0.23   0.23   0.42   2.59   2.04   0.07   0.18   0.87   1.33   0.12   0.14   0.17   0.25   0.95   0.16   0.86   0.17   0.11   0.59   0.27   0.10   36.45   66.34   25.56   100.82   65.93   63.52   45.92   18.02   14.13   4.42   21.69   182.09   618.14   15.55   15.51   86.84   51.82   11.65   146.27   2.36   22.03   66.30   2.81   99.73   7.62   8.12   78.97   62.71   2.13   0.59   1.59   0.42   0.42   1.50   1.86   0.44   0.15   0.41   0.16   0.53   1.04   18.55   0.40   0.19   0.84   2.07   0.53   2.08   0.26   1.01   1.41   0.51   1.71   0.43   0.39   0.62   0.30   0.43   0.41   0.41   0.69   1.08   0.72   0.74   0.48   0.68   0.67   2.06   2.45   1.68   2.81   0.98   0.59   0.71   1.78   0.45   0.82   1.61   0.50   1.22   1.14   1.22   1.90   1.41   0.48   0.88   1.40   225 4.42E-­‐02   4.80E-­‐02   2.22E-­‐06   1.91E-­‐10   8.88E-­‐05   1.86E-­‐05   1.52E-­‐02   4.74E-­‐04   4.75E-­‐05   2.15E-­‐05   0.00E+00   0.00E+00   0.00E+00   8.84E-­‐06   5.75E-­‐03   8.57E-­‐05   1.26E-­‐12   2.74E-­‐02   2.83E-­‐06   1.55E-­‐02   2.15E-­‐02   1.04E-­‐13   4.96E-­‐02   1.20E-­‐13   2.07E-­‐07   9.89E-­‐07   4.23E-­‐04   3.73E-­‐13   2.17E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G23020   AT1G23030   AT1G23140   AT1G23170   AT1G23390   AT1G23440   AT1G23480   AT1G23710   AT1G24100   AT1G24140   AT1G24147   AT1G24150   AT1G24265   AT1G24340   AT1G24350   AT1G24460   AT1G24530   AT1G25220   AT1G25280   AT1G25390   AT1G25400   AT1G25550   AT1G26130   AT1G26270   AT1G26380   AT1G26390   AT1G26420   AT1G27020   AT1G27100     0.92   5.88   21.76   2.25   15.44   47.02   4.12   6.60   32.76   1.07   29.36   5.42   1.77   4.26   19.19   11.27   17.87   7.66   17.68   7.64   1.95   17.05   5.90   18.16   2.49   2.41   0.58   18.94   19.70   0.32   0.05   0.69   0.05   0.57   0.54   0.15   0.24   0.29   0.30   3.05   0.43   0.10   0.07   0.26   0.43   0.37   0.28   0.36   0.13   0.06   0.99   0.18   0.46   0.81   1.65   0.21   1.51   0.04   2.07   8.87   37.15   5.09   41.67   92.47   6.96   13.41   63.52   10.30   149.55   14.65   3.71   9.23   25.97   15.81   26.44   21.53   25.03   16.16   6.52   24.01   10.52   29.43   10.94   41.54   5.66   37.27   30.91   0.14   0.42   0.38   0.20   0.29   0.93   0.35   0.92   0.79   0.42   1.42   0.63   0.12   0.26   0.19   0.64   0.49   0.21   0.34   0.49   0.16   0.77   0.33   0.65   0.67   1.24   0.16   1.25   0.70   1.17   0.59   0.77   1.18   1.43   0.98   0.75   1.02   0.96   3.27   2.35   1.43   1.07   1.12   0.44   0.49   0.57   1.49   0.50   1.08   1.74   0.49   0.84   0.70   2.13   4.11   3.27   0.98   0.65   226 8.78E-­‐03   2.30E-­‐02   3.25E-­‐03   1.23E-­‐04   0.00E+00   1.64E-­‐08   4.12E-­‐03   2.89E-­‐04   4.34E-­‐08   1.85E-­‐14   0.00E+00   2.05E-­‐11   1.88E-­‐02   1.75E-­‐06   3.92E-­‐02   1.11E-­‐03   9.37E-­‐03   1.62E-­‐10   2.21E-­‐03   2.00E-­‐07   3.64E-­‐06   3.11E-­‐02   4.15E-­‐06   1.62E-­‐04   2.81E-­‐13   0.00E+00   1.66E-­‐11   6.62E-­‐07   5.28E-­‐04   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   DOWN   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G27290   AT1G27340   AT1G27730   AT1G27770   AT1G27980   AT1G28010   AT1G28240   AT1G28280   AT1G28370   AT1G28380   AT1G28480   AT1G28680   AT1G29290   AT1G29310   AT1G29690   AT1G30040   AT1G30420   AT1G30450   AT1G30470   AT1G30590   AT1G30720   AT1G30755   AT1G30810   AT1G30850   AT1G31130   AT1G31710   AT1G32100   AT1G32230   AT1G32460     16.41   27.18   9.11   14.85   16.07   1.48   8.42   27.00   5.35   22.00   1.52   12.05   0.77   32.29   5.04   1.26   1.43   8.68   15.22   8.21   0.73   6.87   6.01   0.09   39.25   2.61   1.01   87.88   26.02   0.51   0.64   0.77   0.17   0.55   0.06   0.08   0.39   0.72   0.43   0.54   0.37   0.37   0.45   0.09   0.02   0.11   0.05   0.13   0.38   0.11   0.32   0.17   0.03   0.33   0.33   0.16   0.67   0.27   35.53   36.64   39.18   33.85   23.30   2.97   11.70   48.95   9.51   34.86   29.91   19.91   6.16   65.61   11.90   3.65   4.00   11.61   24.99   13.06   3.14   13.91   9.25   4.89   87.80   6.21   2.84   132.58   47.11   0.16   0.15   0.28   0.60   0.24   0.11   0.29   0.84   0.61   0.81   0.99   0.33   0.29   0.77   0.58   0.22   0.21   0.22   0.50   0.30   0.64   0.64   0.19   0.52   0.46   0.46   0.37   0.41   0.56   1.11   0.43   2.11   1.19   0.54   1.00   0.47   0.86   0.83   0.66   4.30   0.72   2.99   1.02   1.24   1.54   1.49   0.42   0.72   0.67   2.11   1.02   0.62   5.78   1.16   1.25   1.49   0.59   0.86   227 8.73E-­‐08   3.86E-­‐02   0.00E+00   1.85E-­‐12   1.12E-­‐02   1.04E-­‐03   4.92E-­‐02   4.86E-­‐07   3.00E-­‐02   3.79E-­‐04   0.00E+00   8.04E-­‐04   7.65E-­‐05   7.40E-­‐09   3.42E-­‐08   9.63E-­‐04   2.15E-­‐08   1.55E-­‐02   6.31E-­‐05   2.54E-­‐03   2.01E-­‐05   2.76E-­‐06   5.66E-­‐03   1.04E-­‐09   3.59E-­‐12   2.03E-­‐05   1.74E-­‐02   3.61E-­‐07   2.06E-­‐04   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G32640   AT1G32700   AT1G32750   AT1G32928   AT1G32960   AT1G33030   AT1G33240   AT1G33360   AT1G33560   AT1G33700   AT1G33811   AT1G33950   AT1G34180   AT1G34190   AT1G34300   AT1G34420   AT1G34750   AT1G35210   AT1G35220   AT1G35230   AT1G35350   AT1G35580   AT1G35670   AT1G36160   AT1G36370   AT1G37130   AT1G42990   AT1G43710   AT1G47128     20.26   18.62   5.36   1.00   1.74   1.34   12.97   7.30   3.29   0.73   6.68   2.01   3.81   52.30   7.59   0.63   9.81   3.84   7.02   1.60   4.59   22.66   8.89   12.12   15.59   102.67   68.33   39.21   173.90   0.09   0.32   0.17   0.24   0.63   0.40   0.18   0.19   0.15   0.06   0.54   0.24   0.38   0.43   0.13   0.11   0.83   1.01   0.19   0.55   0.11   0.00   0.08   0.30   0.66   0.90   0.49   0.26   1.83   28.10   46.25   7.84   3.72   37.61   11.21   21.71   12.56   10.91   4.13   12.26   30.88   8.79   73.09   13.47   3.69   29.35   12.01   9.60   17.74   9.90   42.71   18.79   18.05   33.71   191.77   197.75   53.39   346.49   0.51   0.18   0.29   0.35   1.51   0.45   0.28   0.10   0.01   0.07   0.76   4.65   0.32   0.84   0.62   0.27   0.28   0.74   0.23   0.67   0.54   0.33   0.20   0.09   1.58   0.18   2.59   1.68   7.44   0.47   1.31   0.55   1.90   4.43   3.06   0.74   0.78   1.73   2.51   0.88   3.94   1.21   0.48   0.83   2.55   1.58   1.64   0.45   3.47   1.11   0.91   1.08   0.57   1.11   0.90   1.53   0.45   0.99   228 2.17E-­‐02   4.90E-­‐12   7.40E-­‐03   1.99E-­‐02   0.00E+00   2.96E-­‐13   8.57E-­‐05   2.66E-­‐04   1.86E-­‐14   0.00E+00   1.15E-­‐03   0.00E+00   3.51E-­‐06   1.35E-­‐02   8.23E-­‐05   1.52E-­‐10   0.00E+00   8.16E-­‐05   4.77E-­‐02   5.86E-­‐11   3.67E-­‐06   1.04E-­‐08   2.74E-­‐07   1.36E-­‐04   3.37E-­‐10   3.64E-­‐06   0.00E+00   2.62E-­‐02   2.91E-­‐07   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G48090   AT1G48120   AT1G48320   AT1G48370   AT1G48490   AT1G48605   AT1G49050   AT1G49450   AT1G49750   AT1G50140   AT1G50740   AT1G51350   AT1G51620   AT1G51660   AT1G51680   AT1G51760   AT1G51790   AT1G51805   AT1G51860   AT1G52030   AT1G52340   AT1G52780   AT1G52800   AT1G53920   AT1G54115   AT1G55210   AT1G55310   AT1G55325   AT1G55350     8.71   2.51   9.72   5.46   5.76   5.47   9.11   8.47   126.99   11.04   15.79   7.22   1.15   15.55   31.66   19.06   0.24   26.84   0.74   5.95   12.27   11.10   0.44   5.93   9.90   120.70   3.99   5.48   10.50   0.41   0.07   0.23   0.04   0.07   0.33   0.74   0.44   0.31   0.25   0.28   0.04   0.29   0.25   1.52   0.33   0.10   0.67   0.20   1.21   0.37   0.21   0.11   0.30   0.20   3.07   0.18   0.11   0.27   12.79   3.67   28.33   8.18   7.78   13.66   24.43   13.78   175.49   14.70   33.18   10.72   3.71   25.44   63.96   50.79   3.31   51.90   2.19   8.86   20.38   19.04   2.88   10.91   15.87   227.86   6.76   8.70   13.42   0.45   0.08   0.63   0.58   0.21   0.45   0.35   0.73   1.10   0.17   0.47   0.22   0.50   0.53   0.51   0.50   0.54   0.54   0.19   1.22   0.79   0.63   0.36   0.13   0.17   4.26   0.08   0.27   0.33   0.56   0.55   1.54   0.58   0.43   1.32   1.42   0.70   0.47   0.41   1.07   0.57   1.69   0.71   1.01   1.41   3.78   0.95   1.56   0.58   0.73   0.78   2.70   0.88   0.68   0.92   0.76   0.67   0.35   229 3.71E-­‐03   4.96E-­‐02   4.12E-­‐09   2.46E-­‐02   4.41E-­‐02   1.68E-­‐03   2.16E-­‐13   2.05E-­‐03   1.98E-­‐02   1.36E-­‐02   8.24E-­‐06   1.61E-­‐02   2.59E-­‐04   4.93E-­‐04   4.40E-­‐11   0.00E+00   4.95E-­‐13   0.00E+00   5.49E-­‐04   2.09E-­‐02   2.28E-­‐03   2.75E-­‐05   3.95E-­‐04   5.11E-­‐03   1.73E-­‐03   6.42E-­‐09   4.53E-­‐02   5.48E-­‐06   4.31E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G55450   AT1G55850   AT1G55910   AT1G55915   AT1G55960   AT1G56060   AT1G56120   AT1G56145   AT1G56150   AT1G56330   AT1G56340   AT1G56460   AT1G56550   AT1G56610   AT1G57990   AT1G58070   AT1G58440   AT1G59580   AT1G59620   AT1G59710   AT1G59870   AT1G59910   AT1G60270   AT1G60420   AT1G60490   AT1G60610   AT1G61100   AT1G61120   AT1G61140     36.73   7.76   20.07   3.87   12.85   1.83   3.27   3.41   3.44   212.73   112.05   19.98   0.45   10.03   2.84   0.69   10.41   10.95   4.73   17.03   166.74   5.40   3.30   25.83   15.97   2.30   12.10   0.25   5.83   1.10   0.27   1.18   0.07   0.27   0.54   0.37   0.12   0.12   1.10   1.76   0.24   0.15   0.36   0.30   0.11   0.39   0.59   0.36   0.20   1.91   0.12   0.13   0.04   0.19   0.74   0.29   0.19   0.23   73.87   19.33   48.57   6.74   23.11   32.49   10.13   6.81   7.98   308.75   246.84   26.83   4.16   14.14   11.19   2.37   15.72   16.37   12.79   38.13   315.07   7.73   5.60   34.77   27.19   4.72   17.64   4.64   8.57   0.94   0.56   0.66   0.05   0.44   1.92   0.08   0.20   0.46   4.47   3.89   0.40   0.36   0.16   0.32   0.27   0.39   0.30   0.53   0.46   5.44   0.32   0.32   0.40   0.40   0.00   0.23   0.33   0.39   1.01   1.32   1.28   0.80   0.85   4.15   1.63   1.00   1.22   0.54   1.14   0.43   3.20   0.50   1.98   1.78   0.59   0.58   1.43   1.16   0.92   0.52   0.76   0.43   0.77   1.04   0.54   4.18   0.56   230 3.64E-­‐14   1.35E-­‐11   1.02E-­‐11   2.00E-­‐02   2.60E-­‐05   9.76E-­‐13   1.84E-­‐13   1.41E-­‐05   4.58E-­‐02   6.42E-­‐03   0.00E+00   3.95E-­‐02   7.62E-­‐09   2.81E-­‐02   3.41E-­‐10   8.54E-­‐03   8.72E-­‐03   7.49E-­‐03   6.19E-­‐12   1.02E-­‐10   1.85E-­‐04   3.68E-­‐02   4.17E-­‐02   3.74E-­‐02   2.54E-­‐05   1.21E-­‐02   6.67E-­‐03   0.00E+00   7.28E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G61250   AT1G61260   AT1G61360   AT1G61370   AT1G61470   AT1G61560   AT1G61660   AT1G62300   AT1G62360   AT1G62422   AT1G62500   AT1G62630   AT1G62660   AT1G62790   AT1G62840   AT1G63010   AT1G63100   AT1G63220   AT1G63670   AT1G63720   AT1G63750   AT1G63840   AT1G64050   AT1G64060   AT1G64280   AT1G64400   AT1G64460   AT1G64470   AT1G64490     28.81   2.53   3.85   1.39   0.63   1.43   19.45   4.75   0.48   4.33   3.68   7.04   1.62   60.31   0.58   22.58   5.42   16.24   19.21   1.93   1.68   8.39   8.49   3.02   17.27   15.23   6.29   6.26   38.19   0.30   0.43   0.06   0.10   0.18   0.00   0.28   0.23   0.07   0.63   0.10   0.10   0.23   1.34   0.23   0.13   0.31   0.35   0.46   0.26   0.04   0.69   0.37   0.06   0.52   0.75   0.44   0.26   0.24   61.10   6.91   7.39   3.25   2.08   4.06   26.03   19.07   2.15   18.22   13.31   12.85   6.23   85.19   3.26   31.26   8.32   35.16   24.89   6.51   3.49   45.30   12.13   7.80   31.50   28.03   16.73   16.82   56.43   0.71   0.29   0.30   0.02   0.14   0.13   0.27   0.78   0.44   0.37   1.12   0.04   0.26   1.05   0.09   0.33   0.29   0.87   0.76   0.04   0.57   1.16   0.37   0.13   0.31   1.06   1.02   0.60   1.51   1.08   1.45   0.94   1.22   1.72   1.50   0.42   2.01   2.16   2.07   1.86   0.87   1.94   0.50   2.48   0.47   0.62   1.11   0.37   1.75   1.06   2.43   0.51   1.37   0.87   0.88   1.41   1.43   0.56   231 5.79E-­‐10   6.66E-­‐05   4.48E-­‐05   1.30E-­‐03   4.40E-­‐02   4.77E-­‐06   2.19E-­‐02   0.00E+00   1.00E-­‐03   4.14E-­‐10   4.37E-­‐11   2.86E-­‐05   3.90E-­‐10   2.54E-­‐03   4.30E-­‐04   4.00E-­‐04   2.62E-­‐02   4.98E-­‐06   1.11E-­‐02   2.40E-­‐06   1.03E-­‐04   0.00E+00   2.04E-­‐02   1.62E-­‐10   8.28E-­‐07   1.79E-­‐06   3.87E-­‐07   4.80E-­‐07   2.26E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G64600   AT1G64620   AT1G65040   AT1G65200   AT1G65481   AT1G65800   AT1G65820   AT1G65845   AT1G66090   AT1G66330   AT1G66760   AT1G66840   AT1G66850   AT1G66910   AT1G66920   AT1G67195   AT1G67260   AT1G67490   AT1G67560   AT1G67800   AT1G67865   AT1G67900   AT1G67920   AT1G68290   AT1G68440   AT1G68670   AT1G68690   AT1G68710   AT1G68840     7.71   1.51   13.10   2.08   0.48   5.50   220.36   13.41   0.50   45.08   5.72   11.73   0.00   3.01   4.87   28.24   2.66   4.47   5.96   23.19   8.39   12.04   9.36   1.28   5.40   9.16   0.98   2.96   14.66   0.30   0.14   0.18   0.04   0.24   0.59   3.01   1.52   0.15   0.34   0.22   0.26   0.00   0.08   0.06   0.46   0.11   0.10   0.14   0.31   1.27   0.25   0.94   0.17   0.07   0.71   0.07   0.06   0.31   11.88   3.22   37.17   3.73   10.67   12.33   302.72   76.46   6.32   62.31   9.08   16.27   6.95   8.41   12.22   62.11   5.37   6.44   13.05   36.85   45.30   20.96   30.74   5.30   23.20   14.72   4.93   4.91   23.43   0.13   0.20   1.55   0.00   1.25   0.15   2.46   3.60   0.20   0.73   1.29   0.41   3.60   0.48   1.06   1.61   3.57   0.08   0.01   0.80   2.21   0.29   0.59   0.84   1.57   0.92   0.04   0.10   1.32   0.62   1.09   1.50   0.85   4.47   1.17   0.46   2.51   3.67   0.47   0.67   0.47   Inf   1.48   1.33   1.14   1.02   0.53   1.13   0.67   2.43   0.80   1.71   2.05   2.10   0.68   2.33   0.73   0.68   232 1.40E-­‐02   2.43E-­‐02   0.00E+00   4.84E-­‐03   7.37E-­‐07   2.47E-­‐08   1.74E-­‐02   0.00E+00   3.27E-­‐12   1.28E-­‐03   1.33E-­‐02   3.91E-­‐02   2.55E-­‐04   1.12E-­‐07   8.40E-­‐10   3.52E-­‐07   2.01E-­‐02   4.23E-­‐02   1.63E-­‐08   4.73E-­‐07   1.10E-­‐10   1.47E-­‐07   6.60E-­‐08   4.01E-­‐05   0.00E+00   4.28E-­‐03   1.96E-­‐12   3.17E-­‐03   2.01E-­‐03   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G68910   AT1G69160   AT1G69440   AT1G69520   AT1G69570   AT1G69572   AT1G69780   AT1G69840   AT1G69900   AT1G69920   AT1G70140   AT1G70160   AT1G70180   AT1G70250   AT1G70290   AT1G70520   AT1G70530   AT1G70620   AT1G70700   AT1G70710   AT1G70740   AT1G70782   AT1G71010   AT1G71030   AT1G71140   AT1G71220   AT1G71390   AT1G71840   AT1G71970     11.17   9.16   1.83   3.39   6.71   9.00   7.58   24.97   0.99   0.56   0.83   60.58   7.76   8.15   4.07   12.40   10.56   12.58   35.34   4.54   3.87   42.74   7.52   61.57   3.76   25.23   0.10   36.83   9.75   0.24   0.08   0.14   0.40   0.08   0.15   0.10   0.20   0.16   0.29   0.10   0.30   0.09   0.40   0.27   0.45   0.45   0.16   0.46   0.15   0.25   0.80   0.10   0.94   0.32   0.68   0.06   0.82   0.20   17.68   19.64   3.48   9.06   12.19   14.98   12.68   60.13   3.16   2.96   3.27   92.33   12.89   14.29   7.71   29.39   19.52   17.19   82.14   7.86   7.69   78.56   12.16   100.03   7.41   41.06   4.21   48.79   15.27   0.10   0.83   0.12   0.57   0.52   0.65   0.80   0.88   0.24   0.85   0.19   0.98   0.06   0.93   0.24   0.63   0.37   0.55   1.30   0.59   0.21   2.38   0.17   0.90   0.55   0.72   0.55   0.97   0.77   0.66   1.10   0.92   1.42   0.86   0.74   0.74   1.27   1.67   2.40   1.99   0.61   0.73   0.81   0.92   1.25   0.89   0.45   1.22   0.79   0.99   0.88   0.69   0.70   0.98   0.70   5.33   0.41   0.65   233 2.01E-­‐04   1.07E-­‐06   3.56E-­‐03   1.55E-­‐03   4.95E-­‐04   1.13E-­‐02   5.68E-­‐03   0.00E+00   2.01E-­‐03   1.33E-­‐03   1.10E-­‐06   1.12E-­‐03   3.89E-­‐04   1.45E-­‐04   6.30E-­‐05   7.05E-­‐12   1.55E-­‐05   3.03E-­‐03   6.92E-­‐13   6.88E-­‐03   6.68E-­‐04   1.05E-­‐06   2.62E-­‐04   9.97E-­‐05   2.33E-­‐03   1.06E-­‐08   5.10E-­‐12   4.99E-­‐02   3.41E-­‐02   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G72150   AT1G72240   AT1G72280   AT1G72450   AT1G72680   AT1G72700   AT1G72770   AT1G72900   AT1G72910   AT1G72920   AT1G72930   AT1G72940   AT1G73030   AT1G73540   AT1G73650   AT1G73740   AT1G74020   AT1G74030   AT1G74080   AT1G74090   AT1G74100   AT1G74210   AT1G74360   AT1G75000   AT1G75130   AT1G75220   AT1G75280   AT1G75380   AT1G75410     54.88   0.96   7.89   28.86   26.54   10.69   15.99   6.70   3.63   1.32   70.95   3.54   72.42   4.91   54.96   5.97   19.30   5.05   0.25   54.16   38.77   5.24   2.32   1.07   6.35   5.01   20.83   52.08   13.39   0.86   0.16   0.83   0.47   0.59   0.29   0.16   0.90   0.55   0.14   0.77   0.20   0.75   0.31   0.23   0.13   0.78   0.28   0.09   0.86   0.58   0.24   0.24   0.06   0.02   0.33   0.30   0.71   0.10   73.07   5.14   29.39   64.54   75.99   25.14   23.55   41.84   16.88   8.66   146.22   13.44   100.93   11.44   71.97   9.95   35.90   8.05   2.77   75.83   96.33   13.56   9.32   5.23   9.87   11.36   29.41   88.51   28.60   1.05   0.61   1.71   0.66   1.46   0.12   0.56   0.76   0.69   0.56   1.42   0.82   1.05   0.66   0.31   0.53   1.25   0.59   0.19   1.79   1.64   0.39   0.06   1.00   0.25   0.05   1.56   0.67   0.55   0.41   2.42   1.90   1.16   1.52   1.23   0.56   2.64   2.22   2.71   1.04   1.92   0.48   1.22   0.39   0.74   0.89   0.67   3.49   0.49   1.31   1.37   2.01   2.28   0.64   1.18   0.50   0.76   1.10   234 4.25E-­‐02   1.78E-­‐03   0.00E+00   5.44E-­‐11   0.00E+00   2.96E-­‐13   7.45E-­‐04   0.00E+00   0.00E+00   2.64E-­‐09   1.22E-­‐10   5.51E-­‐12   1.74E-­‐02   5.28E-­‐04   3.96E-­‐03   7.91E-­‐03   5.42E-­‐06   2.16E-­‐02   2.01E-­‐06   1.64E-­‐02   1.88E-­‐14   1.94E-­‐07   0.00E+00   9.82E-­‐05   2.35E-­‐02   7.79E-­‐07   2.52E-­‐02   1.86E-­‐07   2.74E-­‐10   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G75440   AT1G75960   AT1G76040   AT1G76070   AT1G76090   AT1G76240   AT1G76320   AT1G76390   AT1G76640   AT1G76690   AT1G76760   AT1G76890   AT1G76892   AT1G76980   AT1G77300   AT1G77320   AT1G77380   AT1G77420   AT1G77460   AT1G77770   AT1G77800   AT1G77810   AT1G77990   AT1G78000   AT1G78150   AT1G78200   AT1G78230   AT1G78280   AT1G78460     8.63   0.53   4.58   1.97   4.80   4.44   6.97   6.04   0.18   3.56   4.20   3.09   5.86   14.44   7.22   3.01   1.91   7.25   2.07   9.44   10.18   13.90   3.42   0.66   71.75   35.34   11.55   6.25   38.86   0.68   0.05   0.25   0.24   0.04   0.23   0.09   0.23   0.08   0.41   0.15   0.23   0.29   0.86   0.36   0.16   0.30   0.16   0.63   0.18   0.45   0.36   0.33   0.07   0.57   0.41   0.74   0.04   1.85   17.36   2.22   20.15   4.88   8.15   16.52   9.43   12.52   3.26   6.15   9.80   6.13   9.19   48.59   9.53   4.38   6.72   11.79   3.52   17.39   13.22   28.27   6.50   3.96   102.22   49.03   19.67   10.27   53.12   0.77   0.17   0.60   0.16   0.42   0.32   0.07   0.15   0.73   0.54   0.42   0.25   0.42   1.24   0.16   0.12   0.24   0.21   0.17   0.27   0.34   1.20   0.50   0.50   2.06   0.88   1.06   0.31   1.03   1.01   2.06   2.14   1.31   0.76   1.90   0.43   1.05   4.14   0.79   1.22   0.99   0.65   1.75   0.40   0.54   1.81   0.70   0.76   0.88   0.38   1.02   0.92   2.57   0.51   0.47   0.77   0.72   0.45   235 1.87E-­‐03   2.01E-­‐04   0.00E+00   4.27E-­‐03   2.16E-­‐02   1.63E-­‐10   4.35E-­‐02   9.33E-­‐09   5.42E-­‐03   4.44E-­‐02   3.11E-­‐03   1.32E-­‐03   3.30E-­‐02   0.00E+00   2.41E-­‐02   4.64E-­‐02   4.58E-­‐08   8.11E-­‐03   1.49E-­‐04   2.18E-­‐04   2.97E-­‐02   7.96E-­‐10   1.15E-­‐03   2.84E-­‐11   1.29E-­‐04   4.41E-­‐03   7.53E-­‐05   6.13E-­‐04   4.50E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT1G78510   AT1G78850   AT1G78920   AT1G79000   AT1G79380   AT1G79680   AT1G79830   AT1G79990   AT1G80020   AT1G80280   AT1G80420   AT1G80440   AT1G80460   AT1G80490   AT1G80510   AT1G80610   AT1G80840   AT1G80960   AT2G01180   AT2G01190   AT2G01450   AT2G01650   AT2G01680   AT2G01850   AT2G01860   AT2G02010   AT2G02220   AT2G02810   AT2G02870     48.67   51.94   22.55   8.19   27.31   0.55   27.19   52.87   8.31   8.66   10.74   1.71   21.73   7.49   7.73   1.61   2.27   1.53   4.94   6.00   29.04   11.99   11.26   34.08   14.53   1.38   6.33   13.92   15.85   0.00   1.45   0.29   0.32   0.36   0.20   0.45   0.77   0.22   0.30   0.52   0.35   0.33   0.10   0.03   0.16   0.57   0.16   0.35   0.20   0.39   0.14   0.23   1.42   0.84   0.37   0.34   0.36   0.30   80.65   80.70   36.65   11.65   43.40   4.61   33.59   69.11   11.29   16.01   19.24   7.94   52.88   11.39   11.07   4.11   30.31   4.80   10.49   12.54   55.21   19.87   17.92   47.71   22.19   4.93   16.64   50.41   25.73   0.00   0.76   0.89   0.33   0.34   0.20   0.35   0.86   0.17   0.44   0.38   0.57   0.58   0.26   0.14   0.08   1.33   0.13   0.16   0.42   0.69   0.25   0.29   0.71   0.38   0.57   0.72   1.65   0.48   0.73   0.64   0.70   0.51   0.67   3.06   0.30   0.39   0.44   0.89   0.84   2.21   1.28   0.61   0.52   1.35   3.74   1.65   1.09   1.06   0.93   0.73   0.67   0.49   0.61   1.84   1.39   1.86   0.70   236 4.70E-­‐09   4.07E-­‐04   1.05E-­‐04   5.87E-­‐03   3.25E-­‐04   1.14E-­‐12   6.42E-­‐03   4.45E-­‐03   4.85E-­‐02   5.01E-­‐06   1.09E-­‐05   3.05E-­‐09   1.87E-­‐14   3.81E-­‐04   4.71E-­‐02   8.15E-­‐03   0.00E+00   1.88E-­‐05   1.94E-­‐05   4.32E-­‐07   4.43E-­‐10   7.01E-­‐04   1.42E-­‐03   1.79E-­‐02   3.30E-­‐03   3.56E-­‐06   2.00E-­‐13   0.00E+00   7.78E-­‐05   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G02930   AT2G02990   AT2G03120   AT2G03730   AT2G04160   AT2G04390   AT2G04400   AT2G04430   AT2G04515   AT2G04650   AT2G04780   AT2G05320   AT2G06050   AT2G07180   AT2G11520   AT2G13790   AT2G14740   AT2G15042   AT2G15580   AT2G15760   AT2G16280   AT2G16430   AT2G16500   AT2G16890   AT2G16900   AT2G17130   AT2G17220   AT2G17230   AT2G17290     48.69   2.27   106.40   19.71   16.32   34.13   40.68   5.79   0.44   2.87   19.58   8.68   23.06   37.77   11.05   3.27   7.51   15.65   15.41   1.37   49.01   33.55   104.26   19.67   8.40   39.13   9.88   28.33   16.44   2.21   1.61   1.10   0.45   1.86   0.63   0.67   0.28   0.11   0.09   1.00   0.39   0.72   0.33   0.19   0.29   0.18   0.63   0.26   0.39   1.20   0.38   0.71   0.30   0.13   0.37   0.23   0.49   0.46   157.33   6.85   199.87   26.76   31.34   48.88   81.01   29.57   5.32   8.95   31.59   15.12   36.66   47.71   18.43   8.92   10.52   31.61   32.54   3.12   70.84   65.81   240.53   37.07   39.31   57.83   21.25   42.44   26.81   3.60   1.18   1.76   0.12   1.51   0.91   0.75   0.35   0.47   0.21   1.26   0.46   0.69   0.71   0.14   0.18   0.13   1.29   0.19   0.15   1.54   0.97   1.43   1.18   0.93   0.96   0.22   0.94   0.18   1.69   1.59   0.91   0.44   0.94   0.52   0.99   2.35   3.60   1.64   0.69   0.80   0.67   0.34   0.74   1.45   0.49   1.01   1.08   1.18   0.53   0.97   1.21   0.91   2.23   0.56   1.10   0.58   0.71   237 0.00E+00   5.34E-­‐04   2.55E-­‐07   3.25E-­‐02   4.98E-­‐08   3.32E-­‐02   5.44E-­‐09   0.00E+00   6.56E-­‐07   2.03E-­‐07   3.75E-­‐04   1.11E-­‐03   1.98E-­‐04   3.17E-­‐02   4.21E-­‐04   1.88E-­‐08   4.79E-­‐02   5.08E-­‐08   7.58E-­‐07   3.55E-­‐02   6.05E-­‐03   1.91E-­‐08   1.21E-­‐10   1.41E-­‐07   0.00E+00   1.27E-­‐03   1.16E-­‐10   2.76E-­‐03   1.62E-­‐04   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G17520   AT2G17560   AT2G17710   AT2G17720   AT2G17730   AT2G17740   AT2G17790   AT2G17930   AT2G18160   AT2G18280   AT2G18660   AT2G19130   AT2G19190   AT2G19710   AT2G20010   AT2G20142   AT2G20570   AT2G20610   AT2G20720   AT2G20870   AT2G20960   AT2G21140   AT2G21470   AT2G21500   AT2G22300   AT2G22420   AT2G22480   AT2G22500   AT2G22560     15.79   121.90   20.34   46.21   12.62   4.19   15.64   6.19   22.86   41.81   38.78   8.32   2.31   3.49   7.25   7.52   88.36   60.51   1.86   4.49   17.39   23.67   14.57   8.20   18.06   10.29   10.26   21.82   1.65   0.33   2.40   1.29   0.46   0.15   1.84   0.27   0.18   0.67   0.47   2.20   0.62   0.85   0.06   0.08   0.58   1.33   0.92   0.08   0.46   0.09   0.49   0.17   0.36   0.38   0.16   0.22   1.09   0.09   24.02   148.76   32.91   81.83   25.63   17.39   22.37   9.17   34.43   54.36   135.92   22.48   19.87   9.62   13.07   33.62   122.65   84.20   3.38   9.65   29.38   36.05   21.23   13.05   30.80   15.50   15.30   57.11   4.05   0.55   2.93   0.45   0.77   0.10   1.17   0.23   0.49   0.11   0.50   2.62   0.52   1.54   0.45   0.35   1.95   2.08   1.01   0.44   5.37   0.70   2.73   0.29   0.91   0.45   0.59   0.40   0.55   0.42   0.60   0.29   0.69   0.82   1.02   2.05   0.52   0.57   0.59   0.38   1.81   1.43   3.11   1.46   0.85   2.16   0.47   0.48   0.86   1.10   0.76   0.61   0.54   0.67   0.77   0.59   0.58   1.39   1.29   238 1.58E-­‐03   3.52E-­‐02   2.63E-­‐03   3.46E-­‐06   5.76E-­‐07   3.15E-­‐10   9.85E-­‐03   2.71E-­‐03   5.15E-­‐03   2.68E-­‐02   0.00E+00   1.85E-­‐14   0.00E+00   7.95E-­‐12   1.89E-­‐07   0.00E+00   1.43E-­‐02   8.66E-­‐03   2.90E-­‐02   1.88E-­‐02   4.15E-­‐05   2.90E-­‐03   1.77E-­‐03   2.25E-­‐03   4.77E-­‐09   3.39E-­‐02   1.19E-­‐02   0.00E+00   3.86E-­‐05   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G23140   AT2G23170   AT2G23200   AT2G23270   AT2G23290   AT2G23320   AT2G23380   AT2G23450   AT2G23680   AT2G23770   AT2G23810   AT2G24200   AT2G24360   AT2G24550   AT2G24590   AT2G24600   AT2G24650   AT2G25000   AT2G25110   AT2G25735   AT2G26190   AT2G26300   AT2G26400   AT2G26440   AT2G26530   AT2G26540   AT2G26800   AT2G26980   AT2G27200     6.61   1.31   10.21   0.14   1.71   11.48   7.91   7.44   5.31   1.67   42.29   198.37   62.41   1.52   55.96   2.04   1.79   8.43   48.87   2.79   18.32   18.57   6.87   18.44   4.99   19.68   15.31   8.98   12.49   0.10   0.05   0.46   0.04   0.12   0.54   0.41   0.16   0.92   0.24   0.61   1.31   0.52   0.05   0.72   0.89   0.10   0.34   0.82   0.19   1.15   0.31   1.35   1.48   0.24   0.60   0.47   0.38   0.03   10.60   15.67   17.81   3.59   9.32   31.47   10.83   14.47   12.60   4.82   122.74   287.46   90.15   5.44   77.22   7.22   3.81   20.64   116.75   7.09   45.86   30.66   52.83   35.79   20.76   27.38   23.16   20.12   18.07   0.06   0.62   0.50   0.20   0.34   0.92   0.24   0.10   0.58   0.31   1.44   2.07   1.31   0.15   1.19   0.00   0.11   0.19   1.84   0.26   0.61   0.36   1.90   1.12   0.76   0.16   0.36   0.74   0.19   0.68   3.58   0.80   4.65   2.44   1.45   0.45   0.96   1.25   1.53   1.54   0.54   0.53   1.84   0.46   1.82   1.09   1.29   1.26   1.34   1.32   0.72   2.94   0.96   2.06   0.48   0.60   1.16   0.53   239 6.13E-­‐04   0.00E+00   3.77E-­‐05   2.64E-­‐04   2.49E-­‐09   2.07E-­‐12   4.97E-­‐02   9.48E-­‐08   5.01E-­‐05   4.75E-­‐05   0.00E+00   1.91E-­‐04   4.82E-­‐03   1.13E-­‐04   2.44E-­‐02   9.47E-­‐13   3.43E-­‐04   5.47E-­‐08   2.48E-­‐13   1.36E-­‐02   5.36E-­‐14   1.12E-­‐04   0.00E+00   3.65E-­‐07   0.00E+00   3.22E-­‐02   5.81E-­‐04   2.43E-­‐12   1.69E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   POSITIVE   NO   NO   NO   NO   YES   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G27310   AT2G27350   AT2G27510   AT2G27690   AT2G28190   AT2G28210   AT2G28290   AT2G28570   AT2G28760   AT2G28890   AT2G28910   AT2G28950   AT2G29070   AT2G29110   AT2G29460   AT2G29510   AT2G29720   AT2G29990   AT2G30040   AT2G30140   AT2G30250   AT2G30395   AT2G30520   AT2G30640   AT2G30720   AT2G30750   AT2G30840   AT2G31090   AT2G31110     4.95   20.11   15.98   1.11   280.95   0.38   8.69   1.71   2.25   13.21   104.84   29.43   11.22   0.84   5.67   12.13   15.84   11.48   3.00   9.33   15.25   0.54   38.44   1.79   14.64   3.00   0.88   22.99   2.78   0.27   0.15   0.53   0.06   1.46   0.07   0.36   0.33   0.69   0.72   0.50   0.82   0.27   0.19   1.46   0.17   1.43   0.19   0.32   0.83   0.75   0.07   0.74   0.03   0.11   0.98   0.15   0.51   0.08   10.14   28.01   23.88   3.52   413.77   1.74   11.69   10.48   9.05   22.90   141.36   50.30   16.14   8.37   87.94   16.77   24.09   26.53   5.65   44.26   53.08   2.74   71.03   3.65   21.65   30.63   2.21   41.51   10.16   0.16   0.19   1.07   0.38   6.23   0.24   0.40   0.69   0.46   0.44   0.78   1.83   0.44   1.01   0.69   0.44   0.74   0.57   0.29   0.37   0.64   0.08   1.21   0.12   0.40   1.26   0.11   0.62   0.52   1.03   0.48   0.58   1.67   0.56   2.19   0.43   2.62   2.01   0.79   0.43   0.77   0.52   3.31   3.96   0.47   0.60   1.21   0.91   2.25   1.80   2.34   0.89   1.03   0.56   3.35   1.32   0.85   1.87   240 2.31E-­‐03   3.80E-­‐04   4.88E-­‐02   1.11E-­‐04   4.68E-­‐03   3.39E-­‐02   1.32E-­‐02   2.62E-­‐09   5.59E-­‐11   3.57E-­‐05   2.77E-­‐02   2.62E-­‐05   3.61E-­‐02   0.00E+00   0.00E+00   2.57E-­‐02   5.73E-­‐03   3.27E-­‐10   1.78E-­‐02   0.00E+00   0.00E+00   3.20E-­‐03   3.56E-­‐08   5.81E-­‐03   8.61E-­‐03   0.00E+00   4.45E-­‐02   4.77E-­‐03   2.47E-­‐08   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN     Table A3.4 (cont’d) AT2G31260   AT2G31490   AT2G31570   AT2G31750   AT2G31865   AT2G31870   AT2G31970   AT2G32020   AT2G32030   AT2G32130   AT2G32190   AT2G32210   AT2G32400   AT2G32800   AT2G33170   AT2G33580   AT2G33620   AT2G33770   AT2G34250   AT2G34300   AT2G34780   AT2G34810   AT2G34940   AT2G34960   AT2G35020   AT2G35070   AT2G35110   AT2G35930   AT2G36080     5.08   232.93   99.84   16.83   5.46   4.44   5.06   0.62   1.56   0.61   1.39   6.41   4.96   3.94   9.79   1.28   12.12   29.24   203.22   6.92   4.19   3.30   1.23   4.97   5.21   0.71   8.43   1.33   3.47   0.17   0.66   0.20   0.75   0.71   0.40   0.26   0.17   0.44   0.17   0.60   0.45   0.14   0.33   0.23   0.23   0.41   0.35   2.69   0.24   0.12   0.64   0.24   0.29   0.25   0.12   0.14   0.23   0.31   7.76   313.37   197.37   27.57   30.21   6.89   8.96   2.91   5.87   8.33   18.06   17.48   8.14   12.58   14.35   6.50   16.70   39.34   323.41   12.49   6.11   7.20   5.37   8.13   11.05   3.15   12.54   10.61   13.32   0.15   4.02   2.32   1.80   1.57   0.15   0.25   0.04   0.15   0.35   0.32   0.63   0.23   0.17   0.64   0.21   0.77   0.53   5.27   0.27   0.11   2.19   0.22   0.24   0.15   0.15   0.35   0.88   0.33   0.61   0.43   0.98   0.71   2.47   0.63   0.82   2.22   1.91   3.78   3.69   1.45   0.72   1.68   0.55   2.34   0.46   0.43   0.67   0.85   0.54   1.13   2.12   0.71   1.09   2.14   0.57   3.00   1.94   241 9.51E-­‐03   3.24E-­‐02   3.36E-­‐09   3.02E-­‐04   0.00E+00   3.12E-­‐02   3.92E-­‐05   8.20E-­‐03   4.97E-­‐04   1.95E-­‐07   1.99E-­‐11   3.46E-­‐04   1.55E-­‐03   0.00E+00   6.15E-­‐03   6.53E-­‐12   3.49E-­‐02   3.46E-­‐02   1.33E-­‐03   1.66E-­‐05   6.21E-­‐03   2.04E-­‐04   1.69E-­‐08   1.19E-­‐02   1.24E-­‐05   4.36E-­‐03   3.63E-­‐03   0.00E+00   8.21E-­‐11   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G36310   AT2G36580   AT2G36630   AT2G36792   AT2G36800   AT2G36880   AT2G36895   AT2G37080   AT2G37100   AT2G37110   AT2G37150   AT2G37970   AT2G38250   AT2G38290   AT2G38310   AT2G38360   AT2G38410   AT2G38470   AT2G38750   AT2G38790   AT2G38860   AT2G38940   AT2G39200   AT2G39250   AT2G39350   AT2G39360   AT2G39420   AT2G39518   AT2G39570     20.22   24.04   29.47   0.26   3.45   168.28   43.63   14.74   2.29   68.58   3.93   131.20   0.72   27.76   6.32   8.11   6.05   17.35   5.31   13.08   32.96   4.27   0.92   12.96   0.86   4.85   17.28   1.81   6.28   0.13   0.56   0.40   0.02   0.29   2.63   0.15   0.26   0.03   0.67   0.14   0.47   0.22   0.60   0.34   0.34   0.11   1.37   0.15   0.81   1.10   0.80   0.29   0.13   0.07   0.11   0.87   1.38   0.15   32.47   55.34   40.75   0.97   22.96   282.61   64.98   22.79   5.33   151.89   7.07   303.93   9.21   49.21   17.45   13.92   9.24   77.52   11.45   29.23   106.55   9.96   5.03   21.81   3.34   9.62   35.19   7.65   11.83   0.73   0.70   0.84   0.18   1.13   3.02   0.72   0.58   0.26   1.06   0.41   4.83   0.70   1.17   0.18   0.38   0.09   1.54   1.66   0.84   0.48   0.57   0.58   1.01   0.46   0.14   0.76   1.07   0.52   0.68   1.20   0.47   1.89   2.73   0.75   0.57   0.63   1.22   1.15   0.85   1.21   3.69   0.83   1.47   0.78   0.61   2.16   1.11   1.16   1.69   1.22   2.45   0.75   1.96   0.99   1.03   2.08   0.91   242 6.52E-­‐04   1.32E-­‐12   1.79E-­‐02   1.86E-­‐02   0.00E+00   9.87E-­‐05   5.69E-­‐04   1.34E-­‐03   1.39E-­‐02   3.63E-­‐12   6.30E-­‐04   2.17E-­‐13   1.24E-­‐10   3.34E-­‐06   7.35E-­‐08   1.26E-­‐02   1.23E-­‐02   0.00E+00   9.62E-­‐05   1.41E-­‐07   0.00E+00   2.64E-­‐06   5.89E-­‐09   1.08E-­‐03   1.19E-­‐06   8.57E-­‐06   3.41E-­‐07   3.43E-­‐04   2.22E-­‐04   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G39660   AT2G39770   AT2G39870   AT2G40140   AT2G40330   AT2G40520   AT2G40540   AT2G40570   AT2G40610   AT2G40750   AT2G40940   AT2G41100   AT2G41110   AT2G41220   AT2G41250   AT2G41410   AT2G41430   AT2G41700   AT2G41730   AT2G41835   AT2G41850   AT2G41880   AT2G42330   AT2G42360   AT2G42380   AT2G42490   AT2G42610   AT2G42840   AT2G42890     9.61   139.53   5.40   35.89   1.60   16.28   17.17   4.69   3.34   16.88   29.33   0.00   71.91   6.27   153.48   42.98   342.70   6.15   2.37   4.00   1.65   6.02   2.79   2.46   1.45   16.22   20.53   105.50   11.08   0.41   0.48   0.16   0.65   0.23   0.23   0.43   0.06   0.24   2.29   0.05   4.11   1.07   0.10   1.75   1.35   7.21   0.14   0.31   0.29   1.03   0.25   0.24   0.38   0.10   0.19   0.54   1.30   0.25   21.59   191.92   9.86   93.13   7.25   29.18   27.62   8.78   10.47   48.35   40.32   234.53   186.57   10.50   203.76   137.98   447.09   11.99   26.37   9.50   7.44   10.35   4.24   12.73   3.88   23.33   30.95   143.75   15.79   0.36   4.48   0.63   1.01   0.52   0.21   0.24   0.27   1.40   1.61   0.33   7.88   3.05   0.12   1.22   1.80   2.18   0.90   2.36   0.08   2.86   0.05   0.31   0.92   0.48   0.43   1.34   5.27   0.23   1.17   0.46   0.87   1.38   2.18   0.84   0.69   0.90   1.65   1.52   0.46   3.38   1.38   0.74   0.41   1.68   0.38   0.96   3.48   1.25   2.18   0.78   0.61   2.37   1.42   0.52   0.59   0.45   0.51   243 1.15E-­‐08   1.58E-­‐02   9.48E-­‐04   0.00E+00   1.29E-­‐06   2.38E-­‐08   1.26E-­‐06   4.65E-­‐04   2.35E-­‐06   0.00E+00   1.94E-­‐02   0.00E+00   0.00E+00   7.66E-­‐05   4.59E-­‐02   0.00E+00   4.67E-­‐02   1.43E-­‐07   5.57E-­‐11   1.56E-­‐04   8.32E-­‐09   4.89E-­‐03   3.73E-­‐02   6.41E-­‐10   8.73E-­‐04   7.23E-­‐03   4.23E-­‐03   2.31E-­‐02   1.52E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   DOWN   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G43000   AT2G43140   AT2G43160   AT2G43210   AT2G43320   AT2G43680   AT2G44280   AT2G44370   AT2G44500   AT2G45000   AT2G45010   AT2G45030   AT2G45070   AT2G45170   AT2G45280   AT2G45290   AT2G45540   AT2G45590   AT2G45680   AT2G45700   AT2G45790   AT2G45910   AT2G45960   AT2G46020   AT2G46150   AT2G46270   AT2G46330   AT2G46420   AT2G46500     2.28   0.79   47.80   20.69   7.50   44.11   1.46   0.79   2.01   7.18   19.57   7.17   330.68   18.47   4.72   10.39   9.31   14.98   3.72   7.18   126.47   7.67   239.62   10.43   1.21   1.97   57.26   9.51   15.49   0.58   0.17   0.40   0.37   0.41   0.35   0.03   0.29   0.28   0.26   0.57   0.23   4.89   0.57   0.13   0.51   0.37   0.49   0.19   0.13   0.65   0.10   19.48   0.26   0.17   0.26   0.24   0.30   0.56   15.57   3.14   64.40   28.41   22.48   67.27   4.12   3.02   6.55   10.87   30.58   10.29   498.26   38.79   8.68   14.20   14.32   21.73   9.38   10.18   199.15   11.31   352.32   14.55   7.43   10.45   101.27   17.94   31.20   0.64   0.76   0.29   0.48   0.34   0.24   0.03   0.61   0.30   0.29   0.44   0.17   13.95   0.25   0.36   0.47   0.32   0.37   0.38   0.24   3.30   0.32   26.16   0.34   0.22   0.08   1.02   0.15   0.76   2.77   1.98   0.43   0.46   1.58   0.61   1.49   1.93   1.70   0.60   0.64   0.52   0.59   1.07   0.88   0.45   0.62   0.54   1.33   0.50   0.66   0.56   0.56   0.48   2.61   2.41   0.82   0.92   1.01   244 9.57E-­‐12   1.08E-­‐03   1.34E-­‐04   1.80E-­‐02   0.00E+00   6.84E-­‐07   2.13E-­‐05   2.35E-­‐03   2.71E-­‐09   1.09E-­‐02   1.76E-­‐03   3.00E-­‐02   4.39E-­‐07   7.81E-­‐06   3.15E-­‐03   4.29E-­‐02   1.27E-­‐05   8.51E-­‐03   2.89E-­‐06   3.97E-­‐02   4.11E-­‐04   1.02E-­‐02   4.15E-­‐03   4.60E-­‐04   1.61E-­‐07   0.00E+00   6.51E-­‐06   7.04E-­‐06   1.48E-­‐10   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT2G46600   AT2G47130   AT2G47470   AT2G47730   AT2G47800   AT2G48030   AT3G01290   AT3G01650   AT3G01720   AT3G01830   AT3G02040   AT3G02050   AT3G02170   AT3G02260   AT3G02770   AT3G02840   AT3G02880   AT3G02885   AT3G03440   AT3G03640   AT3G03680   AT3G03990   AT3G04010   AT3G04110   AT3G04210   AT3G04240   AT3G04300   AT3G04580   AT3G04640     146.49   5.18   137.45   154.03   5.79   7.45   32.29   2.23   10.57   2.56   4.01   12.32   3.47   11.00   53.96   0.42   7.25   0.28   13.69   25.52   2.47   30.28   4.60   12.57   64.89   41.06   0.16   11.41   10.01   1.59   1.71   1.77   1.69   0.21   0.41   2.41   0.16   0.05   1.17   0.39   0.12   0.24   0.37   0.57   0.18   0.42   0.12   0.50   1.56   0.04   0.89   0.27   0.21   2.90   0.54   0.02   0.11   1.25   303.78   27.68   319.71   290.59   14.09   15.60   182.61   5.06   19.99   31.33   6.98   22.38   11.01   15.17   86.76   4.45   11.85   1.51   22.16   39.14   3.78   45.27   7.09   17.39   111.38   62.70   1.37   18.27   24.97   2.59   0.47   3.25   0.72   0.09   0.41   1.90   0.05   0.52   1.06   0.61   0.14   0.13   0.43   0.83   0.22   0.44   0.35   0.35   1.96   0.13   0.51   0.45   0.36   0.45   0.47   0.20   0.39   0.98   1.05   2.42   1.22   0.92   1.28   1.07   2.50   1.18   0.92   3.61   0.80   0.86   1.67   0.46   0.69   3.41   0.71   2.44   0.69   0.62   0.61   0.58   0.63   0.47   0.78   0.61   3.10   0.68   1.32   245 1.08E-­‐09   0.00E+00   2.78E-­‐11   4.69E-­‐07   4.04E-­‐13   1.67E-­‐06   0.00E+00   1.12E-­‐03   6.64E-­‐07   0.00E+00   1.76E-­‐02   2.00E-­‐06   0.00E+00   2.22E-­‐02   3.71E-­‐04   1.97E-­‐08   1.48E-­‐03   3.43E-­‐02   1.37E-­‐03   1.54E-­‐03   4.62E-­‐02   4.72E-­‐03   4.98E-­‐02   2.98E-­‐02   8.16E-­‐06   1.22E-­‐03   1.44E-­‐02   7.41E-­‐06   3.40E-­‐05   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   UP   NO   DOWN   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G04670   AT3G04720   AT3G05165   AT3G05320   AT3G05360   AT3G05500   AT3G05680   AT3G05830   AT3G05970   AT3G06070   AT3G06330   AT3G06500   AT3G06890   AT3G07010   AT3G07270   AT3G07340   AT3G07525   AT3G07690   AT3G07700   AT3G07720   AT3G08650   AT3G08670   AT3G08760   AT3G08850   AT3G09010   AT3G09020   AT3G09490   AT3G09520   AT3G09560     14.68   99.75   2.39   1.46   0.77   85.31   5.52   6.18   34.76   4.91   6.38   25.30   3.56   9.44   23.67   1.55   13.45   13.59   37.50   44.16   30.41   7.73   10.23   8.30   5.01   1.63   1.31   1.75   7.92   0.01   5.32   0.12   0.14   0.02   0.90   0.16   0.22   0.35   0.54   0.30   0.45   0.27   0.32   0.13   0.07   0.84   0.12   0.22   0.48   0.22   0.16   0.29   0.25   0.59   0.34   0.26   0.56   2.23   25.52   154.11   6.25   4.82   2.28   125.89   7.39   12.00   51.79   9.65   9.32   51.47   7.96   16.92   39.14   4.29   22.11   20.16   69.92   105.81   42.23   11.36   22.01   11.17   9.78   6.68   4.88   6.04   12.02   0.63   2.36   0.45   0.48   0.08   1.55   0.22   0.25   0.20   0.98   0.37   0.36   0.29   1.17   0.41   0.03   1.20   0.63   0.84   1.02   0.15   0.31   0.20   0.25   0.17   0.17   0.25   0.67   0.30   0.80   0.63   1.39   1.72   1.57   0.56   0.42   0.96   0.58   0.98   0.55   1.02   1.16   0.84   0.73   1.47   0.72   0.57   0.90   1.26   0.47   0.56   1.11   0.43   0.96   2.03   1.90   1.79   0.60   246 2.98E-­‐05   4.27E-­‐04   3.09E-­‐05   7.04E-­‐05   4.99E-­‐04   3.79E-­‐03   4.55E-­‐02   6.76E-­‐04   2.14E-­‐03   2.49E-­‐02   4.73E-­‐02   1.58E-­‐09   2.09E-­‐02   1.54E-­‐04   1.31E-­‐06   1.87E-­‐04   4.13E-­‐03   1.01E-­‐02   1.37E-­‐13   7.04E-­‐14   1.40E-­‐03   2.37E-­‐02   8.30E-­‐09   4.39E-­‐02   3.96E-­‐04   8.38E-­‐07   2.06E-­‐04   2.20E-­‐07   1.76E-­‐04   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G09830   AT3G09920   AT3G10010   AT3G10070   AT3G10130   AT3G10250   AT3G10420   AT3G10500   AT3G10640   AT3G10720   AT3G10740   AT3G10930   AT3G10985   AT3G11090   AT3G11330   AT3G11700   AT3G11820   AT3G11840   AT3G12145   AT3G12150   AT3G12360   AT3G12520   AT3G12600   AT3G12610   AT3G12710   AT3G12740   AT3G12830   AT3G13060   AT3G13061     15.28   12.33   4.55   9.75   25.62   6.05   48.35   6.25   8.42   6.64   9.91   0.39   11.41   8.88   8.64   27.19   18.56   4.31   5.67   3.34   24.84   1.49   15.38   24.66   1.99   42.66   3.60   25.20   7.24   0.22   0.11   0.23   0.47   0.26   0.20   0.45   0.21   0.18   0.28   0.24   0.19   0.41   0.91   0.17   0.55   0.57   0.79   0.37   0.12   0.54   0.10   0.19   0.76   0.27   0.57   0.02   0.12   0.06   30.38   20.52   6.61   13.97   39.01   9.84   84.02   14.72   18.34   11.78   15.48   3.30   37.33   18.11   12.79   36.20   45.73   11.25   10.74   5.59   38.71   4.34   26.10   34.85   5.32   91.40   15.41   33.87   14.99   0.49   0.48   0.18   0.09   0.27   0.17   0.54   0.20   0.29   0.43   0.05   0.29   0.22   0.45   0.35   0.79   0.44   0.17   1.26   0.07   0.48   0.60   0.45   1.91   0.27   0.77   0.43   0.19   0.14   0.99   0.73   0.54   0.52   0.61   0.70   0.80   1.24   1.12   0.83   0.64   3.10   1.71   1.03   0.57   0.41   1.30   1.38   0.92   0.74   0.64   1.54   0.76   0.50   1.42   1.10   2.10   0.43   1.05   247 2.34E-­‐09   9.98E-­‐08   2.27E-­‐02   2.80E-­‐02   4.34E-­‐03   1.45E-­‐02   1.54E-­‐06   1.14E-­‐08   1.08E-­‐05   2.37E-­‐04   2.03E-­‐03   8.10E-­‐04   8.76E-­‐14   6.92E-­‐03   1.84E-­‐02   4.78E-­‐02   0.00E+00   3.03E-­‐07   1.10E-­‐03   4.08E-­‐02   6.05E-­‐04   1.00E-­‐05   5.36E-­‐04   1.71E-­‐02   7.53E-­‐04   3.60E-­‐11   1.05E-­‐09   3.60E-­‐02   5.87E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G13062   32.12   AT3G13080   14.02   AT3G13430   9.49   AT3G13437   0.55   AT3G13445   8.14   AT3G13670   41.70   AT3G13720   18.66   AT3G13810   23.49   AT3G13910   34.83   AT3G14050   14.65   AT3G14060   2.63   AT3G14067   39.58   AT3G14090   5.31   AT3G14230   97.59   AT3G14270   3.95   AT3G14470   2.08   AT3G14610   1.52   AT3G14770   7.69   AT3G14790   16.84   AT3G14840   16.57   AT3G14920   7.72   AT3G15353   1634.45   AT3G15400   2.19   AT3G15430   24.66   AT3G15540   1.88   AT3G15650   1.51   AT3G15760   0.73   AT3G15770   8.41   AT3G15790   30.43     0.55   0.14   0.26   0.25   0.22   0.56   0.87   0.60   0.91   0.54   0.75   0.89   0.06   0.95   0.17   0.09   0.08   0.11   0.46   0.87   0.06   24.92   0.18   0.22   0.12   0.17   0.23   0.53   0.68   47.36   57.64   13.46   2.60   12.02   55.39   27.56   33.83   63.83   24.76   9.81   65.62   8.95   132.75   6.82   5.85   4.09   12.28   30.71   45.40   11.54   2244.20   7.90   37.49   8.58   6.15   3.15   21.36   45.80   0.28   0.39   0.64   0.04   0.19   0.32   0.99   0.38   1.13   0.17   0.47   0.58   0.17   0.65   0.23   0.33   0.08   0.05   0.23   0.44   0.18   9.07   3.71   0.60   0.21   0.28   0.07   0.17   1.02   0.56   2.04   0.50   2.26   0.56   0.41   0.56   0.53   0.87   0.76   1.90   0.73   0.75   0.44   0.79   1.49   1.42   0.68   0.87   1.45   0.58   0.46   1.85   0.60   2.19   2.03   2.10   1.34   0.59   248 2.58E-­‐03   0.00E+00   4.90E-­‐02   4.92E-­‐02   4.32E-­‐02   4.26E-­‐02   2.33E-­‐02   6.62E-­‐04   1.33E-­‐04   5.00E-­‐05   8.58E-­‐04   3.83E-­‐05   2.48E-­‐03   7.01E-­‐03   7.26E-­‐05   7.81E-­‐09   4.78E-­‐04   7.57E-­‐03   1.51E-­‐06   0.00E+00   1.81E-­‐02   3.74E-­‐02   1.92E-­‐07   1.81E-­‐05   6.78E-­‐07   1.71E-­‐05   4.65E-­‐03   1.53E-­‐06   3.14E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G15810   AT3G15820   AT3G15880   AT3G15980   AT3G16230   AT3G16370   AT3G16460   AT3G16720   AT3G16857   AT3G16910   AT3G16950   AT3G17000   AT3G17050   AT3G17205   AT3G17240   AT3G17420   AT3G17609   AT3G17700   AT3G17770   AT3G17800   AT3G17820   AT3G18280   AT3G18773   AT3G18930   AT3G19010   AT3G19260   AT3G19870   AT3G19970   AT3G20250     36.24   4.77   14.80   22.16   12.74   115.85   2.03   2.85   8.09   12.17   3.72   26.38   1.54   12.33   93.11   5.33   75.03   7.52   10.87   81.99   43.96   46.02   0.39   3.10   63.75   5.64   8.64   4.11   14.39   1.14   0.33   0.28   0.48   0.35   0.52   0.22   0.32   0.24   0.25   0.15   0.06   0.19   0.20   1.26   0.11   1.66   0.29   0.63   1.04   0.43   1.13   0.05   0.05   2.07   0.14   0.02   0.20   0.39   77.21   17.65   18.71   47.71   17.54   168.84   5.50   11.68   12.85   19.30   5.87   39.01   5.40   18.11   141.89   11.65   147.59   15.78   17.19   159.86   64.82   104.69   1.24   6.23   169.09   20.03   12.89   9.01   24.04   0.71   0.13   0.33   2.17   0.10   5.24   0.53   0.56   0.05   0.57   0.24   0.50   0.22   0.24   1.73   0.48   0.82   0.60   0.31   1.29   1.68   2.34   0.03   0.17   1.70   0.15   0.35   0.01   0.25   1.09   1.89   0.34   1.11   0.46   0.54   1.44   2.03   0.67   0.67   0.66   0.56   1.81   0.55   0.61   1.13   0.98   1.07   0.66   0.96   0.56   1.19   1.66   1.01   1.41   1.83   0.58   1.13   0.74   249 1.01E-­‐09   9.76E-­‐12   2.85E-­‐02   0.00E+00   2.29E-­‐02   4.27E-­‐03   3.29E-­‐07   1.26E-­‐10   3.17E-­‐04   1.56E-­‐03   2.78E-­‐02   6.32E-­‐03   1.02E-­‐06   1.01E-­‐04   1.08E-­‐03   2.52E-­‐06   6.47E-­‐13   7.74E-­‐08   1.28E-­‐03   1.14E-­‐08   3.66E-­‐03   1.21E-­‐09   4.24E-­‐02   5.63E-­‐04   0.00E+00   1.92E-­‐14   4.61E-­‐03   2.77E-­‐05   5.45E-­‐05   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G20280   AT3G20600   AT3G20830   AT3G21080   AT3G21200   AT3G21230   AT3G21520   AT3G21560   AT3G21630   AT3G21780   AT3G21810   AT3G22060   AT3G22160   AT3G22170   AT3G22190   AT3G22400   AT3G22420   AT3G22460   AT3G22550   AT3G22620   AT3G22890   AT3G23010   AT3G23030   AT3G23050   AT3G23110   AT3G23250   AT3G23280   AT3G23530   AT3G23750     0.88   7.82   1.72   0.83   53.96   1.82   0.31   73.70   11.90   2.70   6.90   18.17   6.19   16.38   28.77   1.01   9.98   0.99   9.48   1.20   222.31   0.22   42.97   33.27   0.94   0.38   63.10   4.24   12.27   0.02   0.93   0.15   0.03   0.97   0.18   0.14   0.41   0.25   0.48   0.26   2.02   0.13   0.07   0.32   0.08   0.32   0.14   0.41   0.15   0.52   0.05   0.75   0.28   0.04   0.27   0.35   0.11   0.35   3.37   20.46   4.17   6.53   73.14   7.79   5.02   106.05   18.51   9.07   13.47   87.21   20.09   23.31   41.33   6.33   26.13   2.66   14.25   10.61   362.29   0.82   73.77   52.85   2.89   2.93   80.83   6.43   16.91   0.06   0.54   0.04   0.13   1.81   0.49   0.56   0.28   0.25   0.26   0.17   3.19   0.76   0.38   0.62   0.09   0.27   0.17   1.06   2.92   1.47   0.09   1.58   2.17   0.30   0.30   0.69   0.10   0.14   1.93   1.39   1.28   2.98   0.44   2.10   4.00   0.52   0.64   1.75   0.96   2.26   1.70   0.51   0.52   2.65   1.39   1.43   0.59   3.14   0.70   1.91   0.78   0.67   1.62   2.94   0.36   0.60   0.46   250 7.54E-­‐08   9.92E-­‐07   7.46E-­‐03   1.61E-­‐08   3.15E-­‐02   6.97E-­‐11   4.86E-­‐07   5.21E-­‐03   2.38E-­‐03   6.53E-­‐08   2.37E-­‐05   0.00E+00   9.72E-­‐11   7.79E-­‐04   4.79E-­‐04   0.00E+00   0.00E+00   2.46E-­‐02   2.02E-­‐02   3.14E-­‐10   4.46E-­‐04   2.77E-­‐02   3.57E-­‐05   4.29E-­‐05   7.62E-­‐05   1.89E-­‐06   9.98E-­‐03   1.62E-­‐02   2.78E-­‐02   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NEGATIVE   NO   NO   POSITIVE   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G24090   AT3G24180   AT3G24480   AT3G24503   AT3G24550   AT3G24560   AT3G25110   AT3G25180   AT3G25220   AT3G25600   AT3G25610   AT3G25780   AT3G25882   AT3G25890   AT3G26210   AT3G26220   AT3G26440   AT3G26500   AT3G26510   AT3G26910   AT3G27020   AT3G28340   AT3G28450   AT3G28500   AT3G28540   AT3G28580   AT3G28850   AT3G28890   AT3G28930     8.20   23.41   34.11   71.74   9.81   4.57   2.69   0.56   73.17   7.95   5.88   4.32   24.94   20.41   15.74   5.11   2.46   3.56   1.63   2.48   6.17   2.59   12.87   1.81   22.33   1.74   1.67   1.26   48.95   0.26   0.08   0.87   0.41   0.34   0.28   0.08   0.37   1.96   0.60   0.36   0.59   3.43   0.53   2.31   0.62   0.44   0.61   0.23   0.20   0.17   0.29   0.23   0.27   1.30   0.61   0.12   0.44   0.51   17.92   32.38   50.20   149.00   16.11   8.69   7.36   2.61   135.94   27.62   18.68   21.18   140.67   28.58   66.62   13.47   8.58   17.18   9.47   5.73   10.57   9.31   23.07   11.02   32.28   27.19   3.89   3.06   92.09   0.66   0.20   0.13   5.00   0.07   0.14   0.35   0.61   2.00   0.74   0.31   0.33   1.76   0.30   0.45   0.48   0.26   1.28   0.92   0.43   0.56   0.78   0.31   4.80   1.27   1.50   0.12   0.43   1.36   1.13   0.47   0.56   1.05   0.72   0.93   1.45   2.22   0.89   1.80   1.67   2.29   2.50   0.49   2.08   1.40   1.80   2.27   2.54   1.21   0.78   1.85   0.84   2.61   0.53   3.97   1.22   1.28   0.91   251 3.04E-­‐08   4.26E-­‐03   3.94E-­‐03   5.55E-­‐10   4.48E-­‐04   5.76E-­‐05   7.62E-­‐06   5.80E-­‐05   9.47E-­‐07   2.23E-­‐09   0.00E+00   5.41E-­‐14   0.00E+00   1.22E-­‐02   0.00E+00   2.01E-­‐08   9.93E-­‐11   0.00E+00   8.31E-­‐12   2.29E-­‐05   5.18E-­‐04   3.06E-­‐09   1.18E-­‐05   1.49E-­‐06   7.34E-­‐03   0.00E+00   8.55E-­‐03   6.52E-­‐04   7.88E-­‐07   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G29000   AT3G29034   AT3G29290   AT3G29400   AT3G44260   AT3G44320   AT3G44350   AT3G44400   AT3G44480   AT3G44720   AT3G45440   AT3G45620   AT3G45640   AT3G45730   AT3G45970   AT3G46080   AT3G46090   AT3G46110   AT3G46190   AT3G46510   AT3G46600   AT3G46620   AT3G46920   AT3G47090   AT3G47250   AT3G47480   AT3G47590   AT3G47780   AT3G47800     2.46   6.96   11.22   8.67   3.59   1.45   1.11   7.81   4.50   12.30   1.71   48.94   81.58   1.90   8.52   1.46   0.79   4.62   0.11   28.10   6.77   29.52   2.15   1.44   44.56   36.53   23.80   2.36   56.55   0.91   0.91   0.32   0.46   0.29   0.13   0.52   0.20   0.11   0.57   0.10   0.69   1.32   0.19   0.79   0.56   0.09   0.41   0.01   0.32   0.20   0.18   0.10   0.18   0.13   2.04   0.34   0.15   0.11   14.32   21.81   17.16   12.73   13.39   7.13   5.79   14.19   7.84   28.97   3.37   64.58   148.74   15.46   22.70   43.03   8.96   9.59   4.45   45.40   20.71   47.10   3.83   5.25   57.74   257.43   34.67   7.42   121.47   0.97   0.99   0.16   0.34   0.24   1.39   0.21   0.34   0.22   0.20   0.12   1.58   1.93   1.04   0.25   1.10   0.15   0.39   0.35   0.32   0.43   0.84   0.16   0.45   0.41   2.68   0.96   0.25   1.05   2.54   1.65   0.61   0.55   1.90   2.30   2.38   0.86   0.80   1.24   0.98   0.40   0.87   3.03   1.41   4.88   3.50   1.05   5.38   0.69   1.61   0.67   0.83   1.86   0.37   2.82   0.54   1.65   1.10   252 2.92E-­‐07   1.49E-­‐05   4.53E-­‐03   1.77E-­‐02   1.93E-­‐08   1.17E-­‐08   2.13E-­‐04   2.89E-­‐06   1.61E-­‐04   1.15E-­‐10   1.87E-­‐02   4.82E-­‐02   5.43E-­‐07   3.43E-­‐09   2.90E-­‐08   0.00E+00   1.49E-­‐07   4.82E-­‐04   1.14E-­‐06   1.10E-­‐04   0.00E+00   3.97E-­‐04   4.03E-­‐03   4.41E-­‐10   5.91E-­‐03   0.00E+00   1.06E-­‐02   7.42E-­‐11   6.15E-­‐11   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G47820   AT3G48090   AT3G48100   AT3G48310   AT3G48320   AT3G48360   AT3G49060   AT3G49120   AT3G49210   AT3G49350   AT3G49530   AT3G49800   AT3G49970   AT3G50340   AT3G50880   AT3G50910   AT3G50930   AT3G50950   AT3G51000   AT3G51130   AT3G51250   AT3G51330   AT3G51370   AT3G51440   AT3G51450   AT3G51550   AT3G51630   AT3G51670   AT3G51770     7.28   29.43   5.82   11.26   21.30   0.71   6.09   220.37   2.25   7.77   11.31   24.10   1.42   2.90   3.62   14.65   2.83   28.96   35.15   18.64   23.60   4.80   65.38   3.76   15.39   39.41   28.76   19.49   6.97   0.50   1.33   0.20   0.13   0.05   0.15   0.11   6.97   0.31   0.29   0.17   0.20   0.25   0.23   0.21   0.45   0.86   1.32   0.44   0.49   0.24   0.56   0.89   0.25   1.50   0.45   0.29   0.28   0.08   15.19   61.61   10.52   24.78   36.37   1.82   9.46   441.30   8.29   13.02   30.26   41.05   3.79   5.57   9.75   23.50   31.13   63.52   48.79   27.82   32.83   23.84   95.13   15.37   31.51   54.75   36.08   38.29   9.32   0.22   0.83   0.51   0.15   0.86   0.21   0.20   8.66   0.19   0.35   0.88   0.62   0.41   0.07   0.28   0.24   0.51   0.51   0.48   0.45   0.60   0.33   0.34   0.25   0.84   0.40   0.10   1.33   0.31   1.06   1.07   0.85   1.14   0.77   1.36   0.64   1.00   1.88   0.74   1.42   0.77   1.41   0.94   1.43   0.68   3.46   1.13   0.47   0.58   0.48   2.31   0.54   2.03   1.03   0.47   0.33   0.97   0.42   253 2.82E-­‐06   2.01E-­‐11   3.88E-­‐03   7.33E-­‐09   4.80E-­‐05   4.12E-­‐02   4.77E-­‐03   3.12E-­‐07   1.61E-­‐09   8.31E-­‐04   1.04E-­‐13   3.26E-­‐05   1.79E-­‐03   1.09E-­‐02   6.00E-­‐05   6.84E-­‐04   0.00E+00   0.00E+00   2.18E-­‐02   5.71E-­‐03   2.19E-­‐02   0.00E+00   3.14E-­‐03   6.28E-­‐12   4.42E-­‐07   1.62E-­‐02   3.73E-­‐02   1.10E-­‐07   3.74E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G51895   AT3G51980   AT3G52430   AT3G52605   AT3G52800   AT3G52930   AT3G53180   AT3G53190   AT3G53230   AT3G53280   AT3G53670   AT3G53710   AT3G54100   AT3G54150   AT3G54250   AT3G54300   AT3G54420   AT3G54640   AT3G54810   AT3G54960   AT3G55130   AT3G55430   AT3G55560   AT3G55840   AT3G55890   AT3G55950   AT3G55960   AT3G55980   AT3G56050     10.62   18.92   18.12   1.28   41.14   188.21   6.18   12.57   11.96   5.88   70.78   17.65   4.44   3.82   9.40   37.42   2.93   51.83   19.70   25.00   26.98   13.48   3.93   0.57   1.62   2.15   23.51   9.43   37.78   0.27   0.75   1.11   0.08   0.60   3.03   0.26   0.46   0.45   0.21   0.71   0.50   0.02   0.91   0.10   0.24   0.41   0.86   0.53   0.24   0.45   0.71   0.21   0.14   0.27   0.21   0.25   0.58   0.36   17.80   50.39   66.65   6.70   64.53   378.52   10.92   17.87   17.55   9.25   96.08   24.25   7.92   13.41   13.62   57.84   14.95   86.62   29.03   67.19   39.42   21.61   7.24   4.49   5.70   5.43   34.53   29.22   51.60   1.28   1.92   0.86   0.07   0.39   6.01   0.62   1.37   0.36   0.41   0.80   0.41   0.35   1.05   0.77   0.85   0.55   0.31   0.61   1.13   1.22   0.57   0.10   0.24   0.88   0.40   0.79   0.80   0.19   0.74   1.41   1.88   2.39   0.65   1.01   0.82   0.51   0.55   0.65   0.44   0.46   0.83   1.81   0.54   0.63   2.35   0.74   0.56   1.43   0.55   0.68   0.88   2.97   1.81   1.34   0.55   1.63   0.45   254 1.79E-­‐04   0.00E+00   0.00E+00   1.49E-­‐07   5.85E-­‐04   2.66E-­‐07   1.25E-­‐04   2.65E-­‐02   6.48E-­‐03   2.20E-­‐02   2.80E-­‐02   8.88E-­‐03   1.17E-­‐03   5.88E-­‐09   4.36E-­‐02   2.81E-­‐04   7.81E-­‐10   2.88E-­‐05   5.22E-­‐03   0.00E+00   4.92E-­‐03   9.38E-­‐04   6.31E-­‐03   2.90E-­‐10   7.45E-­‐03   2.83E-­‐06   7.62E-­‐03   0.00E+00   1.77E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT3G56060   AT3G56170   AT3G56190   AT3G56200   AT3G56410   AT3G56590   AT3G56710   AT3G56880   AT3G56891   AT3G57300   AT3G57330   AT3G57450   AT3G57530   AT3G57550   AT3G57630   AT3G57880   AT3G58110   AT3G58640   AT3G58840   AT3G59080   AT3G59570   AT3G59660   AT3G59700   AT3G60260   AT3G60420   AT3G60520   AT3G60600   AT3G60690   AT3G60860     3.07   15.84   39.81   11.08   4.90   18.37   107.71   33.82   0.45   6.90   14.69   9.23   16.58   22.63   11.29   9.03   21.79   16.97   19.01   2.24   3.25   7.97   1.61   10.80   68.26   123.54   39.89   10.04   11.46   0.10   0.14   0.04   0.47   0.22   0.18   5.21   0.24   0.11   0.36   0.87   0.20   0.06   0.27   0.52   0.26   0.47   0.05   0.56   0.17   0.08   0.42   0.07   0.41   2.09   0.31   0.52   0.12   0.14   5.10   34.15   52.32   36.20   8.90   25.89   201.16   57.32   6.02   9.93   35.97   19.55   27.41   44.64   17.43   13.39   29.36   23.73   25.79   6.16   7.15   18.72   3.61   18.88   254.55   185.59   55.89   21.01   15.87   0.32   0.48   0.84   0.88   0.56   0.33   1.55   0.90   0.37   0.22   0.64   0.74   0.50   0.56   0.29   0.13   0.11   0.30   0.09   0.15   0.18   0.31   0.13   0.76   6.20   2.33   0.72   0.54   0.47   0.73   1.11   0.39   1.71   0.86   0.49   0.90   0.76   3.76   0.53   1.29   1.08   0.73   0.98   0.63   0.57   0.43   0.48   0.44   1.46   1.14   1.23   1.16   0.81   1.90   0.59   0.49   1.07   0.47   255 3.35E-­‐02   5.92E-­‐09   9.06E-­‐03   0.00E+00   5.35E-­‐08   1.98E-­‐03   9.86E-­‐08   8.68E-­‐05   2.61E-­‐05   9.58E-­‐03   3.64E-­‐14   1.06E-­‐03   1.20E-­‐04   7.68E-­‐09   1.32E-­‐04   6.67E-­‐03   3.15E-­‐02   1.03E-­‐03   4.29E-­‐02   1.84E-­‐06   7.32E-­‐06   6.55E-­‐10   2.76E-­‐03   1.06E-­‐04   0.00E+00   1.40E-­‐03   9.07E-­‐03   1.73E-­‐04   1.79E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO     Table A3.4 (cont’d) AT3G61198   AT3G61220   AT3G61240   AT3G61670   AT3G62260   AT3G62422   AT3G62600   AT3G62720   AT3G62860   AT3G63500   AT4G00050   AT4G00750   AT4G00755   AT4G01010   AT4G01320   AT4G01700   AT4G01750   AT4G01950   AT4G02330   AT4G02380   AT4G02520   AT4G03080   AT4G03110   AT4G03190   AT4G03390   AT4G03400   AT4G03550   AT4G03820   AT4G04220     0.15   24.56   36.09   5.34   3.96   26.13   46.82   5.38   0.90   19.36   15.25   6.53   11.10   3.63   61.84   6.86   4.18   5.98   5.86   311.32   537.35   15.06   10.74   5.06   6.37   39.83   12.50   3.78   3.05   0.05   0.50   0.54   0.10   0.14   0.54   0.75   0.17   0.04   0.28   0.13   0.24   0.17   0.32   0.98   0.80   0.08   0.44   0.47   23.01   15.15   0.23   0.38   0.09   0.07   0.31   0.35   0.25   0.13   1.85   54.68   46.17   8.43   10.31   44.30   146.51   8.43   3.03   28.40   23.20   11.37   15.45   10.72   85.85   17.14   11.16   9.75   21.58   975.61   1742.96   24.62   17.16   8.15   13.63   74.63   17.49   9.31   6.56   0.35   0.95   0.48   0.08   0.26   0.29   2.68   0.39   0.09   0.92   0.46   0.56   0.34   0.56   0.68   0.51   0.44   1.14   1.10   23.70   9.96   0.28   0.02   0.29   0.23   0.77   0.46   0.26   0.24   3.67   1.15   0.36   0.66   1.38   0.76   1.65   0.65   1.75   0.55   0.61   0.80   0.48   1.56   0.47   1.32   1.42   0.71   1.88   1.65   1.70   0.71   0.68   0.69   1.10   0.91   0.49   1.30   1.10   256 9.00E-­‐03   9.56E-­‐12   1.40E-­‐02   4.82E-­‐03   1.17E-­‐07   1.06E-­‐04   0.00E+00   1.67E-­‐02   2.03E-­‐03   1.11E-­‐03   6.08E-­‐03   5.43E-­‐04   4.33E-­‐02   4.69E-­‐10   1.60E-­‐02   4.97E-­‐06   4.34E-­‐07   7.44E-­‐03   0.00E+00   0.00E+00   0.00E+00   1.10E-­‐04   1.23E-­‐03   1.08E-­‐02   9.18E-­‐09   1.79E-­‐07   1.26E-­‐02   1.99E-­‐06   4.57E-­‐05   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G04500   AT4G04540   AT4G04570   AT4G04610   AT4G04870   AT4G04880   AT4G04960   AT4G05020   AT4G05390   AT4G08180   AT4G08480   AT4G08555   AT4G08850   AT4G08870   AT4G08950   AT4G09030   AT4G09670   AT4G09770   AT4G10270   AT4G11280   AT4G11290   AT4G11300   AT4G11350   AT4G11360   AT4G11370   AT4G11530   AT4G11800   AT4G11820   AT4G11850     1.07   0.88   20.78   53.48   9.22   5.81   4.55   29.12   7.45   7.21   5.71   2.46   25.71   120.96   4.34   9.13   40.62   0.27   1.53   9.28   0.63   4.43   2.71   16.91   2.79   0.80   5.03   21.47   8.66   0.25   0.35   0.52   0.91   0.45   0.11   0.10   0.44   0.23   0.00   0.26   0.22   0.34   1.75   0.10   0.67   0.51   0.15   0.27   0.99   0.17   0.54   0.16   0.31   0.68   0.10   0.15   0.20   0.30   4.79   9.25   41.11   83.24   14.42   9.88   8.74   50.99   12.24   9.99   8.11   18.58   71.63   167.30   12.59   27.77   65.48   1.93   6.20   29.42   3.08   9.92   7.59   32.72   9.37   3.10   8.90   32.03   20.28   0.43   0.37   0.59   2.23   0.54   0.20   0.17   1.31   0.48   0.01   0.11   1.60   1.69   5.08   0.55   0.48   0.55   0.13   0.49   0.50   0.26   0.20   0.31   0.47   1.00   0.17   0.55   0.28   0.33   2.16   3.39   0.98   0.64   0.65   0.77   0.94   0.81   0.72   0.47   0.50   2.92   1.48   0.47   1.54   1.60   0.69   2.82   2.02   1.67   2.29   1.16   1.48   0.95   1.75   1.95   0.82   0.58   1.23   257 3.77E-­‐08   0.00E+00   2.08E-­‐09   5.21E-­‐04   1.28E-­‐02   1.32E-­‐02   1.24E-­‐04   2.45E-­‐06   6.03E-­‐03   2.04E-­‐02   4.44E-­‐02   3.96E-­‐06   0.00E+00   9.11E-­‐03   2.71E-­‐07   1.28E-­‐06   1.45E-­‐04   1.14E-­‐04   9.53E-­‐03   0.00E+00   2.31E-­‐04   1.05E-­‐04   1.00E-­‐06   1.33E-­‐04   3.51E-­‐04   2.17E-­‐05   1.20E-­‐04   3.10E-­‐03   1.00E-­‐11   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G11890   AT4G12010   AT4G12120   AT4G12490   AT4G12500   AT4G12720   AT4G12730   AT4G12770   AT4G13180   AT4G13340   AT4G13510   AT4G13530   AT4G13540   AT4G14365   AT4G14500   AT4G14550   AT4G15440   AT4G15620   AT4G15680   AT4G15920   AT4G16660   AT4G16780   AT4G16790   AT4G16990   AT4G17070   AT4G17230   AT4G17245   AT4G17250   AT4G17330     11.80   4.93   7.43   31.26   14.79   26.23   17.01   4.87   15.84   6.83   24.86   28.16   1.12   9.19   23.95   2.08   3.11   6.50   3.62   4.43   49.20   3.33   3.66   83.84   12.04   17.69   9.88   3.94   13.24   2.10   0.15   0.16   6.22   5.04   1.12   0.41   0.15   0.51   0.50   1.24   0.26   0.04   1.74   0.25   0.06   0.57   0.05   0.25   0.41   0.23   0.21   0.28   1.22   0.52   0.69   0.51   0.33   0.30   106.43   9.85   21.02   61.22   63.43   76.56   28.94   6.75   30.30   23.79   72.10   41.52   4.57   110.64   33.56   4.81   14.68   14.32   8.93   9.63   128.57   9.86   7.67   121.59   24.63   29.15   20.65   6.39   25.52   2.84   0.52   0.46   0.62   3.93   2.34   1.38   0.19   0.75   0.50   0.37   0.85   0.73   2.11   1.06   0.43   0.36   1.17   0.58   0.60   1.81   0.49   0.25   1.58   0.65   0.63   0.97   0.57   0.27   3.17   1.00   1.50   0.97   2.10   1.55   0.77   0.47   0.94   1.80   1.54   0.56   2.04   3.59   0.49   1.21   2.24   1.14   1.30   1.12   1.39   1.56   1.07   0.54   1.03   0.72   1.06   0.70   0.95   258 0.00E+00   1.03E-­‐06   0.00E+00   1.97E-­‐05   0.00E+00   0.00E+00   1.01E-­‐04   4.55E-­‐02   2.51E-­‐05   0.00E+00   0.00E+00   1.76E-­‐03   1.14E-­‐03   0.00E+00   1.75E-­‐02   3.32E-­‐03   0.00E+00   1.07E-­‐03   1.13E-­‐02   2.26E-­‐03   0.00E+00   5.84E-­‐06   4.45E-­‐04   3.45E-­‐05   1.28E-­‐06   1.59E-­‐04   1.92E-­‐03   1.17E-­‐02   2.89E-­‐08   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G17420   AT4G17490   AT4G17500   AT4G17870   AT4G18280   AT4G18430   AT4G18630   AT4G18760   AT4G18880   AT4G18950   AT4G19170   AT4G19180   AT4G19530   AT4G19660   AT4G19860   AT4G19880   AT4G20000   AT4G20070   AT4G20110   AT4G20320   AT4G20830   AT4G20860   AT4G21380   AT4G21400   AT4G21850   AT4G22305   AT4G22530   AT4G22590   AT4G22980     11.41   0.67   5.11   8.67   25.30   0.95   8.69   4.08   22.63   11.91   77.25   5.69   4.74   6.44   22.14   33.11   0.33   26.44   5.29   1.59   32.08   7.87   4.14   0.32   1.40   5.53   3.01   8.10   2.82   0.32   0.13   0.32   0.35   1.07   0.27   0.31   0.20   0.83   0.25   1.37   0.16   0.42   0.37   0.24   0.15   0.06   0.56   0.45   0.09   1.70   0.70   0.59   0.04   0.32   0.48   0.46   0.07   0.43   19.50   2.31   24.78   14.74   45.12   4.87   12.96   7.92   36.35   18.81   119.25   8.50   7.36   14.11   30.13   42.31   3.66   36.50   17.43   4.39   101.59   21.89   11.48   5.55   33.36   12.90   10.20   19.09   5.25   0.21   0.42   0.34   0.45   1.78   0.32   0.40   0.23   0.50   0.37   1.83   0.14   0.51   0.41   0.23   0.40   0.37   0.44   0.23   0.30   2.34   0.48   0.19   0.48   1.99   0.08   0.64   0.74   0.22   0.77   1.80   2.28   0.77   0.83   2.35   0.58   0.96   0.68   0.66   0.63   0.58   0.64   1.13   0.44   0.35   3.49   0.47   1.72   1.47   1.66   1.48   1.47   4.09   4.58   1.22   1.76   1.24   0.90   259 4.81E-­‐04   1.30E-­‐02   0.00E+00   2.23E-­‐02   2.25E-­‐04   1.07E-­‐04   2.07E-­‐02   5.55E-­‐03   2.65E-­‐04   1.64E-­‐03   6.74E-­‐04   1.71E-­‐02   4.76E-­‐03   1.62E-­‐06   3.44E-­‐02   4.13E-­‐02   2.33E-­‐05   2.12E-­‐02   0.00E+00   5.64E-­‐06   0.00E+00   2.80E-­‐13   5.86E-­‐12   0.00E+00   0.00E+00   1.01E-­‐04   1.03E-­‐06   3.00E-­‐09   1.30E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   DOWN   UP   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G23010   AT4G23030   AT4G23130   AT4G23170   AT4G23180   AT4G23190   AT4G23270   AT4G23310   AT4G23320   AT4G23470   AT4G23610   AT4G23730   AT4G23800   AT4G23850   AT4G24020   AT4G24040   AT4G24230   AT4G24690   AT4G24920   AT4G24970   AT4G25070   AT4G25230   AT4G25290   AT4G25620   AT4G25810   AT4G25880   AT4G25900   AT4G26090   AT4G26120     42.31   0.53   5.00   49.13   15.42   2.37   9.79   0.46   0.73   53.84   2.38   16.44   25.58   77.95   15.56   2.89   5.24   134.45   54.44   1.34   2.15   11.81   12.98   6.25   0.58   13.50   39.54   2.60   3.37   0.73   0.19   0.41   1.57   0.24   0.35   0.50   0.11   0.21   0.52   0.56   0.16   0.58   0.32   0.38   0.16   0.17   1.04   0.82   0.14   0.52   0.16   0.24   0.31   0.40   0.10   0.63   0.40   0.29   61.05   3.42   9.50   118.34   25.48   15.89   16.98   4.90   4.00   119.98   12.04   22.95   34.60   134.24   21.62   7.65   15.00   225.21   104.87   3.77   9.13   17.99   20.89   16.05   7.93   20.83   75.24   4.62   6.75   0.43   0.25   0.43   0.68   0.95   1.09   1.08   0.49   0.22   1.03   0.36   0.87   1.44   0.60   0.33   0.33   0.11   1.97   0.58   0.30   0.40   0.23   0.17   0.06   0.22   0.20   1.08   0.37   0.36   0.53   2.68   0.93   1.27   0.72   2.74   0.79   3.40   2.45   1.16   2.34   0.48   0.44   0.78   0.47   1.40   1.52   0.74   0.95   1.50   2.09   0.61   0.69   1.36   3.78   0.62   0.93   0.83   1.00   260 3.53E-­‐03   2.68E-­‐06   9.06E-­‐05   3.63E-­‐14   1.97E-­‐04   0.00E+00   1.68E-­‐04   3.69E-­‐14   7.51E-­‐06   1.69E-­‐13   6.64E-­‐08   2.84E-­‐02   4.02E-­‐02   1.52E-­‐05   1.90E-­‐02   4.00E-­‐07   2.73E-­‐11   2.38E-­‐04   3.47E-­‐06   7.39E-­‐05   0.00E+00   2.60E-­‐03   4.81E-­‐04   1.45E-­‐10   3.93E-­‐10   7.99E-­‐06   8.51E-­‐08   1.67E-­‐03   1.29E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G26190   AT4G26400   AT4G26470   AT4G26630   AT4G26690   AT4G26910   AT4G26970   AT4G27020   AT4G27040   AT4G27280   AT4G27450   AT4G27470   AT4G27745   AT4G27830   AT4G27860   AT4G27870   AT4G28300   AT4G28390   AT4G28395   AT4G28400   AT4G28490   AT4G28570   AT4G28710   AT4G29160   AT4G29210   AT4G29380   AT4G29440   AT4G29740   AT4G29750     7.52   21.94   0.96   49.19   12.53   27.91   34.88   4.01   7.64   7.30   1.31   7.82   8.23   15.73   2.21   12.68   29.77   5.62   0.00   17.63   6.52   7.90   4.74   36.01   9.94   7.71   4.49   3.33   23.65   0.21   0.51   0.33   0.61   0.38   0.36   0.31   0.27   0.05   1.08   0.26   0.37   0.21   0.17   0.39   0.26   0.33   0.48   0.00   0.20   0.53   0.20   0.25   0.51   0.44   0.06   0.08   0.31   0.27   11.87   29.62   5.40   65.77   21.37   43.80   60.60   9.84   14.12   45.45   8.27   15.60   14.44   25.60   9.17   18.96   56.43   27.38   3.72   40.98   14.29   18.11   7.24   74.53   14.81   11.63   8.85   12.73   31.40   0.08   0.25   0.20   0.01   0.08   0.25   0.21   0.26   0.31   1.26   1.05   0.58   0.40   0.57   1.02   0.22   0.46   0.08   1.30   0.47   0.53   0.11   0.29   0.74   0.65   0.35   0.39   0.90   0.41   0.66   0.43   2.49   0.42   0.77   0.65   0.80   1.29   0.89   2.64   2.66   1.00   0.81   0.70   2.06   0.58   0.92   2.29   Inf   1.22   1.13   1.20   0.61   1.05   0.57   0.59   0.98   1.93   0.41   261 1.11E-­‐03   1.86E-­‐02   5.19E-­‐06   1.44E-­‐03   3.61E-­‐05   1.71E-­‐06   6.01E-­‐06   9.92E-­‐07   3.14E-­‐05   0.00E+00   4.33E-­‐09   3.19E-­‐04   2.11E-­‐02   6.24E-­‐04   0.00E+00   4.01E-­‐03   4.25E-­‐11   0.00E+00   5.84E-­‐05   9.54E-­‐11   5.95E-­‐09   2.10E-­‐10   3.83E-­‐03   3.29E-­‐11   6.00E-­‐03   2.77E-­‐03   2.82E-­‐07   1.03E-­‐13   4.46E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   DOWN   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G29780   AT4G29810   AT4G29900   AT4G29950   AT4G30060   AT4G30190   AT4G30210   AT4G30250   AT4G30270   AT4G30440   AT4G30530   AT4G30640   AT4G31170   AT4G31500   AT4G31510   AT4G31800   AT4G31980   AT4G32030   AT4G32060   AT4G32070   AT4G32285   AT4G32480   AT4G32670   AT4G32760   AT4G33050   AT4G33220   AT4G33240   AT4G33300   AT4G33540     1.72   64.22   15.26   10.45   2.92   77.72   30.92   1.60   88.47   33.47   169.43   0.52   36.58   98.45   11.54   6.95   3.74   11.89   58.70   4.97   29.39   0.69   1.06   27.24   23.67   29.73   9.25   22.57   43.43   0.18   0.59   0.40   0.26   0.05   1.20   0.43   0.18   2.47   0.12   0.64   0.09   1.07   0.92   0.76   0.53   0.23   0.12   0.60   0.19   0.35   0.14   0.08   0.35   2.55   0.49   0.16   0.46   0.66   4.84   87.69   40.90   28.05   7.36   119.44   40.91   3.41   152.43   47.42   283.80   3.17   49.77   264.91   18.55   23.26   6.14   16.92   85.81   7.67   38.47   2.13   3.84   43.31   81.98   41.33   15.30   60.57   124.57   0.04   1.10   0.57   0.15   0.20   2.45   0.29   0.44   1.59   1.18   4.61   0.34   0.47   8.12   0.15   0.22   0.31   0.14   0.18   0.15   0.77   0.12   0.58   0.24   0.99   1.05   0.26   0.87   1.97   1.49   0.45   1.42   1.42   1.33   0.62   0.40   1.09   0.78   0.50   0.74   2.62   0.44   1.43   0.68   1.74   0.72   0.51   0.55   0.62   0.39   1.62   1.86   0.67   1.79   0.48   0.73   1.42   1.52   262 3.18E-­‐05   1.88E-­‐02   0.00E+00   0.00E+00   4.01E-­‐06   3.97E-­‐05   4.24E-­‐02   2.28E-­‐02   1.33E-­‐05   1.04E-­‐02   3.00E-­‐05   3.71E-­‐04   1.32E-­‐03   0.00E+00   3.04E-­‐02   1.20E-­‐13   2.07E-­‐02   4.15E-­‐02   3.71E-­‐03   1.57E-­‐02   1.24E-­‐02   2.11E-­‐02   6.93E-­‐08   4.48E-­‐07   0.00E+00   1.89E-­‐02   3.22E-­‐10   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G33565   AT4G33910   AT4G33920   AT4G34050   AT4G34100   AT4G34135   AT4G34150   AT4G34180   AT4G34480   AT4G34630   AT4G35060   AT4G35180   AT4G35500   AT4G35600   AT4G35810   AT4G36030   AT4G36040   AT4G36220   AT4G36430   AT4G36550   AT4G36670   AT4G36760   AT4G36988   AT4G37010   AT4G37290   AT4G37310   AT4G37390   AT4G37520   AT4G37530     32.20   17.69   36.37   281.78   26.68   22.00   31.66   85.77   23.10   83.64   1.68   1.40   6.22   6.83   0.33   0.70   95.02   69.33   1.48   5.09   15.31   32.53   11.58   0.90   0.29   16.02   1.54   7.80   3.27   0.64   0.14   0.62   4.13   0.40   0.85   0.25   1.04   0.45   0.43   0.44   0.42   0.11   0.52   0.17   0.16   1.16   1.05   0.14   0.27   0.72   0.73   0.78   0.48   0.11   0.63   0.90   1.84   1.08   60.79   33.48   76.81   443.63   36.93   28.86   125.67   122.93   31.90   224.25   4.71   13.14   9.39   22.60   1.77   2.35   198.56   142.64   5.05   9.29   28.94   50.65   55.21   4.07   1.67   39.16   4.90   17.26   13.97   0.91   0.54   0.65   6.28   0.36   3.60   1.34   0.23   0.76   2.05   0.37   0.12   0.06   0.55   0.45   0.35   0.69   1.10   0.35   0.24   2.03   0.09   0.51   0.16   0.26   0.40   0.65   0.46   1.75   0.92   0.92   1.08   0.65   0.47   0.39   1.99   0.52   0.47   1.42   1.48   3.23   0.60   1.73   2.43   1.75   1.06   1.04   1.77   0.87   0.92   0.64   2.25   2.17   2.51   1.29   1.67   1.14   2.10   263 2.56E-­‐07   4.89E-­‐06   1.92E-­‐10   2.31E-­‐07   1.27E-­‐02   3.39E-­‐02   0.00E+00   5.22E-­‐03   2.41E-­‐02   0.00E+00   8.62E-­‐03   0.00E+00   3.31E-­‐02   0.00E+00   1.40E-­‐02   1.79E-­‐04   6.68E-­‐11   1.16E-­‐09   1.47E-­‐04   1.54E-­‐04   2.69E-­‐06   4.41E-­‐04   0.00E+00   8.19E-­‐04   3.63E-­‐02   2.80E-­‐13   2.16E-­‐06   5.50E-­‐06   1.22E-­‐11   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT4G37640   AT4G37870   AT4G37900   AT4G38470   AT4G38480   AT4G38550   AT4G38560   AT4G38620   AT4G39030   AT4G39390   AT4G39580   AT4G39610   AT4G39640   AT4G39670   AT4G39840   AT4G39890   AT4G39950   AT4G40020   AT5G01100   AT5G01210   AT5G01400   AT5G01500   AT5G01540   AT5G01542   AT5G01720   AT5G01770   AT5G01810   AT5G01820   AT5G01830     26.04   39.15   1.07   10.11   3.69   36.80   0.92   11.26   3.74   15.03   2.44   0.52   16.33   3.51   6.39   11.20   24.06   1.46   0.83   3.03   5.65   16.39   0.33   5.18   6.59   1.80   41.09   23.57   0.81   0.36   1.13   0.15   0.37   0.20   1.16   0.37   0.37   0.60   0.20   0.17   0.21   0.30   0.90   0.47   0.64   1.00   0.15   0.23   0.28   0.27   0.28   0.14   0.28   0.30   0.04   1.10   0.48   0.04   48.26   62.11   4.07   17.77   8.49   71.78   12.15   21.77   37.05   21.28   6.20   7.55   32.66   35.79   9.93   18.98   93.40   4.37   4.04   5.17   7.85   24.07   4.05   26.03   12.35   2.90   63.87   54.83   2.20   0.80   0.41   0.15   0.17   0.06   1.56   0.53   0.59   1.19   0.11   0.23   0.37   1.07   0.88   0.13   0.37   0.71   0.10   0.12   0.55   0.26   0.50   0.34   1.40   0.22   0.17   1.28   1.11   0.13   0.89   0.67   1.93   0.81   1.20   0.96   3.73   0.95   3.31   0.50   1.35   3.85   1.00   3.35   0.64   0.76   1.96   1.58   2.28   0.77   0.48   0.55   3.63   2.33   0.91   0.69   0.64   1.22   1.45   264 4.17E-­‐07   2.22E-­‐04   2.14E-­‐07   1.29E-­‐04   6.06E-­‐05   1.36E-­‐08   0.00E+00   2.63E-­‐05   0.00E+00   9.02E-­‐03   1.74E-­‐03   3.97E-­‐11   1.31E-­‐10   0.00E+00   1.50E-­‐02   6.93E-­‐03   0.00E+00   6.09E-­‐05   1.59E-­‐08   3.24E-­‐02   3.22E-­‐02   1.08E-­‐02   1.69E-­‐13   0.00E+00   1.95E-­‐05   2.06E-­‐02   5.61E-­‐04   6.63E-­‐13   1.66E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G01900   AT5G02230   AT5G02270   AT5G02290   AT5G02310   AT5G02620   AT5G02780   AT5G02830   AT5G02880   AT5G03040   AT5G03120   AT5G03160   AT5G03260   AT5G03555   AT5G03610   AT5G04020   AT5G04040   AT5G04160   AT5G04550   AT5G04560   AT5G04720   AT5G04930   AT5G05090   AT5G05100   AT5G05190   AT5G05340   AT5G05410   AT5G05460   AT5G05570     0.30   6.94   35.04   18.83   8.72   17.09   0.92   48.41   9.31   38.90   22.36   23.51   1.36   25.22   9.87   5.30   7.65   5.62   24.32   6.42   11.94   19.45   7.02   16.21   3.62   1.26   9.06   6.51   6.69   0.09   0.55   1.02   0.71   0.13   0.28   0.39   0.46   0.24   0.43   0.60   0.41   0.12   0.66   0.79   0.18   0.26   0.23   0.26   0.31   0.55   0.86   0.51   0.39   0.18   0.89   0.42   0.33   0.05   2.07   14.12   53.66   45.03   11.97   28.86   16.01   64.85   14.38   61.13   45.91   56.61   2.64   40.46   28.49   8.18   11.80   11.18   35.97   10.86   42.09   40.98   11.44   23.58   10.31   4.68   13.13   9.31   10.58   0.24   1.00   0.38   0.49   0.31   0.43   1.73   0.31   0.40   0.17   1.73   0.92   0.25   0.55   0.61   0.73   0.20   0.36   0.31   0.50   0.52   0.42   0.20   0.23   0.25   0.60   0.15   0.11   0.33   2.79   1.03   0.61   1.26   0.46   0.76   4.12   0.42   0.63   0.65   1.04   1.27   0.95   0.68   1.53   0.63   0.62   0.99   0.56   0.76   1.82   1.07   0.70   0.54   1.51   1.89   0.54   0.52   0.66   265 1.24E-­‐03   2.06E-­‐04   6.75E-­‐04   6.63E-­‐13   2.34E-­‐02   8.23E-­‐05   0.00E+00   3.68E-­‐02   9.97E-­‐04   1.11E-­‐06   5.77E-­‐05   4.04E-­‐13   4.83E-­‐02   2.32E-­‐04   7.08E-­‐14   3.26E-­‐03   2.56E-­‐03   6.17E-­‐04   2.82E-­‐03   4.29E-­‐05   0.00E+00   4.04E-­‐10   1.87E-­‐02   1.21E-­‐02   1.20E-­‐10   2.48E-­‐04   4.27E-­‐02   4.75E-­‐02   1.31E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   YES   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G05730   AT5G06120   AT5G06750   AT5G07100   AT5G07180   AT5G07350   AT5G07370   AT5G07710   AT5G07770   AT5G07790   AT5G07870   AT5G08130   AT5G08240   AT5G08450   AT5G08790   AT5G09330   AT5G09670   AT5G10250   AT5G10290   AT5G10300   AT5G10490   AT5G10550   AT5G10770   AT5G11000   AT5G11060   AT5G11250   AT5G11650   AT5G11850   AT5G11920     19.48   7.58   7.34   12.86   1.62   27.22   51.42   3.57   1.76   3.92   4.26   13.92   3.12   17.52   21.50   26.54   10.12   0.67   14.52   2.13   14.09   16.99   3.44   13.55   25.39   4.64   11.35   5.71   2.93   0.75   0.05   0.39   1.34   0.07   0.55   0.68   0.18   0.14   0.20   0.05   0.15   0.28   0.11   0.64   0.56   1.39   0.09   0.13   0.23   0.13   0.17   0.40   0.92   0.47   0.19   0.29   0.16   0.48   43.51   10.11   18.87   20.82   2.90   40.98   72.25   6.95   4.52   7.08   7.76   19.29   8.64   23.93   75.26   35.89   18.84   1.63   24.81   5.71   19.50   23.42   9.43   22.84   35.15   7.67   23.91   8.76   4.91   0.60   0.23   0.34   1.65   0.36   0.66   0.24   0.10   0.38   0.19   0.21   0.60   0.35   0.41   1.28   0.11   0.53   0.16   0.41   0.21   0.20   0.61   0.08   0.44   0.61   0.44   0.21   0.08   0.30   1.16   0.42   1.36   0.70   0.84   0.59   0.49   0.96   1.36   0.85   0.86   0.47   1.47   0.45   1.81   0.44   0.90   1.29   0.77   1.43   0.47   0.46   1.46   0.75   0.47   0.72   1.07   0.62   0.74   266 8.75E-­‐12   1.21E-­‐02   1.16E-­‐10   2.15E-­‐03   1.28E-­‐02   1.23E-­‐05   5.75E-­‐03   1.85E-­‐03   3.34E-­‐06   7.53E-­‐04   6.41E-­‐03   1.18E-­‐03   7.97E-­‐06   6.67E-­‐04   0.00E+00   3.26E-­‐03   8.05E-­‐07   3.80E-­‐02   4.69E-­‐05   3.29E-­‐03   3.66E-­‐03   3.09E-­‐02   2.03E-­‐07   5.70E-­‐04   2.18E-­‐02   1.37E-­‐03   1.19E-­‐07   7.52E-­‐03   3.95E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G11970   AT5G12010   AT5G12300   AT5G12480   AT5G12850   AT5G12940   AT5G12950   AT5G13080   AT5G13190   AT5G13370   AT5G13420   AT5G13490   AT5G13500   AT5G13820   AT5G14120   AT5G14780   AT5G14930   AT5G15850   AT5G15870   AT5G15980   AT5G16170   AT5G16190   AT5G16230   AT5G16510   AT5G16800   AT5G16840   AT5G16880   AT5G17020   AT5G17300     16.93   18.35   4.48   24.60   10.89   2.86   6.06   1.46   29.01   24.41   51.84   4.15   8.17   1.96   6.66   150.00   18.39   76.38   10.05   9.13   1.28   1.17   6.70   37.64   11.32   94.87   46.73   14.44   13.53   0.84   0.68   0.14   0.43   0.43   0.26   0.38   1.01   0.51   0.27   1.35   1.09   0.09   0.18   0.18   1.98   0.55   1.47   0.15   0.16   0.21   0.05   0.23   0.72   0.26   0.37   0.44   0.28   0.34   40.76   32.23   12.83   37.33   16.58   6.46   8.86   21.46   69.41   32.76   71.57   16.33   13.00   4.40   11.77   282.61   33.47   106.37   26.15   12.87   4.81   2.61   14.29   51.90   16.09   117.78   78.62   18.28   28.34   0.80   0.39   0.62   0.20   0.50   0.39   0.25   1.40   0.85   0.32   0.35   1.39   0.13   0.13   0.44   4.38   0.84   0.88   1.00   0.10   0.27   0.26   0.83   1.05   0.18   2.23   1.12   0.29   0.44   1.27   0.81   1.52   0.60   0.61   1.17   0.55   3.88   1.26   0.42   0.47   1.98   0.67   1.17   0.82   0.91   0.86   0.48   1.38   0.50   1.92   1.16   1.09   0.46   0.51   0.31   0.75   0.34   1.07   267 2.15E-­‐07   1.56E-­‐05   1.93E-­‐08   1.42E-­‐03   2.55E-­‐03   2.26E-­‐03   2.01E-­‐02   1.88E-­‐14   1.76E-­‐10   4.28E-­‐02   1.78E-­‐02   1.89E-­‐14   3.54E-­‐03   2.59E-­‐04   1.74E-­‐04   4.57E-­‐07   7.25E-­‐08   1.31E-­‐02   1.53E-­‐13   2.93E-­‐02   3.22E-­‐05   4.14E-­‐02   1.74E-­‐05   1.72E-­‐02   3.19E-­‐02   3.41E-­‐02   5.51E-­‐07   4.60E-­‐02   1.35E-­‐08   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G17640   AT5G17910   AT5G18480   AT5G18490   AT5G18650   AT5G18670   AT5G18780   AT5G19120   AT5G19230   AT5G19290   AT5G19320   AT5G19780   AT5G20350   AT5G20400   AT5G20730   AT5G20960   AT5G21170   AT5G21326   AT5G21940   AT5G22060   AT5G22090   AT5G22380   AT5G22430   AT5G22460   AT5G22510   AT5G22555   AT5G22570   AT5G22580   AT5G22850     5.52   4.82   25.23   7.01   11.27   4.26   3.20   1.13   0.80   32.36   19.14   41.64   24.08   10.76   11.55   1.40   6.73   21.10   7.19   86.47   32.27   0.88   0.04   0.38   25.19   0.47   1.27   59.10   5.51   0.08   0.08   0.12   0.44   0.35   0.13   0.38   0.06   0.37   0.88   0.41   0.68   0.20   0.66   0.15   0.14   0.55   0.31   0.55   0.05   0.71   0.16   0.01   0.09   0.26   0.21   0.63   1.15   0.05   12.44   8.37   46.82   11.20   19.12   14.71   6.74   3.57   4.47   44.93   29.78   58.19   32.74   18.82   16.51   6.67   10.90   31.81   14.14   117.46   54.09   11.33   14.26   3.06   35.74   6.02   7.83   89.04   8.15   0.28   0.30   0.80   0.28   0.49   0.78   0.50   0.05   0.52   1.36   0.38   0.85   0.37   0.53   0.47   0.08   0.13   0.24   0.59   2.01   0.43   0.79   3.68   0.37   0.27   1.08   0.49   2.02   0.04   1.17   0.80   0.89   0.68   0.76   1.79   1.08   1.66   2.49   0.47   0.64   0.48   0.44   0.81   0.52   2.26   0.70   0.59   0.98   0.44   0.75   3.70   8.41   3.02   0.50   3.67   2.62   0.59   0.57   268 8.58E-­‐07   4.01E-­‐05   3.45E-­‐07   5.90E-­‐03   6.37E-­‐04   3.67E-­‐14   7.82E-­‐04   8.07E-­‐04   2.74E-­‐04   2.33E-­‐02   8.63E-­‐04   1.31E-­‐02   3.09E-­‐02   9.58E-­‐04   4.30E-­‐03   0.00E+00   1.70E-­‐02   2.45E-­‐03   4.89E-­‐04   2.62E-­‐02   4.46E-­‐05   4.20E-­‐12   2.18E-­‐07   2.60E-­‐07   8.71E-­‐03   6.89E-­‐05   2.22E-­‐07   3.85E-­‐03   4.53E-­‐02   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO     Table A3.4 (cont’d) AT5G23150   AT5G23510   AT5G23575   AT5G23730   AT5G23860   AT5G24120   AT5G24240   AT5G24590   AT5G24660   AT5G24810   AT5G25130   AT5G25190   AT5G25250   AT5G25265   AT5G25280   AT5G25440   AT5G25770   AT5G25820   AT5G25930   AT5G26040   AT5G26170   AT5G26600   AT5G26920   AT5G27420   AT5G27760   AT5G28540   AT5G28640   AT5G28830   AT5G34850     5.36   11.61   50.90   3.45   6.72   27.43   30.39   9.87   68.17   16.34   3.27   3.72   5.59   47.02   18.40   2.54   0.00   4.24   2.22   4.27   2.04   10.77   8.08   6.14   106.18   66.69   7.62   21.64   39.97   0.15   0.32   0.96   0.27   0.59   1.17   0.23   0.29   1.72   0.50   0.17   0.05   0.77   0.29   0.38   0.67   0.55   0.21   0.43   0.35   0.55   0.13   1.33   1.44   1.09   1.12   0.19   0.16   0.53   8.13   22.53   74.60   5.91   11.26   47.76   42.64   18.51   103.29   38.46   8.32   8.13   21.84   76.78   29.64   9.64   8.01   6.42   13.23   7.13   14.47   15.19   47.83   38.70   196.83   106.15   19.13   38.91   57.82   0.19   0.30   0.74   0.17   0.71   1.56   0.39   0.22   4.04   0.47   0.08   0.73   0.66   0.53   0.40   0.18   0.91   0.15   0.54   0.04   1.18   0.13   0.99   2.04   2.52   2.55   1.97   0.73   1.23   0.60   0.96   0.55   0.78   0.75   0.80   0.49   0.91   0.60   1.23   1.35   1.13   1.97   0.71   0.69   1.92   0.58   0.60   2.58   0.74   2.83   0.50   2.57   2.65   0.89   0.67   1.33   0.85   0.53   269 3.90E-­‐03   4.27E-­‐06   3.59E-­‐03   4.72E-­‐02   1.48E-­‐03   6.62E-­‐06   1.19E-­‐02   1.40E-­‐05   4.73E-­‐03   0.00E+00   7.81E-­‐06   1.13E-­‐02   0.00E+00   7.08E-­‐05   2.48E-­‐04   1.68E-­‐07   4.85E-­‐02   4.32E-­‐02   0.00E+00   3.67E-­‐02   7.48E-­‐07   2.11E-­‐02   0.00E+00   0.00E+00   5.73E-­‐07   3.50E-­‐04   1.03E-­‐07   1.40E-­‐05   5.05E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G34940   AT5G35080   AT5G35200   AT5G35370   AT5G35735   AT5G35950   AT5G35970   AT5G36160   AT5G36880   AT5G36970   AT5G37260   AT5G37540   AT5G37600   AT5G37770   AT5G37830   AT5G38210   AT5G38212   AT5G38310   AT5G38410   AT5G38650   AT5G38900   AT5G38990   AT5G39080   AT5G39110   AT5G39660   AT5G39860   AT5G40010   AT5G40230   AT5G40670     3.32   17.10   19.06   1.32   52.00   1.23   46.00   36.84   30.56   0.36   62.78   1.98   173.68   11.78   13.66   5.38   2.88   0.84   194.55   60.49   18.06   16.12   3.48   0.93   11.05   2.32   0.13   0.60   38.48   0.32   0.05   0.36   0.10   0.89   0.04   0.41   0.63   0.44   0.16   1.03   0.09   2.54   0.57   0.29   0.46   0.15   0.18   3.68   0.19   2.04   0.50   0.21   0.29   0.37   0.33   0.02   0.04   0.33   8.24   33.66   27.50   2.41   101.16   3.53   80.10   50.20   47.60   5.43   87.60   5.50   265.55   24.39   20.05   12.11   7.82   3.85   276.48   81.91   90.89   29.03   9.86   3.06   16.87   6.00   3.12   1.91   59.91   0.14   0.83   0.27   0.09   0.93   0.19   0.64   0.09   0.49   1.74   1.12   0.53   1.91   1.62   0.29   0.70   0.51   0.26   4.42   2.33   2.33   0.30   0.57   0.75   0.37   1.28   0.85   0.26   1.18   1.31   0.98   0.53   0.86   0.96   1.52   0.80   0.45   0.64   3.92   0.48   1.48   0.61   1.05   0.55   1.17   1.44   2.20   0.51   0.44   2.33   0.85   1.50   1.71   0.61   1.37   4.60   1.68   0.64   270 3.32E-­‐06   1.63E-­‐06   7.60E-­‐03   3.46E-­‐02   2.92E-­‐08   2.96E-­‐03   1.05E-­‐05   2.89E-­‐02   6.57E-­‐07   2.25E-­‐06   1.55E-­‐02   1.04E-­‐04   1.73E-­‐03   1.47E-­‐04   3.39E-­‐03   1.46E-­‐07   4.11E-­‐08   6.13E-­‐04   7.43E-­‐03   3.61E-­‐02   0.00E+00   2.57E-­‐06   5.40E-­‐07   4.15E-­‐02   1.56E-­‐03   1.91E-­‐02   7.61E-­‐09   1.57E-­‐02   6.62E-­‐04   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G40690   AT5G40890   AT5G41761   AT5G42010   AT5G42090   AT5G42100   AT5G42250   AT5G42380   AT5G42440   AT5G42760   AT5G42790   AT5G42830   AT5G42870   AT5G42990   AT5G43060   AT5G43380   AT5G43470   AT5G43900   AT5G44070   AT5G44240   AT5G44380   AT5G44390   AT5G44480   AT5G44540   AT5G44585   AT5G44720   AT5G44820   AT5G44860   AT5G44870     1.88   17.01   24.20   2.48   43.55   34.55   12.12   1.30   2.63   22.41   147.36   2.30   8.81   26.96   14.55   3.33   5.52   5.28   21.02   11.73   0.76   5.74   0.72   0.89   5.76   75.31   12.80   9.62   8.04   0.39   1.04   1.79   0.20   0.94   0.76   0.62   0.36   0.35   0.37   0.14   0.39   0.13   0.09   0.89   0.02   0.20   0.24   0.98   0.28   0.33   0.40   0.36   0.16   0.78   1.76   0.46   0.30   0.20   22.81   22.46   42.72   5.95   68.20   51.42   18.85   19.82   5.47   42.02   202.35   14.19   14.18   45.86   40.32   6.00   11.55   12.99   42.36   22.23   1.82   15.56   7.79   4.93   19.45   102.84   22.93   15.88   13.27   1.32   0.27   1.14   0.27   0.56   0.93   0.71   1.20   0.14   0.98   3.53   1.07   0.31   0.74   1.18   0.35   0.06   0.28   0.41   0.26   0.33   0.77   0.11   3.39   1.79   1.44   0.25   0.19   0.66   3.60   0.40   0.82   1.26   0.65   0.57   0.64   3.93   1.06   0.91   0.46   2.63   0.69   0.77   1.47   0.85   1.07   1.30   1.01   0.92   1.25   1.44   3.42   2.48   1.76   0.45   0.84   0.72   0.72   271 0.00E+00   2.60E-­‐02   1.69E-­‐03   1.28E-­‐05   4.06E-­‐04   2.08E-­‐04   6.89E-­‐03   0.00E+00   1.27E-­‐02   4.77E-­‐07   1.90E-­‐02   0.00E+00   1.47E-­‐05   5.46E-­‐04   0.00E+00   1.99E-­‐02   2.01E-­‐09   1.91E-­‐14   1.38E-­‐08   1.81E-­‐07   3.61E-­‐02   2.27E-­‐10   1.27E-­‐11   2.94E-­‐02   1.19E-­‐03   3.54E-­‐03   2.02E-­‐04   3.87E-­‐03   2.53E-­‐04   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G44990   AT5G45000   AT5G45110   AT5G46270   AT5G46330   AT5G46340   AT5G46350   AT5G46450   AT5G46780   AT5G46790   AT5G47050   AT5G47070   AT5G47120   AT5G47220   AT5G47420   AT5G47500   AT5G47560   AT5G47620   AT5G47820   AT5G47960   AT5G48160   AT5G48380   AT5G48540   AT5G48657   AT5G48920   AT5G48930   AT5G49450   AT5G49520   AT5G49555     0.45   2.17   7.49   6.35   7.58   6.74   2.53   11.96   6.27   16.66   2.88   8.90   113.61   47.67   8.91   14.27   45.16   33.22   13.29   2.07   33.08   26.59   34.05   4.97   1.00   42.90   2.80   1.21   25.80   0.24   0.47   0.47   0.21   0.30   0.15   0.71   0.51   0.20   0.36   0.34   0.03   0.35   1.40   0.11   0.58   0.37   0.66   0.44   0.17   0.35   0.44   0.52   0.61   0.10   0.92   0.25   0.05   0.41   9.33   5.34   29.84   10.08   19.24   9.66   15.42   16.70   14.52   28.60   5.14   15.83   277.84   120.04   15.97   21.61   91.77   45.37   17.38   9.10   50.67   67.03   68.48   18.83   3.64   84.59   5.98   5.93   36.93   0.60   0.20   0.74   0.56   0.29   0.25   1.00   0.69   0.41   0.88   0.42   0.84   3.25   2.42   0.45   0.93   0.97   0.59   0.30   0.40   0.27   0.44   1.51   0.91   0.07   0.44   0.19   0.57   0.42   4.39   1.30   1.99   0.67   1.34   0.52   2.61   0.48   1.21   0.78   0.84   0.83   1.29   1.33   0.84   0.60   1.02   0.45   0.39   2.13   0.62   1.33   1.01   1.92   1.87   0.98   1.09   2.29   0.52   272 0.00E+00   9.13E-­‐04   0.00E+00   1.33E-­‐03   1.91E-­‐14   4.44E-­‐02   0.00E+00   2.13E-­‐02   2.61E-­‐05   1.16E-­‐03   2.52E-­‐02   1.74E-­‐04   5.38E-­‐14   5.37E-­‐14   1.07E-­‐03   7.79E-­‐03   1.29E-­‐09   1.61E-­‐03   1.60E-­‐02   2.06E-­‐06   4.81E-­‐04   0.00E+00   5.75E-­‐08   3.95E-­‐10   6.74E-­‐03   7.58E-­‐09   2.96E-­‐02   3.30E-­‐09   8.03E-­‐03   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G49680   AT5G49730   AT5G49740   AT5G49760   AT5G49810   AT5G50110   AT5G50200   AT5G50210   AT5G50460   AT5G51070   AT5G51190   AT5G51290   AT5G51630   AT5G51770   AT5G51830   AT5G52160   AT5G52250   AT5G52390   AT5G52540   AT5G52730   AT5G52760   AT5G52810   AT5G52870   AT5G52900   AT5G53110   AT5G53170   AT5G53370   AT5G53550   AT5G53760     1.40   60.45   12.25   21.93   21.93   20.19   42.67   11.33   160.23   35.89   1.21   10.64   2.22   7.79   45.33   0.00   0.45   2.13   39.48   0.56   3.72   10.61   4.59   5.43   2.65   30.48   63.42   29.86   2.34   0.07   0.74   0.26   0.46   0.26   0.27   1.12   0.10   4.09   0.55   0.01   0.34   0.09   0.30   1.43   0.00   0.10   0.13   0.39   0.23   1.12   0.57   0.05   0.27   0.33   0.40   1.14   0.38   0.03   4.38   85.67   25.84   48.09   32.02   28.53   82.87   18.56   257.73   65.38   4.10   18.05   6.54   12.94   84.19   3.03   2.14   18.68   60.71   4.87   59.88   27.76   8.44   15.64   9.21   41.70   121.27   61.63   4.10   0.39   1.71   0.64   0.14   0.21   0.30   1.79   0.24   4.63   0.80   0.14   0.37   0.51   0.13   1.52   1.77   0.22   3.91   0.36   0.48   1.50   0.85   0.06   0.42   0.25   0.35   1.21   1.26   0.08   1.65   0.50   1.08   1.13   0.55   0.50   0.96   0.71   0.69   0.87   1.76   0.76   1.56   0.73   0.89   Inf   2.26   3.13   0.62   3.11   4.01   1.39   0.88   1.53   1.80   0.45   0.94   1.05   0.81   273 1.93E-­‐14   8.12E-­‐03   1.65E-­‐09   8.72E-­‐12   3.65E-­‐03   4.13E-­‐02   1.28E-­‐07   2.71E-­‐04   2.77E-­‐04   4.15E-­‐07   5.33E-­‐03   1.48E-­‐04   1.89E-­‐14   1.53E-­‐03   2.74E-­‐07   2.67E-­‐03   7.58E-­‐04   0.00E+00   6.92E-­‐04   6.34E-­‐04   0.00E+00   5.32E-­‐11   8.73E-­‐03   1.20E-­‐08   2.15E-­‐07   2.23E-­‐02   5.31E-­‐08   0.00E+00   1.27E-­‐02   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G53830   AT5G53870   AT5G53890   AT5G53970   AT5G54080   AT5G54100   AT5G54160   AT5G54380   AT5G54470   AT5G54490   AT5G54510   AT5G54690   AT5G54710   AT5G54860   AT5G54920   AT5G55100   AT5G55300   AT5G55520   AT5G55600   AT5G55730   AT5G55850   AT5G55860   AT5G56120   AT5G56240   AT5G56250   AT5G56260   AT5G56270   AT5G56350   AT5G56870     2.58   0.84   2.71   18.88   11.61   10.82   45.05   4.77   59.49   6.35   7.54   0.44   26.47   8.76   4.63   11.82   13.31   4.46   12.12   12.41   86.59   10.59   8.99   12.92   12.38   73.75   8.26   63.22   0.74   0.23   0.34   0.08   0.56   0.04   0.35   1.97   0.23   0.39   1.01   0.22   0.05   0.48   0.24   0.09   0.16   0.38   0.15   0.24   0.90   0.66   0.29   0.29   0.21   0.40   0.31   0.25   1.28   0.13   5.33   3.34   4.17   28.31   16.49   19.21   184.13   9.85   96.58   33.22   11.11   1.22   86.02   29.24   7.21   16.67   17.75   6.81   16.49   18.23   133.15   16.58   16.74   20.24   20.21   104.95   11.62   104.84   3.71   0.24   0.52   0.14   0.79   0.16   0.66   0.49   0.24   0.22   0.94   0.74   0.10   1.90   1.00   0.12   0.50   0.20   0.29   0.28   1.41   0.60   0.24   1.08   0.25   0.86   0.76   0.11   1.08   0.28   1.05   2.00   0.62   0.58   0.51   0.83   2.03   1.05   0.70   2.39   0.56   1.46   1.70   1.74   0.64   0.50   0.42   0.61   0.44   0.55   0.62   0.65   0.90   0.65   0.71   0.51   0.49   0.73   2.33   274 4.56E-­‐02   3.25E-­‐04   3.27E-­‐02   4.40E-­‐03   2.86E-­‐02   1.59E-­‐04   0.00E+00   1.55E-­‐06   1.03E-­‐04   2.37E-­‐10   2.05E-­‐02   3.65E-­‐02   0.00E+00   0.00E+00   2.36E-­‐02   2.41E-­‐03   2.75E-­‐02   1.43E-­‐02   9.12E-­‐03   7.60E-­‐03   1.71E-­‐06   1.60E-­‐03   5.07E-­‐04   7.11E-­‐06   3.80E-­‐06   7.93E-­‐03   3.09E-­‐02   4.28E-­‐05   1.18E-­‐08   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G57010   AT5G57035   AT5G57123   AT5G57220   AT5G57480   AT5G57510   AT5G57560   AT5G57710   AT5G57800   AT5G58120   AT5G58220   AT5G58350   AT5G58430   AT5G58710   AT5G58940   AT5G59030   AT5G59070   AT5G59820   AT5G59920   AT5G59960   AT5G60280   AT5G60410   AT5G60800   AT5G60840   AT5G60930   AT5G60950   AT5G61010   AT5G61390   AT5G61560     0.38   9.74   2.77   1.61   2.19   0.69   2.45   24.78   16.76   5.55   35.37   4.95   33.97   192.36   4.04   60.15   0.32   14.00   4.56   14.90   2.60   27.20   6.52   12.25   2.80   10.05   9.72   4.78   3.84   0.11   0.21   0.15   0.32   0.69   0.52   0.79   0.40   0.51   0.26   0.58   0.37   0.10   2.89   0.14   0.45   0.08   0.11   0.33   0.18   0.16   0.11   0.40   0.45   0.07   1.18   1.22   0.04   0.11   2.01   16.64   6.81   7.79   7.98   4.20   17.16   37.24   40.39   8.57   45.46   15.75   47.62   337.11   13.82   89.96   1.72   47.12   9.69   20.62   6.12   35.74   24.58   21.14   4.19   32.43   35.25   7.58   7.88   0.08   0.31   0.95   0.77   0.39   0.85   0.33   0.16   0.74   0.40   0.19   0.37   0.40   4.58   0.83   1.15   0.10   0.66   0.34   0.39   0.27   0.42   1.00   0.97   0.11   0.54   1.48   0.43   0.31   2.41   0.77   1.30   2.27   1.87   2.61   2.81   0.59   1.27   0.63   0.36   1.67   0.49   0.81   1.77   0.58   2.43   1.75   1.09   0.47   1.23   0.39   1.91   0.79   0.58   1.69   1.86   0.67   1.04   275 1.21E-­‐04   7.83E-­‐05   1.59E-­‐02   1.21E-­‐10   4.03E-­‐08   4.04E-­‐02   0.00E+00   1.43E-­‐03   8.78E-­‐14   5.33E-­‐03   4.65E-­‐02   0.00E+00   1.18E-­‐02   6.72E-­‐06   7.17E-­‐12   3.19E-­‐03   5.01E-­‐04   1.85E-­‐14   9.87E-­‐06   4.42E-­‐02   8.51E-­‐05   4.25E-­‐02   0.00E+00   6.24E-­‐03   3.47E-­‐02   3.11E-­‐13   0.00E+00   3.25E-­‐02   9.76E-­‐07   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G61790   AT5G61900   AT5G62000   AT5G62020   AT5G62150   AT5G62390   AT5G62530   AT5G62540   AT5G62560   AT5G62680   AT5G62770   AT5G63225   AT5G63470   AT5G63490   AT5G63530   AT5G63850   AT5G63950   AT5G63970   AT5G64370   AT5G64510   AT5G64570   AT5G64572   AT5G64810   AT5G64905   AT5G65210   AT5G65360   AT5G65440   AT5G65660   AT5G66210     153.94   4.84   80.93   1.36   3.91   59.05   25.69   5.57   6.11   21.72   1.65   0.61   19.09   9.75   22.08   7.68   5.52   4.51   24.77   2.11   10.17   4.10   3.43   0.78   6.23   184.80   6.93   17.87   15.29   2.39   0.10   0.77   0.06   0.59   0.91   0.75   0.25   0.42   0.53   0.30   0.14   0.42   0.20   0.81   0.23   0.17   0.12   0.65   0.11   0.32   0.21   1.51   0.36   0.07   1.71   0.17   0.48   0.17   505.04   15.13   98.93   7.26   19.35   92.43   39.43   16.79   9.77   31.45   4.81   10.03   31.63   15.36   32.01   24.11   7.68   9.46   42.72   12.75   18.18   10.46   45.84   3.45   12.88   262.28   12.86   26.80   29.58   11.60   0.14   0.27   0.17   1.55   0.94   0.53   0.02   0.12   0.57   0.28   0.77   0.88   0.31   0.68   0.61   0.17   0.34   0.93   1.36   0.74   0.67   1.66   0.40   0.54   5.90   0.47   0.74   0.89   1.71   1.64   0.29   2.42   2.31   0.65   0.62   1.59   0.68   0.53   1.55   4.04   0.73   0.66   0.54   1.65   0.48   1.07   0.79   2.59   0.84   1.35   3.74   2.14   1.05   0.51   0.89   0.58   0.95   276 0.00E+00   0.00E+00   9.09E-­‐03   1.19E-­‐09   6.44E-­‐07   4.67E-­‐04   9.37E-­‐04   6.29E-­‐07   9.43E-­‐03   6.06E-­‐03   1.84E-­‐03   3.15E-­‐08   3.05E-­‐04   4.00E-­‐03   4.80E-­‐03   0.00E+00   4.50E-­‐02   8.01E-­‐05   2.39E-­‐05   0.00E+00   1.36E-­‐05   6.33E-­‐06   0.00E+00   3.83E-­‐02   1.32E-­‐07   6.93E-­‐03   2.90E-­‐10   4.00E-­‐02   4.58E-­‐12   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO     Table A3.4 (cont’d) AT5G66250   AT5G66490   AT5G66600   AT5G66650   AT5G66675   AT5G66730   AT5G66850   AT5G66880   AT5G66910   AT5G67080   AT5G67340   AT5G67420   AT5G67450   AT5G67470     18.38   7.94   1.32   0.98   21.61   15.36   9.67   1.88   7.57   0.06   3.51   3.14   0.44   10.22   0.72   0.14   0.03   0.11   0.86   0.30   0.09   0.06   0.33   0.03   0.34   0.18   0.12   0.27   27.67   21.01   2.50   2.48   30.95   25.01   19.04   5.09   15.89   1.01   19.90   7.90   2.20   24.74   1.01   0.21   0.08   0.17   0.43   0.37   0.17   0.04   0.47   0.34   0.38   0.26   0.30   0.53   0.59   1.40   0.93   1.34   0.52   0.70   0.98   1.44   1.07   4.05   2.50   1.33   2.31   1.27   277 5.36E-­‐04   3.30E-­‐04   2.09E-­‐02   2.99E-­‐02   1.09E-­‐02   2.16E-­‐04   1.75E-­‐07   4.42E-­‐04   5.85E-­‐08   7.00E-­‐03   0.00E+00   1.39E-­‐05   1.83E-­‐03   2.95E-­‐13   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   YES   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   Table A3.5. 294 Category 2 FTD Candidate Genes with higher expression in SW at 2 weeks of cold-acclimations. Genes are ordered by their AGI numbers. FPKM values are the average of three biological replicates (n=3). Genes were considered significantly different between SW and IT under non-acclimated conditions if they had a Benjamini-Hochberg corrected p-value (FDR P-VALUE) of <0.05 and a minimum of ≥3FPKM in IT. The differential expression of each gene is also represented by the logarithm (base=2) of the fold change (LOG2.FC). This table also denotes if a gene was previously described as positively or negatively correlating with freezing tolerance (HANNAH;(6)), a transcription factor (TF), a gene previously described as up- or down-regulated by 1 week of cold (COS, (25)), identified as an up- or downregulated component of the CBF regulon (CBF REGULON; (25)). AGI   AT1G01470   AT1G01670   AT1G02270   AT1G03420   AT1G04310   AT1G05320   AT1G05560   AT1G07702   AT1G09350   AT1G10070   AT1G11580   AT1G11840   AT1G12350   AT1G12470   AT1G12550   AT1G13080     SW.1WK   FPKM   860.51   4.42   36.59   54.53   5.13   0.00   25.14   0.00   206.56   31.71   115.76   479.73   39.94   32.76   11.72   8.86   SW.2WK   FPKM   681.04   4.79   39.27   87.54   5.63   12.32   8.34   319.82   355.61   20.16   35.72   534.98   41.62   33.36   12.29   9.67   IT.1WK   FPKM   395.24   1.48   18.62   6.17   2.19   7.15   7.14   646.35   20.52   3.71   30.41   343.20   18.57   8.40   4.21   2.89   IT.2WK   FPKM   310.44   1.77   24.02   6.24   3.21   6.21   4.73   278.55   25.87   7.82   16.33   304.91   20.08   11.38   5.05   2.43   SW.IT.2WK   LOG2FC   -­‐1.13   -­‐1.44   -­‐0.71   -­‐3.81   -­‐0.81   -­‐0.99   -­‐0.82   -­‐0.20   -­‐3.78   -­‐1.37   -­‐1.13   -­‐0.81   -­‐1.05   -­‐1.55   -­‐1.28   -­‐1.99   278 SW.IT.2 WK.  FDR   P-­‐VALUE   HANNAH?   4.71E-­‐11   POSITIVE   1.32E-­‐03   NO   1.62E-­‐04   POSITIVE   0.00E+00   NO   4.32E-­‐03   NO   1.13E-­‐06   NO   5.34E-­‐03   NO   1.64E-­‐02   NO   0.00E+00   POSITIVE   1.67E-­‐12   NO   1.92E-­‐10   NO   0.00E+00   NO   2.72E-­‐08   NO   0.00E+00   NO   2.17E-­‐05   NO   3.65E-­‐11   NO   TF?   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   COS?   POSITIVE   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   DOWN   CBF   REG?   UP   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   Table A3.5 (cont’d) AT1G15125   AT1G16850   AT1G20440   AT1G20450   AT1G20620   AT1G20693   AT1G21130   AT1G22070   AT1G22090   AT1G22360   AT1G22370   AT1G22403   AT1G23120   AT1G23800   AT1G23950   AT1G24370   AT1G24388   AT1G27200   AT1G28330   AT1G28570   AT1G29050   AT1G29395   AT1G33170   AT1G33970   AT1G36180   AT1G42705   AT1G46768   AT1G48100   AT1G48360     90.96   248.49   1586.75   758.00   1910.93   229.74   74.08   40.66   11.46   14.46   4.66   25.06   12.85   30.15   11.82   91.85   266.65   31.50   40.21   5.72   19.81   274.36   19.38   65.18   3.01   4.43   17.71   17.68   16.64   117.61   276.40   1453.90   712.70   1991.20   294.79   94.42   39.26   4.70   15.20   6.20   22.12   8.81   19.22   11.84   88.30   258.07   49.45   50.56   6.05   14.62   455.70   25.16   68.70   2.76   4.66   24.76   21.78   15.13   20.60   117.34   279.94   196.22   845.84   134.25   31.10   21.22   0.06   6.76   0.98   11.54   0.84   8.99   0.91   0.11   0.08   10.57   11.34   1.92   8.38   59.17   10.34   39.41   0.48   0.02   6.34   8.46   3.81   55.00   48.76   254.60   182.55   1107.69   169.80   54.55   27.46   0.00   8.43   1.19   9.38   0.65   9.74   0.93   0.24   0.51   14.74   14.56   2.52   8.27   81.93   9.93   47.81   0.58   0.03   9.53   7.98   3.85   -­‐1.10   -­‐2.50   -­‐2.51   -­‐1.96   -­‐0.85   -­‐0.80   -­‐0.79   -­‐0.52   #NAME?   -­‐0.85   -­‐2.38   -­‐1.24   -­‐3.76   -­‐0.98   -­‐3.68   -­‐8.54   -­‐8.99   -­‐1.75   -­‐1.80   -­‐1.26   -­‐0.82   -­‐2.48   -­‐1.34   -­‐0.52   -­‐2.25   -­‐7.35   -­‐1.38   -­‐1.45   -­‐1.97   279 1.13E-­‐09   0.00E+00   0.00E+00   0.00E+00   8.97E-­‐05   2.94E-­‐09   3.87E-­‐06   9.18E-­‐03   1.01E-­‐09   1.35E-­‐04   2.29E-­‐08   1.13E-­‐07   2.99E-­‐07   9.62E-­‐07   0.00E+00   0.00E+00   0.00E+00   0.00E+00   2.09E-­‐12   8.58E-­‐04   8.25E-­‐04   0.00E+00   1.53E-­‐13   4.51E-­‐04   0.00E+00   6.20E-­‐11   3.00E-­‐07   4.19E-­‐13   0.00E+00   NO   POSITIVE   NO   NO   NO   POSITIVE   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   POSITIVE   POSITIVE   POSITIVE   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   POSITIVE   NO   NO   NO   NO   POSITIVE   NO   NO   NO   UP   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   UP   NO   NO   NO   NO   UP   UP   NO   Table A3.5 (cont’d) AT1G52720   AT1G53230   AT1G53580   AT1G54160   AT1G54890   AT1G56650   AT1G58300   AT1G58602   AT1G59950   AT1G60730   AT1G61800   AT1G62560   AT1G62570   AT1G63350   AT1G64660   AT1G64760   AT1G64780   AT1G64890   AT1G65980   AT1G67265   AT1G68500   AT1G68600   AT1G69720   AT1G69890   AT1G70260   AT1G70645   AT1G70830   AT1G71340   AT1G71360     13.55   63.17   61.84   9.68   5.08   12.38   3.47   14.28   16.46   62.87   93.26   50.21   49.71   4.30   23.05   11.68   8.52   17.73   236.37   3.90   44.40   15.27   23.25   21.94   4.97   14.14   159.30   23.51   13.66   14.24   61.66   51.09   9.02   0.86   10.34   8.24   12.14   5.90   38.04   112.94   31.44   82.13   3.67   17.76   15.90   6.00   21.38   270.97   4.83   56.74   20.41   14.10   20.69   10.70   14.74   82.01   25.02   17.15   4.47   32.14   27.46   3.41   0.30   3.80   0.52   4.84   0.38   12.23   35.70   19.12   14.01   0.01   5.64   5.21   2.43   7.90   105.14   0.87   15.61   2.30   1.43   4.40   0.88   0.00   59.25   2.21   7.05   6.57   43.05   29.98   4.19   0.11   3.04   2.37   8.79   0.37   20.07   48.16   9.55   13.92   0.01   11.60   5.68   1.58   11.83   134.32   1.67   11.79   6.53   1.32   6.55   2.85   0.00   51.81   2.47   9.28   -­‐1.12   -­‐0.52   -­‐0.77   -­‐1.11   -­‐2.95   -­‐1.77   -­‐1.79   -­‐0.47   -­‐3.99   -­‐0.92   -­‐1.23   -­‐1.72   -­‐2.56   -­‐8.37   -­‐0.61   -­‐1.49   -­‐1.93   -­‐0.85   -­‐1.01   -­‐1.54   -­‐2.27   -­‐1.64   -­‐3.41   -­‐1.66   -­‐1.91   #NAME?   -­‐0.66   -­‐3.34   -­‐0.89   280 2.33E-­‐03   6.50E-­‐03   1.92E-­‐07   2.71E-­‐04   2.20E-­‐02   2.03E-­‐05   2.14E-­‐04   1.91E-­‐02   5.27E-­‐07   9.68E-­‐11   7.05E-­‐14   0.00E+00   0.00E+00   2.54E-­‐07   5.86E-­‐03   2.04E-­‐11   1.49E-­‐07   2.63E-­‐05   8.23E-­‐10   3.73E-­‐02   1.04E-­‐13   0.00E+00   0.00E+00   1.41E-­‐11   1.81E-­‐09   0.00E+00   1.27E-­‐02   0.00E+00   6.96E-­‐06   NO   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   UP   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   Table A3.5 (cont’d) AT1G72830   AT1G75370   AT1G76180   AT1G76520   AT1G76530   AT1G76580   AT1G76590   AT1G76820   AT1G77120   AT1G78070   AT1G80130   AT1G80570   AT2G05380   AT2G05510   AT2G12190   AT2G15050   AT2G15970   AT2G16700   AT2G16790   AT2G17280   AT2G17840   AT2G18110   AT2G19810   AT2G21620   AT2G21830   AT2G21860   AT2G22590   AT2G22660   AT2G25510     8.37   50.02   811.66   111.41   3.57   16.90   47.44   2.76   381.80   117.52   293.11   3.70   2196.17   25.45   6.12   98.77   1564.76   140.34   4.99   25.98   143.48   88.39   27.19   384.16   1.05   26.96   7.67   39.60   8645.94   6.31   63.64   855.55   68.90   2.97   20.00   51.12   3.58   355.69   140.91   554.44   4.21   2680.92   8.77   9.06   122.57   1562.47   151.38   3.53   31.01   201.80   80.23   60.23   262.42   1.03   28.21   5.03   42.29   10643.80   1.76   25.50   369.49   34.18   0.89   6.47   18.99   0.16   150.04   45.46   153.50   0.45   529.44   3.06   0.78   23.52   590.27   42.67   0.84   11.91   76.61   19.27   11.08   144.60   0.25   12.70   1.30   14.10   25.11   2.88   36.75   390.72   45.27   1.28   8.75   19.72   0.23   111.48   43.89   213.76   0.98   1140.20   0.87   0.93   17.40   517.44   37.97   0.91   13.10   111.57   18.10   14.26   155.70   0.19   15.56   0.78   21.58   40.26   -­‐1.13   -­‐0.79   -­‐1.13   -­‐0.61   -­‐1.22   -­‐1.19   -­‐1.37   -­‐3.95   -­‐1.67   -­‐1.68   -­‐1.38   -­‐2.10   -­‐1.23   -­‐3.34   -­‐3.29   -­‐2.82   -­‐1.59   -­‐2.00   -­‐1.96   -­‐1.24   -­‐0.86   -­‐2.15   -­‐2.08   -­‐0.75   -­‐2.46   -­‐0.86   -­‐2.68   -­‐0.97   -­‐8.05   281 3.94E-­‐04   9.35E-­‐06   0.00E+00   6.78E-­‐05   2.82E-­‐02   1.13E-­‐11   1.04E-­‐13   0.00E+00   0.00E+00   0.00E+00   3.67E-­‐14   4.21E-­‐07   4.18E-­‐09   5.61E-­‐08   1.87E-­‐14   0.00E+00   0.00E+00   0.00E+00   3.37E-­‐03   3.14E-­‐09   1.10E-­‐06   0.00E+00   0.00E+00   2.19E-­‐05   3.46E-­‐03   8.85E-­‐06   1.13E-­‐07   6.09E-­‐09   0.00E+00   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   POSITIVE   NO   POSITIVE   NO   NO   NO   NO   NO   POSITIVE   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   POSITIVE   POSITIVE   NO   POSITIVE   POSITIVE   POSITIVE   NO   NO   NO   NO   DOWN   POSITIVE   NO   NO   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   UP   UP   NO   NO   UP   UP   NO   NO   NO   NO   NO   UP   UP   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   Table A3.5 (cont’d) AT2G26010   AT2G27360   AT2G28550   AT2G28900   AT2G29420   AT2G33830   AT2G34655   AT2G36750   AT2G39850   AT2G40010   AT2G41010   AT2G43500   AT2G46440   AT2G46640   AT2G47180   AT2G47890   AT3G05660   AT3G05880   AT3G07320   AT3G09030   AT3G12500   AT3G13310   AT3G14150   AT3G14940   AT3G17130   AT3G19000   AT3G20100   AT3G21500   AT3G22121     154.12   16.91   124.43   983.81   69.20   770.26   15.00   18.28   18.21   19.08   21.54   29.93   10.37   13.72   78.66   55.55   12.07   500.06   29.83   10.86   88.88   139.98   26.04   2.95   17.49   10.79   50.62   5.26   284.13   237.06   13.50   155.52   1399.48   29.94   840.49   13.25   13.69   5.47   15.02   23.95   32.48   12.40   11.32   46.04   54.56   17.35   397.98   36.52   11.55   14.23   99.33   33.95   3.54   20.53   9.13   49.57   2.57   267.25   5.34   5.04   79.16   424.69   16.50   310.33   1.64   6.88   9.57   0.08   8.92   7.46   0.12   5.53   32.40   25.76   4.81   255.71   12.44   4.69   2.97   73.66   9.28   0.76   5.51   0.19   9.41   1.11   127.46   66.62   5.83   77.01   418.81   16.50   457.27   3.16   6.50   2.53   0.04   15.99   8.71   0.17   5.66   33.14   26.51   8.32   207.90   16.28   7.64   2.32   67.80   7.60   0.44   4.50   0.11   13.66   1.21   138.18   -­‐1.83   -­‐1.21   -­‐1.01   -­‐1.74   -­‐0.86   -­‐0.88   -­‐2.07   -­‐1.08   -­‐1.11   -­‐8.39   -­‐0.58   -­‐1.90   -­‐6.18   -­‐1.00   -­‐0.47   -­‐1.04   -­‐1.06   -­‐0.94   -­‐1.17   -­‐0.60   -­‐2.62   -­‐0.55   -­‐2.16   -­‐3.02   -­‐2.19   -­‐6.32   -­‐1.86   -­‐1.09   -­‐0.95   282 0.00E+00   3.39E-­‐06   0.00E+00   0.00E+00   2.25E-­‐04   4.66E-­‐07   1.31E-­‐07   2.88E-­‐05   8.11E-­‐05   0.00E+00   2.54E-­‐02   0.00E+00   0.00E+00   5.32E-­‐04   1.80E-­‐02   1.43E-­‐09   9.44E-­‐09   5.80E-­‐08   3.73E-­‐11   2.10E-­‐02   3.08E-­‐12   4.37E-­‐03   0.00E+00   6.29E-­‐12   1.29E-­‐06   7.52E-­‐13   0.00E+00   1.66E-­‐02   7.73E-­‐09   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   POSITIVE   POSITIVE   POSITIVE   NO   NO   NO   NO   NO   NO   NO   POSITIVE   DOWN   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   Table A3.5 (cont’d) AT3G22142   AT3G22840   AT3G26200   AT3G26280   AT3G26460   AT3G26740   AT3G27210   AT3G28050   AT3G28310   AT3G28320   AT3G28740   AT3G28880   AT3G28950   AT3G32230   AT3G42658   AT3G42670   AT3G43270   AT3G43520   AT3G44430   AT3G44620   AT3G44670   AT3G45600   AT3G46370   AT3G46490   AT3G46900   AT3G49130   AT3G50660   AT3G50970   AT3G52180     62.20   290.33   4.37   21.27   8.02   395.85   61.39   38.00   66.01   66.81   51.78   3.16   21.18   1.01   67.13   4.84   17.69   139.84   56.06   49.51   11.85   195.08   5.16   6.08   11.73   6.46   15.34   1609.21   189.78   68.17   502.14   4.70   30.61   14.62   342.98   59.15   36.73   102.25   100.01   22.67   2.37   22.38   0.86   104.06   6.40   14.11   124.49   48.19   51.93   10.44   188.10   5.55   5.35   16.32   0.39   20.44   1188.91   255.58   15.80   155.08   1.26   4.24   1.96   210.41   33.67   15.92   8.89   8.45   13.17   0.66   3.62   0.07   32.11   1.84   8.23   67.75   23.73   17.68   2.07   67.28   0.02   0.64   0.87   0.08   6.46   341.83   105.04   15.85   275.06   2.37   5.35   0.56   232.47   31.14   18.24   19.27   20.41   7.37   0.55   6.36   0.14   42.18   2.54   9.50   58.99   15.08   27.22   3.25   62.81   0.03   0.33   3.25   0.00   5.65   260.26   85.09   -­‐2.10   -­‐0.87   -­‐0.99   -­‐2.52   -­‐4.71   -­‐0.56   -­‐0.93   -­‐1.01   -­‐2.41   -­‐2.29   -­‐1.62   -­‐2.12   -­‐1.82   -­‐2.66   -­‐1.30   -­‐1.34   -­‐0.57   -­‐1.08   -­‐1.68   -­‐0.93   -­‐1.69   -­‐1.58   -­‐7.76   -­‐4.01   -­‐2.33   #NAME?   -­‐1.85   -­‐2.19   -­‐1.59   283 0.00E+00   9.46E-­‐07   1.09E-­‐02   0.00E+00   2.35E-­‐10   1.99E-­‐03   5.83E-­‐07   4.87E-­‐08   5.94E-­‐10   0.00E+00   1.86E-­‐14   2.98E-­‐05   8.41E-­‐10   2.85E-­‐05   5.38E-­‐14   3.09E-­‐09   1.07E-­‐02   1.97E-­‐10   0.00E+00   5.26E-­‐08   0.00E+00   0.00E+00   3.32E-­‐12   3.80E-­‐07   7.17E-­‐07   4.62E-­‐02   0.00E+00   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   UP   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   POSITIVE   UP   NO   NO   Table A3.5 (cont’d) AT3G52340   AT3G53990   AT3G55760   AT3G55940   AT3G57460   AT3G57470   AT3G61010   AT3G62740   AT4G01390   AT4G01500   AT4G01883   AT4G01985   AT4G04020   AT4G04340   AT4G04426   AT4G04925   AT4G05090   AT4G06746   AT4G08390   AT4G09020   AT4G09760   AT4G10955   AT4G11410   AT4G14270   AT4G14580   AT4G15130   AT4G15450   AT4G15910   AT4G16146     33.54   279.86   41.67   9.05   3.48   6.32   5.86   11.79   27.40   3.12   31.74   43.10   140.38   59.98   1.37   35.49   42.87   39.58   366.40   119.34   42.74   0.18   14.71   73.23   49.50   51.50   41.96   324.05   71.67   40.29   311.84   43.58   11.01   1.36   6.45   5.94   8.31   11.43   3.28   28.09   39.78   104.74   72.58   1.62   48.77   49.63   62.79   269.16   208.91   51.58   0.85   15.88   97.47   69.76   63.98   43.04   169.33   76.17   19.25   114.72   13.09   2.48   0.05   0.01   0.04   2.01   2.02   0.47   14.55   22.05   64.25   35.33   0.02   10.47   23.20   7.68   178.17   52.11   20.32   1.88   6.93   22.41   10.86   24.88   16.95   57.83   35.84   19.78   174.88   11.50   3.51   0.06   0.01   0.02   1.13   1.18   0.37   11.71   25.57   46.93   33.33   0.02   14.77   25.25   17.34   195.65   63.04   23.53   0.17   9.76   34.73   13.06   27.19   19.21   28.33   33.41   -­‐1.03   -­‐0.83   -­‐1.92   -­‐1.65   -­‐4.39   -­‐9.02   -­‐8.57   -­‐2.88   -­‐3.27   -­‐3.16   -­‐1.26   -­‐0.64   -­‐1.16   -­‐1.12   -­‐6.36   -­‐1.72   -­‐0.97   -­‐1.86   -­‐0.46   -­‐1.73   -­‐1.13   -­‐2.36   -­‐0.70   -­‐1.49   -­‐2.42   -­‐1.23   -­‐1.16   -­‐2.58   -­‐1.19   284 3.11E-­‐13   1.09E-­‐06   0.00E+00   7.06E-­‐10   6.99E-­‐04   6.31E-­‐11   1.16E-­‐07   2.66E-­‐11   7.05E-­‐10   2.24E-­‐06   7.97E-­‐09   8.44E-­‐04   2.33E-­‐12   7.37E-­‐13   1.32E-­‐05   7.59E-­‐12   4.92E-­‐08   3.68E-­‐14   2.69E-­‐03   0.00E+00   1.89E-­‐14   3.35E-­‐03   5.67E-­‐03   0.00E+00   0.00E+00   3.10E-­‐12   3.94E-­‐10   0.00E+00   2.38E-­‐09   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   POSITIVE   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   UP   UP   UP   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   Table A3.5 (cont’d) AT4G16770   AT4G16860   AT4G16890   AT4G18070   AT4G18422   AT4G19500   AT4G19510   AT4G20310   AT4G23600   AT4G23670   AT4G23920   AT4G24220   AT4G25433   AT4G26255   AT4G27050   AT4G27070   AT4G27300   AT4G27820   AT4G27900   AT4G27970   AT4G28220   AT4G30830   AT4G31620   AT4G33070   AT4G33180   AT4G34530   AT4G35300   AT4G36010   AT4G37000     6.40   10.83   15.95   78.00   432.66   7.58   9.68   6.39   306.42   218.14   23.15   123.54   24.84   13.33   61.39   21.11   38.86   26.99   36.63   7.02   40.47   16.39   2.97   34.90   12.63   7.88   73.80   24.92   51.43   6.99   11.75   17.33   87.26   441.74   8.92   10.76   6.12   161.04   153.99   27.67   159.79   27.81   12.39   51.93   17.59   42.74   25.91   36.02   7.58   50.86   28.21   3.92   33.52   11.48   10.98   100.64   24.96   60.46   1.47   3.69   8.26   44.90   214.21   0.29   0.56   3.00   139.73   116.61   9.43   58.60   4.75   1.06   17.22   6.40   17.91   9.04   14.78   2.42   22.04   2.86   0.92   6.76   4.53   3.13   42.49   12.24   25.14   1.52   5.46   12.80   40.40   128.37   0.28   0.82   2.46   76.29   37.05   7.47   113.38   3.80   1.28   13.57   7.24   22.41   6.87   19.58   2.70   26.26   2.86   1.17   7.35   4.75   1.24   48.10   9.93   27.52   -­‐2.20   -­‐1.11   -­‐0.44   -­‐1.11   -­‐1.78   -­‐5.01   -­‐3.71   -­‐1.31   -­‐1.08   -­‐2.06   -­‐1.89   -­‐0.49   -­‐2.87   -­‐3.28   -­‐1.94   -­‐1.28   -­‐0.93   -­‐1.91   -­‐0.88   -­‐1.49   -­‐0.95   -­‐3.30   -­‐1.74   -­‐2.19   -­‐1.27   -­‐3.14   -­‐1.07   -­‐1.33   -­‐1.14   285 2.16E-­‐06   1.81E-­‐08   3.18E-­‐02   0.00E+00   0.00E+00   0.00E+00   0.00E+00   1.40E-­‐05   0.00E+00   0.00E+00   0.00E+00   9.56E-­‐03   5.41E-­‐13   1.68E-­‐13   0.00E+00   5.37E-­‐10   3.01E-­‐07   0.00E+00   6.66E-­‐07   7.74E-­‐06   2.72E-­‐08   0.00E+00   9.52E-­‐05   0.00E+00   2.32E-­‐05   4.19E-­‐13   0.00E+00   1.82E-­‐11   4.72E-­‐10   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   POSITIVE   NO   NO   POSITIVE   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   UP   NO   Table A3.5 (cont’d) AT4G38580   AT4G39210   AT4G39800   AT5G01600   AT5G02130   AT5G03350   AT5G05270   AT5G07010   AT5G08640   AT5G10140   AT5G13750   AT5G14550   AT5G14920   AT5G17040   AT5G17190   AT5G17460   AT5G17700   AT5G20750   AT5G20830   AT5G23240   AT5G23820   AT5G24160   AT5G24210   AT5G25110   AT5G26770   AT5G26970   AT5G26980   AT5G27930   AT5G28913     141.00   27.59   406.75   335.37   29.98   180.08   96.18   21.10   94.23   145.95   34.18   51.44   299.20   4.53   166.31   126.59   20.09   4.35   281.83   25.83   50.35   17.92   69.59   184.09   18.56   15.23   1.48   28.95   11.63   144.43   45.44   514.02   344.75   29.53   302.99   104.41   36.05   86.83   86.18   18.68   98.97   292.91   5.41   157.65   138.84   20.54   4.06   314.66   35.71   23.88   34.00   52.95   334.24   18.11   18.89   1.97   31.49   12.76   67.73   12.45   199.33   93.33   13.36   56.15   56.56   2.52   42.40   41.81   12.68   32.49   136.81   0.84   75.06   44.65   5.80   0.01   75.39   11.63   15.00   1.27   7.63   11.96   8.91   2.70   0.37   15.44   0.04   78.34   18.03   216.45   147.02   13.82   109.48   46.33   2.44   35.48   28.39   11.19   43.51   112.41   1.17   96.36   37.01   5.38   0.02   107.11   25.31   10.08   3.63   26.16   13.19   11.04   4.84   0.68   14.84   0.04   -­‐0.88   -­‐1.33   -­‐1.25   -­‐1.23   -­‐1.10   -­‐1.47   -­‐1.17   -­‐3.88   -­‐1.29   -­‐1.60   -­‐0.74   -­‐1.19   -­‐1.38   -­‐2.21   -­‐0.71   -­‐1.91   -­‐1.93   -­‐7.84   -­‐1.55   -­‐0.50   -­‐1.24   -­‐3.23   -­‐1.02   -­‐4.66   -­‐0.71   -­‐1.96   -­‐1.53   -­‐1.09   -­‐8.32   286 6.67E-­‐07   0.00E+00   3.00E-­‐10   3.32E-­‐12   1.95E-­‐08   0.00E+00   0.00E+00   0.00E+00   3.61E-­‐14   0.00E+00   9.00E-­‐05   0.00E+00   0.00E+00   2.76E-­‐06   7.05E-­‐05   0.00E+00   0.00E+00   1.84E-­‐10   0.00E+00   1.41E-­‐02   4.12E-­‐06   0.00E+00   2.18E-­‐08   0.00E+00   4.10E-­‐04   7.92E-­‐03   6.74E-­‐03   1.87E-­‐11   3.05E-­‐07   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   DOWN   NO   DOWN   NO   NO   DOWN   POSITIVE   UP   NO   NO   NO   NO   NO   NO   POSITIVE   UP   NO   NO   Table A3.5 (cont’d) AT5G30495   AT5G32460   AT5G35935   AT5G38120   AT5G38250   AT5G39340   AT5G39610   AT5G40390   AT5G41700   AT5G41740   AT5G42570   AT5G42825   AT5G43950   AT5G44400   AT5G44565   AT5G44590   AT5G45470   AT5G45490   AT5G45500   AT5G45510   AT5G47130   AT5G47810   AT5G48490   AT5G48530   AT5G48670   AT5G48880   AT5G49120   AT5G49330   AT5G49630     50.14   4.56   43.62   13.96   2.46   31.94   14.34   33.15   135.29   3.18   107.31   51.11   8.42   41.21   298.94   22.35   10.46   40.54   6.48   47.41   19.94   9.73   550.57   5.30   3.90   73.63   3.78   36.66   42.45   49.94   2.68   53.82   12.38   1.91   37.62   16.65   39.46   140.04   3.23   101.15   100.46   11.05   26.99   294.16   57.70   13.21   63.06   9.78   65.79   15.92   10.00   566.92   2.85   5.05   64.90   6.46   42.76   50.73   28.33   0.26   0.12   5.53   0.12   15.21   4.04   16.34   62.15   0.15   56.93   18.94   3.02   11.16   54.54   8.09   5.33   0.12   2.13   8.21   0.79   0.62   280.57   0.77   0.69   35.35   0.72   16.80   11.09   19.95   0.20   0.13   5.41   0.12   16.11   8.68   17.26   71.14   0.43   72.73   27.90   3.72   10.51   63.70   17.95   9.74   0.25   4.96   13.44   0.87   1.04   166.41   0.63   1.10   33.05   2.12   12.22   26.13   -­‐1.32   -­‐3.77   -­‐8.66   -­‐1.19   -­‐4.03   -­‐1.22   -­‐0.94   -­‐1.19   -­‐0.98   -­‐2.92   -­‐0.48   -­‐1.85   -­‐1.57   -­‐1.36   -­‐2.21   -­‐1.68   -­‐0.44   -­‐7.97   -­‐0.98   -­‐2.29   -­‐4.19   -­‐3.26   -­‐1.77   -­‐2.18   -­‐2.20   -­‐0.97   -­‐1.61   -­‐1.81   -­‐0.96   287 3.11E-­‐13   NO   1.74E-­‐06   NO   0.00E+00   NO   8.37E-­‐07   NO   8.56E-­‐06   NO   8.04E-­‐08   NO   2.08E-­‐04   NO   1.55E-­‐12   NO   1.19E-­‐11   NO   7.04E-­‐14   NO   2.02E-­‐02   POSITIVE   0.00E+00   NO   3.12E-­‐10   NO   9.32E-­‐13   NO   0.00E+00   NO   0.00E+00   NO   4.53E-­‐02   NO   0.00E+00   NO   2.95E-­‐06   NO   0.00E+00   NO   6.92E-­‐09   NO   0.00E+00   NO   0.00E+00   NEGATIVE   1.24E-­‐03   NO   5.75E-­‐05   NO   5.36E-­‐14   NO   8.65E-­‐03   NO   0.00E+00   NO   3.09E-­‐08   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   Table A3.5 (cont’d) AT5G49650   AT5G50950   AT5G52310   AT5G53120   AT5G53420   AT5G54190   AT5G54960   AT5G55250   AT5G57110   AT5G57380   AT5G59130   AT5G59320   AT5G59670   AT5G59732   AT5G65060   AT5G66400   AT5G66480     52.13   18.14   1276.59   34.48   114.21   75.17   95.77   5.55   62.10   1.90   6.94   12.57   5.70   3.72   40.27   77.83   3.41   57.42   21.79   1702.55   25.92   167.27   158.76   129.97   6.28   71.56   3.70   5.31   15.11   9.46   3.84   37.23   11.85   2.61   23.78   8.84   232.93   16.92   40.23   18.54   41.06   1.91   33.91   0.48   1.56   38.55   2.44   0.29   9.41   15.22   0.87   29.79   8.85   238.95   17.75   60.66   29.45   76.58   1.94   38.11   1.45   0.99   7.70   3.32   0.67   9.32   5.96   0.93   -­‐0.95   -­‐1.30   -­‐2.83   -­‐0.55   -­‐1.46   -­‐2.43   -­‐0.76   -­‐1.69   -­‐0.91   -­‐1.36   -­‐2.42   -­‐0.97   -­‐1.51   -­‐2.51   -­‐2.00   -­‐0.99   -­‐1.48   288 3.32E-­‐08   1.35E-­‐11   0.00E+00   2.62E-­‐03   0.00E+00   0.00E+00   1.27E-­‐05   2.09E-­‐05   2.43E-­‐07   4.74E-­‐04   4.11E-­‐11   1.20E-­‐02   2.96E-­‐11   4.11E-­‐06   0.00E+00   5.83E-­‐03   6.49E-­‐03   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO       Table A3.6. 504 Category 2 FTD Candidate Genes with higher expression in IT at 2 weeks of cold-acclimations. Genes are ordered by their AGI numbers. FPKM values are the average of three biological replicates (n=3). Genes were considered significantly different between SW and IT under non-acclimated conditions if they had a Benjamini-Hochberg corrected p-value (FDR P-VALUE) of <0.05 and a minimum of ≥3FPKM in IT. The differential expression of each gene is also represented by the logarithm (base=2) of the fold change (LOG2.FC). This table also denotes if a gene was previously described as positively or negatively correlating with freezing tolerance (HANNAH;(6)), a transcription factor (TF), a gene previously described as up- or down-regulated by 1 week of cold (COS, (25)), identified as an up- or downregulated component of the CBF regulon (CBF REGULON; (25)).   AGI   AT1G01480   AT1G01620   AT1G03870   AT1G03905   AT1G03940   AT1G04250   AT1G04680   AT1G05550   AT1G05680   AT1G07570   AT1G07610   AT1G08610   AT1G09420   AT1G09932   AT1G10340   AT1G10470   AT1G10585     SW.1WK FPKM   8.34   42.30   11.15   6.63   0.28   15.91   15.13   0.60   27.57   14.32   23.26   4.53   2.62   10.59   0.04   45.17   31.14   SW.2WK   FPKM   0.32   57.78   9.32   4.45   0.09   13.52   17.36   0.33   0.28   14.56   13.71   3.76   2.00   3.04   0.04   41.35   2.18   IT.1WKF PKM   1.91   123.45   27.33   16.31   1.36   33.42   33.66   2.30   1.84   28.20   96.25   11.30   15.93   47.32   3.04   85.36   7.09   IT.2WK   FPKM   2.39   111.61   20.77   19.80   0.74   24.95   27.90   2.38   4.26   31.08   59.98   7.03   16.47   52.92   9.81   65.58   9.90   SW.IT.2WK.   SW.IT.2WK FDR  P-­‐ LOG2FC   VALUE   HANNAH?   2.91   2.24E-­‐07   NO   0.95   2.17E-­‐08   NEGATIVE   1.16   4.74E-­‐06   NO   2.15   3.64E-­‐14   NO   3.01   3.42E-­‐03   NO   0.88   1.22E-­‐04   NO   0.68   3.65E-­‐04   NO   2.85   2.94E-­‐05   NO   3.92   1.85E-­‐09   NO   1.09   1.53E-­‐13   NO   2.13   9.30E-­‐12   NO   0.90   1.37E-­‐03   NO   3.04   0.00E+00   NO   4.12   0.00E+00   NO   8.12   6.32E-­‐13   NO   0.67   3.68E-­‐04   NO   2.18   5.22E-­‐06   NO   289 TF?   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   COS?   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   CBF   REG?   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT1G11175   AT1G12220   AT1G13350   AT1G13470   AT1G13650   AT1G14700   AT1G14880   AT1G15010   AT1G15870   AT1G15960   AT1G15980   AT1G16510   AT1G16820   AT1G17170   AT1G17180   AT1G17190   AT1G17665   AT1G17880   AT1G18330   AT1G18910   AT1G20160   AT1G20190   AT1G20350   AT1G21270   AT1G21750   AT1G22400   AT1G23410   AT1G23850   AT1G27330     0.05   0.02   1.96   23.26   0.03   0.54   191.11   7.71   3.63   0.44   64.43   4.82   0.08   315.23   14.44   4.46   20.11   132.66   0.26   1.28   1.77   16.22   0.04   1.19   153.45   14.75   14.20   1.37   71.88   0.00   0.01   1.54   23.68   0.00   0.55   286.21   0.94   2.49   0.89   79.74   5.13   0.27   64.23   0.44   8.13   20.86   115.11   0.27   1.02   1.00   27.07   0.05   1.76   119.07   2.31   10.85   0.14   64.60   6.19   4.47   11.14   79.09   2.10   6.19   548.89   1.47   12.68   2.06   121.81   19.10   2.50   71.61   2.03   18.90   39.85   303.81   1.78   5.61   7.05   40.89   4.15   40.11   255.57   6.24   41.72   11.26   146.63   9.83   5.55   10.91   164.60   0.85   4.43   980.29   5.08   6.98   3.23   118.60   22.54   4.25   137.50   4.20   17.12   35.92   185.93   1.73   10.00   5.82   47.79   1.85   69.59   386.01   21.78   19.68   16.17   205.94   Inf   8.94   2.82   2.80   Inf   3.02   1.78   2.44   1.48   1.86   0.57   2.13   4.00   1.10   3.24   1.07   0.78   0.69   2.67   3.30   2.55   0.82   5.32   5.30   1.70   3.24   0.86   6.82   1.67   290 2.31E-­‐11   4.47E-­‐08   0.00E+00   0.00E+00   3.36E-­‐03   3.28E-­‐09   0.00E+00   4.41E-­‐03   8.34E-­‐04   1.74E-­‐05   1.89E-­‐03   5.36E-­‐12   1.92E-­‐03   1.94E-­‐10   1.72E-­‐05   2.03E-­‐04   1.20E-­‐04   7.84E-­‐05   1.93E-­‐04   0.00E+00   1.04E-­‐13   1.66E-­‐05   3.62E-­‐03   0.00E+00   0.00E+00   0.00E+00   1.83E-­‐02   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NEGATIVE   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT1G28670   AT1G30700   AT1G30730   AT1G30900   AT1G31540   AT1G32920   AT1G32940   AT1G32950   AT1G33610   AT1G33960   AT1G34575   AT1G35710   AT1G36622   AT1G43910   AT1G44350   AT1G44750   AT1G45201   AT1G47370   AT1G47510   AT1G47760   AT1G48260   AT1G48460   AT1G48750   AT1G50110   AT1G50520   AT1G51270   AT1G51430   AT1G51890   AT1G52040     0.56   19.20   3.43   8.43   5.28   30.56   24.51   4.44   1.57   74.61   0.06   9.96   13.65   12.36   12.96   15.10   16.08   0.05   16.58   0.08   0.83   20.68   13.04   1.20   0.02   0.08   3.33   4.82   47.33   1.06   2.23   0.96   5.76   4.94   7.65   4.85   0.13   0.97   17.50   0.09   5.45   2.44   9.22   2.31   15.20   5.38   0.07   1.04   0.09   0.80   16.50   11.81   1.50   0.03   0.08   4.41   1.05   50.83   3.04   2.83   0.84   20.12   14.14   9.64   8.65   0.89   12.98   18.95   22.63   39.22   51.22   5.52   5.40   30.81   53.25   1.52   3.00   7.72   3.71   45.61   58.82   7.93   4.99   6.76   16.09   10.50   162.86   3.42   8.76   2.90   25.78   23.47   26.23   17.27   1.24   18.93   220.93   25.60   53.32   73.32   26.96   8.98   38.79   26.96   1.98   3.72   5.50   4.54   22.96   40.33   4.44   10.09   17.01   14.93   17.17   233.88   1.69   1.98   1.60   2.16   2.25   1.78   1.83   3.28   4.29   3.66   8.17   3.29   4.91   1.55   1.96   1.35   2.32   4.82   1.83   5.98   2.51   0.48   1.77   1.57   8.59   7.78   1.76   4.03   2.20   291 9.07E-­‐04   3.35E-­‐10   1.38E-­‐03   0.00E+00   0.00E+00   6.51E-­‐06   0.00E+00   1.69E-­‐05   0.00E+00   0.00E+00   0.00E+00   0.00E+00   0.00E+00   0.00E+00   2.32E-­‐11   0.00E+00   0.00E+00   1.25E-­‐04   2.24E-­‐04   6.93E-­‐04   6.56E-­‐08   4.56E-­‐02   4.61E-­‐09   7.73E-­‐04   4.26E-­‐10   0.00E+00   2.87E-­‐07   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT1G52200   AT1G52290   AT1G52342   AT1G52400   AT1G53480   AT1G55330   AT1G56510   AT1G56520   AT1G56660   AT1G57630   AT1G57650   AT1G57660   AT1G58225   AT1G58380   AT1G58420   AT1G60190   AT1G61820   AT1G61980   AT1G61990   AT1G62355   AT1G62540   AT1G63160   AT1G63780   AT1G63860   AT1G65150   AT1G65490   AT1G65500   AT1G65790   AT1G66725     176.46   6.40   3.58   329.62   8.94   56.02   0.19   0.08   6.04   0.00   0.12   30.17   43.95   21.08   5.45   7.31   3.18   4.57   8.42   0.00   0.06   11.31   32.67   0.31   0.35   49.55   34.66   6.35   10.13   78.73   4.56   3.12   218.65   5.43   42.22   0.17   0.07   4.80   0.00   0.09   28.98   12.15   16.63   1.60   15.05   0.41   4.07   7.38   0.00   0.10   9.71   21.83   0.30   0.34   62.83   23.58   5.29   9.69   79.71   17.61   14.61   1020.56   83.13   128.20   2.21   4.64   20.21   1.28   2.63   105.56   187.81   61.75   16.96   17.37   0.72   13.74   23.26   10.98   0.86   28.43   62.18   4.47   3.18   178.33   134.03   18.60   43.58   124.75   21.66   19.68   974.36   61.00   71.21   7.04   7.70   48.80   4.69   7.97   69.70   104.62   39.80   12.78   20.99   1.65   8.60   16.90   5.62   0.77   20.83   31.93   7.27   1.32   229.20   294.68   20.61   88.10   0.66   2.25   2.66   2.16   3.49   0.75   5.34   6.80   3.35   Inf   6.51   1.27   3.11   1.26   3.00   0.48   2.03   1.08   1.20   Inf   2.88   1.10   0.55   4.62   1.97   1.87   3.64   1.96   3.19   292 2.38E-­‐04   0.00E+00   7.26E-­‐08   0.00E+00   0.00E+00   4.96E-­‐03   0.00E+00   0.00E+00   0.00E+00   5.67E-­‐04   0.00E+00   8.39E-­‐10   0.00E+00   1.84E-­‐10   2.39E-­‐09   2.17E-­‐02   1.34E-­‐03   3.17E-­‐04   3.80E-­‐08   3.39E-­‐13   4.11E-­‐03   1.59E-­‐06   1.24E-­‐02   0.00E+00   1.67E-­‐02   0.00E+00   0.00E+00   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO         Table A3.6 (cont’d) AT1G66880   14.14   AT1G66960   0.02   AT1G67365   0.00   AT1G67590   0.27   AT1G67980   40.86   AT1G68620   8.64   AT1G69530   19.91   AT1G69880   19.88   AT1G70090   22.24   AT1G70200   62.82   AT1G70420   18.09   AT1G70985   2.74   AT1G71880   26.41   AT1G72130   4.75   AT1G72430   8.93   AT1G72520   8.00   AT1G72970   11.58   AT1G73260   24.81   AT1G73805   10.37   AT1G74670   14.88   AT1G74710   6.67   AT1G75030   2.70   AT1G75040   1396.09   AT1G76470   30.54   AT1G76650   20.07   AT1G76930   461.90   AT1G76955   22.27   AT1G76970   8.86   AT1G77510   64.03     8.80   0.05   0.05   0.44   11.03   0.87   34.29   2.06   14.81   75.02   22.79   2.78   21.58   3.86   2.64   3.85   12.72   24.99   12.01   8.58   6.29   2.13   359.53   0.06   4.03   155.62   27.04   6.20   31.34   31.95   7.84   6.89   5.65   16.60   22.47   88.43   2.93   40.78   129.16   79.36   10.64   67.52   12.48   36.31   3.01   26.39   72.52   27.81   70.40   14.45   9.56   8818.64   0.08   8.36   122.20   55.22   20.65   213.11   48.11   10.15   11.59   5.86   43.41   24.40   97.64   7.52   28.19   106.71   58.81   8.66   62.98   12.73   30.88   7.99   20.79   97.23   86.07   51.51   27.71   6.90   8119.78   0.53   27.88   624.22   43.05   23.37   281.48   2.45   7.80   7.96   3.73   1.98   4.81   1.51   1.87   0.93   0.51   1.37   1.64   1.54   1.72   3.55   1.06   0.71   1.96   2.84   2.59   2.14   1.69   4.50   3.13   2.79   2.00   0.67   1.92   3.17   293 0.00E+00   0.00E+00   0.00E+00   3.79E-­‐11   0.00E+00   0.00E+00   0.00E+00   1.81E-­‐03   2.41E-­‐08   9.22E-­‐03   3.66E-­‐14   2.90E-­‐03   0.00E+00   1.04E-­‐13   0.00E+00   4.87E-­‐06   2.86E-­‐04   0.00E+00   0.00E+00   0.00E+00   0.00E+00   3.81E-­‐04   0.00E+00   3.81E-­‐02   0.00E+00   0.00E+00   9.31E-­‐03   0.00E+00   0.00E+00   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT1G77630   6.48   AT1G77760   23.40   AT1G78080   14.51   AT1G78290   20.46   AT1G78410   16.77   AT2G01670   28.44   AT2G01890   1.94   AT2G03750   15.05   AT2G04040   27.39   AT2G04450   38.95   AT2G04495   3.49   AT2G07750   0.06   AT2G10940   641.34   AT2G14580   2.21   AT2G14610   1484.66   AT2G15128   1.25   AT2G16676   0.02   AT2G16680   0.01   AT2G17040   3.95   AT2G17250   19.27   AT2G18193   3.54   AT2G18300   46.62   AT2G18560   2.17   AT2G18680   3.92   AT2G22330   19.52   AT2G22980   3.01   AT2G23000   1.92   AT2G23110   0.36   AT2G23600   23.61     4.41   20.68   18.21   23.90   6.68   13.42   0.81   20.08   3.36   17.47   1.14   0.06   427.76   2.81   1091.57   0.97   0.00   0.00   3.31   12.76   0.49   43.56   1.67   0.05   18.36   2.88   1.64   0.07   23.39   15.64   42.37   27.88   49.12   43.36   79.08   15.20   54.35   13.81   14.49   15.43   4.89   1405.64   17.58   4492.87   383.42   1.37   0.99   11.63   38.86   0.38   69.80   7.20   12.71   44.94   11.91   6.58   4.54   119.48   9.92   43.76   39.48   60.90   106.49   68.28   9.88   47.16   23.64   112.95   14.71   3.62   689.21   7.73   15144.50   430.07   1.91   1.62   24.66   20.78   1.42   64.40   6.35   9.74   30.13   11.57   4.63   5.90   91.89   1.17   1.08   1.12   1.35   4.00   2.35   3.60   1.23   2.82   2.69   3.69   5.99   0.69   1.46   3.79   8.79   Inf   Inf   2.90   0.70   1.54   0.56   1.93   7.59   0.72   2.01   1.50   6.42   1.97   294 1.64E-­‐05   7.77E-­‐11   3.76E-­‐10   0.00E+00   0.00E+00   0.00E+00   1.91E-­‐14   4.26E-­‐11   0.00E+00   0.00E+00   0.00E+00   0.00E+00   1.39E-­‐03   7.67E-­‐03   0.00E+00   4.88E-­‐08   6.92E-­‐07   7.01E-­‐12   0.00E+00   4.67E-­‐04   2.51E-­‐02   5.40E-­‐06   6.26E-­‐08   7.89E-­‐08   2.14E-­‐04   0.00E+00   3.93E-­‐04   2.31E-­‐04   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT2G24850   AT2G25440   AT2G27420   AT2G27660   AT2G29310   AT2G29350   AT2G29470   AT2G30010   AT2G30400   AT2G30550   AT2G30770   AT2G31420   AT2G31880   AT2G32530   AT2G32560   AT2G32680   AT2G32870   AT2G32880   AT2G33400   AT2G33710   AT2G34070   AT2G35035   AT2G36780   AT2G36790   AT2G36830   AT2G37710   AT2G37760   AT2G37770   AT2G38240     23.62   0.25   1.88   1.09   6.91   72.51   30.68   26.75   8.45   11.60   45.39   4.01   45.23   0.13   8.98   9.55   1.51   2.03   2.52   3.87   25.41   0.60   3.82   2.66   34.90   60.27   48.93   37.03   31.81   4.61   0.10   0.93   0.45   6.48   15.31   3.33   27.35   0.42   8.20   2.42   0.12   25.29   0.06   8.27   3.84   0.17   0.35   5.10   0.19   10.98   1.07   0.59   0.45   29.60   51.54   17.62   7.05   3.82   141.32   2.92   7.85   6.04   19.69   176.40   8.74   60.60   1.38   26.05   16.80   0.60   132.42   1.37   22.32   25.99   10.11   9.76   7.12   1.11   10.84   3.46   0.64   0.29   127.65   175.02   20.27   16.65   9.22   133.81   5.06   2.98   10.82   13.33   300.97   25.58   56.11   2.53   32.67   13.56   1.38   190.79   0.96   19.90   35.79   2.84   3.80   10.05   1.20   18.19   4.60   2.02   1.23   74.42   280.31   23.51   24.94   15.75   4.86   5.68   1.67   4.60   1.04   4.30   2.94   1.04   2.60   1.99   2.49   3.56   2.92   3.90   1.27   3.22   4.02   3.46   0.98   2.63   0.73   2.11   1.78   1.46   1.33   2.44   0.42   1.82   2.04   295 0.00E+00   3.68E-­‐14   3.05E-­‐03   0.00E+00   1.16E-­‐03   0.00E+00   0.00E+00   3.90E-­‐09   6.79E-­‐04   0.00E+00   0.00E+00   2.54E-­‐02   0.00E+00   4.23E-­‐05   2.86E-­‐08   0.00E+00   2.69E-­‐08   1.65E-­‐07   3.97E-­‐04   4.83E-­‐03   1.60E-­‐03   9.23E-­‐05   2.76E-­‐03   4.72E-­‐02   5.37E-­‐14   0.00E+00   4.69E-­‐02   1.86E-­‐14   1.53E-­‐11   NO   NO   NO   NO   NEGATIVE   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT2G38340   4.95   AT2G39030   120.09   AT2G39210   15.16   AT2G39330   24.36   AT2G39390   181.59   AT2G39710   16.28   AT2G40000   29.40   AT2G40205   2047.97   AT2G40740   0.92   AT2G41120   3.40   AT2G42170   0.30   AT2G43150   228.88   AT2G43530   96.10   AT2G43535   11.04   AT2G43570   99.62   AT2G43820   69.95   AT2G44240   0.06   AT2G44290   5.60   AT2G44790   27.68   AT2G45570   27.02   AT2G45660   25.94   AT2G45710   31.00   AT2G46430   10.76   AT2G46710   5.21   AT2G47000   14.07   AT2G47950   4.51   AT3G02790   9.84   AT3G03030   1.41   AT3G03450   3.81     0.62   6.08   10.10   5.93   172.65   17.94   5.70   2835.29   0.43   3.64   0.36   58.20   36.88   4.35   10.79   4.43   0.09   7.85   2.66   1.64   34.57   24.68   11.01   6.62   3.81   0.27   1.94   1.65   3.74   1.28   16.25   30.93   11.79   345.16   43.86   11.23   8695.17   3.94   18.19   10.52   77.25   220.75   59.24   737.05   11.09   30.21   60.45   7.65   4.69   64.51   92.21   27.25   19.78   2.86   0.85   57.45   5.78   9.49   4.92   16.77   47.34   10.93   231.96   36.63   22.99   6573.37   3.31   21.68   7.22   103.87   189.27   36.47   418.54   17.38   55.03   74.46   8.86   7.43   89.14   46.41   46.16   16.23   9.76   1.56   17.65   3.91   8.81   2.98   1.46   2.23   0.88   0.43   1.03   2.01   1.21   2.93   2.57   4.31   0.84   2.36   3.07   5.28   1.97   9.29   3.25   1.74   2.18   1.37   0.91   2.07   1.29   1.36   2.53   3.19   1.25   1.24   296 3.48E-­‐06   1.43E-­‐06   0.00E+00   8.43E-­‐04   3.18E-­‐02   2.55E-­‐08   0.00E+00   5.05E-­‐05   1.24E-­‐03   0.00E+00   3.64E-­‐14   1.13E-­‐06   0.00E+00   0.00E+00   0.00E+00   0.00E+00   0.00E+00   0.00E+00   3.10E-­‐05   8.59E-­‐10   0.00E+00   4.71E-­‐04   0.00E+00   4.92E-­‐09   5.29E-­‐12   3.52E-­‐02   5.75E-­‐10   2.16E-­‐03   1.87E-­‐06   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   NO   NO   POSITIVE   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT3G03850   AT3G04480   AT3G06145   AT3G06200   AT3G06530   AT3G07520   AT3G08870   AT3G08970   AT3G09270   AT3G09940   AT3G11000   AT3G11010   AT3G11080   AT3G11280   AT3G11340   AT3G13130   AT3G13433   AT3G13560   AT3G13610   AT3G13750   AT3G14990   AT3G15536   AT3G16240   AT3G16470   AT3G17510   AT3G18830   AT3G19515   AT3G19520   AT3G21150     1.39   8.82   2.58   31.59   17.72   4.41   1.25   3.41   70.18   2.43   0.08   0.09   0.98   14.36   13.22   2.29   0.92   17.13   28.30   18.94   181.64   8.13   25.22   139.59   14.85   86.28   4.82   10.49   2.99   1.14   13.11   2.47   26.41   10.29   3.62   0.73   3.13   6.61   0.38   0.05   0.04   0.81   10.47   0.49   1.08   0.67   15.52   1.63   24.75   53.71   2.70   31.18   35.68   14.91   49.21   5.09   9.28   3.14   8.42   17.47   16.48   62.95   29.68   12.42   6.59   10.83   8.98   17.79   1.87   6.18   11.97   42.95   3.40   8.94   19.18   34.68   12.07   43.02   86.39   72.57   67.21   53.09   37.44   40.80   22.10   23.00   10.78   4.20   19.68   9.97   44.21   13.63   14.34   10.34   23.42   22.97   33.00   0.81   9.82   16.48   33.84   9.67   8.64   21.88   34.00   50.87   57.22   135.78   73.19   54.88   55.60   40.54   66.53   23.36   27.82   20.55   1.89   0.59   2.01   0.74   0.41   1.98   3.82   2.90   1.80   6.43   3.88   7.88   4.35   1.69   4.29   3.00   5.02   1.13   4.96   1.21   1.34   4.76   0.82   0.64   1.44   0.44   2.20   1.58   2.71   297 1.14E-­‐02   3.69E-­‐03   2.38E-­‐05   1.03E-­‐04   3.48E-­‐03   0.00E+00   0.00E+00   0.00E+00   1.16E-­‐09   0.00E+00   8.26E-­‐05   0.00E+00   0.00E+00   0.00E+00   0.00E+00   2.21E-­‐08   1.53E-­‐10   3.42E-­‐13   0.00E+00   2.17E-­‐13   0.00E+00   0.00E+00   1.18E-­‐05   3.12E-­‐04   0.00E+00   2.70E-­‐02   0.00E+00   5.37E-­‐14   1.92E-­‐14   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT3G22370   AT3G22930   AT3G23080   AT3G23120   AT3G23240   AT3G23480   AT3G23620   AT3G23730   AT3G23890   AT3G24516   AT3G24517   AT3G24518   AT3G25010   AT3G25020   AT3G25250   AT3G25572   AT3G26170   AT3G26240   AT3G26520   AT3G26830   AT3G28270   AT3G28510   AT3G29240   AT3G29575   AT3G29590   AT3G43670   AT3G43800   AT3G44215   AT3G44300     55.20   0.29   10.53   1.82   7.29   0.93   88.22   7.47   2.31   2.27   0.47   1.08   1.46   0.07   5.77   0.77   2.97   0.04   46.81   119.27   0.19   0.26   41.24   3.71   2.27   10.19   37.23   0.02   144.08   22.25   0.13   9.99   1.87   0.86   0.91   68.95   10.56   4.21   1.54   0.41   1.54   0.65   0.06   0.49   0.88   1.95   0.04   45.98   10.37   0.16   0.11   43.43   2.33   1.42   11.54   42.60   0.01   24.36   27.15   2.64   20.20   6.77   1.77   6.09   204.42   21.25   4.84   273.52   74.93   15.14   8.62   6.74   1.43   3.52   8.83   2.61   235.68   42.52   9.21   3.30   79.69   13.02   6.97   20.39   100.26   7.10   28.96   49.59   4.95   19.10   9.93   2.93   6.18   94.61   18.52   6.38   216.80   50.31   9.72   9.03   9.49   4.01   2.78   10.78   2.42   208.37   76.07   1.85   7.75   110.08   16.42   4.02   23.48   116.26   10.24   79.80   1.16   5.30   0.94   2.41   1.77   2.76   0.46   0.81   0.60   7.13   6.93   2.65   3.80   7.36   3.05   1.67   2.47   6.07   2.18   2.88   3.50   6.11   1.34   2.82   1.51   1.02   1.45   9.75   1.71   298 9.80E-­‐11   7.52E-­‐07   2.09E-­‐06   0.00E+00   9.62E-­‐03   3.67E-­‐11   2.30E-­‐02   1.75E-­‐03   1.71E-­‐03   2.15E-­‐05   0.00E+00   1.36E-­‐11   0.00E+00   0.00E+00   6.66E-­‐08   1.04E-­‐03   1.91E-­‐14   3.73E-­‐13   0.00E+00   0.00E+00   3.46E-­‐07   0.00E+00   0.00E+00   0.00E+00   1.71E-­‐03   3.15E-­‐08   0.00E+00   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT3G44310   AT3G44970   AT3G45030   AT3G45290   AT3G45390   AT3G45443   AT3G45446   AT3G45860   AT3G46650   AT3G46668   AT3G47560   AT3G48080   AT3G48640   AT3G48850   AT3G49180   AT3G49340   AT3G49780   AT3G51230   AT3G51860   AT3G53150   AT3G54390   AT3G54830   AT3G56400   AT3G57040   AT3G57260   AT3G57490   AT3G58120   AT3G59140   AT3G60140     114.22   0.04   161.26   5.34   0.36   1.73   0.02   20.46   0.46   0.00   4.22   4.12   23.78   12.06   15.81   0.50   59.43   0.68   25.18   3.12   1.69   10.87   22.07   7.44   552.31   39.33   4.42   14.00   19.06   128.73   0.05   131.07   3.34   0.73   2.37   0.02   3.93   0.35   0.00   4.25   8.01   14.19   0.70   13.64   0.16   26.52   1.92   5.37   0.44   1.39   7.84   20.16   9.34   123.31   25.39   3.29   4.31   2.17   280.91   2.68   307.10   17.72   1.88   7.43   2.33   71.63   4.56   3.18   12.31   11.64   66.49   3.25   37.59   47.82   168.86   5.41   87.42   11.31   5.95   45.73   52.03   17.78   3942.03   74.37   11.94   4.15   1.51   332.51   1.20   186.51   9.90   2.63   7.13   4.81   51.93   1.42   2.83   12.45   24.86   218.10   4.78   21.10   34.60   171.89   8.82   68.76   8.86   4.50   27.79   125.10   14.46   2454.12   35.80   9.16   6.20   19.34   1.37   4.57   0.51   1.57   1.86   1.59   8.18   3.72   2.01   Inf   1.55   1.63   3.94   2.78   0.63   7.74   2.70   2.20   3.68   4.32   1.69   1.83   2.63   0.63   4.31   0.50   1.48   0.53   3.16   299 0.00E+00   3.53E-­‐04   1.06E-­‐02   8.92E-­‐08   2.56E-­‐04   3.13E-­‐04   5.25E-­‐07   0.00E+00   8.76E-­‐03   1.64E-­‐03   8.82E-­‐07   0.00E+00   0.00E+00   1.30E-­‐06   4.15E-­‐03   0.00E+00   0.00E+00   3.46E-­‐07   0.00E+00   0.00E+00   2.53E-­‐04   0.00E+00   0.00E+00   1.73E-­‐02   0.00E+00   2.77E-­‐02   8.90E-­‐07   2.41E-­‐02   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   YES   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT3G60170   AT3G60500   AT3G60540   AT3G62890   AT4G00700   AT4G01490   AT4G01525   AT4G01720   AT4G01870   AT4G03320   AT4G03450   AT4G04510   AT4G05050   AT4G08470   AT4G08770   AT4G08780   AT4G10120   AT4G10500   AT4G10860   AT4G11000   AT4G11170   AT4G11211   AT4G11650   AT4G12320   AT4G12420   AT4G12735   AT4G12880   AT4G13810   AT4G13820     0.01   16.25   9.28   2.87   4.63   0.03   0.51   2.37   388.35   3.39   5.12   0.77   98.34   5.82   42.17   11.41   23.44   3.49   0.81   1.02   0.07   3.02   8.31   12.34   22.21   24.95   40.19   0.65   0.31   0.01   13.62   5.86   2.37   1.01   0.02   0.56   1.42   46.12   0.43   2.70   0.32   83.62   6.68   0.59   0.12   39.04   0.55   0.37   0.83   0.06   3.01   2.14   10.46   28.47   1.97   42.77   0.49   0.16   1.66   27.72   29.19   9.15   15.88   8.29   146.37   6.86   85.28   0.92   12.63   3.89   204.45   21.66   6.17   1.85   42.77   50.01   20.01   14.62   2.91   13.03   2.75   35.67   41.77   3.93   82.31   3.13   2.12   2.80   18.73   36.38   5.30   16.67   10.52   247.03   8.32   137.91   1.32   22.77   6.23   315.81   33.85   8.50   2.89   65.83   41.30   81.53   26.50   7.09   17.52   4.55   38.43   39.89   8.14   69.19   5.05   2.50   7.70   0.46   2.63   1.16   4.05   9.03   8.79   2.55   1.58   1.61   3.08   4.28   1.92   2.34   3.84   4.57   0.75   6.24   7.78   4.99   6.98   2.54   1.09   1.88   0.49   2.05   0.69   3.35   3.93   300 4.35E-­‐10   2.09E-­‐02   3.36E-­‐12   8.80E-­‐04   0.00E+00   0.00E+00   0.00E+00   5.56E-­‐13   0.00E+00   5.00E-­‐02   0.00E+00   0.00E+00   0.00E+00   0.00E+00   1.07E-­‐12   1.74E-­‐06   1.74E-­‐09   0.00E+00   0.00E+00   0.00E+00   0.00E+00   1.36E-­‐06   4.10E-­‐02   0.00E+00   1.37E-­‐02   1.97E-­‐03   1.06E-­‐04   8.78E-­‐14   2.53E-­‐11   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT4G13890   AT4G14090   AT4G14390   AT4G14400   AT4G14640   AT4G14750   AT4G15248   AT4G15530   AT4G16000   AT4G16760   AT4G17340   AT4G18010   AT4G18170   AT4G18970   AT4G19370   AT4G19420   AT4G19630   AT4G19990   AT4G20960   AT4G21390   AT4G21640   AT4G21680   AT4G21750   AT4G21870   AT4G22485   AT4G22505   AT4G22570   AT4G23150   AT4G23160     0.22   7.42   0.16   75.26   1.09   1.40   6.30   81.86   1.31   61.24   1.92   19.10   15.76   4.08   2.91   8.35   0.32   4.98   16.54   3.99   0.00   25.71   10.66   1.30   22.65   23.62   24.92   8.73   2.48   0.11   4.04   0.16   86.66   0.98   2.61   4.53   47.74   0.12   29.43   0.72   11.47   2.40   4.94   0.79   8.56   1.07   5.53   18.25   0.88   0.02   2.15   11.42   1.55   15.17   15.78   33.82   2.59   0.81   8.62   20.43   30.89   196.22   6.83   6.10   24.70   47.85   9.07   28.21   18.70   31.58   4.86   15.09   10.29   16.19   11.99   9.94   36.04   1.37   2.05   1.19   19.72   19.68   47.77   46.45   80.97   35.84   11.42   12.83   15.37   43.04   281.37   7.44   8.91   16.45   81.65   4.30   38.33   9.50   50.12   7.16   12.78   17.89   19.29   8.38   14.76   36.11   2.88   1.98   4.43   22.57   15.79   42.46   32.43   60.33   43.22   15.01   6.87   1.93   8.06   1.70   2.93   1.77   1.86   0.77   5.18   0.38   3.73   2.13   1.58   1.37   4.51   1.17   2.96   1.42   0.98   1.72   7.03   1.04   0.98   3.35   1.48   1.04   0.84   4.06   4.22   301 0.00E+00   1.85E-­‐14   0.00E+00   0.00E+00   1.58E-­‐05   4.16E-­‐08   7.17E-­‐05   0.00E+00   1.45E-­‐04   4.35E-­‐02   5.12E-­‐12   0.00E+00   5.15E-­‐05   3.76E-­‐07   0.00E+00   7.18E-­‐10   1.37E-­‐06   0.00E+00   1.80E-­‐07   1.51E-­‐05   2.98E-­‐05   2.78E-­‐03   7.15E-­‐09   1.63E-­‐10   0.00E+00   4.58E-­‐08   1.86E-­‐05   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT4G23200   AT4G23210   AT4G23220   AT4G23230   AT4G23260   AT4G23810   AT4G23880   AT4G24780   AT4G25000   AT4G25110   AT4G26760   AT4G29520   AT4G29700   AT4G29905   AT4G30720   AT4G31790   AT4G31870   AT4G32340   AT4G33355   AT4G33360   AT4G33560   AT4G34131   AT4G34760   AT4G34930   AT4G36130   AT4G37150   AT4G37370   AT4G37800   AT4G37990     1.57   0.97   0.13   0.37   8.05   6.69   13.60   22.92   2.39   0.33   4.22   49.07   3.35   55.47   35.61   28.25   0.15   52.99   0.35   1.50   2.57   10.82   20.41   0.30   168.11   0.37   21.47   13.58   54.06   0.18   0.47   0.16   0.13   8.04   3.47   9.62   36.43   1.11   0.30   6.75   32.64   1.29   48.47   41.66   16.79   0.10   63.93   1.30   1.27   3.57   2.79   30.15   0.28   135.32   0.33   4.72   16.03   1.88   5.09   4.55   22.00   4.68   16.25   26.46   38.28   52.97   34.11   8.48   9.89   96.39   9.68   148.11   191.54   49.15   8.52   104.36   4.66   28.27   16.73   2.27   50.28   1.68   340.21   8.36   7.09   28.25   6.82   10.00   16.91   33.28   6.03   31.92   34.17   24.62   55.53   16.94   17.10   11.74   118.75   12.68   71.06   154.76   25.75   18.24   105.77   4.65   35.06   22.73   4.75   46.20   1.20   189.28   7.53   19.85   26.50   13.51   5.83   5.16   7.73   5.56   1.99   3.30   1.36   0.61   3.93   5.85   0.80   1.86   3.29   0.55   1.89   0.62   7.48   0.73   1.84   4.79   2.67   0.77   0.62   2.10   0.48   4.53   2.07   0.73   2.84   302 0.00E+00   0.00E+00   0.00E+00   0.00E+00   0.00E+00   0.00E+00   1.67E-­‐06   1.13E-­‐03   0.00E+00   0.00E+00   4.03E-­‐04   0.00E+00   0.00E+00   1.33E-­‐02   0.00E+00   1.89E-­‐03   1.76E-­‐09   7.26E-­‐05   1.93E-­‐03   0.00E+00   5.96E-­‐09   4.29E-­‐02   9.34E-­‐03   7.51E-­‐03   1.31E-­‐02   2.38E-­‐10   0.00E+00   1.36E-­‐03   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO         Table A3.6 (cont’d) AT4G38540   AT4G38840   AT4G38850   AT4G39830   AT4G40060   AT5G01290   AT5G01550   AT5G02020   AT5G03380   AT5G03760   AT5G05600   AT5G06860   AT5G06870   AT5G07440   AT5G08760   AT5G09290   AT5G10380   AT5G10572   AT5G10760   AT5G11580   AT5G11930   AT5G13330   AT5G14800   AT5G15350   AT5G15948   AT5G15950   AT5G16250   AT5G16960   AT5G17220     45.54   44.11   2.78   3.48   4.53   9.47   0.41   35.83   9.37   28.57   34.73   111.41   6.34   95.06   37.63   0.10   28.68   98.89   176.34   22.24   1.44   18.68   53.65   24.99   2.17   209.86   9.47   10.89   13.42   19.59   46.52   5.66   2.22   4.79   7.84   0.16   0.66   5.61   24.06   6.61   24.82   2.96   24.31   27.22   0.07   11.78   110.74   39.35   27.12   2.45   1.39   58.66   24.67   1.90   167.81   16.30   0.52   10.00   15.45   90.62   14.53   10.73   22.83   21.66   2.78   3.40   26.68   54.54   13.01   24.92   31.45   29.15   141.56   4.08   77.88   760.83   736.26   47.35   6.86   2.58   134.11   50.17   7.38   524.89   28.16   1.16   31.84   27.95   77.15   18.83   20.43   23.79   16.99   4.75   2.87   36.04   35.95   16.77   36.68   33.70   33.46   191.60   10.26   95.21   397.41   639.62   49.09   9.30   5.29   113.66   39.89   7.70   521.42   38.53   2.74   20.41   0.51   0.73   1.73   3.20   2.31   1.12   4.91   2.13   2.68   0.58   1.34   0.56   3.51   0.46   2.82   7.25   3.01   1.84   4.02   0.86   1.92   1.93   0.95   0.69   2.02   1.64   1.24   2.40   1.03   303 1.83E-­‐02   7.53E-­‐04   1.05E-­‐04   0.00E+00   0.00E+00   1.53E-­‐08   3.63E-­‐14   7.64E-­‐04   0.00E+00   2.22E-­‐03   4.96E-­‐08   8.38E-­‐03   0.00E+00   3.45E-­‐03   0.00E+00   3.40E-­‐11   0.00E+00   2.04E-­‐02   0.00E+00   1.27E-­‐06   1.11E-­‐04   9.54E-­‐05   1.61E-­‐08   2.26E-­‐03   8.79E-­‐10   0.00E+00   1.49E-­‐08   1.45E-­‐04   5.70E-­‐04   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT5G17330   AT5G17760   AT5G17860   AT5G18130   AT5G18470   AT5G19190   AT5G19240   AT5G19880   AT5G20480   AT5G21060   AT5G22300   AT5G22320   AT5G22330   AT5G22390   AT5G22520   AT5G23010   AT5G24110   AT5G24150   AT5G24200   AT5G24530   AT5G24655   AT5G24770   AT5G25180   AT5G25220   AT5G25460   AT5G26340   AT5G28770   AT5G33290   AT5G35790     0.19   27.54   9.24   42.01   26.63   11.17   144.36   0.40   5.04   8.49   62.12   12.95   27.69   11.93   0.00   254.95   6.01   0.18   16.84   36.09   1.12   1.54   0.03   14.99   29.36   83.69   14.11   32.79   28.51   0.09   20.50   2.12   17.91   16.32   9.65   94.13   0.20   3.18   11.15   19.15   11.74   22.99   15.89   0.15   112.20   1.92   0.19   5.63   12.93   1.74   0.57   0.01   15.06   38.13   28.78   10.86   19.84   33.15   1.44   13.33   3.47   22.50   100.75   34.82   348.53   3.05   2.10   38.30   12.16   27.48   55.09   36.79   4.27   598.88   26.95   7.90   2.09   267.38   14.39   6.75   3.54   38.23   0.00   33.14   26.57   15.41   55.80   3.30   31.07   7.35   30.90   112.36   32.59   448.98   6.69   4.76   35.90   29.39   21.53   38.23   28.81   25.53   236.47   22.99   6.41   10.66   257.60   32.58   3.00   3.95   41.50   70.33   59.47   18.54   26.90   54.01   5.17   0.60   1.80   0.79   2.78   1.76   2.25   5.06   0.58   1.69   0.62   0.87   0.73   0.86   7.38   1.08   3.58   5.08   0.92   4.32   4.22   2.40   8.41   1.46   0.88   1.05   0.77   0.44   0.70   304 3.60E-­‐10   1.72E-­‐03   8.77E-­‐09   4.64E-­‐05   0.00E+00   1.42E-­‐09   0.00E+00   1.03E-­‐10   4.25E-­‐02   0.00E+00   3.25E-­‐03   9.98E-­‐06   8.43E-­‐05   7.94E-­‐05   6.83E-­‐11   1.77E-­‐08   0.00E+00   0.00E+00   1.53E-­‐03   0.00E+00   0.00E+00   3.65E-­‐05   0.00E+00   0.00E+00   3.72E-­‐07   4.10E-­‐10   3.20E-­‐04   3.94E-­‐02   8.49E-­‐05   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   POSITIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT5G36930   AT5G38150   AT5G38430   AT5G38940   AT5G39050   AT5G39100   AT5G40340   AT5G41590   AT5G42020   AT5G43440   AT5G43450   AT5G43870   AT5G44020   AT5G44420   AT5G44430   AT5G44568   AT5G44570   AT5G45030   AT5G45100   AT5G45380   AT5G45670   AT5G45820   AT5G45950   AT5G46050   AT5G46260   AT5G46490   AT5G46500   AT5G47630   AT5G48110     0.03   2.82   502.18   2.44   39.29   4.64   9.70   8.78   49.68   5.34   45.30   4.66   14.42   247.91   65.29   7.92   1.01   7.76   14.53   6.08   6.08   5.13   19.00   10.26   4.72   0.06   0.38   49.93   0.73   0.04   3.04   303.86   1.36   14.02   2.45   8.56   6.79   38.33   4.46   28.89   5.91   18.81   152.80   16.67   7.74   1.66   9.69   12.18   5.31   7.28   3.95   19.65   4.17   5.35   0.03   0.30   41.40   1.84   9.88   7.40   1114.11   8.85   17.57   15.28   18.04   30.90   97.39   19.91   20.52   11.83   68.46   38.75   13.26   91.63   70.24   15.09   32.52   14.72   16.26   24.64   37.98   40.63   10.60   3.09   30.99   96.89   8.17   14.82   10.21   450.18   5.87   24.86   13.82   18.67   28.87   165.70   27.97   42.35   14.23   53.72   439.65   143.59   213.40   205.43   20.65   33.33   24.19   16.66   14.53   31.20   49.94   18.57   5.71   56.23   59.28   13.05   8.58   1.75   0.57   2.11   0.83   2.50   1.13   2.09   2.11   2.65   0.55   1.27   1.51   1.52   3.11   4.78   6.95   1.09   1.45   2.19   1.19   1.88   0.67   3.58   1.80   7.41   7.57   0.52   2.83   305 0.00E+00   6.41E-­‐10   5.81E-­‐03   2.71E-­‐04   5.97E-­‐05   1.86E-­‐09   7.32E-­‐10   7.83E-­‐13   0.00E+00   0.00E+00   6.21E-­‐03   1.37E-­‐07   0.00E+00   8.73E-­‐14   0.00E+00   0.00E+00   0.00E+00   5.39E-­‐14   9.92E-­‐13   0.00E+00   3.23E-­‐07   1.30E-­‐12   1.05E-­‐03   0.00E+00   0.00E+00   0.00E+00   0.00E+00   2.95E-­‐02   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   UP   NO   NO   NO   NO   NO         Table A3.6 (cont’d) AT5G48620   AT5G48960   AT5G49360   AT5G50915   AT5G51460   AT5G52750   AT5G53460   AT5G54610   AT5G54720   AT5G55450   AT5G55460   AT5G55510   AT5G57180   AT5G57700   AT5G60900   AT5G62630   AT5G63120   AT5G64000   AT5G65730   AT5G66052   AT5G66053   AT5G66630   AT5G66690   4.61   4.94   0.96   3.20   3.26   18.24   50.53   10.49   3.54   429.86   21.38   17.65   20.19   2.23   7.85   14.13   16.93   10.02   39.44   0.05   4.51   0.08   0.38   4.51   3.71   1.14   2.03   3.50   12.67   36.75   10.41   3.20   232.46   17.30   15.66   19.02   3.73   6.90   16.33   12.95   2.43   44.38   0.05   0.00   0.14   0.12   10.10   32.53   4.55   8.64   6.92   47.35   87.25   65.06   17.20   1212.13   50.62   45.22   55.08   6.77   16.51   35.41   36.74   23.80   79.15   10.64   120.50   2.07   10.26   14.20   23.14   6.88   6.95   7.05   76.53   63.80   95.39   37.96   1217.23   48.44   27.38   41.30   9.50   30.42   40.32   26.45   17.27   68.68   13.49   146.67   4.36   9.50   1.66   2.64   2.60   1.77   1.01   2.59   0.80   3.20   3.57   2.39   1.48   0.81   1.12   1.35   2.14   1.30   1.03   2.83   0.63   8.05   Inf   4.97   6.28       306 0.00E+00   0.00E+00   0.00E+00   7.91E-­‐07   4.99E-­‐04   0.00E+00   1.38E-­‐07   0.00E+00   0.00E+00   0.00E+00   1.09E-­‐07   1.27E-­‐03   0.00E+00   1.92E-­‐06   0.00E+00   3.66E-­‐14   4.00E-­‐09   0.00E+00   1.16E-­‐03   8.75E-­‐07   3.87E-­‐03   0.00E+00   0.00E+00   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NEGATIVE   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   NO   NO   NO   NO   NO   YES   NO   NO   NO   DOWN   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   NO   Table A3.7. Primer pairs used for qRT-PCR and sequencing. QRT-PCR Primers used in this study were designed with Primer Express 3.0. EQTL  PRIMERS       AGI   NAME   AT1G27730   ZAT10   AT1G29390   COR314     AT2G42540   COR15A   AT3G55980   STZ1     AT5G59820   ZAT12   AT1G20440   COR47   AT1G20450   ERD10   AT1G75040   PR5   AT4G15910   DI21     AT5G52310   COR78   AT3G02780   IPP2   AT2G41100   TCH3       FW   TCGAGCACTGGACAAAGGGTAAGC   CCAGGTGAACTTGAGCTTCCA   CGTTGATCTACGCCGCTAAAG   TGTCCCCTGTCCCGAGTTC   GTGCGAGTCACAAGAAGCCTAACA   CGGTACCAGTGTCGGAGAGT   TTCCAGGCCACAGCAAGAA   AACGGCGGCGGAGTTC   TGGCCGCTCGTTCACTCT   GAAAGGAGGAGGAGGAATGG   ATTTGCCCATCGTCCTCTGT   CAATATCATCGCTTCGCCAAT       SEQUENCING  PRIMERS       RV   CCTGAGTGAGGTTTTGGTGGTGGA   TGATATGGCGCCACAATCA   TGGCCTCGTTGAGGTCATC   CGTGAGCATACTCGCAAGAATC   GCGACGACGTTTTCACCTTCTTCA   ACAGCTGGTGAATCCTCTGC   CAACCAGCGGTGTGGTGTT   CGCCATCGCCTACTAGAGTGA   TGCGGCGGAGCAAAGA   AACCAGCCAGATGATTTTGG   GAGAAAGCACGAAAATTCGGTAA   ATAACAGCGCTTCGAACAAATCT                   AGI   NAME   FW   RV   AT4G25490   CBF1   GGGATTGCTTCGCTATGTACTATACACGTGTC   TCCACATATTTTGCCTTGAAAGCAACTACAATATGAC   AT4G25470   CBF2   ATCCAAATATCTTCCGGCCAATATAAACAGCAAGCT   AGCAGAAAGGCAGAAACTGTTGCTATCAACCAAC   AT4G25480   CBF3   CCGCCATTATAAAACAGCATGCTCTCACTCC   GCTGAATCGGTTGTTTCGGTTTTATACTCTAATTTAAAC   AT2G42540   COR15A   GGCCTGAAAAGAACGAACAGAAACTCGGTAATG   GGTACAACAATGTATCATACCAATTCGATGTCATAC   AT1G20440   COR47   GTTACCTCCGCGTTGGCCTGGACCCTTCTC   CCTAAGAAGCCAGATGGCGAAATATTGAAATATTCG   AT5G52310   COR78   CCCTCCTCTGTTTTACTCACAAATATGCAAACTAG   CTTTAACGATAGAGACAACACCTCAACAAGTCAC         307 Table A3.8. Number of reads passing the Illumina purity filter for each RNA-seq sample. The lane name represents the lane in which each sample was run. ‘PF clusters’ indicates reads passing Illumina purity filters, while ‘Raw Clusters’ denotes all reads. For the sample names: ‘SW’ indicates the Swedish accession; ‘IT’ indicates the Italian accession; ‘NONACC’ indicates the samples not cold-acclimated, ‘1WK’ indicates samples cold-acclimated for 1 week; ‘2WK’ indicates samples cold-acclimated for 2 weeks; the last number in the sample name indicates biological replicate number. Lane   1   4       1   4       1   4       1   4       1   4       1   4       2   5       2     Sample   SW-­‐NONACC-­‐1   SW-­‐NONACC-­‐1   SW-­‐NONACC-­‐1  Total   SW-­‐NONACC-­‐2   SW-­‐NONACC-­‐2   SW-­‐NONACC-­‐2  Total   SW-­‐NONACC-­‐3   SW-­‐NONACC-­‐3   SW-­‐NONACC-­‐3  Total   IT-­‐NONACC-­‐1   IT-­‐NONACC-­‐1   IT-­‐NONACC-­‐1  Total   IT-­‐NONACC-­‐2   IT-­‐NONACC-­‐2   IT-­‐NONACC-­‐2  Total   IT-­‐NONACC-­‐3   IT-­‐NONACC-­‐3   IT-­‐NONACC-­‐3  Total   SW-­‐1WK-­‐1   SW-­‐1WK-­‐1   SW-­‐1WK  Total   SW-­‐1WK-­‐2   Raw  Clusters   29,958,443   29,918,355   59,876,798   25,139,472   25,085,948   50,225,420   24,918,522   24,880,981   49,799,503   28,636,037   28,552,106   57,188,143   24,842,595   24,815,959   49,658,554   31,151,487   31,118,564   62,270,051   28,952,674   28,715,485   57,668,159   26,129,644   308 PF  Clusters   24,697,162   24,249,872   48,947,034   20,662,571   20,259,178   40,921,749   20,505,290   20,140,545   40,645,835   23,420,339   22,949,660   46,369,999   20,467,125   20,088,847   40,555,972   25,609,805   25,143,267   50,753,072   23,350,344   22,777,897   46,128,241   21,279,472   Sample  %  PF   82.4%   81.1%   81.7%   82.2%   80.8%   81.5%   82.3%   80.9%   81.6%   81.8%   80.4%   81.1%   82.4%   81.0%   81.7%   82.2%   80.8%   81.5%   80.7%   79.3%   80.0%   81.4%       Table A3.8 cont’d 5       2   5       2   5       2   5       2   5       3   6       3   6       3   6       3   6       3   6     SW-­‐1WK-­‐2   SW-­‐1WK-­‐2  Total   SW-­‐1WK-­‐3   SW-­‐1WK-­‐3   SW-­‐1WK-­‐3  Total   IT-­‐1WK-­‐1   IT-­‐1WK-­‐1   IT-­‐1WK-­‐1  Total   IT-­‐1WK-­‐2   IT-­‐1WK-­‐2   IT-­‐1WK-­‐2  Total   IT-­‐1WK-­‐3   IT-­‐1WK-­‐3   IT-­‐1WK-­‐3  Total   SW-­‐2WK-­‐1   SW-­‐2WK-­‐1   SW-­‐2WK-­‐1  Total   SW-­‐2WK-­‐2   SW-­‐2WK-­‐2   SW-­‐2WK-­‐2  Total   SW-­‐2WK-­‐3   SW-­‐2WK-­‐3   SW-­‐2WK-­‐3  Total   IT-­‐2WK-­‐1   IT-­‐2WK-­‐1   IT-­‐2WK-­‐1  Total   IT-­‐2WK-­‐2   IT-­‐2WK-­‐2   25,897,791   52,027,435   29,760,111   29,565,270   59,325,381   29,677,878   29,401,956   59,079,834   29,822,085   29,615,504   59,437,589   23,782,918   23,506,197   47,289,115   28,637,922   28,377,699   57,015,621   25,168,550   24,935,016   50,103,566   25,484,480   25,236,381   50,720,861   32,233,028   31,955,554   64,188,582   26,054,424   25,813,016   309 20,772,596   42,052,068   24,072,758   23,523,986   47,596,744   24,017,917   23,424,329   47,442,246   24,213,801   23,659,830   47,873,631   19,711,843   19,220,083   38,931,926   23,936,002   23,775,237   47,711,239   21,001,016   20,853,600   41,854,616   21,280,683   21,125,228   42,405,911   26,752,432   26,583,643   53,336,075   21,749,785   21,599,147   80.2%   80.8%   80.9%   79.6%   80.2%   80.9%   79.7%   80.3%   81.2%   79.9%   80.5%   82.9%   81.8%   82.3%   83.6%   83.8%   83.7%   83.4%   83.6%   83.5%   83.5%   83.7%   83.6%   83.0%   83.2%   83.1%   83.5%   83.7%       Table A3.8 cont’d     3   6               IT-­‐2WK-­‐2  Total   IT-­‐2WK-­‐3   IT-­‐2WK-­‐3   IT-­‐2WK-­‐3  Total   Grand  Total   51,867,440   28,738,532   28,468,237   57,206,769   994,948,821     310 43,348,932   23,962,836   23,789,534   47,752,370   814,627,660   83.6%   83.4%   83.6%   83.5%   81.9%       Table A3.9. Summary of SW and IT RNA-seq samples. ‘Average PF Clusters’ indicates the average number of reads passing the Illumina purity filters for each sample. ‘# Genes’ indicates the number of genes in each sample. ‘Average R2’ indicates the average correlation of genes for each biological replicate when all comparisons are made (i.e. replicate 1 to 2 2, replicate 2 to 3, and replicate 1 to 3) ± the standard error. While calculating the correlation (R ) for each sample genes that were outliers were removed, none of the outliers removed from cold samples were genes found to be cold regulated and none of the outliers removed from non-acclimated samples were included in the list of genes differentially regulated between SW and IT. The total number of genes removed from calculating correlation was small (18 genes total) in comparison to the number of genes analyzed (‘# Genes’). Genes removed were: AT1G15800, AT1G26240, AT1G50310, AT1G71070, AT2G19790, AT2G23740, AT2G30370, AT3G13061, AT4G08109, AT4G15020, AT4G15620, AT5G07720, AT5G08480, AT5G20620, AT5G27450, AT5G35900, AT5G47450, and AT5G58787. Sample   SW-­‐NONACC  Average   IT-­‐NONACC  Average   SW-­‐1WK  Average   IT-­‐1WK  Average   SW-­‐2WK  Average   IT-­‐2WK  Average   Average  PF   Clusters   43,504,873   45,893,014   45,259,018   44,749,268   43,990,589   48,145,792   #  GENES   Average  R2   Maximum  FPKM   23,127   23,429   23,303   23,123   22,818   23,785   0.90  ±  0.015   0.90  ±  0.032   0.88  ±  0.022   0.91  ±  0.012   0.90  ±  0.015   0.90  ±  0.013   28,114   24,553   42,983   18,584   35,927   20,597       311 LITERATURE CITED   312 LITERATURE CITED 1. Fournier-Level A, Korte A, Cooper MD, Nordborg M, Schmitt J, & Wilczek AM (2011) A Map Of Local Adaptation In Arabidopsis Thaliana. Science 334(6052):86-89. 2. 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Conference 1:5531-5534.     319 CHAPTER FOUR INTERESTING OBSERVATIONS AND FUTURE DIRECTIONS   320 CHAPTER FOUR INTERESTING OBSERVATIONS AND FUTURE DIRECTIONS SUMMARY The following represents results and preliminary results from several experiments that were conducted during the course of research described in Chapters Two and Three. These experiments produced interesting results that require further research and may be of value to continue as full focused projects. Preliminary results for additional mechanisms of circadian and gated cold-induced expression are described for the CBFs and the CBF-independent gene, COR27. Temperatureentrained circadian and gated cold-induced expression of CBF genes are also examined in this section, as well as the disruption of the circadian clock by extended cold temperatures. Lastly, morphological differences between SW and IT ecotypes used in Chapter Three are documented, and future construction of a low-temperature network using eQTL and RNA-seq data is discussed.   321 RESULTS AND DISCUSSION Regulation of CBF genes by the REVEILLES, CK2, and CAMTAS, and circadian regulation of CBF-independent gene, COR27. In Chapter Two of this study, we show that EE-binding transcription factors CCA1 and LHY are major positive regulators of CBF genes under both circadian and gated (circadian with cold-treatment) conditions (Fig 2.1 and 2.4). ChIP experiments seem to suggest that this interaction is direct. CBF-target gene expression is also affected by mutations in CCA1 and LHY and electrolyte leakage assays indicate that these central clock genes impact freezing tolerance. CCA1 and LHY are thus components of the CBF pathway upstream of the CBFs. Doherty et al. 2009 determined that CAMTA3 is a transcription factor that also positively regulates CBF genes (1). In Chapter Two we present a model that suggests, CCA1, LHY and CATMA transcription factors (1) work synergistically to increase expression of CBF genes (Fig 2.5). The CAMTAs are hypothesized to be a connection between calcium and CBF signaling (1). Cellular calcium levels also oscillate with circadian rhythms (2). Therefore, the CAMTAs may be a second connection between the clock and CBF genes. As a first step CAMTA mutants were tested to see if they affect basal circadian and gated cold-induction of CBF genes. There are a total of six CAMTAs in Arabidopsis, some of which have been shown to have overlapping function ((1) and Kim and Thomashow, unpublished). Preliminary results indicate that peak expression of CBF1 and 2 is reduced under circadian conditions in a camta3/camta2 double mutant (mutant constructed by Kim and Thomashow, unpublished), which is consistent with the   322 CAMTAs acting as activators of CBF1 and 2 (1). This also suggests that CAMTAs influence basal expression of CBF genes. The reduction of CBF expression in camta3/camta2 was not as severe as in cca1-11/lhy-21 mutants. Gated cold-induced expression of CBF1-3 also appears to be reduced though the reduction is greater for CBF1 and CBF2 than CBF3. The reduction of gated CBF expression in camta3/camta2 was not as severe as in cca1-11/lhy-21 mutants. This result needs to be repeated, but does support the model proposed in Chapter Two. Although CBF1 and CBF3 expression is essentially knocked-out under both circadian and gated conditions in the cca1-11/lhy-21 double mutant, there is some residual (~20%) expression of CBF2 (Fig. 2.1 and 2.4). In Chapter Two we suggest that the CCA1-like REVEILLE (RVE) genes may play a role in the remaining induction of CBF2. Like CCA1 and LHY, the RVEs bind to the EE and are circadian regulated. Preliminary results indicate that rve2, rve3, rve4, and an additional EE-binding tcp2 mutant do not significantly affect CBF expression, under both circadian and gated conditions, though higher order mutants would need to be constructed since there may be redundancy between these genes. Preliminary results show that the rve8-1 mutant may have altered CBF expression in comparison to wild-type. However, CBF expression appears to be elevated in rve8-1 rather than reduced. This would suggest that RVE8 functions as a repressor rather than an activator of CBF expression like CCA1 and LHY. RVE8 transcripts peak at the same time as CCA1 and LHY, but Rawat et al. 2011 demonstrated that the RVE8 protein does not accumulate until after subjective noon (3),   323 when CBF expression levels drop off (Fig. 2.1). Therefore, circadian oscillations of RVE8 are consistent with RVE8 functioning as a repressor of CBF expression. Interestingly, RVE8 was recently found to promote expression of PRR5 (3), which has previously been implicated as a repressor of CBF expression (4). Therefore, RVE8 may potentially acts as a direct or indirect regulator of CBF expression. CCA1 and LHY are controlled by transcriptional as well as post-translational mechanisms (5, 6). Phosphorylation of CCA1 and LHY by CASEIN KINASE 2 (CK2) is necessary for normal clock function and can affect DNA binding (6, 7). Therefore, CK2 was tested to determine if it is necessary for induction of the CBF genes. Preliminary time-course experiments using the ck2β3 mutant indicate that circadian expression, as well as gated cold-induction of CBF1-3, and CBF-target genes are not strongly affected. This suggests that post-translational modification of CCA1 and LHY is not necessary for binding to the CBF locus, but experiments need to be repeated. The CBF regulon only comprises 12% of cold-regulated genes (8). Therefore, it is of interest to determine if CCA1 and LHY also regulate CBF-independent pathways of cold-signaling. The cold regulated gene, COR27, is independent of the CBF pathway, and also gated by the circadian clock (9). Promoter analysis of COR27 demonstrated that the EE has a role in the cold-induction of COR27 (9). However, the transcription factors mediating cold-induction through the COR27-EEs have yet to be identified (9). EE-binding transcription factors CCA1 and LHY seem like especially good candidates because of their role in the induction of CBF genes (10).   324 Under circadian conditions, peak COR27 expression in the cca1-11/lhy-21 double mutant is not reduced nearly as much as that of the CBF genes (Fig 4.1; Fig A4.1; Fig. 2.1). Although more biological replicates may be necessary, five independent biological replicates of ChIP experiments did not find statistically significant enrichment of CCA1 on the COR27 promoter or coding region (Fig 4.2). Gated coldinduced expression of COR27 is also not strongly reduced in cca1-11/lhy-21 in comparison to wild-type (Fig. 4.1). Therefore, CCA1 and LHY do not appear to play a major role in circadian regulation or gated cold-induction of COR27 (Fig. 4.1). This suggests that there is specificity for CCA1 and LHY in the regulation of CBF signaling and that there are multiple mechanisms for gating cold gene expression. Contrary to previous reports that CBF and COR27 genes have similar peaks and troughs of expression (9), data from this study suggests that COR27 peaks in expression a few hours after CBF genes (Fig 4.1; Fig 2.1 and 2.4). The shift in peak expression is most likely due to the greater number of time-points used in this study. The difference in expression pattern between CBF and COR27 seems significant and may be the foundation for differing modes of circadian regulation. Further work must be conducted in order to determine the major mechanism of circadian and gated coldexpression of COR27. The previously mentioned RVE genes are known to bind to the EE (11). Many of these genes peak in expression before COR27, and thus are good first candidates for regulating COR27 expression.   325 WS" cca1$11/lhy$21* RELATIVE"EXPRESSION" A.(COR27( 1.20" 1.00" 0.80" 0.60" 0.40" 0.20" 0.00" 4" 12" 20" 28" 36" 44" TIME"(ZT)" B.(COR27( 1.40" 1.20" 1.00" 0.80" 0.60" 0.40" 0.20" 0.00" 2" 10" 18" 26" 34" 42" TIME"TRANSFERRED"TO"COLD"(ZT)" Figure 4.1. Effects of the cca1-11/lhy-21double mutation on circadian and gated coldinduction of COR27. Expression for COR27 was relative to one wild-type sample (WS) set to a value of 1 in each biological replicate. Values are averages from three independent experiments (n=3); error bars indicate ± SEM. (A) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were harvested every 2 h and the transcript levels for COR27 were determined by qRTPCR. Gene expression was normalized to IPP2 for each sample. (B) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were transferred to cold temperature (4⁰C) for 2 h, every 2 h, for 48 h at the start of constant light conditions and the transcript levels for COR27 were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were dropped to cold temperature. Gene expression was normalized to UBQ10 for each sample. See Table A4.1 for primer pair sequences.   326 1"KB" A." AT5G42905" COR27" RELATIVE(IP( (NORMALIZED(TO(INPUT)( A" B" *" 20" 15" 10" 5" 0" C" COR27" 1"KB" B." AT5G42905" A" RELATIVE(IP( (NORMALIZED(TO(INPUT)( D" COR27" B" C" D" 20" 15" 10" 5" 0" Figure 4.2. Binding of CCA1 on COR27. cca1-1 and cca1-1 CCA1p:CCA1-GFP plants were grown at 22⁰C under a 12 h photoperiod to the four leaf stage. Tissue was fixed at ZT4 and ChIP was performed using an anti-GFP (A) or mock IgG (B) antibody. Immunoprecipitated DNA was quantified by qRT-PCR with primers on COR27 (boxes A through D). Levels of immunoprecipitated DNA were normalized to input DNA. Immunoprecipitation in cca1-1 CCA1p:CCA1-GFP plants (black bars) is relative to cca11 plants (gray bars) set to 1. Primers TOC1-3’UTR, ACTIN7 (ACTIN) and UBIQUITIN10 (UBQ10) were negative controls. TOC1-EE was a positive control. Values are the average of five independent experiments (n=5). Error bars indicate ± SEM. *=p<0.05 using a paired, one-tailed t-test. In the COR27 diagram, the transcribed regions are indicted with white boxes and the approximate positions of CBS (AATCT), and EE (AAAATATCT) motifs are indicated by gray circles, and white squares respectively.   327 Temperature-entrained circadian regulation and gated cold-induction of CBF genes. Both light and temperature cues entrain the circadian clock to its external environment (12, 13). CBF2, CBF3 and the CBF-independent cold-regulated gene, COR27, all oscillate under temperature-entrained circadian conditions (Fig. 4.3). Temperature-entrained circadian oscillations of CBF1 do not appear regular and therefore are not as robust as under light-entrained circadian conditions (Fig 4.3; Fig 2.1). This is interesting and may suggest that CBF1 is distinctively regulated by light in comparison to CBF2 and CBF3. In Chapter Two we establish that CCA1 and LHY play a major role in the induction of CBF genes under light-entrained circadian conditions. Consequently, it is of interest to determine if CCA1 and LHY also play a major role in the induction of CBF genes under temperature-entrained circadian conditions. Peak expression of CBF1-3 is significantly reduced in the cca1-11/lhy-21 double mutant under temperature-entrained circadian conditions (Fig 4.3). CBF-target genes, COR15A, COR47 and COR78 also have reduced peak expression in cca1-11/lhy-21 in comparison to wild-type (Fig 4.4). This suggests that CCA1 and LHY play a major role in the basal induction of CBF genes under temperature-entrained circadian conditions. This also would suggest a similar mechanistic model for circadian regulation under temperature-entrained and light entrained conditions (Fig 2.5). However, analysis of clock and cycling genes reveals that there may be a more widespread reduction of rhythmicity in cca1-11/lhy-21 under temperature-entrained circadian conditions in comparison to light entrained conditions.   328 COR27 expression is significantly reduced in cca1-11/lhy-21 under temperatureentrained conditions (Fig 4.3), which is interesting considering that COR27 expression is not strongly reduced in cca1-11/lhy-21 under light-entrained circadian conditions (Fig. 4.1 and 4.3). Moreover, cycling control gene, LHCB1.4, also has significantly reduced peak expression in the cca1-11/lhy-21 under temperature-entrained conditions (Fig. 4.4), which is not the case under light-entrained conditions (Fig A2.3). Therefore expression of clock components, TOC1, PRR3, PRR5, PRR7 and PRR9, were further tested in cca1-11/lhy-21 under temperature-entrained circadian conditions (Fig. 4.5). Previous literature has shown that the cca1-11/lhy-21 mutant has robust oscillations of TOC1 under light-entrained conditions (14). Under temperature-entrained circadian conditions TOC1, PRR3, PRR5, and PRR7 have significantly reduced peak expression in cca1-11/lhy-21 in comparison to WT (Fig 4.5). Conversely, PRR9 maintains robust oscillations in cca1-11/lhy-21 under temperature-entrained conditions (Fig. 4.5). Previous literature has shown robust cycling of clock-output genes CAB2 (15, 16) and CCR2 (14) in cca1-11/lhy-21, under light-entrained conditions. Therefore, expression of cycling control genes, CAB2 and CCR2, were tested under temperature-entrained circadian conditions. CAB2 and CCR2, did show robust oscillations in cca1-11/lhy-21, with peak levels similar to wild-type (WS-2) under temperature-entrained conditions (Fig 4.4). Therefore, not all oscillations are knocked-out in cca1-11/lhy-21 under temperature-entrained conditions. Consequently, there is some specificity for the reduction in CBF gene expression under temperature-entrained conditions by CCA1 and LHY, in support of a mechanistic model similar to light-entrained conditions (Fig   329 2.5). But a more general clock question also seems to emerge from this data. To what extent to do CCA1 and LHY function differently under light- and temperature-entrained circadian conditions? To determine the extent of these differences, light and temperature cycling time-course microarrays or RNA-seq data would have to be performed for cca1-11/lhy-21 and wild-type (WS-2) plants. This type of data for cca111/lhy-21 does not appear to be publicly available, though there are light and temperature-entrained arrays publically available for wild-type plants. Significant progress has been made in determining the targets of light input into the clock (17-21), and temperature entrainment of the circadian clock is well established (12). However, mechanisms underlying temperature entrainment are still largely unknown (13, 22). Previous literature suggests that PRR7 and PRR9 are important for temperature-entrainment (22). This was shown through arrhythmic CCA1 and TOC1 expression as well as arrhythmic hypocotyl movement in prr7-3/prr9-1 under temperature-entrained conditions (22). The cca1-11/lhy-21 mutant also fails to maintain circadian oscillations of several clock genes, as well as cycling control gene LHCB1.4 under temperature-entrained conditions. CCA1 and LHY have been previously connected to temperature compensation of the clock (23). But data from this study suggests that CCA1 and LHY are also important for temperature entrainment of the circadian clock. This hypothesis should be further tested using other clock-output measurements such as hypocotyl movement. Fowler et al. 2005 demonstrated that cold-induced expression of CBF genes is gated by a light-entrained circadian clock (24). Therefore cold-induced gene expression   330 was tested under temperature-entrained circadian conditions to see if it is gated as well. Indeed, cold-induced expression of CBF1-3, CBF-target genes and COR27 is gated under temperature-entrained circadian conditions (Fig 4.6 and 4.7). In Chapter Two we show that CCA1 and LHY play a major role in the gated cold-induced expression of CBF genes. Therefore, it is of interest to determine if CCA1 and LHY influence gated coldinduction of CBF genes under temperature-entrained circadian conditions. The peak cold-induced expression of CBF1-3 and CBF-target genes is significantly reduced in cca1-11/lhy-21 under temperature-entrained conditions (Fig. 4.6 and 4.7), similar to light-entrained conditions (Fig 2.4), which suggests that CCA1 and LHY also are vital for the gated cold-induction of CBF genes under temperature-entrained conditions and might imply a similar gating mechanism to the one proposed for gated cold-induced CBF expression under light-entrained circadian conditions (Fig. 2.5). However, there again appears to be a general reduction in rhythmicity as well as expression level in cca1-11/lhy-21 under gated temperature-entrained conditions. Under both basal temperature-entrained circadian conditions and gated temperature-entrained conditions the cca1-11/lhy-21 double mutant appears to have a more generalized reduction in gene expression in comparison to light-entrained conditions. This sensitivity seems to be intensified with the addition of a cold treatment (gated conditions) since COR27 and all cycling control genes had significantly reduced peak expression in comparison to wild-type (Fig 4.7). Clock genes TOC1, PRR3, PRR5, and PRR7, are arrhythmic in cca1-11/lhy-21 under gated temperature-entrained circadian conditions, similar to basal temperature-entrained circadian conditions.   331 However, PRR9 is also arrhythmic under gated temperature-entrained circadian conditions (Fig 4.8), which was not the case under basal temperature-entrained circadian conditions (Fig. 4.5). CCA1 and LHY have previously been shown to be important for temperature compensation of the clock under light-entrained conditions (23). The increased arrhythmia seen with the addition of a cold treatment under temperature-entrained conditions may be due to the role of CCA1 and LHY in temperature compensation of the clock under temperature-entrained conditions. However, further experiments would need to be conducted to support this claim.   332 CBF1( RELATIVE"EXPRESSION" 3" WS" CBF2( cca1G11/lhyG21" 1.5" 2" 1" 1" 0.5" 0" 0" 4" 8" 12"16"20"24"28"32"36"40"44"48" 4" 8" 12"16"20"24"28"32"36"40"44"48" CBF3( COR27( 3" 1.5" 2" 1" 1" 0.5" 0" 4" 8" 12"16"20"24"28"32"36"40"44"48" 0" 4" 8" 12"16"20"24"28"32"36"40"44"48" TIME"(ZT)" Figure 4.3. Effects of the cca1-11/lhy-21double mutation on temperature-entrained circadian regulation of CBF1, CBF2, CBF3 and COR27. Wild-type Ws-2 (WS) and cca111/lhy-21 double mutant plants were grown at constant light under a 12 h temperature period (22⁰C day/ 12⁰C night) to the four leaf stage and transferred to constant temperature (22⁰C) at ZT0. Plants were harvested every 4 h and the transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from four independent biological experiments (n=4). Error bars indicate ±SEM. Please see Table A4.1 for primer pair sequences.   333 COR15A( RELATIVE"EXPRESSION" 1.5" WS" COR47( cca1G11/lhyG21" 1.5" 1" COR78( 2" 1.5" 1" 1" 0.5" 0.5" 0" 0" 4" 12" 20" 28" 36" 44" 0.5" 0" 4" 12" 20" 28" 36" 44" LHCB1.4( CAB2( 1.5" 3" 1" 2" 4" 12" 20" 28" 36" 44" CCR2( 2" 1.5" 1" 0.5" 1" 0" 0" 4" 12" 20" 28" 36" 44" 0.5" 0" 4" 12" 20" 28" 36" 44" 4" 12" 20" 28" 36" 44" TIME"(ZT)" Figure 4.4. Effects of the cca1-11/lhy-21double mutation on temperature-entrained circadian regulation of CBF target genes, COR15A, COR47 and COR78, and cycling control genes LHCB1.4, CAB2 and CCR2. Wild-type Ws-2 (WS) and cca111/lhy-21 double mutant plants were grown at constant light under a 12 h temperature period (22⁰C day/ 12⁰C night) to the four leaf stage and transferred to constant temperature (22⁰C) at ZT0. Plants were harvested every 4 h and the transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from four independent biological experiments (n=4). Error bars indicate ±SEM. Please see Table A4.1 for primer pair sequences.   334 TOC1( 2" WS" PRR3( cca1G11/lhyG21" 1.5" RELATIVE"EXPRESSION" 1.5" PRR5( 2" 1.5" 1" 1" 1" 0.5" 0.5" 0" 0.5" 0" 4" 12" 20" 28" 36" 44" 0" 4" 12" 20" 28" 36" 44" PRR7( 4" 12" 20" 28" 36" 44" PRR9( 1.5" 2" 1.5" 1" 1" 0.5" 0.5" 0" 0" 4" 12" 20" 28" 36" 44" 4" 12" 20" 28" 36" 44" TIME"(ZT)" Figure 4.5. Effects of the cca1-11/lhy-21double mutation on temperature-entrained circadian regulation of clock genes TOC1, PRR3, PRR5, PRR7 and PRR9. Wild-type Ws-2 (WS) and cca111/lhy-21 double mutant plants were grown at constant light under a 12 h temperature period (22⁰C day/ 12⁰C night) to the four leaf stage and transferred to constant temperature (22⁰C) at ZT0. Plants were harvested every 4 h and the transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from four independent biological experiments (n=4). Error bars indicate ±SEM. Please see Table A4.1 for primer pair sequences.   335 CBF1( RELATIVE"EXPRESSION" 2" WS" CBF2( cca1G11/lhyG21"1.5" 1" 1" 0.5" 0" 0" 4" 8" 12"16"20"24"28"32"36"40"44"48" 4" 8" 12"16"20"24"28"32"36"40"44"48" CBF3( COR27( 1.5" 1.5" 1" 1" 0.5" 0.5" 0" 4" 8" 12"16"20"24"28"32"36"40"44"48" 0" 4" 8" 12"16"20"24"28"32"36"40"44"48" TIME"TRANSFERRED"TO"COLD"(ZT)" Figure 4.6. Effects of the cca1-11/lhy-21double mutation on temperature-entrained cold-induction of CBF1, CBF2, CBF3 and COR27. Wild-type Ws-2 (WS) and cca111/lhy-21 double mutant plants were grown under constant light with a 12 h temperature period (22⁰C day/ 12⁰C night) to the four leaf stage and transferred to constant light at ZT0. Plants were transferred to cold temperature (4⁰C) for 2 h, every 4 h at the start of constant temperature conditions. Transcript levels for the indicated genes were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were transferred to cold temperature. Gene expression was normalized to UBQ10 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from four independent biological experiments (n=3). Error bars indicate ±SEM. Please see Table A4.1 for primer pair sequences.   336 COR15A( RELATIVE"EXPRESSION" 1.5" WS" COR47( cca1G11/lhyG21"1.5" COR78( 1.5" 1" 1" 1" 0.5" 0.5" 0.5" 0" 0" 4" 12" 20" 28" 36" 44" 0" 4" 12" 20" 28" 36" 44" LHCB1.4( CAB2( 1.5" 1.5" 1" 1" 0.5" 0.5" 0" 0" 4" 12" 20" 28" 36" 44" CCR2( 1" 0.5" 4" 12" 20" 28" 36" 44" 0" 4" 12" 20" 28" 36" 44" 4" 12" 20" 28" 36" 44" TIME"TRANSFERRED"TO"COLD"(ZT)" Figure 4.7. Effects of the cca1-11/lhy-21double mutation on temperature-entrained cold-induction of CBF target genes, COR15A, COR47 and COR78, and cycling control genes LHCB1.4, CAB2 and CCR2. Wild-type Ws-2 (WS) and cca1-11/lhy-21 double mutant plants were grown under constant light with a 12 h temperature period (22⁰C day/ 12⁰C night) to the four leaf stage and transferred to constant light at ZT0. Plants were transferred to cold temperature (4⁰C) for 2 h, every 4 h at the start of constant temperature conditions. Transcript levels for the indicated genes were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were transferred to cold temperature. Gene expression was normalized to UBQ10 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from four independent biological experiments (n=3). Error bars indicate ±SEM. Please see Table A4.1 for primer pair sequences.   337 TOC1( RELATIVE"EXPRESSION" 1.5" WS" PRR3( cca1G11/lhyG21" 1" PRR5( 1.5" 1" 1" 0.5" 0.5" 0.5" 0" 0" 4" 12" 20" 28" 36" 44" 0" 4" 12" 20" 28" 36" 44" PRR7( 4" 12" 20" 28" 36" 44" PRR9( 2" 1.5" 1.5" 1" 1" 0.5" 0.5" 0" 0" 4" 12" 20" 28" 36" 44" 4" 12" 20" 28" 36" 44" TIME"TRANSFERRED"TO"COLD"(ZT)" Figure 4.8. Effects of the cca1-11/lhy-21double mutation on temperature-entrained cold-induction of PRR clock genes TOC1, PRR3, PRR5, PRR7 and PRR9. Wild-type Ws-2 (WS) and cca1-11/lhy-21 double mutant plants were grown under constant light with a 12 h temperature period (22⁰C day/ 12⁰C night) to the four leaf stage and transferred to constant light at ZT0. Plants were transferred to cold temperature (4⁰C) for 2 h, every 4 h at the start of constant temperature conditions. Transcript levels for the indicated genes were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were transferred to cold temperature. Gene expression was normalized to UBQ10 for each sample. Gene expression is relative to one wild-type sample set to a value of 1 for each biological replicate. Values are averages from four independent biological experiments (n=3). Error bars indicate ±SEM. Please see Table A4.1 for primer pair sequences.   338 Disruption of the circadian clock by low-temperature. In 2005, Ramos et al. concluded that the chestnut tree circadian clock was disrupted with extended cold treatment (25). Bieniawska et al. 2008 further explored the disruption of circadian and diurnal oscillations by extended cold in Arabidopsis (26). They showed that extended cold treatment disrupts circadian and diurnal oscillations of all clock output genes (CCR1, CCR2, CAB2, CAT3), clock components (PRR3, PRR5, PRR7, PRR9, ELF3, ELF4, GI, TOC1, CCA1, LHY), and cold-genes (CBF1-3, COR15A, COR47, COR78) tested (26). These results are very interesting, but the reported induction pattern of CBF1-3 genes was also surprising (26). Bieniawska et al. 2008 show CBF expression quickly increasing within 2 h of cold treatment then staying at this maximal level for over 2 days (26). In previous experiments performed under constant-light conditions in constant-light grown plants, CBF genes also consistently reached maximal levels of cold-induced expression by 2 h (27-29). However, this maximum CBF expression level is then reduced significantly (though still higher than warm expression levels) by 24 h in cold (27-29) and remains down for at least 3 weeks (28). Experiments were conducted similar to Bieniawska et al. 2008 to determine if cold-induced CBF kinetics in plants grown under entrained conditions is significantly different from previously reported constant-light experiments (27-29). Preliminary experiments (2 biological reps; Fig. 4.9) indicate very different cold-induced CBF kinetics from Bieniawska et al. 2008 (26), but similar kinetics to previous constant-light experiments (27-29). There are some differences experimental set-up in comparison to Bieniawska et al. 2008. For example, plants were entrained under a 12 h photoperiod rather than long day condition (26)   339 before release into constant light conditions. Plants were also transferred to cold temperature at ZT4, rather than at ZT14 (26). These differences in experimental set up could be significant so future experiments should be conducted exactly as they were described in Bieniawska et al. 2008. Yet, more significant might be technical differences in the amount of RNA used for qRT-PCR. Bieniawska et al. 2008, used 2500ng of RNA to synthesize cDNA for expression analysis of CBF genes by qRT-PCR. The 25ng-200ng of RNA, used in this study, fell well into the dynamic range of CBF genes, whereas 2500ng did not. Therefore, it is possible that differences seen in kinetics of CBF genes are the result of technical rather than biological differences, but further experiments are required. Ramos et al. 2005 used northern blots to show that circadian oscillations of clock genes, TOC1 and LHY, were disrupted with extended cold treatment in chestnut trees (25). These experiments used plants grown in natural winter conditions with both cycling light and temperature (25). Consequently, Ramos et al. concluded that circadian clocks do not function during winter months (25). Although this may be the case in chestnut trees, Arabidopsis carrying null mutations in either TOC1 (30) or LHY(31) can continue to have robust rhythms. Furthermore, Arabidopsis carrying null mutations in both TOC1 and LHY also have continued rhythmicity (32). In a more extensive analysis of clock gene disruption by cold, Bieniawska et al. 2008 also saw interrupted oscillations of many clock genes (26). However, clock component LUX continued to cycle under diurnal conditions (26). This indicates that there is continued rhythmicity under extended cold and thus the possibility of a functioning ‘low-temperature’ oscillator.   340 Furthermore, LUX may not be the only cold oscillating gene in the Arabidopsis transcriptome (26). Therefore, the extent to which genes oscillate under these lowtemperature conditions should continue to be examined. Moreover, Ramos et al. 2005 sampled plants in natural conditions with both diurnal cycles and changing day and night temperature, whereas Bieniawska et al. 2008 sampled plants only under circadian and diurnal conditions (25, 26). Both light and temperature entrain the circadian clock and temperature oscillations could be especially important under low-temperature conditions. Accordingly, it is of interest to determine if the disruption of clock gene oscillations by cold also occurs in plants sampled under temperature cycles (comparable to a diurnal treatment; Fig. 4.10). In preliminary experiments, robust rhythms of central clock component, CCA1, were observed in samples without cold treatment (‘WARM’), similar to Bieniawska et al. 2008. Disrupted rhythmicity of CCA1 with extended cold treatment (‘COLD’) was also observed, similar to Bieniawska et al. 2008 (Fig 4.10). Interestingly, plants sampled under thermocycles (‘TEMP CYCLING’), continued to show regular though widened oscillations (Fig. 4.10). However one caveat for these experiments is that plants were grown under 12 h photoperiods then transferred to12 h thermocycles (9⁰C day and 4⁰C night) for sampling (Fig. 4.10). Previously, 8⁰C treatments have been used for chillingstress in peppers (33), 7⁰C treatments for wheat (34), 6⁰C treatments for soybean (35), and 5⁰C treatments for tomato (36). Consequently, future experiments should be set up using colder temperature cycles such as a 6⁰C day with 2⁰C night, or 4⁰C day with 0⁰C night. Nevertheless, preliminary results suggest the possibility that the circadian clock   341 functions under low-temperature conditions, which should be further explored in the future.   342 RELATIVE"EXPRESSION" CBF2( CBF2( 1.2" 1.2" 1" 1" 0.8" 0.8" 0.6" 0.6" 0.4" 0.4" 0.2" 0.2" 0" 0" 6" 10"14"18"22"26"30"34"38"42"46"50" 6" 10"14"18"22"26"30"34"38"42"46"50" TIME"(ZT)" Figure 4.9. Cycling of CBF2 expression in plants grown under light-entrained conditions then sampled in constant-cold conditions. Wild-type, Ws-2, plants were grown at 22⁰C with a 12 h photoperiod to the four leaf stage and then transferred to constant light and constant cold (4⁰C) conditions at ZT4. Transcript levels for the indicated genes were determined by qRT-PCR. Gene expression was normalized to UBQ10 for each sample. Gene expression is relative to one sample set to a value of 1 in each experiment. Two biological replications are shown (left graph and right graph). Please see Table A4.1 for primer pair sequences.   343 RELATIVE"EXPRESSION" CCA1( WARM" 5" TEMP"CYCLING" COLD" 4" 3" 2" 1" 0" 6" 10"14"18"22"26"30"34"38"42"46"50" TIME"(ZT)" Figure 4.10. Cycling of CCA1 under warm, temperature cycling or constant-cold conditions. Wild-type, Ws-2, plants were grown at 22⁰C with a 12 h photoperiod to the four leaf stage and then transferred to one of three conditions at ZT4. ‘WARM’ plants were transferred to constant light and constant temperature (22⁰C) conditions. ‘TEMP CYLING’ plants were transferred to constant light with a 12 h cold temperature period (9⁰C day/ 4⁰C night). ‘COLD’ plants were transferred to constant light and constant cold (4⁰C) conditions. Transcript levels for CCA1 were determined by qRT-PCR. Gene expression was normalized to UBQ10 for each sample. Gene expression is relative to one sample set to a value of 1 for each condition. Values are from one experiment (n=1). Please see Table A4.1 for primer pair sequences.   344 Morphological differences between SW and IT. Morphological differences between the SW and IT ecotypes, used in Chapter Three of this study, were observed but not quantified. SW leaf edges were rounded, whereas IT leaf edges were variegated. SW and IT plants also appear to grow at different rates, since IT has more leaves than SW at the same number of days in age. SW leaves seem to be thicker than IT at all time-points examined (non-acclimated, 1 week, 2 weeks and 3 weeks coldacclimated). Interestingly, plants overexpressing CBF genes have thicker leaves than wild-type plants and are constitutively freezing tolerant, (37). There is also a strong positive correlation between leaf thickness and photosynthetic capacity (38). Increased photosynthetic capacity is also associated with cold-acclimation (39). This is especially interesting because RNA-seq data in Chapter Three shows that the majority of differentially expressed photosynthesis genes have higher expression in SW (Fig. 3.33). SW leaf thickness also seemed to increase with cold, which is a change associated with cold-acclimation (40-43) and cold developed leaves (43). However, these morphological changes and differences between SW and IT will have to be measured experimentally. Low-temperature network construction using SW and IT RILs. In Chapter Three of this study, eQTL mapped for genes associated with differences in freezing tolerance (FTD genes), which also fall under fitness QTL, were detailed. The gene hypothesized to be under the FTD eQTL on chromosome 4, which overlaps with a fitness QTL, is CBF2. The FTD eQTL on chromosome 5, overlapping with a fitness   345 QTL, may be a cis-eQTL, implicating COR78 as the underlying gene. Alternatively, the eQTL on chromosome 5 may be a trans-eQTL, which is postulated to be CBF4. As stated in Chapter Three, near-isogenic lines and complementation lines will need to be made for candidate genes underlying these eQTL, to establish their importance for differential expression of cold-regulated genes in SW and IT. These lines will also be employed to ascertain the role of these genes in freezing tolerance and fitness. There were also eQTL mapped for genes associated with differences in freezing tolerance that did not fall under fitness QTL (Table 3.18 and 3.19). These genes are not included in the CBF-regulon and for most of these genes specific roles in coldacclimation are yet to be established. Many of the eQTL were located trans- to the gene they were mapped for and thus potentially represent regulatory loci for CBFindependent cold-regulated genes (Table 3.18 and 3.19), which is especially interesting because relatively little is known about CBF-independent pathways of cold acclimation (44). Hundreds of genes underlie these trans-eQTL. However, RNA-seq data described in detail in Chapter Three can be used to define a smaller set of candidate regulatory genes. For example, SALT-INDUCIBLE ZINC FINGER 1 (SZF1) is a coldregulated transcription factor previously described as down-regulated at 1 week of coldacclimation in Arabidopsis (29). Indeed, RNA-seq data from this study also shows SW and IT both down-regulating SZF1 with 1 week of cold-acclimation (Fig 4.11). But SW and IT have significantly different SZF1 expression at 2 weeks of cold-acclimation, when eQTL were mapped (Fig. 4.11). The trans-eQTL mapped for SZF1 on chromosome 5   346 encompasses 805 genes. Of these 805 genes, 108 are differentially regulated between SW and IT at 2 weeks of cold-acclimation, which is when SZF1 expression is significantly different between SW and IT (Fig. 4.11). There are limitations to this candidate gene list, for instance it would not include genes that have differences in posttranscriptional regulation between SW and IT. Nevertheless, these 108 genes seem like a good starting point for regulation of SZF1. Since this is a trans-eQTL, the transcription factors in this list of 108 genes were determined. There were a total of 11 transcription factors among these 108 candidate regulatory genes (Table 4.1). Using the RNA-seq data from all time points, expression patterns for SZF1 and the 11 transcription factors can be compared (Fig. 4.11). Based on these data, AT5G26170 (WRKY50; Table 4.1) is the transcription factor with an expression pattern that best parallels SZF1 expression and analysis of the SZF1-500 bp promoter reveals two known WRKY binding sites (Fig. 4.11). Again, near-isogenic lines and complementation lines will need to made for WRKY50 to establish its importance in regulation of SZF1 expression. These lines would also be tested for differences in freezing tolerance. This method of network construction is a rather tedious process, especially for a single gene that may or may not largely contribute to differences in freezing tolerance. Consequently, future efforts for low-temperature network construction should include transcriptomic analysis of the RILs used for mapping. It is fiscally daunting to do microarray or RNA-seq eQTL analysis on 544 RILs. However, the minimum number of randomly selected RILs necessary to get similar eQTL results to 544 RILs can be determined through analysis of eQTL mapped for the   347 10 FTD genes in 544 RILs (described in Chapter Three). The same RIL RNA samples used in eQTL analysis can be submitted for transcriptomic analysis by either RNA-seq or Affymetrix arrays. Genome-wide eQTL would thus be mapped for this subset of RILs and trans-regulatory hotspots (45-48) can be identified at a time-point where there are differences in freezing tolerance (2 weeks of cold-acclimation; Fig 3.2). Accordingly, efforts to find the genes responsible for these trans-eQTL will be focused on major regulatory hubs. It is clear that there is much future effort needed to determine the genes responsible for differences in freezing tolerance between the SW and IT ecotypes, especially for CBF-independent pathways. Nevertheless, data from this study provides a necessary backbone for future studies.   348 Table 4.1. Candidate cold-regulated transcription factors for SZF1 trans-QTL on chromosome 5 (between RIL markers 20.0 and 36.7). The SeqViewer tool on TAIR (www.arabidopsis.org) was used to determine within SZF1 trans-QTL on chromosome 5. From this list SW and IT cold-regulated transcription factors were identified using merge function in R. The comparison of SW and IT expression at 2 weeks of cold-acclimation for each gene, was measured by RNA-seq and is represented by the logarithm (base=2) of the fold change (LOG2FC). The FDR corrected P-value is given for each comparison. The results presented are based on averages from three independent experiments (n=3). The shaded transcription factor is located closest to the peak RIL marker for the SZF1-QTL (marker 22.0). AGI   SHORT  DESCRIPTION   2WK.SW.IT   2WK.LOG2FC   FDR  P-­‐VALUE   AT5G24110   WRKY  DNA-­‐BINDING  PROTEIN  30   ITHIGH   3.58   0.00E+00   AT5G24590   TCV-­‐INTERACTING  PROTEIN   ITHIGH   0.91   1.40E-­‐05   AT5G25190   INTEGRASE-­‐TYPE  DNA-­‐BINDING   ITHIGH   1.13   1.13E-­‐02   AT5G25390   INTEGRASE-­‐TYPE  DNA-­‐BINDING   SWHIGH   -­‐1.20   5.15E-­‐05   AT5G25810   INTEGRASE-­‐TYPE  DNA-­‐BINDING   SWHIGH   -­‐1.92   2.21E-­‐03   AT5G25890   INDOLE-­‐3-­‐ACETIC  ACID  INDUCIBLE  28   SWHIGH   -­‐0.59   3.32E-­‐02   AT5G26170   WRKY  DNA-­‐BINDING  PROTEIN  50   ITHIGH   2.83   7.48E-­‐07   AT5G27610   DIRP;  MYB-­‐LIKE  DNA-­‐BINDING  DOMAIN   ITHIGH   0.74   2.60E-­‐02   AT5G28300   SUPERFAMILY  PROTEIN   SWHIGH   -­‐0.87   4.43E-­‐07   AT5G28640   SSXT  FAMILY  PROTEIN   ITHIGH   1.33   1.03E-­‐07   AT5G28770   BZIP  TRANSCRIPTION  FACTOR  FAMILY   ITHIGH   0.77   3.20E-­‐04       349 30" IT" 20" 10" 20" 6" FPKM( 10" NON" 1WK" 2WK" AT5G25390( 10" 0" NON" 1WK" 2WK" AT5G25890( AT5G26170( 20" 10" 0" 0" NON" 1WK" 2WK" 30" 0" AT5G28640( 20" 10" 0" NON" 1WK" 2WK" NON" 1WK" 2WK" 40" FPKM( FPKM( 0" AT5G27610( 4" NON" 1WK" 2WK" AT5G28300( 20" NON" 1WK" 2WK" 2" 10" 40" 2" FPKM( 20" 4" NON" 1WK" 2WK" 30" AT5G25810( 0" FPKM( FPKM( 0" FPKM( FPKM( AT5G25190( 20" 60" 10" NON" 1WK" 2WK" 0" FPKM( 20" 20" NON" 1WK" 2WK" 30" AT5G24590( 0" 0" 30" AT5G24110( FPKM( SW" FPKM( FPKM( 40" SZF1( AT5G28770( 20" 0" NON" 1WK" 2WK" NON" 1WK" 2WK" Figure 4.11. SZF1-regulation candidate gene expression. IT samples are shown in red, and SW in blue. Gene expression was measured by RNA-seq (FPKM). The results presented are average values from three independent experiments (n=3). Error bars indicate ±SEM. Plants were grown at 22⁰C under a 12 h photoperiod and sampled directly or transferred to 4⁰C for 1 or 2 weeks under a 12 h photoperiod then sampled.   350 MATERIALS AND METHODS Regulation of CBF genes by the REVEILLES, CKβ3, and CAMTAS and circadian regulation of CBF-independent gene, COR27: Plants were grown and experiments were conducted as described in Chapter Two of this study. Please see Table A4.1and A4.2 for primers. Temperature-entrained circadian regulation and gated cold-induction of CBF genes: Plants were grown and experiments were conducted as described in Chapter Two of this study, except that instead of entrainment with a 12 h photoperiod, plants were entrained with 12 h thermocycles (22⁰C day/ 12⁰C night) under constant light, then sampled under constant light and constant temperature conditions (22⁰C). Please see Table A4.1 for primers. Disruption of the circadian clock by low-temperature: Experiments were conducted as described in Chapter Two of this study. Wild-type Ws-2 plants were grown at 22⁰C with a 12 h photoperiod to the four leaf stage and then transferred to one of three conditions at ZT4: ‘WARM’ plants were transferred to constant light and constant temperature (22⁰C) conditions. ‘TEMP CYLING’ plants were transferred to constant light with a 12 h cold temperature period (9⁰C day/ 4⁰C night). ‘COLD’ plants were transferred to constant light and constant cold (4⁰C) conditions. Please see Table A4.1 for primers.   351 Low-temperature network construction using SW and IT RILs: Experiments were conducted as described in Chapter Three of this study.   352 APPENDIX   353 WS" cca1$11* lhy$21* A.(COR27( 1.50" RELATIVE"EXPRESSION" 1.00" 0.50" 0.00" 4" 12" 20" 28" 36" 44" TIME"(ZT)" B.(COR27( 4.00 3.00 2.00 TIME"(ZT)" 1.00 0.00 2 10 18 26 34 42 TIME"TRANSFERRED"TO"COLD"(ZT)" Figure A4.1. Effects of the cca1-11 and lhy-21 single mutants on circadian and gated cold-induction of COR27. Expression for COR27 was relative to one wild-type sample WS-2 (WS) set to a value of 1 in each biological replicate. Values for WS-2 are averages from three independent experiments (n=3). Values for cca1-11 and lhy-21 are averages from two independent experiments (n=2). Error bars indicate ± SEM. (A) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were harvested every 2 h and the transcript levels for COR27 were determined by qRT-PCR. Gene expression was normalized to IPP2 for each sample. (B) Plants were grown with a 12 h photoperiod at 22⁰C to the four leaf stage and transferred to constant light at ZT0 (subjective day and night are indicated by white and gray bars, respectively). Plants were transferred to cold temperature (4⁰C) for 2 h, every 2 h, for 48 h at the start of constant light conditions and the transcript levels for COR27 were determined by qRT-PCR. Horizontal axis represents time (ZT) when plants were dropped to cold temperature. Gene expression was normalized to UBQ10 for each sample. Please see Table A4.1 for primer pair sequences.     354 Table A4.1. Primer pairs used for qRT-PCR. Primers used in this study were designed with Primer Express 3.0. qRT-­‐PCR  Primers     Name   Forward   CAB2   CCGGAAAGGCTGTGAACCT   CBF1   GGAGACAATGTTTGGGATGC   CBF2   CGACGGATGCTCATGGTCTT   CBF2   CGACGGATGCTCATGGTCTT   CBF3   TTCCGTCCGTACAGTGGAAT   CCA1   CCGCAACTTTCGCCTCAT   CCR2   TAGGGCGACGTTATTGATTCC   COR15A   GAAAAAAACAGTGAAACCGCAGAT   COR27   GCTCTGGCTCAGCCTCTAGTCT   COR47   CGGTACCAGTGTCGGAGAGT   COR78   GAAAGGAGGAGGAGGAATGG   IPP2   ATTTGCCCATCGTCCTCTGT   LHCB1.4   GCCTTCGCTACCAACTTCGTC   PRR3   GTGGGAGTAGTGGTGGTTTGAGTA   PRR5   CGAGAAGCCGCTTTAACCAA   PRR7   CCACGAGCGGTATCTCTATGG   PRR9   GCCAGAGAGAAGCTGCATTGA   TOC1   TCTTCGCAGAATCCCTGTGAT   UBQ10   GGCCTTGTATAATCCCTGATGAATAAG       Reverse   CACACGGCCGCTTCCA   CGACTATCGAATATTAGTAACTCC   TCTTCATCCATATAAAACGCATCTTG   TCTTCATCCATATAAAACGCATCTTG   AACTCCATAACGATACGTCGTC   GCCAGATTCGGAGGTGAGTTC   CCCTCAATCGCATCCTTCA   CCACATACGCCGCAGCTT   GGTCGTGGTCACGCGAAT   ACAGCTGGTGAATCCTCTGC   AACCAGCCAGATGATTTTGG   GAGAAAGCACGAAAATTCGGTAA   AACCGGATACACACAACTCGATC   TTTGTCCAAGAACTCTGAGTTCCA   CGGCTCTCGTAACGAACCTT   ACTTGGAAACTCAGGGTTAGAA   CCTGCTCTGGTACCGAACCTT   GCTGCACCTAGCTTCAAGCA   AAAGAGATAACAGGAACGGAAACATAGT   355   ATG   AT1G29920   AT4G25490   AT4G25470   AT4G25470   AT4G25480   AT2G46830   AT1G06820   AT2G42540   AT5G42900   AT1G20440   AT5G52310   AT3G02780   AT2G34430   AT5G60100   AT5G24470   AT5G02810   AT2G46790   AT5G61380   AT4G05320   Table A4.2. Primer pairs used for ChIP qRT-PCR. Primers used in this study were designed with Primer Express 3.0. ChIP  PRIMERS       Name   ACTIN2   COR27-­‐A   COR27-­‐B   COR27-­‐C   COR27-­‐D   TOC1   TOC1  3'UTR   UBQ10     Forward   CGTTTCGCTTTCCTTAGTGTTA   TTCGCATGGGAGAAGAAGGT   AGTTACAACAATCAATAAGCA   TCTTCCAAAAGAGACATGTAATAGTCAA   GCTCTGGCTCAGCCTCTAGTCT   TTTTATGGCCTGCACTTTTTATTG   GCTACAGCCAAAAAAACATCGA   TCCAGGACAAGGAGGTATTCCTCCG       Reverse   AGCGAACGGATCTAGAGACTC   CCACTATCGCCTCTGAATCCA   GAGAAGGGTGAGTAATTATGTGAATATACAA   TGGGACGATTCACTTTTCTTATTAGA   GGTCGTGGTCACGCGAAT   GGTGGGACTTGGGATATTTTAGG   GAGCCGCAAGAGCCAACAT   CCACCAAAGTTTTACATGAAACGAA       356     ATG   AT3G18780   AT5G42900   AT5G42900   AT5G42900   AT5G42900   AT5G61380     AT5G61380     AT4G05320       LITERATURE CITED   357 LITERATURE CITED 1. Doherty CJ, Van Buskirk HA, Myers SJ, & Thomashow MF (2009) Roles For Arabidopsis CAMTA Transcription Factors In Cold-Regulated Gene Expression And Freezing Tolerance. 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