TRANSCRIPTIONAL REGULATION OF COLD ACCLIMATION IN ARABIDOPSIS THALIANA By Chin-Mei Lee A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Biochemistry and Molecular Biology 2012 ABSTRACT TRANSCRIPTIONAL REGULATION OF COLD ACCLIMATION IN ARABIDOPSIS THALIANA By Chin-Mei Lee Low temperature is a major environmental factor that affects the yield and quality of food and bioenergy crop plants. To cope with low temperature stress, many plants can acquire freezing tolerance through cold acclimation, in which remodeling of the metabolome and biological pathways leads to physiological adaptation to low temperatures. Many of these adjustments are brought about by intensively reconfiguring the expression of cold-regulated (COR) genes during cold acclimation. Some of the COR genes are regulated by the well-studied CBF pathway and its parallel ZAT12 pathway. However, the regulation and functions of other CBF-independent pathways that are involved in COR gene regulation are not well understood. Freezing tolerance in plants is affected by external signals as well as the growth conditions of plants themselves, but cross talk between cold and other signals has not been fully elucidated. The goal of this dissertation is to understand how freezing tolerance is established during cold acclimation in plants by addressing two key questions: first, how do plants perceive and integrate photoperiod as seasonal signals to modulate the expression of COR genes and subsequent freezing tolerance; second, what are the CBF-independent pathways that have major roles in COR gene expression and their roles in tolerance to low temperatures? In the first part of the dissertation, the photoperiodic regulation of the coldresponsive CBF pathway and freezing tolerance was demonstrated in Arabidopsis. The CBF transcript levels in short-day (SD) plants were higher than in long-day (LD) plants. Genetic analysis indicated that phytochrome B (PHYB) functions with two phytochrome interacting factors, PIF4 and PIF7, to down-regulate the CBF pathway and freezing tolerance under LD conditions. Down-regulation of the CBF pathway in LD plants correlated with higher PIF4 and PIF7 transcript levels and greater stability of the PIF4 and PIF7 proteins under LD conditions. These findings provide a mechanism of how plants sense and integrate photoperiod as seasonal signals to regulate cold responsive pathways and freezing tolerance. To address the second question, fifteen early cold-induced transcription factors (TF) sharing similar cold-induction kinetics with CBFs and ZAT12 were identified as regulators of CBF-independent pathways in parallel with the CBF and ZAT12 pathways. These TFs regulate about 29% of the COR genes; they regulate different sets of COR genes but with substantial overlap, which indicates that highly co-regulation of COR genes exists. Functional enrichment analysis of the COR genes regulated by each TF indicated that they are involved in diverse but overlapping biological pathways during cold acclimation. My results shed light on the regulation of COR genes by CBF- independent pathways and also on interactions between different biological pathways in cold acclimation. In summary, this dissertation provides substantial contributions to the understanding of gene regulatory networks in cold acclimation by integrating photoperiod signals into cold responsive pathways, and by dissecting the coldregulatory pathways. In addition, the knowledge can be potentially applied for crop engineering and improvements. Copyright by CHIN-MEI LEE 2012 ACKNOWLEDGEMENTS I would like to express my deepest appreciation to my graduate research committee, Dr. Michael Thomashow, Dr. Min-Hao Kuo, Dr. David Arnosti, Dr. Steven Triezenberg, and Dr. Gregg Howe for their guidance and advice throughout my doctoral study. I want to especially thank Dr. Min-Hao Kuo for always encouraging and inspiring me. It is with immense gratitude that I acknowledge the support and help of my advisor, Dr. Michael Thomashow. This dissertation would not have been possible unless Dr. Sarah Gilmour helped for reading and editing the dissertation and Dr. Sunchung Park collaborated with me on the work presented in chapter 3. I would like to share the credit of my work with Susan Myer, Cynthia Collings, Sarah Olaniyan, Jordan Stubleski, Josh Polito, and Linda Yuen, who helped me for preparing experimental materials. In addition, I would like to give my acknowledgements to Dr. Beronda Motgomery for discussing the research related to light signaling, to Sankalpi Warnasooriya and Michael Ruckle for helping me setting up monochromatic light chambers. I appreciate the help and discussion from Dr. Eva Farre, Tiffany Liu, Linsey Newton, and Dr. Malia Dong for the ChIP assays. It is my great honor to work with former and current members in the Thomashow lab and to discuss my research with them. As part of PRL study group, I want to acknowledge Kyaw Aung, Yani Chen, Christine Shyu, Jeongwoon Kim, and Nobuko Sugimoto as companies during the entire PhD program to discuss research and as friends to share with happiness and sadness v of life. In addition, I would like to thank my dear friends, Grace, Kai-Chun, Ying-Chou, Chih-Shia, Joyce, Yin-Phan and Kuolin, who always help and encourage me. Finally, without the support from my family in Taiwan and my husband, Mao-Ning, I would not have accomplished the PhD degree. vi TABLE OF CONTENTS LIST OF TABLES………………………………………………………………..…….……...viii LIST OF FIGURES……………………………………………………………………………..ix KEY TO ABBREVIATIONS…………………………………………………………………...xi CHAPTER 1 LITERATURE REVIEW: COLD ACCLIMATION IN PLANTS…………………………...1 Overview of Low Temperature Stress and Cold Acclimation in Plants…………2 CBF Pathway in Cold Acclimation………………………………………………….4 Regulation of the CBF Pathway in Response to Low Temperature…………….5 Interplay between Cold and the Circadian Clock………………….……………...8 Regulation of Freezing Tolerance by Light………………………..……………..10 Regulation of Freezing Tolerance by Seasonal Signals in Plants……………..11 CBF-independent Pathways in Cold Acclimation and Freezing Tolerance…...13 Gene Regulatory Network of Cold Acclimation…………………………….………15 CHAPTER 2 PHOTOPERIODIC REGULATION OF CBF COLD ACCLIMATION PATHWAY AND FREEZING TOLERANCE IN ARABIDOPSIS THALIANA……………………………...18 Abstract………………………………………………………………………………19 Introduction……………………………………………………………………………. 20 Results……………………………………………………………………………….23 Discussion…………………………………………………………………………...30 Materials and Methods…………………………………………………………......55 Acknowledgements…………………………………………………………………58 CHAPTER 3 TRANSCRIPTIONAL REGULATORY NETWORKS OF COLD ACCLIMATION IN ARABIDOPSIS THALIANA………………………………………..……………………….59 Preface……………………………………………………………………………...60 Abstract………………………………………………………………………………61 Introduction…………………………………………………………………………..62 Results………………………………………………………………………………..65 Discussion……………………………………………………………………………78 Materials and Methods…………………………………………………………….114 APPPEDIX…………………………………………………………………………………..118 REFERENCES………………………………………………………………………………206 vii LIST OF TABLES Table 2.1 List of primers for qRT-PCR and ChIP assays………………………………51 Table 2.2 List of primers for cloning and EMSA…………………………………………54 Table 3.1 List of transcription factors up-regulated at low temperature……………….98 Table 3.2 Summary of differentially expressed genes (DEG) in each TF-OX line….104 Table 3.3 GO analysis of COR genes and TF-regulons………………………………106 Table 3.4 Genes in circadian or starch degradation pathways affected by overexpressing ZF…………………………………………………………………………108 Table 3.5 List of primers for qRT-PCR………………………………………………….109 Table 3.6 List of primers for cloning……………………………………………………..113 Table A1. List of COR genes and the regulons of each overexpressed TF…………119 viii LIST OF FIGURES Figure 2.1 Arabidopsis freezing tolerance is regulated by photoperiod………………35 Figure 2.2 The CBF pathway is regulated by photoperiod…………………………….36 Figure 2.3 The CBF pathway is regulated by photoperiod…………………………….38 Figure 2.4 A G-box motif within the CBF2 promoter confers photoperiod-regulated gene expression………………………………………………………………………………………39 Figure 2.5 PIF4 and PIF7 are required for repression of the CBF pathway under LD conditions…………………………………………………………………………………….41 Figure 2.6 PIF4 and PIF7 are expressed at higher levels, and their proteins are more stable under LD conditions.…………………………………………………………………43 Figure 2.7 Overexpression of PIF4 or PIF7 represses CBF2 expression.……………44 Figure 2.8 PIF4 and PIF7 bind to CBF1, CBF2 and CBF3 promoters through G-box and E-box motifs.…………………………………………………………………………………46 Figure 2.9 PIF7 binds at the CBF locus.…………………………………………………47 Figure 2.10 PHYB is required for photoperiod regulation of the CBF pathway and freezing tolerance.…………………………………………………………………………..48 Figure 2.11 Model of photoperiodic regulation of the CBF pathway and freezing tolerance.…………………………………………………………………………................50 Figure 3.1 Temporal expression of the COR genes…………………………………….82 Figure 3.2 Cluster analysis of cold-induced transcription factors……………………...83 ix Figure 3.3 Relative transcript levels of cold-induced transcription factors of cluster 3 in Figure 3.2 and Table 3.1……………………………………………………………………84 Figure 3.4 Spearman correlation of the expression of COR genes regulated by each TF and that in WT at each time point………………………………………………………….86 Figure 3.5 The co-regulatory network of the TF-regulons……………………………...87 Figure 3.6 Phenotypes of the transgenic plants overexpressing early cold-induced TFs..............................................................................................................................89 Figure 3.7 Freezing tolerance test of the TF-overexpressing lines under non-cold acclimated (NAc) conditions……………………………………………………………….90 Figure 3.8 Freezing tolerance test of the TF-overexpressing lines under cold acclimated (Ac) condition………………………………………………………………….91 Figure 3.9 Expression of the enzymes involved in the starch degradation pathway are affected by low temperature………………………………………………………………..92 Figure 3.10 The expression of key enzymes in the starch degradation pathway is affected by overexpression of ZF………………………………………………………….93 Figure 3.11 Model of plant circadian clock………………………………………………94 Figure 3.12 Phase enrichment of the ZF regulon……………………………………….95 Figure 3.13 The effects of overexpressing ZF on the expression of circadian regulators and output genes in warm conditions……………………………………………………..96 x KEY TO ABBREVIATIONS ABA: abscisic acid AP2: apetala2 bHLH: basic helix-loop-helix bp: base pair bZIP: basic-leucine zipper CaMV: cauliflower mosaic virus CAMTA: calmodulin binding transcriptional activator CBF: c-repeat/ drought-responsive element binding factor CCA1: circadian clock-associated 1 CFP: cyan fluorescent protein ChIP: chromatin Immunoprecipitation CM: conserved motif COR: cold-regulated CRT/DRE: c-repeat/drought-responsive element Cvi: Cape Verde Islands DEAR1: DREB and EAR motif protein 1 DNA: deoxyribonucleic acid DREB: dehydration responsive element binding factor DOF: DNA binding with one finger EL50: the temperature at which freezing damage results in leakage of 50% of the total cellular electrolytes xi ELA: electrolyte leakage assay EMAS: electrophoretic mobility shift assay ERF: ethylene response factor FR: far-red light GA: gibberellins GOLS: galactinol synthase GUS: beta-glucuronidase HOS: high expression of osmotically responsive gene HSF: heat-shock factor HSP: heat-shock protein ICE: inducer of CBF expression ICEr: Inducer of CBF expression region LD: long-day LHY: late elongated hypocotyl HY5: elongated hypocotyl 5 LOS1: low expression of osmotically responsive genes 1 LUC: luciferase P5CS: delta 1-pyrroline-5-carboxylate synthase qRT-PCR: quantitative reverse transcription polymerase chain reaction R: red light RD29A: responsive to dessication 29a; cor78 PR: plant pathogen-related PIF: phytochrome interacting factor xii PHY: phytochrome PRR: pseudo-response regulator PS: photosystem QTL: quantitative trait locus RIL: recombinant inbred lines ROS: reactive oxygen species SA: salicylic acid SD: short-day TAIR: The Arabidopsis Information Resource TF: transcription factor TOC1: timing of cab expression 1 TF-OX: overexpression line of transcription factor WT: wild type ZF: zinc-finger transcription factor, at4g29190 ZT: zeitgeber time xiii CHAPTER 1 LITERATURE REVIEW: COLD ACCLIMATION IN PLANTS 1 Overview of Low Temperature Stress and Cold Acclimation in Plants Low temperature is one of the environmental factors that regulate multiple developmental processes in plants, but it also has adverse effects on plant growth and survival. In agriculture, chilling (> 0°C) and freezing (< 0°C) stresses cause major economic loss by affecting the production, quality, and biomass of crops. Therefore, understanding how plants cope with low temperature stress has been an important research area for crop improvement. Under natural selection, many plants have evolved the ability to develop freezing tolerance when exposed to low, non-freezing temperatures, a process known as cold acclimation (1, 2). However, not all plant species have the same ability to survive freezing temperatures after cold acclimation. Fully cold-acclimated red osier dogwood can survive temperatures lower than -50°C, and rye to -30°C, whereas the model plant Arabidopsis thaliana is killed at -10°C (3, 4). The freezing tolerance within plant species can also vary, depending on its developmental stage, other environmental signals, and the temperature itself during cold acclimation. The different levels of freezing tolerance result from differences in the transcriptional activity and metabolic changes during cold acclimation (5, 6). Transcriptional and metabolic adjustments are critical for survival at freezing temperatures (7, 8). The primary site of freezing injury to the plant cell is the plasma membrane, caused by severe cellular dehydration (9). When plant tissues are frozen, water in the extracellular spaces freezes, resulting in lower water potential. As a result, intracellular water moves into the extracellular spaces causing cell dehydration. The 2 consequences of cellular dehydration and extracellular ice crystal formation are the rupture of membranes, cell wall lesion, and eventually cell death (10). One of the major functions of cold acclimation is to increase the cellular solutes and to stabilize cell membranes to prevent cellular damage. It has been reported that accumulation of cryoprotectants (sucrose, raffinose, and proline), membrane stabilizing proteins (coldregulated proteins (CORs), dehydrins, and heat shock proteins (HSPs)), and induction of plant pathogen-related (PR) proteins prevent dehydration and ice nucleation during cold acclimation (1, 2, 11). In addition, an increase in the unsaturated fatty acid content of membranes, lipid remodeling, and loosening of cell walls occur to maintain membrane integrity at low temperatures (12-14). Secondary freezing damage is induced by cellular dehydration and changes in temperature-sensitive reactions in cells (1, 15). Part of the cold acclimation process is to relieve the stresses that accompany cold. Drought-responsive and ABA-responsive genes are induced to cope with water deprivation (16, 17). Stress caused by cold-induced reactive oxygen species (ROS) can be relieved by increases in the antioxidation enzymes (superoxide dismutase, glutathione peroxide, and glutathione reductase) and antioxidants (glutathione and ascorbic acid) (11, 18). Accumulation of flavonoid and anthocyanin, reduction in components of the light harvesting complexes (LHC) and plastocyanin can mitigate photo-oxidative stress induced by excessive excitation of Photosystem II (PSII) and excessive electron transfer to PSI under low temperatures (19, 20). Plants also establish physiological adaptation to long-term cold. Remodeling of the photosynthetic capacity has been shown to be an important factor for perennial plants to overwinter (21, 22). Maintaining energy homeostasis by reconfiguration of 3 energy flow and growth cessation is part of the strategy for plant survival (22, 23). Low temperatures also modulate hormone signaling pathways, such as those of gibberellins (GA), salicylic acid (SA), and auxin, to regulate plant growth (24-26). CBF Pathway in Cold Acclimation Many of aforementioned metabolic adjustments and physiological adaptations are brought about by reprogramming the transcription of cold-regulated (COR) genes during cold acclimation (27-30). The CBF (CRT/DRE Binding Factor) pathway is the best studied gene regulatory pathway in cold acclimation (31). In Arabidopsis, CBF1, CBF2, and CBF3 genes (also known as DREB1B, DREB1C, and DREB1A, respectively) form a tandem repeat on chromosome 4, and their transcripts are induced within 15 minutes of exposure to cold (32). The CBF genes encode members of AP2/ERF transcriptional activators, and specifically interact with the CRT/DRE (C-repeat/droughtresponsive element, RCCGAC) elements in the promoters of their target genes (33, 34). Overexpression of CBF affects the expression of approximately 140 COR genes, termed the “CBF regulon”, under warm conditions, and the transgenic lines confer freezing and drought tolerance (35, 36). Results from the metabolomic analysis of the CBF-overexpressing lines agree with transcriptome data in that CBF affects multiple key pathways in cold acclimation (5). Some COR genes directly regulated by CBFs, such as COR6.6, and COR15a, encode membrane stabilizing proteins (33, 37). Other CBF target genes, such as GALACTINOL SYNTHASE 3 (GOLS3) and DELTA 1PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) encode key enzymes in raffinose and proline biosynthesis for cryoprotectant production (35, 38). 4 CBF pathways are conserved among many plant species (39-42). Ectopic expression of Arabidopsis CBFs in chilling-sensitive species, such as potato, or rice, increases their freezing tolerance (40, 41, 43). Quantitative trait locus (QTL) analysis of recombinant inbred lines (RIL) from two parental lines with different freezing tolerance ability in Arabidopsis, barley and wheat identified a major locus responsible for freezing tolerance, which is co-localized to the CBF locus (44, 45). In Arabidopsis Cape Verde Islands (Cvi), an accession originating from the subtropics, lower freezing tolerance is exhibited compared to other accessions. Cvi has a deletion of 1.6 kbp in the CBF2 promoter, which greatly decreases the cold induction of CBFs and hence the CBF regulon (5, 44). In addition, the changes in metabolites induced by overexpression of CBF3 are largely depleted in Cvi (5). These studies suggest a prominent role of the CBF pathway in cold acclimation. Regulation of the CBF Pathway in Response to Low Temperature It is still not understood how plants perceive low temperature, but extensive studies in both cis-acting elements and trans-acting regulators have shed light on the regulation of CBFs in response to low temperatures (28, 31, 46). The transcripts of CBFs are induced within minutes at 4°C, peak at around 3 h, and then drop off again in cold. Moreover, the transcripts of CBFs are degraded rapidly soon after returning to warm temperature (47). Promoter deletion analysis identified a 125 bp promoter region in the CBF2 promoter that is sufficient to impart cold-induction of CBF2 expression (47). The 125 bp CBF2 promoter region contains cold-responsive elements, ICEr1 and ICEr2 (Inducer of CBF expression region 1 and 2), which are conserved among Arabidopsis 5 CBF1, CBF2, and CBF3 (47, 48). Further analysis of this promoter region defined 7 conserved motifs (CM1-7) shared between CBF2 and another cold-induced transcription factor, ZAT12 (49). CM6 and CM7 partially overlap with ICEr1, and CM2 partially overlaps with ICEr2. CM6 and CM4 have negative regulatory activities, and CM2 functions as both a positive and negative regulatory element. These results indicate that CBF2 is tightly regulated through different elements. The trans-acting regulators of CBFs were largely identified by a genetic screen of the CBF promoter fused to a LUC reporter (50). INDUCER OF CBF EXPRESSION 1 (ICE1), a MYC-like bHLH transcription factor, was identified as interacting with the CANNTG element in the CBF3 promoter by this strategy (51). Mutation in ICE1 affects cold induction of CBF3 but not of CBF1 and CFB2. The transcriptional activation activity of ICE1 is post-translationally regulated by HOS1 and SIZ1 (52, 53). HOS1 (HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE 1) encodes a RING type E3 ligase for ubiquitination of K403 in ICE1 and its degradation in cold (52, 54). A SUMO E3 ligase, SIZ1, on the other hand, functions as a positive regulator of ICE1 via sumoylation of ICE1 to antagonize ubiquitination of ICE1 by HOS1 (53). ICE1 is also found in wheat and Camellia sinensis, suggesting it is a conserved regulator of cold acclimation (55, 56). An ICE1 homolog, ICE2, appears to affect the expression of CBF1, but has been less studied (57). In addition to ICE1, MYB15 has been identified from a mutant screen as a coldinduced transcription factor that negatively regulates CBF1, CBF2, and CBF3 by interacting with their promoters (58). Rather than being involved in cold induction of the CBFs, the myb15 mutant showed higher transcript levels of the CBFs than WT after 6h 6 in cold, indicating that MYB15 down-regulates the CBFs after cold induction. There is crosstalk between MYB15 and ICE1 in regulating the CBFs. At the protein level, MYB15 interacts with ICE1, and the MYB15 protein is more stable in the ice1 mutant (58). In addition, disruption of SIZ1-mediated ubiquitination of ICE1 increases the expression of MYB15 (59). Another positive regulator of CBF1 and CBF2 is CALMODULIN BINDING TRANSCRIPTIONAL ACTIVATOR 3 (CAMTA3) (49). CAMTA3 interacts with the CG-1 element (CGCG) in the CM2 motif of the CBF2 promoter. In the camta3 mutant, the expression of CBF1, CBF2, and GOLS3 were reduced about 50% at 0°C with no obvious effect on freezing tolerance. The camta1/ camta3 double mutant, however, is compromised in freezing tolerance. This finding provides a potential link between the CBF pathway and calcium signaling, which is thought to be an early signaling transduction pathway in cold (49, 60). The transcription of CBFs is tightly regulated. Mutation of CBF2 enhances the expression of CBF1 and CBF3 in the cold, suggesting that CBF2 has a role in the negative regulation of CBF1 and CBF3 (61). Surprisingly, freezing tolerance was found to be decreased in the cbf2 mutant. Constitutive expression of several cold-induced transcription factors has been shown to negatively regulate CBFs. ZAT12 is a coldinduced transcription factor sharing similar expression profile to the CBFs in cold. Overexpression of ZAT12 showed repression of CBFs in the cold (62). DEAR1 is another cold-induced AP2 transcription factor that negatively regulates the CBFs (63). The function of these negative regulation could be similar to that of MYB15 in that they prevent overexpression of CBFs, which gives rise to stunted plants (35, 36). 7 Interplay between Cold and the Circadian Clock The main trigger for cold acclimation is low temperature, but other environmental factors have impacts on acquired freezing tolerance (3, 4). Harmer et al. (64) showed that the expression of CBFs is circadian regulated at warm temperatures. The transcript levels of CBFs oscillate with a peak around 8h after dawn (zeitgeber time 8; ZT8) and a trough around ZT20. It has been demonstrated that the peak expression pattern is regulated by two central clock regulators, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), which bind to the promoters of the CBFs at ZT8 (65). The oscillation of CBF1 and CBF3 were eliminated in the cca1/ lhy double mutant. The cycling expression of CBF2 is largely reduced in the cca1/ lhy double mutant, but with some residual activity. The trough is partially regulated by PHYTOCHROME INTERACTING FACTOR 7 (PIF7), a bHLH transcription factor which binds to the G-box, and which overlaps with the CM6 motif in the CBF2 promoter. The expression of CBFs is elevated around ZT15 in the pif7-2 mutant under circadian conditions (66). The cold responsiveness of the CBFs is “gated” by the circadian clock (67). This means that the CBF transcript levels are time-dependent during the day; for example, cold treatment given at ZT4 induces higher CBF expression than at ZT16. Mutation in both CCA1 and LHY disrupts the gating effects on the CBFs and impairs freezing tolerance (65). Another line of evidence supporting the importance of the clock on freezing tolerance was obtained using the prr5/prr7/prr9 triple mutant (d975) (68). In the d975 mutant, the oscillated expression of CCA1 and LHY is disrupted, but with 8 intermediate expression levels. Up-regulation of the CBF pathway and accumulation of galactinol, raffinose, and proline in this mutant lead to an increase in freezing tolerance. Conversely, low temperature has an impact on circadian rhythm (69, 70). Transcription profiling of plants undergoing cold stress and other abiotic stresses showed that the cold-responsive genes lost their oscillating expression patterns in the cold, which is a unique phenomenon among abiotic stresses (71). In chestnut, the expression of several central clock regulators, including CCA1, LHY, TOC1, and PRRs stop their oscillated expression pattern in winter (70, 72). Recently, Bieniaawska et al. (69) surveyed the clock genes in Arabidopsis and showed that the oscillation of many central clock regulators are dampened in the cold under diurnal conditions, and that they are arrhythmic in the cold under continuous light. The exception to this is LUX ARRHYTHMO (LUX), whose expression continues to cycle under both conditions. Transcriptome and metabolome analyses further showed that cold affects major clockregulated metabolic pathways, such as carbohydrate metabolism and amino acid biosynthesis (30). In the cold under diurnal conditions, approximately 80% of the tested metabolites are still cycling but their phase is shifted. The oscillated expression of genes encoding enzymes involved in biosynthetic pathways of the selected metabolites is largely dampened. Most of the tested metabolites and the expression of genes regulating their biosynthesis are arrhythmic at low temperature under continuous light conditions. Mutations in both CCA1 and LHY have a negative impact on freezing tolerance; however, the biological significance of disrupting the cycling expression of central clock regulators is not understood (73). The circadian clock regulates major metabolic pathways (64, 74), and disruption of the clock may provide an efficient 9 remodeling of metabolism in the cold. Some metabolites, however, still cycle in the cold, and this finding raises a question as to whether the circadian clock is totally disrupted by the cold. There may be another set of regulators for maintaining the circadian clock in the cold. Regulation of Freezing Tolerance by Light Light can regulate freezing tolerance in many different ways. High light treatment has been shown to increase freezing tolerance and induces COR genes in winter wheat and rye without cold treatment (75, 76). This phenomenon is due to photoinhibition, a process whereby light energy absorbed in the photosynthetic light processes exceeds the energy demand of the dark processes, which then leads to over-reduction of PS II and subsequent inhibition of the photosynthetic capacity (77). Photoinhinition and the ROS stress that accompanies it are common mechanisms shared by cold acclimation and high light acclimation (22). Cold, in conjunction with light treatment, is required for full-strength freezing tolerance in Arabidopsis (78). Transcriptome profiling has shown that twice as many genes are cold-induced in the light as in the dark (79). The major differences in upregulated genes in the light and dark are the oxidative stress-related genes, photosynthesis-related genes, ABA biosynthesis pathway genes, and genes involved in the production of protective molecules, such as phenylpropanoids. How does the light signal integrate into the cold pathway to regulate COR genes? Recently, Catala et al. (80) identified HY5, a master regulator of light signaling, as being involved in regulating about 100 COR genes independent of the CBF pathway under low temperature 10 conditions in the light. The expression of HY5 is induced by cold, and low temperature stabilizes the HY5 protein by preventing its proteasome-mediated protein degradation. The hy5 mutant displays a freezing sensitive phenotype, which is due to a decrease in anthocyanin and antioxidant production. Regulation of Freezing Tolerance by Seasonal Signals in Plants In nature, plants perceive seasonal signals in addition to drop in temperature before the onset of winter. Annual changes in light quality, in particular red (R) and farred (FR) light, occur during the twilight period when the solar elevation is less than 10°. A decrease in R light, or low R/FR ratio, in autumn has been suggested to be a signal in which plants anticipate winter (81). In addition, shortening day-length in autumn has been reported to prepare plants for overwintering (3). It is well-established in woody species that the acquired freezing tolerance is obtained first by a shortening of photoperiod, and thereafter by a decrease in temperature (82, 83). Plants sense light signals through photoreceptors. R and FR light are perceived through a set of phytochrome photoreceptors, PHYTOCHROME A to E (PHYA to PHYE) (84, 85). The phytochromes are dimers of two chromoproteins consisting of polypeptide subunits that carry a tetrapyrrole chromophore in each chromoprotein. They exist as two inter-convertible forms: the biologically inactive R-absorbing Pr and the active FRabsorbing Pfr. R light converts Pr to Pfr, whereas FR converts Pfr back to Pr. Phytochromes regulate a wide range of biological pathways through interacting with other proteins, including PHYTOCHROME INTERACTING FACTORS, or PIFs, a subfamily of bHLH transcription factors. The functions of phytochromes are more than 11 just sensing changes in light quality; PHYA and PHYB have also been shown to be involved in photoperiodic regulation of flowering time, dormancy, bud set, and tuberization in plants (86). Historically, the effects of photoperiod on freezing tolerance were first documented in woody species. It has been demonstrated that a short-day (SD) photoperiod and low temperature are more effective in establishing freezing tolerance than are long-day (LD) and cold in many woody species, including red osier dogwood, aspen, and birch (3, 87). The SD-induced increase in ABA content and the subsequent induction of dehydrins have been shown to enhance freezing tolerance (88, 89). Interestingly, the effects of SD on freezing tolerance were hampered by 15 minutes of R light treatment, and these inhibitory effects are reversible by subsequent FR light treatment (87, 90, 91). These results suggest that phytochromes are involved in photoperiodic regulation of freezing tolerance in woody species. It has been shown that overexpression of oat PHYA in aspen leads to a SD-insensitive phenotype, including loss of dormancy and growth cessation (88). However, overexpression of PHYA in Arabidopsis has both positive and negative effects on pathways mediated by other phytochromes, including PHYB (92). In potato, overexpression of PHYA resulted in a photoperiod-insensitive tuberization response, which was caused by disruption of the circadian clock. Therefore, the mechanism of how plants sense photoperiod through phytochrome in regulating freezing tolerance is far from being completely understood. The effect of SD on freezing tolerance is not limited to woody species. Similar phenomena have been reported in spring wheat, barley, Arabidopsis and Gaura coccinea (44, 93-95). Studies in barley and wheat suggested that SD- and cold-induced 12 vernalization can affect expression of CBFs or COR genes, which may be responsible for the freezing tolerance phenotype (93, 96). QTL mapping of RILs from Ler (Landsberg erecta) and Cvi showed that the QTLs responsible for freezing tolerance are different under SD and LD conditions, except for the CBF locus (44). The photoperiodic regulation of freezing tolerance was observed in both woody species and herbaceous species. Is this response regulated by the same mechanism? How does photoperiod regulate freezing tolerance? These important questions remain to be elucidated. Franklin and Whitelam first demonstrated that light quality alone (12h light and 12h dark photoperiod, a neutral day-length condition) affects freezing tolerance in Arabidopsis at ambient temperature (81). They showed that the expression of several CBF regulon genes, COR15a, COR47, COR78, and KIN1, are induced under low R/FR ratio at 16°C but not at 22°C, a response mediated by PHYB and PHYD. Furthermore, low R/FR ratio induced higher expression of CBF1-3 than high R/FR ratidid o under circadian conditions. Low R/FR ratio treatment at 16°C is sufficient to increase freezing tolerance in wild type Arabidopsis (81). These results showed that light quality regulates the CBF pathway and therefore freezing tolerance. It further indicated that the CBF pathway could be an integration point for light signals. CBF-independent Pathways in Cold Acclimation and Freezing Tolerance Freezing tolerance in plants is regulated by multiple pathways in addition to the CBFs (38). Overexpression of CBF at warm temperatures is sufficient for development of freezing tolerance, but it cannot lead to full-strength freezing tolerance induced by cold acclimation (35, 36). In addition, transcription profiling has shown that 13 overexpression of CBF affects about 140 COR genes, while there are thousands of COR genes that are affected by cold (28, 38, 62). These findings indicate that other cold-regulatory pathways in addition to CBF pathway exist. Several regulators have been identified which regulate CBF-independent pathways. The esk1 (eskimo1) mutant was identified through a mutant screen. esk1 showed constitutive freezing tolerance, which is due to the accumulation of high levels of proline (97). Transcriptome analysis indicated that about 130 COR genes were affected in esk1, and around 40 of them overlap with the CBF regulon (98). ESKMO1 encodes a DUF231 domain protein of unknown function; it is currently not understood how ESKMO1 regulates COR genes (98). The hos9 mutant was identified as constitutively expressing RD29A in mutant screens of cold-induced RD29A promoterreporter lines, and the mutant displayed an increase in freezing tolerance (99). HOS9 encodes a putative homeodomain transcription factor. Transcription profiling has shown that hos9 affects around 40 COR genes, none of which overlaps with the CBF regulon. As described in the previous section, HY5 mediates the light regulation of around 100 COR genes, but these genes do not overlap with the CBF regulon either (100). A coldinduced nuclear protein, GIGANTEA (GI), has pleiotropic effects on the circadian clock and flowering (101, 102). The gi mutant exhibits a freezing sensitive phenotype, but expression of the CBFs or the CBF regulon are not affected in the mutant (103). Further analysis showed that failure to accumulate soluble sugar as a cryoprotectant in the gi mutant may account for the phenotype (104). These results reveal that the regulation of COR genes is interdigiteted between CBF-dependent and CBFindependent pathways. 14 Gene Regulatory Network of Cold Acclimation Plant response to low temperature is a complex process: integration of multiple external signals to establish freezing tolerance, intensive interactions between biological pathways in cold acclimation, and multiple levels of regulation within a pathway. Understanding the dynamics of cold responses in plants needs to be in the context of systems biology. Ideker et al. (105) proposed systems biology as a framework for modeling biological systems by integrating genome-wide measurements to understand the properties of biological systems: from gene to protein and metabolites, their functions, pathways and then their response to changing environments. For the first step of the systems approach, the components of the interaction within the network have to be identified and characterized in a global manner. The next step is to perturb the system and monitor its responses on a genome-scale. Finally, the data are collected to model the system and novel interactions can be inferred and examined (105). Top-down strategies have accumulated a wealth of genomic-scale data, such as transcriptome, proteome, and metabolome analyses in wild type and mutants, which facilitate the discovery of complex biological pathways in cold stress responses. Comparison of cold-responsive genes to those regulated by other conditions has revealed novel interaction between pathways, such as cold and the clock (106). Association network modeling based on the co-expression analysis of microarray data across various conditions has predicted several regulatory modules in different biological processes (107, 108). Whether the predicted regulators and pathways are involved in cold stress responses still awaits experimental validation. Vogel et al. (62) 15 analyzed the expression kinetics of COR genes with cold transcriptome data and identified ZAT12 as a cold-induced transcription factor in addition to the CBF pathway. Overexpression of ZAT12 confers freezing tolerance in a whole plant freeze test, which may result from an increase in tolerance to ROS stress (109). Chawade et al. (110) combined the time-dependent expression information and consensus transcription factor binding sites to model gene regulatory networks in the cold. This strategy may give several false positive interactions. For example, not all the motifs in the promoter are involved in controlling cold responses. Additionally, the consensus binding sites can be bound by multiple transcription factors within the same family or in different transcription factor families. The bottom-up strategy to identify components of gene regulatory networks is limited, partly due to the lack of systematic strategies to capture the cold responses and to identify the components in these responses. Mutant screens have been a popular genetic strategy to identify regulators in biological pathways. However, current methods, such as survival tests and electrolyte leakage assays are laborious and not quantitative. Ehlert and Hincha (111) reported a new strategy that measures chlorophyll florescence. It has been shown that chlorophyll florescence is correlated to leaf damage. With this method, a large-scale screen would be feasible, but this method cannot capture all the cold responses. Insights have been gained into regulation of the CBF pathway by targeted genetic screens with the CBF or RD29A promoter fused to a reporter gene, but fewer advances have been made with the CBF-independent pathways (50). From transcription profiling experiments, more than a thousand COR genes have been identified (38, 62, 112-115). The expression of these COR genes revealed a 16 series of transient waves, suggesting transcriptional regulation cascades (28, 38, 62). Our understanding of the regulation of the COR genes by early cold-induced transcription factors is limited to CBF1-3 and ZAT12 (62). To understand the regulation of biological pathways and the interactions between pathways during cold acclimation, new regulators for each pathway in cold acclimation will need to be identified, and their effects on COR genes will have to be studied systematically. 17 CHAPTER 2 PHOTOPERIODIC REGULATION OF THE CBF COLD ACCLIMATION PATHWAY AND FREEZING TOLERANCE IN ARABIDOPSIS THALIANA 18 Abstract The CBF pathway has a major role in plant cold acclimation, the process whereby certain plants increase in freezing tolerance in response to low nonfreezing temperatures. In Arabidopsis thaliana, this pathway is characterized by rapid cold induction of CBF1, CBF2 and CBF3, which encode transcriptional activators, followed by induction of CBF-targeted genes that impart freezing tolerance. At warm temperature, CBF transcript levels are low, but oscillate due to circadian regulation with peak expression occurring at 8 h after dawn (ZT8). Here we establish that the CBF pathway is also regulated by photoperiod at warm temperature. At ZT8, CBF transcript levels in short-day (SD; 8 h photoperiod) plants were 3-5 fold higher than in long-day plants (LD; 16 h photoperiod). Moreover, the freezing tolerance of SD plants was greater than that of LD plants. Genetic analysis indicated that phytochrome B (PHYB) functions with two phytochrome interacting factors, PIF4 and PIF7, to down-regulate the CBF pathway and freezing tolerance under LD conditions. Down-regulation of the CBF pathway in LD plants correlated with higher PIF4 and PIF7 transcript levels and greater stability of the PIF4 and PIF7 proteins under LD conditions. Our results indicate that during the warm LD growing season, the CBF pathway is actively repressed by PHYB, PIF4 and PIF7 thus mitigating allocation of energy and nutrient resources towards unneeded frost protection, and that this repression is relieved by shortening day-length resulting in up-regulation of the CBF pathway and increased freezing tolerance in preparation for coming cold temperatures. 19 Introduction Plants vary greatly in their ability to survive freezing temperatures. Whereas plants from tropical and subtropical regions are generally killed by the slightest freeze, plants from temperate regions exhibit varying degrees of freezing tolerance (4). For instance, Arabidopsis thaliana (hereafter referred to as Arabidopsis) and wheat have a maximum freezing tolerance of about -10°C and -20°C, respectively, and hardy deciduous trees can survive freezing below -50°C. However, the freezing tolerance of frost hardy plants is not a constant property; it changes over the course of the year in response to changing environmental conditions. The primary factor is low temperature (3, 4). When winter rye is grown at warm temperature, plants are killed upon freezing at about -5°C, but upon exposure to low nonfreezing temperatures, they can survive freezing below -20°C. The molecular basis for this phenomenon, known as cold acclimation, is not completely understood, but includes changes in membrane cryobehavior, the production of cryoprotective proteins, and the biosynthesis of low molecular weight cryoprotectants such as sucrose, raffinose and proline (1, 116). Many of the biochemical and metabolic changes that occur in response to low temperature and contribute to an increase in freezing tolerance involve changes in gene expression (28, 29, 117). The best understood cold regulatory pathway with a role in freezing tolerance is the CBF pathway of Arabidopsis (28, 31, 118). CBF1, CBF2 and CBF3 (also known as DREB1B, DREB1C, and DREB1A, respectively) encode closely related members of the AP2/ERF family of DNA binding proteins that recognize the CRT/DRE DNA regulatory element, RCCGAC (33, 34). Within minutes of transferring 20 Arabidopsis plants to low temperature, CBF1, CBF2, and CBF3 are induced followed at about 3 h by induction of CBF-targeted cold-regulated (COR) genes, referred to as the CBF regulon (28). Constitutive overexpression of CBF1, CBF2, or CBF3 at warm temperature leads to constitutive expression of the CBF regulon and a marked increase in freezing tolerance (34, 36, 119). Although the CBF pathway is not as well studied in other plant species, it has been established that cold-inducible CBF genes are highly conserved among higher plants and that CBF overexpression increases the freezing tolerance in plants ranging from closely related canola to distantly related poplar and wheat (120-123). Photoperiod is another environmental factor that regulates freezing tolerance, a phenomenon that is well documented in woody deciduous trees (3, 83). As summer turns to fall, these plants sense the shortening day-length and initiate developmental programs that result in the cessation of growth and an increase in freezing tolerance that can be more than 10°C in some hardy species. As the season continues to progress and the temperatures become cold, the plants sense the low nonfreezing temperatures and increase an additional 40°C or more in freezing tolerance (3, 124). The molecular basis for photoperiodic regulation of freezing tolerance is not well understood. However, the increase in freezing tolerance that occurs in response to short-day in red-osier dogwood and other perennial woody tree species is prevented if the plants are briefly exposed to red (R) light during the nighttime, but not if the R light exposure is followed by brief exposure to far-red (FR) light (87, 125). These are classic indicators of a phytochrome-mediated response (126). Phytochromes are proteins that have a tetrapyrrole chromophore that exists in two interchangeable forms covalently 21 linked to their N-terminal end: the R-light absorbing form, designated Pr, and the FRlight adsorbing form, designated Pfr. The Pr form, which is inactive, is converted to the active Pfr form by exposure R light, and is converted back to the inactive Pr form by exposure to FR light. The fact that the freezing tolerance of short-day grown red-osier dogwood is reduced when the plants are exposed to R light during the night suggests that an active Pfr phytochrome represses freezing tolerance. Whereas photoperiodic regulation of freezing tolerance is recognized as a fundamental feature of cold acclimation in woody plants, there is little evidence for the phenomenon in herbaceous plants (3). Pietsch et al. (95) found that the freezing tolerance of Gaura coccinea, a perennial herbaceous species, increases about 3°C when plants are exposed to short-day photoperiods, but beyond this, photoperiodic regulation of freezing tolerance at warm growth temperature is poorly documented in perennial and annual herbaceous species. However, similar to what has been reported in woody plants, Franklin and Whitelam (81) showed that phytochromes have a role in regulating freezing tolerance in Arabidopsis. When plants were grown at 16°C (but not at 22°C) under a 12 h photoperiod, the freezing tolerance and transcript levels for three CBF target genes—COR15a, COR15b and KIN1—were greater in plants exposed to a low R/FR light ratio than if they were exposed to a high R/FR light ratio. Also, transferring plants to constant light and exposing them to a low R/FR light ratio for 2 h in the morning resulted in increased transcript levels for CBF1, CBF2 and CBF3. These results suggested that a Pfr form of one or more phytochromes repressed the expression of the CBF pathway. Indeed, at 16°C, under high R/F light, both phyB and 22 phyD mutations resulted in increased transcript levels for COR15a, a CBF target gene, and the phyD mutation resulted in greater freezing tolerance (the effects of a phyB mutation on freezing tolerance were not reported). Arabidopsis has proven to be a powerful model plant to study the regulation of freezing tolerance by low temperature (28, 127). Here we show that it is also a powerful model to study photoperiodic regulation of freezing tolerance. Our results indicate that Arabidopsis plants increase in freezing tolerance in response to a short-day photoperiod, that this regulation involves photoperiodic regulation of the CBF pathway, and that this regulation is mediated by the PHYB photoreceptor and two PIF transcription factors with which PHYB physically interacts, PIF4 and PIF7 (128, 129). Results Freezing Tolerance is Regulated by Photoperiod To determine whether the freezing tolerance of Arabidopsis (Col-0) is regulated by photoperiod, we grew plants under short-days (SD; 8 h light, 16 h dark) and longdays (LD; 16 h light, 8 h dark) and compared their freezing tolerance using the electrolyte leakage assay. The results indicated that the freezing tolerance of the SD plants was greater than that of the LD plants; whereas the EL50 (the temperature at which freezing damage results in leakage of 50% of the total cellular electrolytes) of the LD plants was about -3°C, the SD plants had an EL50 of about -5.5°C (Fig. 2.1A). In these experiments, the SD and LD plants were tested at the point that they each had about 8 true leaves. However, to produce this number of leaves, the SD and 23 LD plants were grown for 5 weeks and 3 weeks, respectively. To address the possibility that differences in age were the cause of the observed differences in freezing tolerance, we grew plants under SD or LD conditions and then switched their photoperiod and tested their freezing tolerance; all plants again had about 8 true leaves. When plants were grown under SD for 3 weeks and transferred to LD for 2 weeks, they had the same freezing tolerance as plants grown for 3 weeks under LD (Fig. 2.1B). When plants were grown under LD for 2 weeks and transferred to SD for 2 weeks, they had the same freezing tolerance as plants grown under SD for 5 weeks (Fig. 2.1B). Thus, regardless of the direction of the day-length shift or total age of the plants, the freezing tolerance of the plants was determined by the final 2-week treatment; SD to LD produced the same freezing tolerance as constant LD treatment, and LD to SD produced the same freezing tolerance as constant SD treatment. From these results, we concluded that the freezing tolerance of Arabidopsis is regulated by photoperiod. The CBF Pathway is Regulated by Photoperiod Given the prominent role of the CBF pathway in cold acclimation, we asked whether the CBF genes were expressed at different levels in SD and LD plants. Previous studies (64, 66, 130) established that CBF1, CBF2 and CBF3 are regulated by the circadian clock and that the transcript levels for each gene peaks at about 8 h after dawn, a time referred to as ZT8 (zeitgeber time 8). Our results were consistent with these findings; the transcript levels for each CBF gene peaked at about ZT8 under both SD and LD conditions (Fig. 2.2). However, the CBF transcript levels at ZT8 were about 3 to 5 fold higher in the SD plants as compared to the LD plants. The transcript levels 24 for two CBF regulon genes, COR15a and GOLS3, also oscillated, having peak expression between ZT8 and ZT12, and at their peak the transcript levels for these two genes were about 5 fold higher in the SD plants (Fig. 2.2). To address the possibility that differences in plant age accounted for the differences in CBF and CBF regulon gene transcript levels in the SD and LD plants, we transferred LD plants to SD conditions, and SD plants to LD conditions, and determined their transcript levels. First, we determined the transcript levels of CBF2 at ZT8 at 0, 1, 3, 5, and 7 d after the switch in photoperiod. The results indicated that over the course of the week, the lower level of CBF2 transcripts initially observed in the LD plants rose to the level observed in the SD plants, and that the higher level of CBF2 transcripts initially observed in the SD plants, decreased to the level observed in the LD plants (Fig. 2.3A). We then determined the transcript levels of CBF1, CBF2, CBF3 and two CBF regulon genes, COR15a, and GOLS3, at ZT8 in SD and LD plants, in SD plants transferred to LD for 7 d, and in LD plants transferred to SD for 7 d. The results indicated that with each gene, the lower transcript levels initially observed in the LD plants rose to those observed in the SD plants after transfer to SD, and that the higher transcript levels initially observed in the SD plants, fell to those observed in the LD plants after transfer to LD (Fig. 2.3B). These results indicated that the CBF pathway is regulated by photoperiod and that the greater freezing tolerance of the SD plants was due, at least in part, to greater expression of the CBF pathway under SD conditions. The results also showed that the difference in CBF pathway expression produced by SD and LD conditions was reversible during vegetative growth. 25 Photoperiodic Regulation of CBF2 Involves a G-box Motif within the CBF2 Promoter The photoperiodic control of CBF transcript levels could involve either transcriptional or posttranscriptional regulatory mechanisms or both. To determine whether transcriptional mechanisms were involved, we asked whether the CBF2 promoter included DNA regulatory elements that were responsive to photoperiod. Previous studies showed that the region of the CBF2 promoter from -189 to -35 relative to the transcription start site (this region is numbered -207 to -53 in the current TAIR10 database; hereafter we use TAIR10 designations for sequence locations) included elements that could impart both cold (47, 49) and circadian (67) regulation when fused the GUS reporter gene. We therefore tested this region for photoperiodic regulation. We fused the CBF2 promoter region from -207 to +134 to the GUS reporter gene (WTpro) (Fig. 2.4A), transformed the construct into Arabidopsis, and determined the level of GUS transcripts in SD and LD grown plants (Fig. 2.4B). The results indicated that the WT-pro construct produced peak levels of GUS transcripts at ZT8 in both SD and LD grown plants, but that the peak was about 3 fold greater in the SD plants. These results were consistent with the -207 to + 134 CBF2 promoter fragment including a regulatory motif that was responsive to photoperiod. Previous studies showed that the CBF2 promoter region between -207 and -53 bp included a G-box motif, CACGTG (-112 to -107) that imparted negative regulation in plants that were grown at warm temperature and in plants that were exposed to low temperature (49, 66). Therefore, we asked whether this motif was also involved in photoperiodic regulation. We mutated the G-box sequence within the WT-pro construct 26 (Gmut-pro) (Fig. 2.4A), transformed the construct into Arabidopsis, and determined the GUS transcript levels under SD and LD conditions. As with the WT-pro construct, the GUS levels for the Gmut-pro construct peaked at ZT8 in both SD and LD grown plants (Fig. 2.4B). However, whereas the GUS transcript levels produced by the WT-pro construct were greater in SD plants, the levels produced by the Gmut-pro construct were approximately the same in the SD and LD grown plants (Fig. 2.4B). These results were consistent with the G-box having a role in photoperiod regulation of CBF2. To confirm this result, we determined the GUS transcript levels at ZT8 for the WT-pro and Gmut-pro constructs in 8 independent transgenic lines grown under SD and LD conditions (Fig. 2.4C). The results indicated that the GUS transcript levels obtained with the WT-pro construct were, on average, 2-fold higher in the SD plants as compared to LD plants, and that this difference was eliminated when the G-box was mutated (Fig. 2.4D). In addition, the results indicated that mutation of the G-box resulted in higherlevel expression of the reporter gene in both SD and LD grown plants (Fig. 2.4E), a finding that was consistent with the element having a repressive effect. PIF4 and PIF7 Repress Expression of the CBF Pathway under LD Conditions Kidokoro et al. (66) found that the PIF7 transcription factor binds in vitro to the Gbox within the CBF2 promoter (the G-box present in the WT-pro GUS fusion described above) and represses expression of the CBF genes during the subjective night phase in circadian regulation experiments (i.e., the plants were shifted from a day-night cycle to constant light and gene expression was determined). Thus, we considered PIF7 to be a candidate for mediating photoperiodic control of CBF2 expression. In addition, we 27 considered PIF4 a candidate as it has been reported to physically interact with PIF7 (66) and like other PIFs, it binds to G-box and related E-box (CANNTG) motifs (128). To test whether PIF4 or PIF7 were involved in photoperiodic regulation of CBF2, we asked whether pif4 or pif7 null mutations affected the patterns of CBF2 expression under LD or SD conditions. Our results indicated that neither of the single mutations had an effect; CBF2 transcript levels peaked at ZT8 in WT, pif4 and pif7 plants and were about 3-fold higher in SD plants than in LD plants (Fig. 2.5 A and B). However, the pif4 pif7 double mutation eliminated the photoperiodic regulation of CBF2; at ZT8, CBF2 transcript levels in pif4 pif7 double mutant plants grown under LD conditions were the same as in WT plants grown under SD conditions (Fig. 2.5C). The pif4 pif7 double mutation also eliminated the differences in transcripts levels at ZT8 observed for CBF1, CBF3, COR15a and GOLS3 in WT plants grown under SD and LD conditions (Fig. 2.5D). These results indicated that PIF4 and PIF7 function redundantly to repress expression of the CBF pathway under LD conditions. The lower level of CBF expression under LD conditions could have resulted from higher-level expression of PIF4 and PIF7 in plants grown under LD. Indeed, we found that the transcript levels for PIF4 and PIF7 oscillated over the course of the day under both LD and SD conditions, peaking at ZT8 and ZT4 respectively, and that the levels for both genes were higher under LD conditions (Fig. 2.6A). In addition, the steady-state accumulation of the PIF4 and PIF7 proteins was slightly greater under LD conditions. This was determined by examining the accumulation of TAP-tagged PIF4 and CFPtagged PIF7 in transgenic plants carrying these protein fusions placed under control of the CaMV 35S promoter. Whereas the transcript levels for the two transgenes were not 28 affected by photoperiod (Fig. 2.6C and D), the protein levels of both PIF4-TAP and PIF7-CFP at ZT8 were about two-fold higher in LD plants as compared to SD plants (Fig. 2.6B). Additional experiments indicated that PIF4-TAP and PIF7-CFP proteins were functional repressors; in the higher expressing lines, both PIF4-TAP and PIF7CFP reduced CBF2 transcript levels by about 50% at ZT8 under SD conditions (Fig. 2.7 A and B); similar results were obtained testing CBF2 expression over a 24 h growth period (Fig. 2.7C-F). Under LD conditions, PIF4-TAP also reduced CBF2 transcript levels by about 50%, whereas PIF7-CFP had little or no effect suggesting that the endogenous PIF7 levels were saturating in regard to CBF2 repression under LD conditions. Finally, using the electromobility shift assay we established that both PIF4 and PIF7 could bind to G-box motifs in the CBF1 and CBF2 promoters and the E-box motif in the CBF3 promoter (Fig. 2.8). Moreover, the results of chromatin immunoprecipitation (ChIP) experiments indicated that the PIF7-CFP protein bound at the CBF locus at ZT8 in LD plants. In test experiments using antibody against CFP, significant enrichment of PIF7-CFP was detected throughout the CBF locus (Fig. 2.9A); in mock experiments using non-immune serum, no enrichment was observed (Fig. 2.9B). PHYB is Required for Photoperiodic Regulation of the CBF Pathway and Freezing Tolerance PHYB is known to physically interact with both PIF4 and PIF7 (131, 132). Thus, we asked whether PHYB was required for photoperiodic regulation of the CBF pathway. 29 Our results indicated that it was. Under LD conditions, the transcript levels for CBF2 were about 3-fold higher at ZT8 in plants carrying a phyB null mutation than they were in WT plants, and matched the CBF2 transcript levels observed in WT plants grown under SD (Fig. 2.10A). This was also true for CBF1, CBF3 and the CBF regulon genes COR15a and GOLS3; under LD conditions at ZT8, the transcript levels for these genes were about 3-fold higher in phyB plants than in WT plants and approximated the transcript levels in WT plants grown under SD (Fig. 2.10B). Consistent with these results, the phyB mutation eliminated the photoperiodic regulation of freezing tolerance; the freezing tolerance of phyB plants grown under LD conditions was equal to that of WT plants grown under SD (Fig. 2.10C). Discussion Here we show that Arabidopsis, like woody perennial trees, can sense shortening day-length as a harbinger of coming cold temperatures and respond by increasing in freezing tolerance. Although the increase that we observed, about 2°C, is modest in comparison to the increase that typically occurs in woody tree species, it is about the same as that reported for the perennial herbaceous species G. coccinea (95). Moreover, it is a considerable portion of the maximum increase in freezing tolerance that occurs in Arabidopsis in response to low temperature, which is about 6°C (133, 134). Thus, it is likely that the SD-induced increase in freezing tolerance that occurs in Arabidopsis has adaptive value in nature protecting plants against sudden early autumn frosts. It will be of interest to determine whether there is significant natural variation in 30 photoperiodic regulation of freezing tolerance among Arabidopsis accessions and if there is, to understand the relationship of these differences to the environmental conditions that characterize the geographical locations from where the accessions originate. It will also be of interest to determine whether photoperiodic regulation of freezing tolerance has been overlooked as a common feature of cold acclimation in frost hardy herbaceous plants. Our results indicate that the increase in freezing tolerance that occurs in response to SD conditions is due to a loss of negative regulation of freezing tolerance under LD conditions. In particular, our results show that PHYB mediates repression of freezing tolerance under LD conditions and that this is due, at least in part, to PHYBmediated repression of the CBF pathway. The key findings supporting these conclusions are two-fold. First, whereas the freezing tolerance of LD WT plants was about 2°C less than that of SD WT plants, the freezing tolerance of LD phyB plants was equal to that of SD WT plants (Fig. 2.10C). Second, whereas the transcript levels for CBF1, CBF2, and CBF3 and downstream CBF regulon genes were lower in LD WT plants than they were in SD WT plants, they were about the same in LD phyB plants and SD WT plants (Fig. 2.10A and B). This down-regulation of the CBF pathway under LD conditions could have adaptive value in at least two ways. One is that it would diminish the allocation of energy and nutrient resources towards unneeded frost protection during the active growing season. In addition, it would mitigate CBF-induced retardation of growth during the growing season. Achard et al. (24) have shown that activation of the CBF pathway results in up-regulation of gibberellin 2-oxidase genes causing a decrease in the levels of active gibberellins. This, in turn, results in an 31 increase in the levels of DELLA proteins, a small family of regulatory proteins that inhibit growth (135). Achard et al. (24) have presented evidence that the CBF-programmed repression of growth caused by the DELLA proteins contributes to the increase in freezing tolerance that occurs with cold acclimation. How does PHYB mediate repression of the CBF genes? A simple working model is suggested from what is known about phytochrome signal transduction and our results indicating a role of PIF4 and PIF7 in regulating the CBF pathway. It is well established that exposure of plants to white light converts the inactive Pr form of PHYB to the active Pfr form, which rapidly moves from the cytoplasm into the nucleus where it interacts with PIF transcription factors to alter gene expression (128, 129). We propose that PHYB is converted to the Pfr form in the morning, moves into the nucleus where it interacts with either homo- or heterodimers of PIF4 and PIF7 (or both), and that the PHYB-PIF4/PIF7 complexes bind to the promoters of the CBF genes at the G-box and E-box motifs and down-regulate their transcription. This model (Fig. 2.11) is supported by multiple findings. First, it has been shown that PIF4 and PIF7 form homo- and heterodimers (66, 136) and that they physically interact with the Pfr form of PHYB (131, 132). Second, our results indicate that PIF4 and PIF7 repress expression of the CBF genes; whereas the peak transcript levels for CBF1, CBF2, and CBF3 and downstream CBF regulon genes were about 3-5 fold lower in LD WT plants than in SD WT plants, they were about the same in LD pif4 pif7 double mutant plants and SD WT plants (Fig. 2.5). Single pif4 and pif7 mutations did not affect expression of the CBF genes indicating that PIF4 and PIF7 act redundantly to repress expression of the CBF pathway (Fig. 2.5). This conclusion is further supported by our observations that: constitutive 32 overexpression of PIF4-TAP and PIF7-CFP reduced the transcript levels of CBF2 at ZT8 in SD plants (Fig. 2.7); both PIF4 and PIF7 bind in vitro to G-box and E-box motifs within the CBF locus (Fig. 2.8); the G-box at position -112 to -107 of the CBF2 promoter is involved in photoperiod-regulated transcription of CBF2 (Fig. 2.4); and PIF7 can bind in vivo throughout the CBF locus at ZT8 under LD conditions (Fig. 2.9). Although this working model is strongly supported by our results, a key aspect of it—that the Pfr form of PHYB interacts with PIF4 to form a complex that represses expression of the CBF genes—would appear to be in conflict with results indicating that the interaction of PHYB-Pfr with PIF4 results in degradation of PIF4 (137). It is true that the interaction of PHYB-Pfr with PIF7 does not lead to degradation of PIF7 (131), and thus, interaction of PHYB-Pfr with heterodimers of PIF4 and PIF7 might result in stable protein complexes. However, this would not explain our observed repression of the CBF genes in the pif7 mutant (Fig. 2.5). In our model, this repression would involve interaction of PHYB-Pfr with PIF4. A detailed analysis of the PHYB and PIF protein complexes that are physically present at the CBF locus under SD and LD photoperiods will be required to resolve this issue. Whereas our results clearly establish a role for PHYB, PIF4 and PIF7 in repressing the expression of CBF1, CBF2 and CBF3, they only provide a partial explanation for why the CBF pathway is repressed to a greater extent under LD conditions than under SD conditions. Our results indicate that PIF4 and PIF7 transcripts peak at about a two-fold higher level under LD conditions (Fig. 2.6A) and that the PIF4 and PIF7 proteins are about two-fold more stable under LD conditions (Fig. 2.6B). These results suggest that greater levels of the PIF4 and PIF7 proteins account, 33 at least in part, for the greater repression of the CBF pathway under LD conditions. However, the molecular basis for the greater expression of PIF4 and PIF7 under LD conditions is unknown. Additional study will be required to address this issue. A final note regards the near overlap in peak expression of PIF4 and PIF7 with that of the CBF genes. If PIF4 and PIF7 were expressed at high levels when expression of the CBF genes was low (at ZT16, for instance), their effects on the CBF pathway would be minimal. A key facet of PIF4 and PIF7 control of CBF expression is the concordant expression of these genes. The CBF genes are regulated by the circadian clock in plants exposed to normal warm growth temperature (64, 66, 130) and their peak expression is driven largely by the Myb transcription factors CCA1 and LHY (65), key components of the core circadian regulatory loop (138, 139). The CCA1 and LHY proteins, which have peak levels in the morning, bind to the Evening Element (EE) and related DNA regulatory motifs present within the CBF locus and induce high-level expression of the CBF genes at ZT8 (65). One simple model would be that PIF4 and PIF7 are also circadian-regulated and timed to peak in the morning hours. Indeed, the oscillation in PIF4 transcript levels is disrupted by constitutive overexpression of CCA1 (140), a classic indicator of circadian regulation. In addition, the promoters of PIF4 and PIF7 genes have EE motifs that could potentially drive their circadian regulation. Future experiments will be directed at testing this hypothesis and how output from the clock is integrated with photoperiodic regulation of the CBF pathway to condition freezing tolerance. 34 Figure 2.1 Arabidopsis freezing tolerance is regulated by photoperiod. (A) WT plants were grown under SD or LD conditions for 5 weeks and 3 weeks, respectively, and tested for freezing tolerance using the electrolyte leakage assay. (B) Plants grown under four conditions were tested for freezing tolerance: SD for 5 weeks (SD); LD for 3 weeks (LD); SD for 3 weeks and transferred to LD for 2 weeks (SD to LD); and LD for 2 weeks and transferred to SD for 2 weeks (LD to SD). The results are mean values from three independent experiments (error bars indicate SEM). (For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation) 35 Figure 2.2 The CBF pathway is regulated by photoperiod. WT plants were grown under SD or LD conditions and the transcript levels for CBF1, CBF2, CBF3, and two CBF regulon genes, COR15a and GOLS3, were determined at the indicated times. The expression values were normalized with internal control gene, IPP2. The results are mean values from three independent experiments (error bars indicate SEM). 36 Figure 2.2 (Continued) 37 Figure 2.3 The CBF pathway is regulated by photoperiod. (A) Plants were grown under SD or LD conditions, further grown under the same photoperiod or shifted from SD to LD or LD to SD for the indicated number of days, and the transcript levels for CBF2 were determined at ZT8. (B) Plants were grown as in (A) and the transcript levels for CBF1, CBF2, CBF3, COR15a, and GOLS3 were determined at ZT8 (photoperiod shift was for 7 d). The results are mean values from three independent experiments (error bars indicate SEM). 38 Figure 2.4 A G-box motif within the CBF2 promoter confers photoperiod-regulated gene expression. (A) Diagram of CBF2::GUS reporter fusions used to test role of the G-box (-112 to -107 bp) in photoperiod-regulated gene expression. (B) Transgenic plants carrying the WT-pro and Gmut-pro constructs were grown under SD or LD conditions and GUS transcript levels were determined at the indicated times. (C) The relative GUS transcript levels normalized against IPP2 were determined at ZT8 in 8 independent transgenic lines for each construct are presented. The results are means from two independent experiments (error bars indicate SEM). (D) Ratio of GUS transcript levels at ZT8 in transgenic plants carrying the WT-pro (WT) or Gmut-pro (Gmut) constructs grown under SD or LD conditions. Values are mean ratios from 8 independent transgenic lines in (C) (Student’s t-test, p<0.001) (e) Relative expression levels of the WT-pro and Gmutpro constructs in the experiments described in (D) (Student’s t-test, p<0.001). 39 Figure 2.4 (Continued) 40 Figure 2.5 PIF4 and PIF7 are required for repression of the CBF pathway under LD conditions. (A, B, C) Plants were grown under SD or LD conditions and the transcript levels for CBF2 were determined at the indicated times in WT plants and in pif4, pif7, and pif4 pif7 mutant plants as indicated. (D) Plants were grown under SD or LD conditions and the transcript levels for CBF1, CBF2, CBF3, COR15a, and GOLS3 were determined at ZT8. The results are mean values from three independent experiments (error bars indicate SEM). 41 Figure 2.5 (Continued) 42 Figure 2.6 PIF4 and PIF7 are expressed at higher levels, and their proteins are more stable under LD conditions. (A) WT plants were grown under SD or LD conditions and the transcript levels for PIF4 and PIF7 were determined at the indicated times. (B) The steady-state accumulation of PIF4-TAP (PIF4-OX) and PIF7-CFP (PIF7-OX) in SD or LD transgenic plants at ZT8 were detected with anti-myc or anti-GFP antibodies respectively. Histone H3 protein was used as loading control and detected using rabbit anti-Histone H3 antibodies. n.s. indicates non-specific signals. The results presented are representative of three experiments. (C abd D) The relative transcript levels for the PIF4-TAP (PIF4OX-1 and -2) and PIF7-CFP (PIF7OX-1 and -2) gene fusions were determined at ZT8 in transgenic plants grown under SD or LD conditions. The expression levels were normalized to IPP2. Transcripts for the transgenes were nondetectable (ND) in WT plants. The results are mean values from three independent experiments (error bars indicate SEM). 43 Figure 2.7 Overexpression of PIF4 or PIF7 represses CBF2 expression. Relative levels of CBF2 transcripts in a 24 hour period (C to F) and at ZT8 (A and B) in transgenic plants overexpressing PIF4-TAP or PIF7-CFP grown under LD and SD conditions were determined. The results are mean values from three independent experiments (error * ** bars are SEM). (Student’s t-test, p<0.05; p<0.01) 44 Figure 2.7 (Continued) 45 Figure 2.8 PIF4 and PIF7 bind to CBF1, CBF2 and CBF3 promoters through G-box and E-box motifs. The production of GST-PIF4 and GST-PIF7 recombinant proteins and information of how the EMSAs were performed are described in the Materials and Methods. The probes used are indicated at the top of the gels. The observed binding was specific as it decreased in response to addition of 20-fold and 100-fold G-box (CACGTG) or E-box (CATGTG) sequence as competitor, but not mutated G-box (ggtacc). The sequences of competitors are listed in Table 2.2. 46 Figure 2.9 PIF7 binds at the CBF locus. The CBF locus is comprised CBF1, CBF3, and CBF2 genes in tandem repeat, and the white boxes represent transcribed regions. The location of G-box (CACGTG, circle) and E-box (CANNTG, triangle) were labeled across CBF locus. WT plants and transgenic plants overexpressing PIF7-CFP were grown under LD conditions, tissue was harvested at ZT8, and ChIP assays were performed using (A) )anti-GFP antibody (IP) or (B) IgG. Precipitated DNA sequences were quantified using primer sets across CBF locus (boxes A through N). DNA sequences from ACTIN7 and UBQ10 were used as negative controls. The fold enrichment of precipitated DNA for each primer set in PIF7-OX (PIF7-IP in (A) or PIF7-mock in (B), black bars) samples are relative to the level in the WT samples (WT-IP in (A) or WTmock in (B), open bars). The locations and sequences of primer sets are listed in Table 2.1. Data are presented as mean ± SEM; n=4 (* p<0.05 and ** p<0.01, paired t-test). The location of G-box (CACGTG, circle) and E-box (CANNTG, triangle) motifs are indicated. 47 Figure 2.10 PHYB is required for photoperiod regulation of the CBF pathway and freezing tolerance. (A) WT and phyB plants were grown under SD or LD conditions and the relative transcript levels for CBF2 were determined at the indicated times. (B) WT and phyB plants were grown as in (A) and the relative expression levels of CBF1, CBF2, CBF3, COR15a, and GOLS3 were determined at ZT8. (C) WT and phyB plants were grown under SD and LD and tested for freezing tolerance using the electrolyte leakage assay. The results are mean values from three independent experiments (error bars indicate SEM). 48 Figure 2.10 (Continued) 49 Figure 2.11 Model of photoperiodic regulation of the CBF pathway and freezing tolerance. 50 Table 2.1 List of primers for qRT-PCR and ChIP assays. Name Locus primer (Fw or Rv) qRT-PCR CBF1 AT4g25490 CBF2 AT4g25470 CBF3 AT4g25480 COR15a AT2g42540 GOLS3 AT1g09350 IPP2 AT3g02780 PIF4 AT2g43010 PIF7 AT5g61270 Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv CCGCCGTCTGTTCAATGGAATCAT TCCAAAGCGACACGTCACCATCTC CGACGGATGCTCATGGTCTT TCTTCATCCATATAAAACGCATCTTG TTCCGTCCGTACAGTGGAAT AACTCCATAACGATACGTCGTC GAAAAAAACAGTGAAACCGCAGAT CCACATACGCCGCAGCTT CTGACGAGCGAGGTTCTTGTC AACAAATTCTAAGTAAACATCACCAGTT ATTTGCCCATCGTCCTCTGT GAGAAAGCACGAAAATTCGGTAA TCTCCGACCGGTTTGCTAGA CGCGGCCTGCATGTGT CAAGTGCGAGTGGTACCAATATG TTCAAGCTCCGACCGGATT TGGCCTGGCAGGAGAAACT CGTATCCACGCCGTATTCG TAP (myc) Fw TGCAGCCTAGGGATTACGATATC CFP Rv Fw Rv GGCCCCTGGAACAGAACTTC GTCCGCCCTGAGCAAAGA TCCAGCAGGACCATGTGATC GUS primer sequence 51 position + 734 + 774 + 562 + 630 + 694 + 741 + 704 + 750 + 1090 + 1137 + 115 + 155 + 1360 + 1397 + 484 + 523 Table 2.1 (Continued) Name ChIP A Locus primer (Fw or Rv) AT4g25490 B AT4g25490 C AT4g25490 D AT4g25490 E AT4g25480 F AT4g25480 G AT4g25480 H AT4g25480 I AT4g25480 J AT4g25470 K AT4g25470 L AT4g25470 M AT4g25470 Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv primer sequence TGCTTTCAAGGCCGAATGAT CGTTCTCATTCCACGTGTGATG TTACCACTCTTTTTTTCCCTCTTTG CTCGCTCTCACGTTATTGACATTT TCTTTACAAGGGTCAAAGGACACA GCGAAGCAATCCCACGAT CCGCCGTCTGTTCAATGGAATCAT TCCAAAGCGACACGTCACCATCTC AGTTCTATCGGACTAATTCTTGGCTTA GATGATCAAGCGTAATTGCTTTGT TGACTAAGGACGTGGTGGTTGA AGCGCACTTCCTTCTCACTCA TGTTACATTTGATCATTCACCCAAA CGTATATAAGCACGTAAGTCACCAAGT CGTGGCATTACCAGAGACACA GCGGAAGATATTTTAGAGGCAAAA TTCCGTCCGTACAGTGGAAT AACTCCATAACGATACGTCGTC CAAGAGAGCACTGTCCGTAGCTT TGGTTACAAGAGGAGCCACGTA TTTGCCGGAAAACTCAACTCA CCTTCTTTTTGGTCTGAAA GAGAGATGCTGGAAATTGTGATCA AAATATGGTAAGTGGTTAGGCGAAA GGGTCAAAGGACACATGTCAG GAACGCGGAGTTTCTGTCTC 52 position - 1312 - 1247 - 845 - 801 - 186 - 142 + 734 + 774 - 1859 - 1752 - 1235 - 1178 - 604 - 550 - 124 - 83 + 694 + 741 - 1851 - 1811 - 1147 - 1108 - 943 - 897 - 201 - 102 Table 2.1 (Continued) Name ChIP N Locus primer (Fw or Rv) AT4g25470 Actin7 AT5g09810 UBQ10 AT4g05320 Fw Rv Fw Rv Fw Rv primer sequence CGACGGATGCTCATGGTCTT TCTTCATCCATATAAAACGCATCTTG CGTTTCGCTTTCCTTAGTGTTA AGCGAACGGATCTAGAGACTC TCCAGGACAAGGAGGTATTCCTCCG CCACCAAAGTTTTACATGAAACGAA 53 position + 562 + 630 + 54 + 167 + 1616 + 1796 Table 2.2 List of primers for cloning and EMSA Name Locus Cloning CBF2-pro AT4g25470 CBF2-pro-Gmut AT4g25470 PIF4 AT2g43010 PIF7 AT5g61270 EMSA CBF1-pro AT4g25490 CBF2-pro AT4g25470 CBF3-pro AT4g25480 6X G-box 6X E-box 6X G-box mutated Primer (Fw or Rv) Primer sequences position Fw Rv Fw Rv Fw Rv Fw Rv CAAGATGGGTCAAAGGACACATGTCAGATT TGATCAGAAGAGTACTCTGTTTCAAGAAACTGGA TTAGCTGTTTCTTATCggtaccGCATTCACAGAGACAGA TCTGTCTCTGTGAATGCggtaccGATAAGAAACAGCTAA ATGGAACACCAAGGTTGGAGTTTTGAGGAGAA CGCGGCCTGCATGTGT CAAGTGCGAGTGGTACCAATATG TTCAAGCTCCGACCGGATT - 189 -1 - 133 -95 + 234 + 1397 + 484 + 523 Fw Rv Fw Rv Fw Rv AAGAACTCATAAAGGTTAACGAGTGAAGAGTCAAAAG TGTGTAGTTAGTATAAAAAGTGAGAGTGAGAATTGGT CAAGATGGGTCAAAGGACACATGTCAGATT TGATCAGAAGAGTACTCTGTTTCAAGAAACTGGA ACGGTTACCCTACACCTAGTACACTAAATCCT ACGGAGTTTGTGTCTCTGGTAATGCCACGT CACGTGCACGTGCACGTGCACGTGCACGTGCACGTG CATGTGCATGTGCATGTGCATGTGCATGTGCATGTG GGTACCGGTACCGGTACCGGTACCGGTACCGGTACC - 189 -1 - 224 -1 - 316 - 96 54 Materials and Methods Plant Materials and Growth Conditions Arabidopsis thaliana Columbia-0 WT and mutant derivatives were used in all experiments. Plants carrying the pif7-1, pif4-2, pif4-2 pif7-1, and phyb-9 null mutant alleles were kindly provided by Dr. Peter Quail (University of California, Berkeley, CA) (131). Seeds were stratified for 3 to 5 d at 4°C in the dark and then grown under either SD (8h light, 16h dark) or LD (16h light, 8h dark) conditions. For gene expression studies, plants were grown under SD (~12 d) or LD (~10 d) conditions on sterilized Gamborg’s B5 medium (Caisson Laboratories). For freezing tolerance studies, plants were grown in soil as described (49) under SD (~ 5 weeks) or LD (~ 3 weeks) conditions 2 -1 unless indicated otherwise. All plants were grown at 22°C under ~100 µmol m- s fluorescent white light. Determination of Transcript Levels Transcript levels were determined using real-time quantitative RT-PCR (qRTPCR). Plant sample collection, RNA extraction, and qRT-PCR were performed as described (49), except that 200 ng of total RNA were used for 20 µl reverse transcription reaction, and 2 µl of 10-fold diluted cDNA was used as template in a 10 µl reaction for qRT-PCR.In the SD and LD experiments, the 8 h and 16 h time points, respectively, were taken during the light phase; in both experiments, the 24 h sample was taken during the light phase. IPP2 (ISOPENTENYL PYROPHOSPHATE-DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2) was used as the reference gene. Primers are listed in Table 2.1. 55 CBF2 Promoter Reporter Lines The CBF2 promoter fragment from -207 bp to + 134 (just upstream of the ATG) was first cloned into pCR®8⁄GW⁄TOPO® vector (Invitrogen). The G-box at -112 to -107 bp, CACGTG, was converted to GGTACC by site-directed mutagenesis using the Quick Change kit (Stratagene). The WT and mutagenized promoter fragments were fused to the GUS reporter in the pMDC164 vector (141) using a recombination reaction (Invitrogen), and then transformed to WT Arabidopsis using the floral dip method (142). Transgenic lines in the T3 generation were used for experiments. The primers used for cloning are listed in Table 2.2. Transgenic Lines Overexpressing PIF7 or PIF4 For 35S::PIF7-CFP-HA, the open-reading frame (ORF) sequence of PIF7 was cloned into the pCR®8⁄GW⁄TOPO® vector (Invitrogen), transferred to the plant binary vector pEarleyGate102 (143) by recombination, and then transformed into WT Arabidopsis plants. The cloning primers are listed in Table 2.2. For 35S::PIF4-TAP lines, a plant binary vector containing the ORF of PIF4 fused to the TAP-tag (DKLAT2G43010) was obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu) and transformed into WT Arabidopsis plants. Electromobility Shift Assay (EMSA) To prepare recombinant PIF4 and PIF7 protein for EMSA, the ORF of PIF4 or PIF7 was first cloned into pCR®8⁄GW⁄TOPO® vector (Invitrogen), and then moved to pET-60-DEST vector (Stratagene) through recombination reactions (Invitrogen). The 56 recombinant proteins were induced in E. coli BL21-Gold (DE3) strain by addition of 0.1 mM of IPTG at 30°C for 6 h. The crude protein was extracted with B-PER Protein Extraction Reagent (Pierce), and quantified with BCA Protein Assays (Pierce). The probes for EMSA were prepared by amplification of CBF1 (-224 to -1), CBF2 (-189 to -1), and CBF3 (-316 to -94) promoter regions by PCR from the genomic DNA, and then end-labeled with gamma 32 P. The WT and G-box mutated competitors were made by annealing of G-box (CACGTG), E-box (CATGTG) or mutated G-box (ggtacc) primers. The primers are listed in Table S2. The EMSAs were performed as described (49), except 20 ng of crude protein extract, 4 fmole of probes, and 20-fold and 100-fold competitors were used in the binding reactions. The samples were resolved on 5% polyacrylamide as described (144). Protein Extraction and Immunoblots Protein from Arabidopsis seedlings was obtained by heating samples at 70°C for 10 minutes in extraction buffer (60 mM Tris-HCl, pH8.5; 2% SDS; 2.5% Glycerol; 0.13 mM EDTA, pH8.0; protease inhibitor cocktail (Roche)). The soluble protein was quantified with DC Protein Assay (Bio-Rad). 100 μg of total soluble protein with 5% βmercaptoethanol was separated on 4-12% NuPAGE® SDS-PAGE (Invitrogen) followed by western blotting analysis. Immunodetection of PIF4-TAP and PIF7-CFP-HA was done using rabbit anti-myc monoclonal antibodies (71D10, Cell Signaling) and mouse anti-GFP antibodies (11814460001, Roche), respectively. Histone H3 was detected with rabbit anti-Histone H3 antibodies (07-690, Millipore). Corresponding secondary 57 antibodies conjugated with horseradish peroxidase (Thermo Scientific) and SuperSignal West Pico or Femto Chemiluminescent Substrate kits (Thermo Scientific) were used for detection. Chromatin Immunoprecipitation (ChIP) The ChIP assays were performed as described previously (65) with minor modifications. The 35S::PIF7-CFP-HA line and wild type (WT) 14-day-old seedlings were grown under LD and harvested at ZT8. For each biological replicate, IP and mock samples were normalized to the total input for each line, and the fold enrichment was relative to WT. Paired t-test was applied to test the statistical significance of fold enrichment for each primer sets. The primers for qRT-PCR are listed in Table 2.1. Acknowledgments We thank Peter Quail for providing the pif7, pif4, pif4 pif7, and phyB mutants, Tiffany Liu for providing primers for ChIP, and Sarah Gilmour for helping prepare the manuscript. This research was funded primarily by Grant DBI 0701709 from the National Science Foundation Plant Genome Project, but included infrastructure support provided by Grant DE-FG02-91ER20021 from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy and the Michigan Agricultural Experiment Station. 58 CHAPTER 3 TRANSCRIPTIONAL REGULATORY NETWORKS OF COLD ACCLIMATION IN ARABIDOPSIS THALIANA 59 PREFACE The work presented in Chapter 3 was in collaboration with Dr. Sunchung Park. Dr. Park was responsible for the cluster analysis of cold stress transcriptome data and selection of cold-induced transcription factors for further experiments. The expression kinetics of CBF1, CBF2, CBF3, ZF, CZF1, RAV1, CZF2, MYB73, ZAT12, DOF1.10, DOF1.3, DEAR2, ZAT10, ANAC62, AZF2, and RVE2 with qRT–PCR was examined by the author of this dissertation, and Dr. Park was responsible for HSFC1, DEAR1, MYB44, ERF5, ERF6, CRF2, CRF3, WRKY33, ATHB2, AXR5, WRKY22, WRKY40, ERF11, and MYB7. For generation of transgenic lines, characterization of phenotypes, and microarray experiments, the author of this dissertation was responsible for the overexpression lines of CZF1, CZF2, MYB73, RAV1, ZAT10, ANAC62, and ZF, and Dr. Park worked on the overexpression lines of HSFC1, MYB44, DEAR1, ERF5, WRKY33, ERF6, CRF3, and CRF2. Further the transcriptome analysis, functional analysis and verification with qRT-PCR in the ZF-OX lines were performed by the author of this dissertation. 60 Abstract Low temperatures profoundly affect growth, development, and survival of plants. Many temperate plants can acquire freezing tolerance via exposure to low, non-freezing temperature, a process known as cold acclimation. Extensive alterations of biochemical and metabolic pathways occur during cold acclimation that help plants adapt to low temperatures. Many of these changes are brought about through reprogramming transcription. The expression of thousands of cold-regulated (COR) genes are affected by cold, and their expression occurs in successive waves, suggesting hierarchical regulation. The well-studied CBF pathway and its parallel ZAT12 pathway, however, account for the expression of less than 10% of these COR genes. In this study, fifteen transcription factors (TF) sharing a similar expression pattern to that of the CBFs and ZAT12 in the cold were identified. Additionally, transcriptome analysis was performed to understand the regulation of the COR genes. The results showed that these TFs likely regulate about 29% of the COR genes, and they regulate different sets of COR genes with substantial overlap, which indicated that extensive crosstalk and functional redundancy in COR gene expression. Functional enrichment analysis of the COR genes regulated by each TF indicated that they regulate diverse, but overlapping, biological pathways in cold acclimation. Among these pathways regulated by these cold-induced TFs, the COR genes that are regulated by a zinc-finger transcription factor (ZF, AT4G29190) exclusively enriched in starch metabolism and circadian rhythm pathways, which have been established to be strongly affected by cold. These results shed light on the regulation of COR genes by CBF-independent pathways and their potential roles in freezing tolerance. 61 Introduction Low temperature is one of the adverse environmental factors that limit land usage and affect crop production. To cope with cold stress, many temperate plants have evolved the ability to acquire freezing tolerance through exposure to low, nonfreezing temperatures, a process known as cold acclimation (1). This process is associated with the alteration of biochemical processes and metabolic pathways (27, 28, 145). Several major metabolic adjustments not only prevent cell damage from ice formation, but also allow the development of resistance to other stresses, such as drought, osmotic, oxidative, and photo-oxidative stresses associated with cold stress (7, 11, 29). Long-term adaptations, such as changing plant architecture by modulating hormone pathways and adjustment of energy utilization are also established during cold acclimation (22, 25, 117). Recently, it was demonstrated that low temperatures have global effects on major metabolic pathways through regulating the circadian clock; however, the mechanism for this is not well understood (30, 64). The metabolic adjustments and physiological adaptations associated with cold acclimation are largely caused by extensive reprogramming of transcription during cold acclimation (27-30). Hundreds to thousands of cold-regulated (COR) genes were identified as changing their expression during cold acclimation depending on the conditions of plant growth and treatments and the expression profiling platforms used (38, 62, 112-115). Although the functions of the COR genes are not fully understood, Gene Ontology (GO) analysis of COR genes and co-expression analysis have revealed that COR genes are involved in diverse biological pathways (38, 113, 115, 146). The expression kinetics of the COR genes reveals successive waves in time-series 62 expression profiling experiments, suggesting hierarchical regulation of the COR genes, i.e., the early cold-induced genes regulate the ones that respond later (28, 38, 62). In Arabidopsis, a group of early cold-induced C-REPEAT BINDING FACTORS, or CBF1, CBF2, and CBF3, play prominent roles in regulating COR genes and cold acclimation (5, 32). They encode transcriptional activators of the AP2/ERF transcription factor family and bind specifically to CRT/DRE (RCCGAC) elements in the promoters of their target genes. (33, 34, 62). Overexpression of CBFs at warm affects expression of about 140 COR genes, or the “CBF regulon”, confers freezing and drought tolerance and results in small stature of the plants (36, 62, 119). The functions of CBF regulon genes are not fully understood. Some of them encode enzymes involved in biosynthesis of sugars or proline and membrane stabilizing proteins (35, 147). Others encode late cold-induced transcription factors, such as RAP2.1, which negatively regulates some CBF regulon genes involved in cold or associated osmotic stresses (62, 148) Transcriptome and quantitative trait locus (QTL) studies have revealed multiple cold regulatory pathways besides the CBF pathway (44, 98, 99, 149). ZAT12 represents a cold regulatory pathway parallel to the CBF pathway, but is much less well-studied (62). ZAT12 encodes a C2H2 zinc-finger transcription factor with a expression pattern similar to that of the CBFs in the cold under continuous light. Overexpression of ZAT12 increases resistance to cold and oxidative stress (62, 150). Transcriptome analysis of transgenic lines overexpressing ZAT12 showed that ZAT12 regulates around 70 COR genes, termed the “ZAT12 regulon”, which has a small overlap with the CBF regulon (62). Interestingly, ZAT12 negatively regulates the CBFs, 63 which indicates multiple pathways and a complex interaction between pathways in the regulation of COR genes during cold acclimation. In addition to ZAT12, GIGANTEA (GI) is the only cold-induced transcription factor identified as regulating a CBF-independent pathway, although the transcriptome was not examined (103). Deciphering transcriptional regulatory networks that regulate cold acclimation provides information for the molecular basis of cold acclimation. This knowledge has the potential to help find molecular tools for engineering crops to improve crop quality (151). Chawade et al. applied computational modeling based on co-expression analysis and transcription factor binding sites to predicted transcription factors (TF) regulating COR genes in Arabidopsis thaliana (110). Several cold-regulated TFs were identified as putative regulators; however, there is largely a lack of experimental validation. One limitation in validating the predicted results and identifying new regulators is the functional redundancy of TFs in Arabidopsis. The goal of this study was to systematically identify early cold-upregulated TFs that regulate COR genes in CBF-independent pathways, and to dissect the biological pathways regulated by these TFs. Fifteen selected TFs were ectopically expressed in Arabidopsis thaliana and subjected to transcriptome analysis with Arabidopsis ATH1 GeneChips. The regulons of seven tested TFs, ZAT10, HSFC1, CZF1, ZF, MYB44, DEAR1, and ERF5, were compared to the CBF and ZAT12 regulons, and the results showed considerable overlap of COR genes, suggesting the COR genes are tightly regulated by multiple regulators. Functional analysis of these TFs indicated that they regulate different biological and metabolic pathways in cold acclimation, with some overlap of functions. In the transgenic plants overexpressing a zinc-finger transcription 64 factor (ZF, AT4G29190), the expression of genes associated with the circadian clock and carbohydrate metabolic pathways were affected. The function of ZF in cold acclimation is discussed. Results Expression Kinetics of COR Genes Reveals Successive Waves of Transcription in Cold Acclimation From previous studies, the number of the cold-regulated (COR) genes can vary depending on the experimental settings (38, 62, 112-115). To define the COR genes in this study, Arabidopsis low temperature time course expression profiling experiments (GSE5621 and GSE5620) (113) from ATGenExpress were analyzed. In these datasets, the WT Arabidopsis plants were grown in long-day conditions (16 h light and 8 h dark), and the tissue was harvested 3 h after dawn either with or without cold treatment for 0, 0.5, 1, 3, 6, 12, and 24 h. The expression of 2397 probe sets, which corresponds to 2455 genes based on the TAIR10 database (Appendix Table A1), was found to be changed more than 4-fold (FDR< 0.01) at one or more time points at 4°C by comparison of cold-treated samples (GSE5621) and warm samples (GSE5620). These COR genes were clustered into 6 groups (G1 to G6, Fig. 3.1A) based on their temporal expression patterns in the cold with the k-mean clustering (k=6) algorithm. The expression of these 6 groups (G1 to G6) of COR genes revealed successive waves of transcription, with 1091 genes mostly down-regulated (G1 and G2) and 1364 genes up-regulated (G3 to G6) sequentially in a 24h cold-treatment period. 65 The expression in cold samples was compared to that in warm samples at each time point rather than only at 0h because it has been demonstrated that COR genes are regulated by the circadian clock and have daily cycling expression patterns at warm temperatures (69, 71). However, it is possible that the COR genes selected by these criteria may be “apparently” affected by cold rather than “actually” regulated by cold. For example, the expression of genes possibly remains constant in the cold, but the expression cycles at warm temperature, thus resulting in up- or down-regulation when cold and warm samples are compared. To test this possibility, the expression of coldregulated genes at each time point compared to 0h for the COR genes defined in the previous section (cold/warm) were plotted (Fig. 3.1B), and this comparison was defined as “cold/0h”. The overall expression patterns of the previously defined cold/warm and cold/0h are very similar (Fig. 3.1 A and B). The primary differences are in G3, G4 and G6. The induction levels of many genes at 1, 3, 6, 12, and 24h in G3 and at 6 and 12h in G4 in cold/0h are much lower compared to that in cold/warm. Conversely, expression is considerably repressed at 3, 6, and 12h in G6. To test whether these differences are due to diurnal expression in the warm, the expressions of warm samples compared to 0h for the COR genes defined by cold/warm was plotted (Fig. 3.1C), and this comparison was defined as “warm/0h”. Indeed, the expression level of G3 genes at 1, 3, and 6h and that of G4 genes at 6 and 12h were decreased compared to 0h (Fig 3.1C). In G6, the peak expression of some genes at 3, 6, and 12h in warm/0h resulted in repression at 3 and 6h without much induction at 12h in cold/warm compared to cold/0h (Fig 3.1 A-C). Although the expression levels are not identical in cold/warm and cold/0h, 66 it does not change the general expression kinetics for each cluster. Therefore, the cold/warm comparison was used to define COR gene sets for further analysis. Identification of Early Cold-induced TFs in the Same Cluster as CBFs and ZAT12 The sequential waves of expression patterns of the COR genes suggested hierarchical regulation. To identify regulators parallel to the CBF and ZAT12 pathway, we first identified 174 transcription factors up-regulated in cold from G3 to G6 in Fig. 3.1A based on Gene Ontology (GO) (Table 3.1). These 174 TFs were subjected to kmean clustering analysis (k=7) to find the TFs sharing similar expression kinetics to that of CBFs and ZAT12. Among seven clusters (TFC1 to TFC7 in Fig. 3.2), CBFs and ZAT12 are members of TFC3; there are 26 additional transcription factors in the same cluster. We first confirmed that these 26 transcription factors are induced in cold. Plants were grown under the same photoperiodic conditions and harvested at the same time points as in the ATGenExpress experiments (113), and the transcript levels were examined by quantitative RT-PCR (qRT-PCR). The expression kinetics of the TFs in the cold were compared to the expression in the warm (cold/warm, blue solid lines in Fig 3.3) or at 0h (cold/0h, red dashed lines). Of the 26 transcription factors examined, 24 of which were induced to different levels in cold/warm, which agrees with the results from transcription profiling experiments, except for MYB7 and ERF11. However, the expression levels were generally lower than those observed in the array experiments. A possible cause for this is the difference in growth conditions, such as light intensity and growth media. 67 The general expression patterns of TFs in the TFC3 peak at 3 or 6h after cold treatment, and even persist at 12 and 24h (Fig. 3.2). The CBFs were highly induced at 3h in the cold and then the expression dropped (Fig. 3.2). Some TFs, including ZF, RAV1, MYB73, ZAT10, DEAR1, ERF5, CRF2, WRKY33, ERF6, CRF3, RVE2, and WRKY 40, showed a similar expression pattern to the CBFs. The expression of ZAT12 peaked at 3h cold, decreased at 6h and then increased again at 12 and 24h, which is different from a previous report under continuous light (62). The differences between the expression patterns of the CBFs and ZAT12 is likely due to the diurnal conditions in this experiment; this is supported by comparing the cold/warm to cold/0h patterns in Fig. 3.3. Several TFs, including DOF1.10, DOF1.3, CZF1, CZF2, HSFC1, MYB44, and ANAC62 followed a similar expression pattern to ZAT12. When the patterns of cold/warm and cold/0h were compared, CBF1, CBF2, CBF3, ZF, CZF1, RAV1, MYB73, DOF1.10, DEAR2, DOF1.3, AZF2, RVE2, AXR5, ATHB2, and WRKY40 showed very different patterns, which was due to strong cycling of these TFs under warm conditions. Finally, TFs with similar expression patterns to those of the CBFs or ZAT12 were chosen to study the regulation of the COR genes. Transgenic lines ectopically expressing these TFs driven by the CaMV 35S promoter were generated (listed in Table 3.2) with the exception of WRKY40, RVE2, DOF1.3, and DOF1.10, which we failed to generate lines for further experiments. Early Cold-induced TFs Regulate Approximately 29% of COR Genes To identify the COR genes regulated by the 15 early cold-induced TFs described in the section above, two independent transgenic lines overexpressing each TF along 68 with WT were grown under continuous light at 22°C for 14d and their RNA hybridized to Arabidopsis ATH1 GeneChips. The continuous light condition was chosen to reduce any circadian or diurnal effects. We first identified differentially expressed genes (DEG) in TF-overexpressing lines (TF-OX) by comparing transgenic lines to WT using a criterion of more than 2-fold change (p-value< 0.01) (Table 3.2). The DEG were subsequently compared to the defined COR genes to find the COR genes regulated by each TF, which were defined as their regulons (Table 3.2 and Table A1). Combined with the previously published transcription profiling datasets of CBF2-OX and ZAT12OX lines (62), the regulons of these TFs covered around 29% of the COR genes defined in this study. Of these 17 TF-OX lines, some affected expression of more than 50 genes as well as COR genes, whereas the rest of the TF-OX lines had mild effects on gene expression; this result could be due to the low expression of transgenes for the CZF2-OX and MYB73-OX lines (data not shown), or the requirement for posttranslational modification and/or cofactors at low temperature. Transgenic lines overexpressing CBF2, ZAT12, HSFC1, ZF, ZAT10, CZF1, MYB44, DEAR1, and ERF5, were selected for further analysis because the COR genes are over-represented in their DEG (p-value< 1E-8, fisher’s exact test), suggesting these TFs have significant roles in regulating the COR genes. The regulons of each TF are significantly distributed in multiple groups, except for the CBF regulon. The majority of the CBF regulon falls into G5 (Fisher’s exact test, p< 0.01), whose expression patterns are mainly induced after 12h and persist at 24h, consistent with the role of the CBFs functioning as activators. The known CBF direct targets, including COR15a, COR78, COR47, and GOLS3 (35, 152), belong to G5. In 69 contrast, the rest of the TF-OX lines affect both down- (G1 or G2) and up-regulated (G3 to G6) genes (Table 3.2). The regulons for each TF are mainly found in G1, G2, G5, and G6 (p-value< 0.01), whose expression patterns are either repressed or induced after 12h, similar to the induction kinetics of CBF targets. Some DEAR1 regulon genes were significantly distributed in G4, and their expression was repressed by overexpressing DEAR1, which suggests DEAR1 may have role in negatively feedback regulation of these COR genes. We further examined whether the COR genes up-regulated by the TFs are also up-regulated in cold, and vice versa. The correlation of the expression patterns (upand down- regulation) of TF regulons to those of the COR genes at different time points were analyzed (Fig. 3.4). The expression patterns of the CBF, HSFC1, and ZAT10 regulons show a general positive correlation at most of the time points. The strongest positive correlation is only at 12 and 24h for the CBF regulon (spearman’s correlation coefficient, rho> 0.8, p-value < 0.01). The positive correlations suggest CBF, HSFC1, and ZAT10 may have a positive effect in regulating COR genes and therefore freezing tolerance. In the case of MYB44, ERF5, DEAR1, and ZF, however, only a mild negative correlation was found, suggesting that overexpression of these TFs resulted in misregulation of the COR genes. Early Cold-induced TFs Co-regulate a Substantial Number of COR Genes Previous studies have suggested that multiple pathways regulate COR genes (44, 98, 99, 149); we therefore investigated the relationships of the TF regulons identified 70 here. The TF regulons were visualized with Cytoscape (http://www.cytoscape.org/) (153) shown in Fig. 3.5A. Each TF apparently regulates a different set of COR genes represented by an individual circle connected to each TF, while they also co-regulate COR genes with other TFs. Of 696 COR genes regulated by the TFs in this study, 70% of them were regulated by a single TF (Fig. 3.5A and B). However, this number should be interpreted with caution. In this study, only 17 cold-induced TFs were examined, while there are 174 TFs induced in cold. The number of COR genes co-regulated by multiple TFs may increase when more TFs are examined. In addition, several coregulated genes could be missed in this study if they require more than one TF to activate or repress their expression. Another result which supports the high likelihood of co-regulation of COR genes is that around 50% of the TF regulon genes are also regulated by other TFs (Fig. 3.5 C). In the ERF5 and DEAR1 regulons, there are close to 80% co-regulated COR genes. The CBF regulon has fewer co-regulated genes, which is around 35% of its regulon (Fig 3.5C). These results indicate the regulation of COR genes is tightly coordinated among these TFs. Among the co-regulated genes in these TF regulons, there is a significant number of COR genes co-regulated by ZF and HSFC1 (more than 20%, p-value< 0.01, fisher’s exact test) (Fig. 3.5D). It is possible that they are involved in regulating the same biological pathways, such as carbohydrate metabolism shown in Table 3.3. Phenotypic Analysis of the TF-overexpressing Lines It has been demonstrated that low temperatures can modulate the GA and SA pathways to inhibit plant growth (24, 26). Accumulation of SA and its glucosylated 71 derivatives is associated with growth inhibition at low temperatures, although the mechanism has not been studied (26). Overexpression of CBF1 induces expression of GA-inactivating GA-2 oxidase (GA2ox), which leads to the accumulation of the growth inhibiting DELLA proteins and subsequent growth retardation (24). Similar to the CBFoverexpressing lines, when the ZAT12 was overexpressed in Arabidopsis, the plants also exhibited stunted growth (36, 62). We therefore set out to study the growth phenotype and freezing tolerance of the TF-OX lines. WT and two transgenic lines for each TF were grown at 22°C in long-day (16h light and 8h dark) for 19 days. Similar to CBF-OX and ZAT12-OX, most of the TF-OX lines showed dwarf phenotypes except for ERF5-OX (Fig. 3.6). To test whether the dwarfism was caused by changing the GA and/or SA pathways, the expression of genes encoding enzymes involved in SA biosynthesis or its glycosylation, including ICS1, ICS2, PAL1, PAL2, PAL3, PAL4, UGT74F1, and UGT74F2/SGT1, and biosynthesis and inactivation of GA, such as GA20ox, GA3ox, and GA2ox, were investigated in the TF-OX lines. The expression of genes encoding GA inactivating enzymes, GA2ox2 (AT1G30040) and GA2ox6 (AT1G02400), and enzymes in the SA biosynthetic pathway, PAL2 (AT3G53260) and PAL3 (AT5G04230), were down-regulated in HSFC1-OX; whereas no significant difference in other TF-OX lines was observed. These results indicate that the dwarfism could be due to the adverse effects of overexpressing TFs rather than alteration of GA and/or SA pathways. Overexpression of CBFs or ZAT12 enhances freezing tolerance of plants. To test the freezing tolerance of the TF-OX lines, electrolyte leakage assays (ELA) were performed, in which damage to leaves caused by freezing is measured by ion leakage 72 from the leaves. Only HSFC1-OX was slightly increased in freezing tolerance compared to WT in both non-acclimated (Fig. 3.7) and cold-acclimated (Fig. 3.8) conditions. These results are consistent with the positive correlation of expression patterns of COR genes and the HSFC1 regulon (Fig. 3.4). Most of the transgenic lines, however, showed different levels of decrease in freezing tolerance, except for the ERF5-OX under non-cold acclimated condition. The CZF1-OX and DEAR1-OX showed significant reduction in freezing tolerance in both conditions, and chilling sensitive phenotypes were observed as the cold-acclimated transgenic lines had higher ion leakage even without freezing treatments. These phenotypes can be partially explained by negative regulation of CBFs and/or their downstream targets by overexpressing CZF1 or DEAR1 in the cold (63, 154). Another possible reason could be a growth defect when TFs are constitutively expressed. The expression of COR genes is regulated coordinately and expression changed during cold acclimation (Fig. 3.1A). Constitutive induction or repression when TFs were overexpressed may affect the normal expression pattern of COR genes and the freezing tolerance of the plants (Fig 3.4). Gene Ontology Analysis Reveals Diverse Pathways Regulated by Early Coldinduced TFs To investigate the functions of the early cold-induced TFs in the complex cold acclimation process, we analyzed the enrichment of the Gene Ontology (GO) categories of COR genes and the TF regulons (Table 3.3). The GO analysis of COR genes reveals that GO categories of abiotic stresses, biotic stresses, hormone responses, 73 carbohydrate metabolism, circadian rhythm, cell wall modification, pigment metabolism, and chlorophyll metabolism are significantly enriched (p-value< 10E-2, Fisher’s exact test). Although the regulons for each TF are over-represented in different GO categories, they have substantial overlaps between TFs. The CBF regulon is significantly enriched in response to abiotic stresses and ABA responses, which are largely associated with osmotic and drought stresses along with cold stress. Among the other regulons, the HSFC1 and ZAT10 regulons are also enriched in response to abiotic stresses; however, they are associated with water deprivation stress. It may appear contradictory to observe that the GO categories of abiotic stress and defense responses are over-represented in the COR genes; however, previous research has shown that low temperatures induce a set of defense responsive genes, including PATHOGEN-RELATED GENE 1 (PR1), PR2, and PR5 . In rye, several PR genes encode short cysteine-rich peptides that may function as antifreeze proteins (155), but the function of the Arabidopsis PR genes in cold acclimation has not been studied. Arabidopsis PR1, PR2 and PR5 are also induced by SA (156). The induction of PR genes is observed within 24 h, suggesting the regulation of these genes is by CBF, ZF, and HSFC1 as they accumulate early in the cold response, rather than by SA, which does not accumulate until after 7 days in the cold (26). Both the ZAT10 and ZF regulons are enriched in genes involved in pigment biosynthesis; however, they regulate different pathways. ZAT10 has reduced expression of TOCOPHEROL CYCLASE (AT4G32770), a key enzyme in the tocopherol biosynthetic pathway (157). The rapid reduction of TOCOPHEROL CYCLASE expression has been associated with a decrease in tocopherol content in leaf tissue and 74 accumulation of anthocyanin in low temperatures, which may explain the purple color in the adxial leaf of ZAT10-OX lines (158). Overexpression of ZF repressed several genes involved in chlorophyll biosynthesis, including HEMA1 (AT1G58290), GUN4 (AT3G59400), and CHLOROPHYLL A OXYGENASE (AT3G59400). It has been suggested that reduction of chlorophyll biosynthesis has a protective role in relieving photo-oxidative stress at low temperatures (29). The GO enrichment of carbohydrate metabolism in COR genes has long been recognized as being involved in the production of sugars which have a cryoprotective function in freezing tolerance (5, 11, 29). Accumulation of galactinol and raffinose occurs as a result of induction of a group of galactinol synthase genes (GOLS) (38, 159). Taji et al showed that Arabidopsis GOLS3 (AT4g23990) is induced by cold through the CBF pathway, while GOLS1 (AT2g47180) and GOLS2 (AT1g56600) are induced by salt and drought stresses (159). However, transcriptome analysis in previous studies (8, 38) and our study showed that GOLS1 and GOLS2 were also induced in cold. Overexpression of HSFC1 increases the expression of GOLS1 and GOLS2, but not GOLS3. This result may explain the increase of freezing tolerance in HSFC1-OX. The Role of ZF in Starch Degradation and Circadian Rhythm Starch degradation provides a source of sucrose during cold acclimation (160). Starch degradation is initiated by phosphorylation of amylose and amylopectin in starch granules by phosphoglucan water dikinase (PWD) and glucan water dikinase (GWD). It is then further linearized to glucans by α-amylase (AMY) and isoamylase (ISA) (Fig. 3.9A). The linear glucans can be converted to maltose through the action of β-amylase 75 (BAM) or glucose by disproportionating enzyme (DPE1). Glucose and maltose are transported to the cytosol, and maltose can be converted to glucose through transglucosidase (DPE2). It has been demonstrated that BAM2, BAM3, ISA3, DPE2 are induced by cold (161). In addition, a mutation in the cold-inducible GWD1 was found to impair freezing tolerance (160). Interestingly, overexpression of ZF increased transcript levels of GWD1, GWD3, ISA3, DPE1, and DPE2 (Table 3.4). We further verified these results by qRT-PCR. Consistent with previous reports (162) these genes showed diurnal expression at warm temperature (Fig. 3.9B) in a 12h light and 12h dark photoperiod with peak expression at ZT8 (or 8h after dawn), prior to starch degradation, which occurs at night. When cold treatment was started at 4h after dawn, the expression of all the genes was induced except for DPE1. We then verified the effects of overexpressing ZF by examining the expression of these genes at ZT8 in both WT and two ZF-OX lines (Fig. 3.10). Consistent with the array data, the transcript levels of these genes induced 3 to 4-fold in the ZF-overexpressing lines. The enrichment of starch degradation genes in the ZF regulon is unique in the TFs we tested (Table 3.3), which suggested that ZF might play an important role in regulating the starch degradation pathway in cold acclimation. Further analysis of starch metabolism and expression of the genes involved in this pathway in ZF mutants or knockdown lines in the cold may elucidate the function of ZF in cold acclimation. Another unique GO term enriched in the ZF-OX lines is circadian rhythm (Table 3.3). In plants, the central clock components form three interlocking loops to regulate each other (Fig. 3.11) (139, 163, 164). Briefly, the morning-phased CCA1 and LHY are induced in the morning when they repress the evening-phased TOC1, and accumulation 76 of TOC1 at night in turn represses CCA1 and LHY. TOC1 can also activate the expression of CCA1 and LHY before dawn, possibly through interacting with other regulator(s). CCA1 and LHY also induce PRR7 and PRR9 in the morning and their proteins form a negative feedback loop to regulate CCA1 and LHY. The stability of the TOC1 protein is post-translationally regulated by PRR5 and PRR3 to prevent ZTLmediated degradation. GI indirectly affects the stability of the TOC1 protein by stabilizing the ZTL protein in the light. Recently, an evening-phased JMJD5 was identified as positively regulating CCA1 and LHY. It has been established that low temperatures either dampen or disrupt cycling patterns of many central clock genes, but the regulators linking the cold signal to the clock have not been identified (69). Overexpression of ZF repressed the morning-phased genes, CCA1 and LHY, while it induced evening-phased genes, including TOC1, PRR5, PRR3, GI, and JMJD5 (Table 3.4). This tendency was extended to all the ZF regulon (Fig 3.12). The phase enrichment analysis of the ZF regulon revealed that up-regulated genes are enriched around ZT12, while down-regulated genes are enriched around ZT2. This result suggests that overexpression of ZF may have global effects on the cycling patterns of circadian- regulated genes through regulating central clock components. To test this hypothesis, WT and ZF-OX lines were entrained in 12h light and 12h dark for 12d and then moved to continuous light at ZT4 for free-run experiments. Samples were harvested at 4h intervals for 48h, and the transcript levels were examined (Figure 3.13). The rhythm of the central clock genes, including CCA1, LHY, PRR9, PRR7, PRR5, PRR3, TOC1, JMJD5, and GI, and a classic clock output gene, CAB2, were largely unaffected in ZF-overexpressing lines. However, the transcript levels of PPR3 and 77 CAB2, are significantly induced and repressed, respectively, in ZF-OX plants. In addition, overexpression of ZF has mild effects on repression of CCA1 and LHY after dawn (ZT24 and ZT28), and induction of GI, whereas the expression of PRR9, PRR7, TOC1, and JMJD5 was not affected. This result supports the hypothesis that ZF regulates central clock genes. Further global analysis of circadian-regulated genes under circadian conditions could help elucidate the function of ZF in regulating the circadian clock during cold acclimation. Discussion Cold acclimation is a process that brings about complex remodeling of biological and metabolic pathways through extensive changes in gene expression. The objective of this study was to determine how the expression of COR genes is configured by novel regulators in addition to the previously identified CBF and ZAT12 pathways of cold acclimation. To this end, 15 early cold-induced TFs with similar expression patterns to those of the CBFs or ZAT12 were chosen for ectopic expression and transcriptome analysis. The results presented here increase our understanding of the regulation of about 29% of the COR genes defined in this study (Table 3.2). The actual size of the regulon could be larger than we observed in this study in which the effects of overexpression lines were examined in individual TF-OX lines. It is possible that plants have a requirement for regulating COR genes coordinately by more than one TF in the cold, or for post-translational modifications to activate their functions (165). In addition, the repressive effects on the cold-induced COR genes by TFs in the cold could have 78 been missed from our data (63). Further transcriptome analysis of overexpression lines or higher order mutants in cold conditions could complement our current data. In addition, ChIP experiments could further dissect the direct or indirect regulation of the TFs to construct gene regulatory networks. The TFs described here regulate different sets of COR genes, but a substantial number of COR genes are co-regulated by multiple TFs, indicating an intensive coregulation of the COR genes shared between these TF regulatory pathways in cold acclimation (Fig. 3.5 and Table 3.2). The co-regulation of the COR genes may integrate different environmental signals and regulators to fine-tune expression of some key pathways in cold acclimation, such as carbohydrate metabolism (Table 3.3). On the other hand, subsets of COR genes are only affected by a single transcription factor, which suggests that these TFs also have different biological functions in cold acclimation. The CBF pathway is unique among gene regulatory pathways in cold acclimation in several different ways. The correlation of the expression pattern of the CBF regulon and COR genes indicated that overexpression of CBF has a positive effect on regulatory gene expression; for example, the COR genes up-regulated in the cold are also induced by overexpressing CBF (Fig. 3.4). This could explain the increase in freezing tolerance phenotype of CBF-OX lines (36, 119). In addition, the CBF regulon genes are mainly involved in coping with cold, osmotic, and drought stresses in preventing the cellular damage (Table 3.3), which are critical for plant survival at freezing temperatures. The HSFC1 regulon also showed positive correlation to the COR genes at most time points. Consistent with this observation, the HSFC1-OX lines 79 increased in freezing tolerance as determined by ELA. The potential cause could be the induction of GOLS1 and GOLS2 resulting in the accumulation of raffinose as cryoprotectant (Figure 3.7, Fig. 3.8, and Table A1). Most of the transgenic plants overexpressing early cold-induced TFs have dwarf phenotypes (Fig. 3.6). It is unlikely that changes in biosynthesis or degradation of GA and SA are the cause of this phenotype as reported in CBF1-OX. The adverse growth effects when overexpressing these TFs may cause a decrease in freezing tolerance in general (Fig. 3.7 and Fig. 3.8). The possibility that these TFs positively regulate cold acclimation cannot be totally excluded. In ZF-OX and HSFC1-OX, induction of the genes involved in starch metabolism and raffinose synthesis (Table3.3 and Table 3.4) can protect plants from damage by freezing temperatures. However, the effect of overexpressing ZF in mis-regulation of COR genes may in turn have a negative impact on freezing tolerance (Fig. 3.4). Recently, Bieniawska et al. (69) demonstrated that the number of COR genes and their strength of expression during short term cold treatment are greatly dependent on the time of day, a phenomenon termed “gating”. Dong et al. (65) further showed that CCA1 and LHY regulate the gating response of the CBF pathway through interacting with the CBF promoters. Low temperature has broad effects on the circadian rhythm, which is unique among abiotic stresses (69, 71). Many central clock regulators in Arabidopsis and chestnuts, including CCA1, LHY, TOC1, PRRs, GI, and ELFs, are either dampened or arrhythmic in the cold under diurnal or circadian conditions (69, 70, 72). From the transcriptome analysis of ZF overexpressing lines, the up-regulated regulon genes mostly peak during the evening, while the down-regulated regulon genes 80 are enriched in the morning (Fig. 3.12). This effect was seen in several central clock regulators, including CCA1, LHY, TOC1, PRR5, PRR3, GI, and JMJD5 in the array experiment (Table 3.4). Among these genes, the transcript levels of PRR3 and CAB2 are considerably affected in ZF-OX lines, whereas a mild reduction of CCA1 and LHY was observed under circadian conditions (Fig. 3.13). Based on the current clock model (139, 163, 164) and our data, it is possible that ZF regulates PRR3 to modulate its expression in the cold: increased expression of PRR3 can stabilize TOC1 protein to repress the expression of CCA1 and LHY. However, this scenario does not explain the reduction of CAB2 expression, because the effects on the expression of CCA1 and LHY are minimal in the ZF-OX lines. The other explanation is that overexpression of ZF affects genes involved in chlorophyll biosynthesis, which is shown in the GO enrichment analysis (Table 3.3). In addition, overexpression of ZF induced expression of GWD1, GWD3, ISA3, DPE1, and DPE2, which encode key enzymes involved in starch degradation used as a source of sucrose in the cold (Table 3.4 and Fig.3.10). The function of ZF in modulation of the circadian clock and starch degradation pathway needs to be further elucidated using zf mutants. 81 Figure 3.1 Temporal expression of the COR genes. The 2455 cold-regulated (COR) genes were selected with criteria of more than 4-fold change and FDR of <0.01 at any time point in comparison between cold and warm samples (cold/warm). (A) K-mean clustering (k=6) of COR genes form 6 groups of COR genes according to their expression patterns. (B) The relative expression of the COR genes when the transcript levels in the cold at each time point are compared to 0h (cold/0h). (C) The relative expression of the COR genes when the transcript levels in warm at each time point are compared to 0h (warm/0h).The signals are shown as log2-fold change. 82    Figure 3.2 Cluster analysis of cold-induced transcription factors. The174 cold-induced transcription factors in G3 to G6 in Figure 3.1A were subjected to k-mean clustering (k=7). The signals are shown as log2-fold change. 83    Figure 3.3 Relative transcript levels of cold-induced transcription factors of cluster 3 in Figure 3.2 and Table 3.1. WT Arabidopsis was grown under LD (16h light and 8h dark) for 14d and harvested starting at 3h after dawn with or without cold treatment (4°C) for 0, 0.5, 1, 3, 6, 12, and 24h. The transcript levels were quantified by qRT-PCR, normalized with IPP2, and then compared to corresponding warm samples (blue solid line) or 0h samples (red dashed line). The data are presented as mean ± SEM (n=3). 84    Figure 3.3 (Continued) 85    Figure 3.4 Spearman correlation of the expression of COR genes regulated by each TF and that in WT at each time point. The y-axis represents the Spearman’s correlation coefficiency (rho), and the x-axis indicates duration of cold treatments. * P-value < 0.05. ** P-value < 0.01. 86    Figure 3.5 The co-regulatory network of the TF-regulons. (A) The regulon in G1 and G2 (green dots) and G3 to G6 (red dots) of COR genes for each TF (yellow circles) are visualized with Cytoscape. The COR genes from individual circles connected to TFs are the COR genes regulated by a single TF. Other COR genes forming the two circles in the middle represent COR genes regulated by two (outer) or more than two (inner) TFs. The connectivity of the 696 COR genes in (A) and each TF-regulon are represented in (B) and (C). The numbers of the co-regulated genes between two TFs are shown in (D). 87    Figure 3.5 (Continued) 88    Figure 3.6 Phenotypes of the transgenic plants overexpressing early cold-induced TFs. Plants were grown in long day (16h light, 8h dark) for 19 days. 89    Figure 3.7 Freezing tolerance test of the TF-overexpressing lines under non-cold acclimated (NAc) conditions. Two independent transgenic lines and WT were grown at 22°C in 12h light/12h dark for 21d and subjected to a freezing tolerance test using electrolyte leakage assays. 90    Figure 3.8 Freezing tolerance test of the TF-overexpressing lines under cold acclimated (Ac) condition. Two independent transgenic lines and WT were grown at 22°C in 12h light/12h dark for 21d, and then transferred to 4°C for 7d for cold acclimation before being subjected to electrolyte leakage assays. 91    Figure 3.9 Expression of enzymes involved in the starch degradation pathway are affected by low temperature. (A) The starch degradation pathway is mediated by several key enzymes, including glucan water dikinase (GWD), phosphoglucan water dikinase (PWD), α-amylase (AMY), isoamylase3 (ISA3), β-amylase (BAM), disproportionating enzyme (DPE1), and transglucosidase (DPE2) as described in the text. (B) The expression profiles of GWD1, GWD3, ISA3, DPE1, and DPE2 were measured using qRT-PCR in WT Arabidopsis grown in 12h light/12h dark for 12d and treated without (warm) or with cold (4°C) starting from 4h after dawn. The data are presented as mean ± SEM (n=3). 92    Figure 3.10 The expression of key enzymes in the starch degradation pathway is affected by overexpression of ZF. WT and two ZF-OX lines were grown at 22°C in 12h light/12h dark for 13d and the samples were harvest at 8h after dawn. The transcript levels were normalized with IPP2, and compared to the WT warm sample (set as 1). The data are presented as mean ± SEM (n=3). 93    Figure 3.11 Model of plant circadian clock. The circadian clock is regulated by several central clock regulators, including CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1), PSEUDO-RESPONSE REGULATOR (PRR), GIGANTEA (GI), JUMONJI DOMAIN CONTAINING 5 (JMJD5), and ZEITLUPE (ZTL) as described in the text. 94    Figure 3.12 Phase enrichment of the ZF regulon. The up- and down-regulated ZF regulon (Table A1) was subjected to PHASER to compare the enrichment of its phase to the whole circadian datasets (LL12_LDHH). 95    Figure 3.13 The effects of overexpressing ZF on the expression of circadian regulators and output genes in warm conditions. WT and two ZF-OX lines were entrained in 12h light/12h dark at 22°C for 12d and then shifted to continuous light starting at 4h after dawn (ZT4). The samples were harvested every 4h in a 48h-period, and the transcript levels were quantified with qRT-PCR. The relative expression levels were first normalized with IPP2, and then compared to WT samples at ZT8, which was set as 1. The data are presented as mean ± SEM (n=3). 96    Figure 3.13 (Continued) 97    Table 3.1 List of transcription factors up-regulated at low temperature. Cluster C1 C1 C1 C1 C1 Affy ID 261569_at 259751_at 260380_at 263739_at 263549_at Locus AT1G01060 AT1G71030 AT1G73870 AT2G21320 AT2G21650 C1 C1 C1 C1 C1 C1 C1 C1 C1 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 C2 263252_at 265248_at 266719_at 258497_at 258723_at 252917_at 249769_at 245925_at 248306_at 260956_at 255937_at 262028_at 263735_s_at 263584_at 263253_at 267515_at 263783_at 259293_at 257262_at 251745_at 254592_at 254016_at AT2G31380 AT2G43010 AT2G46830 AT3G02380 AT3G09600 AT4G38960 AT5G24120 AT5G28770 AT5G52830 AT1G06040 AT1G12610 AT1G35560 AT1G60040 AT2G17040 AT2G31370 AT2G45680 AT2G46400 AT3G11580 AT3G21890 AT3G55980 AT4G18880 AT4G26150 C2 253405_at AT4G32800 C2 C2 C2 C2 C2 250099_at 249415_at 248611_at 248389_at 247455_at AT5G17300 AT5G39660 AT5G49520 AT5G51990 AT5G62470 Gene Name LHY MYBL2 (ARABIDOPSIS MYB-LIKE 2) zinc finger (B-box type) family protein zinc finger (B-box type) family protein MEE3 (MATERNAL EFFECT EMBRYO ARREST 3) STH PIF4 (phytochrome interacting factor 4) CCA1 COL2 (constans-like 2) myb family transcription factor zinc finger (B-box type) family protein SIGE (SIGMA FACTOR E) BZO2H3 WRKY27 STO (SALT TOLERANCE) DDF1 TCP family transcription factor, putative AGL49 (AGAMOUS-LIKE 49) ANAC036 bZIP transcription factor (POSF21) TCP family transcription factor, putative WRKY46 DNA-binding protein, putative zinc finger (B-box type) family protein SZF1 (SALT-INDUCIBLE ZINC FINGER 1) AT-HSFA4A CGA1 (CYTOKININ-RESPONSIVE GATA FACTOR 1) AP2 domain-containing transcription factor TINY, putative myb family transcription factor CDF2 (CYCLING DOF FACTOR 2) WRKY48 CBF4 (C- REPEAT-BINDING FACTOR 4) MYB96 (myb domain protein 96) 98    Table 3.1 (Continued) Cluster C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 C3 Affy ID 259364_at 261263_at 261648_at 261470_at 259834_at 261892_at 265359_at 267028_at 263379_at 257022_at 258139_at 252278_at 252214_at Locus AT1G13260 AT1G26790 AT1G27730 AT1G28370 AT1G69570 AT1G80840 AT2G16720 AT2G38470 AT2G40140 AT3G19580 AT3G24520 AT3G49530 AT3G50260 C3 C3 255568_at 245397_at AT4G01250 AT4G14560 C3 C3 C3 245276_at 245250_at 254235_at AT4G16780 AT4G17490 AT4G23750 C3 C3 C3 C3 C3 C3 C3 C3 C3 254075_at 254066_at 254074_at 253722_at 246253_at 245711_at 249606_at 248799_at 248253_at AT4G25470 AT4G25480 AT4G25490 AT4G29190 AT4G37260 AT5G04340 AT5G37260 AT5G47230 AT5G53290 C3 C3 247655_at 247029_at AT5G59820 AT5G67190 C3 246987_at AT5G67300 Gene Name RAV1 DOF1.3 ZAT10; STZ (salt tolerance zinc finger) ERF11 (ERF DOMAIN PROTEIN 11) DOF1.10 WRKY40 MYB7 (MYB DOMAIN PROTEIN 7) WRKY33 CZF1 AZF2 AT-HSFC1 ANAC062 DEAR1; CEJ1 (COOPERATIVELY REGULATED BY ETHYLENE AND JASMONATE 1) WRKY22 AXR5; IAA1 (INDOLE-3-ACETIC ACID INDUCIBLE) ATHB-2 ATERF6 CRF2 (CYTOKININ RESPONSE FACTOR 2) CBF2 CBF3 CBF1 zinc finger (CCCH-type) family protein MYB73 (MYB DOMAIN PROTEIN 73) CZF2; ZAT6 RVE2 (REVEILLE 2); CIR2 ERF5 CRF3 (CYTOKININ RESPONSE FACTOR 3) ZAT12 DEAR2, DREB; AP2 domain-containing transcription factor, putative MYB44; MYBR1 99    Figure 3.1 (Continued) Cluster C4 C4 Affy ID 260203_at 256149_at Locus AT1G52890 AT1G55110 C4 C4 C4 259992_at 263797_at 245635_at AT1G67970 AT2G24570 AT1G25250 C4 C4 C4 C4 267246_at 263963_at 266555_at 259129_at AT2G30250 AT2G36080 AT2G46270 AT3G02150 C4 C4 C4 C4 C4 258742_at 259244_at 256430_at 257053_at 251282_at AT3G05800 AT3G07650 AT3G11020 AT3G15210 AT3G61630 C4 C4 C4 C4 C4 C4 C4 C4 C5 C5 C5 C5 C5 C5 245247_at 254159_at 253535_at 253485_at 250781_at 249746_at 248744_at 247452_at 263656_at 261766_at 261663_at 259971_at 262136_at 245078_at AT4G17230 AT4G24240 AT4G31550 AT4G31800 AT5G05410 AT5G24590 AT5G48250 AT5G62430 AT1G04240 AT1G15580 AT1G18330 AT1G76580 AT1G77850 AT2G23340 C5 C5 C5 C5 264057_at 265842_at 260540_at 253799_at AT2G28550 AT2G35700 AT2G43500 AT4G28140 C5 246217_at AT4G36920 Gene Name ANAC019 AtIDD7 (Arabidopsis thaliana Indeterminate(ID)-Domain 7) AT-HSFA8 WRKY17 AtIDD16 (Arabidopsis thaliana Indeterminate(ID)-Domain 16) WRKY25 DNA-binding protein, putative GBF3 (G-BOX BINDING FACTOR 3) PTF1 (PLASTID TRANSCRIPTION FACTOR 1) transcription factor COL9 (CONSTANS-LIKE 9) DREB2B ERF4 CRF6 (CYTOKININ RESPONSE FACTOR 6) SCL13 (Scarecrow-like 13) WRKY7 WRKY11 WRKY18 DREB2A TIP (TCV-INTERACTING PROTEIN) zinc finger (B-box type) family protein CDF1 (CYCLING DOF FACTOR 1) SHY2 (SHORT HYPOCOTYL 2) IAA5 EPR1 transcription factor ARF17 (AUXIN RESPONSE FACTOR 17) AP2 domain-containing transcription factor, putative RAP2.7 (RELATED TO AP2.7) ERF38 RWP-RK domain-containing protein AP2 domain-containing transcription factor, putative AP2 (APETALA 2) 100    Table 3.1 (Continued) Cluster C5 Affy ID 263128_at Locus AT1G78600 C5 C5 C5 C5 C5 C5 C5 C5 C5 C5 C5 C5 250694_at 250379_at 246523_at 246911_at 249422_at 249087_at 248366_at 248328_at 248160_at 247945_at 247921_at 247707_at AT5G06710 AT5G11590 AT5G15850 AT5G25810 AT5G39760 AT5G44210 AT5G52510 AT5G52660 AT5G54470 AT5G57150 AT5G57660 AT5G59450 C5 C5 C5 247601_at 247519_at 266820_at AT5G60850 AT5G61430 AT2G44940 C5 C5 C5 C5 266125_at 258813_at 258325_at 257643_at AT2G45050 AT3G04060 AT3G22830 AT3G25730 C5 C5 252210_at 252175_at AT3G50410 AT3G50700 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 259417_at 261254_at 260776_at 262843_at 256091_at 256092_at 259595_at 261192_at 245807_at 261610_at 261613_at 264190_at AT1G02340 AT1G05805 AT1G14580 AT1G14687 AT1G20693 AT1G20696 AT1G28050 AT1G32870 AT1G46768 AT1G49560 AT1G49720 AT1G54830 Gene Name LZF1 (LIGHT-REGULATED ZINC FINGER PROTEIN 1) HAT14 TINY2 (TINY2) COL1 (constans-like 1) tny (TINY) AtHB23 ERF9 scarecrow-like transcription factor 8 (SCL8) myb family transcription factor zinc finger (B-box type) family protein basic helix-loop-helix (bHLH) family protein zinc finger (B-box type) family protein scarecrow-like transcription factor 11 (SCL11) OBP4 ANAC100 AP2 domain-containing transcription factor TINY, putative zinc finger (GATA type) family protein ANAC046 AT-HSFA6B AP2 domain-containing transcription factor, putative OBP1 (OBF BINDING PROTEIN 1) AtIDD2 (Arabidopsis thaliana Indeterminate(ID)-Domain 2) HFR1 (LONG HYPOCOTYL IN FAR-RED) basic helix-loop-helix (bHLH) family protein zinc finger (C2H2 type) family protein AtHB32 HMGB2 (HIGH MOBILITY GROUP B 2) HMGB3 (HIGH MOBILITY GROUP B 3) zinc finger (B-box type) family protein ANAC13 RAP2.1 (related to AP2 1) myb family transcription factor ABF1 NF-YC3 (NUCLEAR FACTOR Y, SUBUNIT C3) 101    Table 3.1 (Continued) Cluster C6 Affy ID 260627_at Locus AT1G62310 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 262166_at 259711_at 265333_at 266695_at 265662_at 267509_at 266514_at 258603_at 258809_at 258395_at 252573_at 252429_at 251623_at 255625_at 255585_at 254778_at AT1G74840 AT1G77570 AT2G18350 AT2G19810 AT2G24500 AT2G45660 AT2G47890 AT3G02990 AT3G04070 AT3G15500 AT3G45260 AT3G47500 AT3G57390 AT4G01120 AT4G01550 AT4G12750 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 C6 254670_at 253872_at 253603_at 253423_at 253245_at 246222_at 251132_at 250155_at 246432_at 249944_at 246939_at 249065_at AT4G18390 AT4G27410 AT4G30935 AT4G32280 AT4G34590 AT4G36900 AT5G01200 AT5G15160 AT5G17490 AT5G22290 AT5G25390 AT5G44260 Gene Name transcription factor jumonji (jmjC) domaincontaining protein myb family transcription factor DNA binding / transcription factor AtHB24 zinc finger (CCCH-type) family protein FZF (C2H2 zinc finger protein) AGL20 (AGAMOUS-LIKE 20) zinc finger (B-box type) family protein ATHSFA1E ANAC047 ANAC055 zinc finger (C2H2 type) family protein CDF3 (CYCLING DOF FACTOR 3) AGL18 GBF2 (G-BOX BINDING FACTOR 2) ANAC069 sequence-specific DNA binding / transcription factor TCP family transcription factor, putative RD26 WRKY32 IAA29 GBF6 (G-BOX BINDING FACTOR 6) RAP2.10 (related to AP2 10) myb family transcription factor bHLH family protein RGL3 (RGA-LIKE PROTEIN 3) ANAC089 SHN2 (shine2) zinc finger (CCCH-type) family protein 102    Table 3.1 (Continued) Cluster C7 C7 C7 C7 C7 C7 C7 C7 C7 C7 Affy ID 261564_at 255953_at 259626_at 259705_at 263295_at 266839_at 266841_at 267534_at 266327_at 257610_at Locus AT1G01720 AT1G22070 AT1G42990 AT1G77450 AT2G14210 AT2G25930 AT2G26150 AT2G41900 AT2G46680 AT3G13810 C7 C7 258157_at 257985_at AT3G18100 AT3G20810 C7 C7 C7 251272_at 250858_at 247795_at AT3G61890 AT5G04760 AT5G58620 Gene Name ATAF1 TGA3 ATBZIP60 ANAC032 AGL44 (AGAMOUS-LIKE 44) ELF3 (EARLY FLOWERING 3) ATHSFA2 zinc finger (CCCH-type) family protein ATHB-7 AtIDD11 (Arabidopsis thaliana Indeterminate(ID)-Domain 11) MYB4R1 (myb domain protein 4R1) transcription factor; jumonji (jmjC) domaincontaining protein ATHB-12 myb family transcription factor zinc finger (CCCH-type) family protein 103    Table 3.2 Summary of differentially expressed genes (DEG) in each TF-OX line.  TF Locus HSFC1 AT3G24520 Zinc-finger (ZF) AT4G29190 DEG CORs (% of 2455 CORs) p-value G1 **(512) G2 **(579) G3 **(298) G4 **(276) G5 **(586) G6 **(204) 632 210 (8.55) <2.2E-16 51 54 21 22 40 22 460 167 (6.80) <2.2E-16 30 46 14 21 26 30 146 (5.95) 145 (5.91) 108 (4.4) <2.2E-16 <2.2E-16 <2.2E-16 14 30 17 13 45 17 7 16 14 8 17 16 101 26 24 3 11 20 CBF2* ZAT10 CZF1 AT4G25470 AT1G27730 AT2G40140 238 364 213 ZAT12* MYB44 DEAR1 ERF5 WRKY33 ERF6 ANAC62 CRF3 RAV1 CRF2 CZF2 MYB73 AT5G59820 AT5G67300 AT3G50260 AT5G47230 AT2G38470 AT4G17490 AT3G49530 AT5G53290 AT1G13260 AT4G23750 AT5G04340 AT4G37260 335 188 70 59 18 12 13 33 19 14 7 9 72 (2.97) <2.2E-16 18 24 13 5 11 1 58 (2.36) 2.04E-9 13 17 7 4 9 8 37 (1.51) 1.0E-14 7 8 3 9 4 7 24 (0.98) 2.2E-16 7 2 4 3 2 6 5 11 (0.45) 1.09E-8 2 1 2 0 1 8 (0.33) 2.09E-7 1 1 1 2 1 2 5 (0.20) 4.10E-6 1 0 2 1 1 0 2 0 1 0 0 4 (0.16) 7.14E-3 1 2 0 0 1 1 4 (0.16) 0.77 0 3 (0.12) 0.12 1 0 1 0 1 0 2 (0.08) 0.17 1 1 0 0 0 0 1 (0.04) 0.16 1 0 0 0 0 0 696 (28.35) The DEG are defined by comparing transgenic lines to WT using a criterion of more than 2-fold change (p-value< 0.01). The significance of enrichments of COR genes in DEG for each TF were tested with Fisher’s exact test, and the p-values were presented in table. The significance enrichments of COR genes regulated by each TF in each group of COR genes defined in Table A.1 and Figure 3.1 were tested with Fisher’s exact test. The red block represents the p-value< 0.01, and the yellow block represents p-value< 0.05.   104    Table 3.2 (Continued) * The CBF2-OX and ZAT12-OX data were analyzed from GSE5536 and GSE5742 microarray experiments. ** The number in parentheses represents the number of genes in each group. 105    Table 3.3 GO analysis of COR genes and TF-regulons Gene Counts Description ‡ CORs p-value P response to abiotic stimulus P response to temperature stimulus P response to cold P response to water deprivation P response to osmotic stress P response to salt stress P response to heat P response to hormone stimulus P response to abscisic acid stimulus P response to salicylic acid stimulus P response to ethylene stimulus P response to wounding 311 1.03E-40 31 12 24 19 16 38 85 4.91E-27 20 4 13 6 7 63 49 5.65E-25 1.56E-21 19 16 4 3 8 5 5 4 63 6.20E-20 14 2 7 54 1.19E-16 10 2 23 144 2.30E-05 2.46E-27 0 18 72 4.60E-24 35 CBF ZAT12 ZAT10 ZF CZF1 HSF MYB44 (146) (72) (145) (167) (108) (210) (58) ERF5 (24) DEAR1 (37) 10 6 8 14 1 0 3 4 3 9 9 0 2 0 0 1 1 3 2 5 3 1 0 5 2 2 5 3 1 0 2 7 2 14 1 5 4 8 6 18 0 5 0 4 2 1 15 3 9 3 3 11 2 1 1 2.35E-10 3 1 3 1 3 2 2 2 3 36 7.91E-10 1 0 2 1 2 6 3 4 0 64 1.71E-19 6 5 6 2 1 6 3 2 0 106    Table 3.3 (Continued) Gene Counts Description ‡ CORs p-value CBF (146) 8 ZAT12 (72) 8 ZAT10 (145) 19 ZF (167) 13 CZF1 (108) 7 HSF (210) 22 MYB44 (58) 6 ERF5 (24) 4 P response to biotic 163 2.40E-23 stimulus P response to other 107 7.56E-21 5 7 14 9 4 15 5 4 organism P defense response 133 4.04E-20 7 5 12 7 7 17 5 4 4 3 0 1 P defense response 13 3.89E-03 1 1 2 0 to fungi P circadian rhythm 15 3.91E-08 0 1 3 6 0 3 0 1 15 7 20 5 0 P carbohydrate 117 7.51E-11 8 4 11 metabolism P starch metabolism 10 6.51E-05 0 0 1 6 0 3 0 0 7 1 6 1 0 P glucan metabolism 20 9.34E-05 0 0 3 P polysaccharide 28 1.32E-05 1 0 4 7 2 8 2 0 metabolism P cell wall loosening 12 2.14E-05 3 1 3 2 2 3 1 0 C cell wall 41 4.25E-05 3 1 2 4 9 8 4 1 3 1 3 2 2 3 1 0 P cell wall 12 5.62E-05 modification P pigment 21 4.16E-05 0 0 5 5 0 4 0 0 metabolism P chlorophyll 9 3.66E-03 0 0 2 3 0 3 0 0 metabolism F oxidoreductase 21 2.92E-03 1 0 5 2 0 2 1 0 activity 1. ‡: P, Biological Process; C, Celluler Component; F, Molecular Function 2. The numbers in parentheses below the TFs represent the number of TF-regulon genes. 3. The red and yellow colors indicate a p-value less than 0.01 and 0.05 respectively with Fisher's exact test. 107    DEAR1 (37) 2 2 1 0 0 5 0 1 2 0 2 0 1 0 2 Table 3.4 Genes in circadian or starch degradation pathways affected by overexpressing ZF Locus Gene CIRCADIAN RHYTHM AT2G46830 CIRCADIAN CLOCK ASSOCIATED (CCA1) AT1G01060 LATE ELONGATED HYPOCOTYL (LHY) AT1G29920 CAB2 AT5G61380 TIMING OF CAB EXPRESSION 1 (TOC1) AT5G24470 PSEUDO-RESPONSE REGULATOR 5 (PRR5) AT5G60100 PSEUDO-RESPONSE REGULATOR 3 (PRR3) AT1G22770 GIGANTEA (GI) AT3G20810 JUMONJI DOMAIN CONTAINING 5 (JMJD5) STARCH DEGRADATION AT1G10760 GLUCAN, WATER DIKINASE; STARCH EXCESS 1 (GWD1; SEX1) AT5G26570 PHOSPHOGLUCAN, WATER DIKINASE (GWD3) AT3G52180 DUAL-SPECIFICITY PROTEIN PHOSPHATASE 4; STARCH-EXCESS 4 (DSP4, SEX4) AT4G09020 ISOAMYLASE 3 (ISA3) AT5G64860 α-1,4 GLUCANOTRANSFERASE (DPE1) AT2G40840 TRANSGLICOSIDASE (DPE2) 108    log2 (FC) -1.15 -1.83 -0.42 1.52 1.71 2.84 2.11 2.19 1.28 1.54 1.76 1.5 1.66 1.06 Table 3.5 List of primers for qRT-PCR Gene Locus WRKY33 AT2G38470 HSFC1 AT3G24520 ERF6 AT4G17490 CRF2 AT4G23750 ERF5 AT5G47230 CRF3 AT5G53290 DEAR1 AT3G50260 MYB44 AT5G67300 ZAT10 AT1g27730 CZF1 AT2g40140 ZF AT4G29190 RAV1 At1g13260 ANAC62 AT3g49530 Primer (Fw or Rv) Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Primer sequences CGATGTTCCTGCAGCTCGTGG TGTGGTGCTCTGTTTGTGGCG CCGGATGGGTGGATTGTTCCTATGAC GTTGAATTCGAGAGCATCGACTTCGC CTACTACTGCCACCACCAATCGATGG AAAATCTCCGGTTTGGGAGTGACGAG AAAATGGGCGGCGGAGATAAGAGATC GAGAAATTAGTCAGAGCGTCGGGACC GGCGACTCCTAACGAAGTATCTGCAC CATGGATCTGTAGCCACAGGAGAAGC TCCGGCGTCGAGTCGTCAAC ATCTCCGCCGCCCATTTCCC GCTGACGTGGCAGTCAAAATGAAGAG TGAACGCTTGTTGGGTTCTCGAATCT AATGGGGAAGTCTTTTCCCGGTAACG CATTGTTCCGTTGCATCTCCGTCATG TCGAGCACTGGACAAAGGGTAAGC CCTCAGTGAGGTTTTGGTGGTGGA GCCTTGTCCCGAGTTTCGTA TGCGCGTACTCACACGAATC TCATTTCCTCGTAACAATCCTTTATTC CGGTGTTGTAGGCAGAGACTGA TAGATGCGGGTCGGGTTTT GTTTCTTGAACTCTCCGGTGAAA GCAGTTGACATGAGCAATGATGT TGTGCCTGGCGACTCTTAATC 109    Table 3.5 (Continued) Gene Locus MYB73 AT4g37260 CZF2 AT5g04340 AZF2 AT3G19580 DEAR2 AT5G67190 AXR5 AT4G14560 WRKY22 AT4G01250 ATHB2 AT4G16780 ERF11 AT1G28370 WRKY40 AT1G80840 DOF1.3 AT1G26790 RVE2 AT5G37260 DOF1.10 AT1G69570 MYB7 AT2G16720 Primer (Fw or Rv) Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Primer sequences TGAGGAGTTACATGGCGGATT CGCCGCCAGAACTACTACCA TCGCGACGGAGATAGAAACC GCAGAGGAGGTGAAGACGAAGA TACGAAGGCAACCTCGGCGG CGTGCTCGACACGCTTCCAC GGAGGTCGGTGCTCAGGTT CGGTGGCGGTTATTTTGC TGGATGGCCTCCAGTGAGATCTAACC GGAGCTCCGTCCATACTCACTTTCAC TGAGGATCATCTAGCGGTGGGAGATC ATCGCTAACCACCGTATCCGACAAAG GACGAAGACGCTGGAGTATCTTCACC TCCCTGTAGAGCTTGAGACTGTACTGT ACAGAGGAGTGAGGAAGAGACCATGG CTTCAGGAGTGTCGAAAGTACCGAGC GAGGACGCATTCAGCTGCGC GAGCTACTCTCCGACACTCCGC CCCAACAACTTCCAAGGGTTAC AGGCCAAGGAGGCGAAAC CGCCTCCAAGGCCAAAG TTGCATCAGGAATCACAAGCTT CCAACGGGTCCTAACGCTAGT GGGTATATCGGAACCGTGCAT TCGCTGCGGTAAAAGCTGCC AGACCACTTGTTGCCTAGGAGGC 110    Table 3.5 (Continued) Gene Locus CBF1 AT4G25490 CBF2 AT4G25470 CBF3 AT4G25480 ZAT12 AT5G59820 IPP2 AT3G02780 CCA1 AT2G46830 LHY AT1G01060 TOC1 AT5G61380 PRR3 AT5G60100 PRR5 AT5G24470 PRR7 AT5G02810 PRR9 AT2G46790 JMJD5 AT3G20810 Primer (Fw or Rv) Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Primer sequences CCGCCGTCTGTTCAATGGAATCAT TCCAAAGCGACACGTCACCATCTC CGACGGATGCTCATGGTCTT TCTTCATCCATATAAAACGCATCTTG TTCCGTCCGTACAGTGGAAT AACTCCATAACGATACGTCGTC GTGCGAGTCACAAGAAGCCTAACA GCGACGACGTTTTCACCTTCTTCA ATTTGCCCATCGTCCTCTGT GAGAAAGCACGAAAATTCGGTAA TCTGTGTCTGACGAGGGTCGAATT ACTTTGCGGCAATACCTCTCTGG CTGCCGCTGTGCATGACT GGATGAGCGGTCCACAAGAT GCTGCACCTAGCTTCAAGCA TCTTCGCAGAATCCCTGTGAT GGACAAAAAGAGCCAGTGATACTAAGA GGGTGCATCGGGAAATTG CGAGAAGCCGCTTTAACCAA CGGCTCTCGTAACGAACCTT AAAAGCTGTGGATGTTGATGACA GTGCTATCAACTCGGTCCCATAG GCCAGAGAGAAGCTGCATTGA CCTGCTCTGGTACCGAACCTT CGGAGACCACAACAACCTCCGTC ATAAGCGTAACCCCCTTCAGCAGAAA 111    Table 3.5 (Continued) Gene Locus GI AT1G22770 CAB2 AT1G29920 ISA3 AT4G09020 DPE1 AT5G64860 DPE2 AT2G40840 GWD1 AT1G10760 GWD3 AT5G26570 Primer (Fw or Rv) Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv Primer sequences GTATCTGCAACGCCAGCGA GCAACTCCCTTTCAGCCTGA CCGGAAAGGCTGTGAACCT CACACGGCCGCTTCCA GGTGGCCGTGACATCTATGTG TGGAATCAGAGCCTTGACAAAG CAGAGGATTTGCAGGGTTTTG CGTCCAACCATGGCAACTTT CCTGCAACAGAGGAGACAATCA CGTGTACGCGGTATCTCCAGTA AGTACAGAGAACTTTTGCGGATGAT TTGCCCGACATCTCCTTCA TTCAGATTCGGTTTGCCAAGT GCTCTACGGCTTAGAACAGCTCTT 112    Table 3.6 List of primers for cloning Gene Locus ZAT10 AT1g27730 CZF1 AT2g40140 RAV1 At1g13260 MYB73 AT4g37260 CZF2 AT5g04340 Primer (Fw or Rv) Fw Rv Fw Rv Fw Rv Fw Rv Fw Rv primer sequences ATGGCGCTCGAGGCTCTTACATCACCAA TTAAAGTTGAAGTTTGACCGGAAAGTCAAACCGA ATGTGCGGTGCAAAGAGCAACCTTTGCT TTATGCCACAATCTGCTGCTCATGGTCTATATA ATGGAATCGAGTAGCGTTGATGAGAGT TTACGAGGCGTGAAAGATGCGTTGCTTCT ATGTCAAACCCGACCCGTAAGAATATGGA CTACTCCATCTTCCCAATTCCGATTTGGT ATGGCACTTGAAACTCTTACTTCTCCAAGA TTAGGGTTTCTCCGGGAAGTCAAACCGGA-       113    Materials and Methods Plant Material and Growth Conditions Wild type Arabidopsis thaliana (Col-0) and transgenic lines overexpressing TF were stratified at 4°C in the dark for 3 to 5 days prior to growth. For gene expression studies and Affymetrix GeneChip experiments, plants were grown on sterilized Gamborg’s B5 medium (Caisson Laboratories) and 8% phytoagar (Caisson Laboratories) without sucrose. For freezing tolerance studies, plants were grown in soil -2 -1 as described (49). All plants were grown at 22°C under ~100 µmol m s fluorescent white light. Abiotic Gene Expression Datasets and Cluster The ATGenExpress Abiotic time course experiment series (113) was downloaded from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). The accession numbers of the cold stress time series and its control are GSE5621and GSE5621, respectively. Only the datasets from shoot tissues were normalized and analyzed with GCRMA (166) and LIMMA (167) packages from Bioconductor (http://www.bioconductor.org/) in R (http://www.R-project.org). Differentially expressed genes were selected by more than 4-fold change and FDR <0.01 at at least one time point by comparison of cold-treated samples to control samples at the same time point. For the fold-change of cold/0h and warm (compared to 0h), GCRMA and LIMMA were applied. K-mean clustering was performed with normalized expression data using CLUSTER, and the results were visualized with TREEVIEW (168). 114 Phase Enrichment Analysis The enrichment of phase (time of the peak expression of circadian genes) was analyzed with PHASER (haystack.cgrb.oregonstate.edu/) as previously described (169). The enrichment score was calculated as the ratio of genes at a specific phase within a given list divided by the ratio of the circadian regulated genes at a specific phase within GeneChip. The LL12_LDHH dataset was used for phase analysis in this experiment. Briefly, the dataset was collected from plants entrained in 12 h light/12 h dark and moved to continuous light starting at ZT0, and samples were harvested at 4 h intervals for 24 h. The graphs were plotted over a 48h period for visualization. Determination of Gene Expression Transcript levels were determined using real-time quantitative RT-PCR (qRTPCR). Plant sample collection, RNA extraction, and qRT-PCR were performed as described (49), except that 100 ng of total RNA was used in a 20 µl reverse transcription reaction, and 2 µl of 10-fold diluted cDNA was used as template in a 10 µl reaction for qRT-PCR. IPP2 (ISOPENTENYL PYROPHOSPHATE-DIMETHYLALLYL PYROPHOSPHATE ISOMERASE 2) was used as the reference gene. Primers are listed in Table 3.5. Generation of Transgenic Lines Overexpressing TFs For transgenic lines overexpressing ZAT10, CZF1, RAV1, CZF2, and MYB73, the coding regions were amplified from WT Arabidopsis cDNA and cloned into the pCR®8⁄GW⁄TOPO® vector (Invitrogen). For CRF3, DEAR1, MYB44, ZF, and ANAC62, 115 the coding region in gateway pENTR223 entry vectors were obtained from the Arabidopsis Biological Resource Center (ABRC) (https://abrc.osu.edu). The cloned sequences were transferred to the plant gateway binary pEarleyGate100 vector (143) through recombination reactions (Invitrogen). For ERF5, ERF6, and CRF2, the plant binary vector pYL436 containing the coding regions of these TFs were obtained from ABRC. The floral dip method was used to transform genes into WT Arabidopsis (142). The expression levels of transgenes were examined using qRT-PCR and two independent T3 homozygous lines were selected for further experiments. The primer sequences are listed in Table 3.6. The clones requested from ABRC were: WRKY33, DKLAT2G38470; ERF6, DKLAT4G17490; ERF5, DKLAT5G47230; CRF2, DKLAT4G23750; HSFC1, G14335; CRF3, G68286; DEAR1, G13196; MYB44, G09033. Affymetrix GeneChip Experiments WT and two independent transgenic lines for each TF were grown under continuous light for 14 days on plates. Samples were collected, and RNA was extracted with the RNeasy Plant Mini Kit following the manufacture’s protocol (Qiagen). Biotinylated cRNA was prepared from 200 ng of total RNA using MessageAmp™ Premier RNA Amplification Kit (Ambion), and hybridized to the Affymetrix Arabidopsis ATH1 GeneChip. 116 Affymetrix GeneChip Data Analysis The CEL files from WT and transgenic lines overexpressing TFs (TF-OX) from the ATH1 GeneChip experiments were normalized and processed as described in the previous section. The differentially expressed genes (DEG) in the TF-OX lines were selected by comparison of TF-OX and WT with more than 2-fold change (p-vale< 0.01). The TF-regulon was defined by the genes’ overlap between the DEG and the COR genes for each TF. For correlation analysis of gene expression in the COR genes and the TFregulon at each time point, Spearman’s correlation efficiency (rho) was calculated. The Gene Ontology (GO) analysis was performed using Classification SuperViewer Tool (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi ) with 1000 times bootstrap. Enrichment of the GO terms for the COR genes was calculated using all the genes on the Arabidopsis ATH1 GeneChips as background. For the analysis of TF-regulons, the COR genes were used as background. Electrolyte Leakage Assays (ELA) WT and transgenic plants were grown at 22°C under a 12 h-photoperiod (12 h light, 12 h dark) for three weeks prior to ELA as described (49). For cold acclimation, -2 -1 plants were moved to 4°C under a 12 h-photoperiod with ~35 µmol m s white light for 7 d. 117 fluorescent APPENDIX 118 Table A1. List of COR genes and the regulons of each overexpressed TF The numbers represent log2 fold-change in the transgenic lines overexpressing TF. Affy ID 245094_at 245129_at 245176_at 245215_at 245253_at 245265_at 245266_at 245296_at 245325_at 245326_at 245334_at 245341_at 245362_at 245386_at 245422_at 245463_at 245592_at 245642_at 245672_at 245692_at 245760_s_at 245776_at 245800_at Locus AT2G40840 AT2G45350 AT2G47440 AT1G67830 AT4G15440 AT4G14400 AT4G17070 AT4G16370 AT4G14130 AT4G14100 AT4G15800 AT4G16447 AT4G17460 AT4G14010 AT4G17470 AT4G17030 AT4G14540 AT1G25275 AT1G56710 AT5G04150 AT1G66920; AT1G66910 AT1G30260 AT1G46264 group g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 CBF2 ZAT12 HSFC1 ZF 1.58 ZAT10 -1.12 CZF1 MYB44 -4.16 DEAR1 ERF5 1.69 2.27 -1.76 -2.29 2.53 -1.89 -1.96 -1.24 1.30 1.67 1.71 1.77 119 Table A.1 (Continued) 245881_at AT5G09462; AT5G09460; AT5G09463; AT5G09461 246004_at AT5G20630 246011_at AT5G08330 246079_at AT5G20450 246159_at AT5G20935 246200_at AT4G37240 246231_at AT4G37080 246236_at AT4G36470 246260_at AT1G31820 246275_at AT4G36540 246296_at AT3G56750 246518_at AT5G15770 246573_at AT1G31690 246584_at AT5G14730 246603_at AT1G31690 246607_at AT5G35370 246620_at AT5G36220 246832_at AT5G26600 246901_at AT5G25630 246948_at AT5G25130 246996_at AT5G67420 247037_at AT5G67070 247072_at AT5G66490 247100_at AT5G66520 247213_at AT5G64900 247216_at AT5G64860 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.26 -1.11 2.91 1.63 1.93 1.66 120 Table A.1 (Continued) 247263_at AT5G64470 247377_at AT5G63180 247385_at AT5G63420 247386_at AT5G63420 247388_s_at AT5G63470; AT3G48590 247396_at AT5G62930 247443_at AT5G62720 247447_at AT5G62730 247486_at AT5G62140 247524_at AT5G61440 247529_at AT5G61520 247540_at AT5G61590 247549_at AT5G61420 247600_at AT5G60890 247628_at AT5G60400 247638_at AT5G60490 247684_at AT5G59670 247800_at AT5G58570 247814_at AT5G58310 247880_at AT5G57780 247882_at AT5G57785 248046_at AT5G56040 248169_at AT5G54610 248186_at AT5G53880 248199_at AT5G54170 248230_at AT5G53830 248270_at AT5G53450 248291_at AT5G53020 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.48 2.65 -1.28 2.24 -1.53 1.74 -2.57 -1.86 1.19 121 1.65 Table A.1 (Continued) 248329_at AT5G52780 248330_at AT5G52810 248336_at AT5G52420 248348_at AT5G52190 248353_at AT5G52320 248385_at AT5G51910 248606_at AT5G49450; AT5G49448 248685_at AT5G48500 248764_at AT5G47640 248807_at AT5G47500 248839_at AT5G46690 248865_at AT5G46790 248867_at AT5G46830 248868_at AT5G46780 248873_at AT5G46450 248969_at AT5G45310 248994_at AT5G45250 249006_at AT5G44660 249008_at AT5G44680 249047_at AT5G44410 249052_at AT5G44420 249072_at AT5G44060 249140_at AT5G43190 249144_at AT5G43270 249190_at AT5G42750 249209_at AT5G42620 249211_at AT5G42680 249279_at AT5G41920 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.90 1.23 -1.84 -3.85 5.87 122 Table A.1 (Continued) 249320_at AT5G40910 249325_at AT5G40850 249408_at AT5G40330 249410_at AT5G40380 249480_s_at AT5G38990; AT5G39000 249482_at AT5G38980 249486_at AT5G39030 249493_at AT5G39080 249515_at AT5G38530 249542_at AT5G38140 249639_at AT5G36930 249728_at AT5G24390 249800_at AT5G23660 249810_at AT5G23920 249869_at AT5G23050 249872_at AT5G23130 249904_at AT5G22700 249923_at AT5G19120 249941_at AT5G22270 250002_at AT5G18690 250063_at AT5G17880 250132_at AT5G16560 250160_at AT5G15210 250167_at AT5G15310 250216_at AT5G14090 250255_at AT5G13730 250265_at AT5G12900 250424_at AT5G10550 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.34 2.12 -1.46 1.27 1.77 123 Table A.1 (Continued) 250487_at AT5G09690 250503_at AT5G09820 250550_at AT5G07870 250582_at AT5G07580 250689_at AT5G06610 250704_at AT5G06265 250777_at AT5G05440 250828_at AT5G05250 250844_at AT5G04470 250942_at AT5G03350 250975_at AT5G03050 250981_at AT5G03140 251017_at AT5G02760 251155_at AT3G63160 251169_at AT3G63210 251199_at AT3G62980 251218_at AT3G62410 251230_at no_match 251299_at AT3G61950 251342_at AT3G60690 251427_at AT3G60130 251509_at AT3G59010 251575_at AT3G58120 251586_at AT3G58070 251661_at AT3G56950 251677_at AT3G56980 251704_at AT3G56360 251705_at AT3G56400 251746_at AT3G56060 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 1.61 1.45 -2.23 1.45 -1.62 -1.62 1.17 4.79 1.87 -1.54 3.70 -1.85 124 2.38 1.13 Table A.1 (Continued) 251919_at AT3G53800 251931_at AT3G53950 252040_at AT3G52060 252050_at AT3G52550 252117_at AT3G51430 252168_at AT3G50440 252170_at AT3G50480 252173_at AT3G50650 252365_at AT3G48350 252367_at AT3G48360 252427_at AT3G47640 252478_at AT3G46540 252485_at AT3G46530 252534_at AT3G46130 252549_at AT3G45860 252615_at AT3G45230 252618_at AT3G45140 252692_at AT3G43960 252698_at AT3G43670 252701_at AT3G43700 252712_at AT3G43800 252736_at AT3G43210 252958_at AT4G38620 252965_at AT4G38860 252970_at AT4G38850 252972_at AT4G38840 252992_at AT4G38520 253022_at AT4G38060 253043_at AT4G37540 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 2.34 -1.65 -1.13 -1.42 -1.13 -1.15 1.20 125 -1.42 Table A.1 (Continued) 253050_at AT4G37450 253061_at AT4G37610 253220_s_at AT4G34930; AT4G34920 253228_at AT4G34630 253255_at AT4G34760 253268_s_at AT4G34131; AT4G34135 253326_at AT4G33440 253411_at AT4G32980 253553_at AT4G31050 253580_at AT4G30400 253597_at AT4G30690 253636_at AT4G30500 253697_at AT4G29700 253708_at AT4G29210 253709_at AT4G29220 253729_at AT4G29360 253779_at AT4G28490 254024_at AT4G25780 254032_at AT4G25940 254133_at AT4G24810 254163_s_at AT4G24340; AT4G24350 254251_at AT4G23300 254424_at AT4G21510 254512_at AT4G20230 254532_at AT4G19660 254533_at AT4G19670 g1 g1 g1 -2.00 1.29 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.76 -1.77 -1.47 2.81 1.33 1.31 1.74 1.18 -1.30 -1.27 -1.27 -1.34 126 -1.33 Table A.1 (Continued) 254547_at AT4G19860 254697_at AT4G17970 254794_at AT4G12970 254803_at AT4G13100 254851_at AT4G12010 254870_at no_match 254878_at AT4G11660 254931_at AT4G11460 254982_at AT4G10470 255064_at AT4G08950 255294_at AT4G04750 255306_at AT4G04740 255437_at AT4G03060 255438_at AT4G03070 255484_at AT4G02540 255579_at AT4G01460 255740_at AT1G25390 255786_at AT1G19670 255794_at AT2G33480 255857_at AT1G67080 255908_s_at AT1G18010; AT1G18000 255926_at AT1G22190 255943_at AT1G22370 255962_at AT1G22330 255969_at AT1G22330 256222_at AT1G56210 256237_at AT3G12610 256281_at AT3G12560 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 2.16 1.18 -1.34 -1.74 1.95 2.83 3.14 -1.45 1.56 -1.90 1.62 -2.79 127 -1.32 Table A.1 (Continued) 256332_at AT1G76890 256383_at AT1G66820 256400_at AT3G06140 256427_at AT3G11090 256446_at AT3G11110 256489_at AT1G31550 256518_at AT1G66080 256598_at AT3G30180 256599_at AT3G14760 256617_at AT3G22240 256622_at AT3G28920 256664_at AT3G12040 256698_at AT3G20680 256721_at AT2G34150 256746_at AT3G29320 256766_at AT3G22231 256796_at AT3G22210 256828_at AT3G22970; AT3G22968 256869_at AT3G26420; AT3G26430 256914_at AT3G23880 256948_at AT3G18930 256980_at AT3G26932 257051_at AT3G15270 257175_s_at AT3G23480; AT3G23470 257203_at AT3G23730 257272_at AT3G28130 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.44 1.24 1.44 -1.85 -1.70 1.17 -3.44 -1.96 g1 g1 g1 g1 g1 g1 g1 g1 1.39 -1.43 128 Table A.1 (Continued) 257474_at AT1G80850 257502_at AT1G78110 257504_at AT1G52250 257507_at AT1G29600 257615_at AT3G26510 257625_at AT3G26230 257634_s_at AT3G26170; AT3G26180 257709_at AT3G27325 257748_at AT3G18710 257750_at AT3G18800 257763_s_at AT3G23120; AT3G23110 257815_at AT3G25130 257832_at AT3G26740 257900_at AT3G28420 257923_at AT3G23160 257964_at AT3G19850 258063_at AT3G14620 258133_at AT3G24500 258156_at AT3G18050 258225_at AT3G15630 258334_at AT3G16010 258432_at AT3G16570 258528_at AT3G06770 258530_at AT3G06840 258537_at AT3G04210 258609_at AT3G02910 258787_at AT3G11840 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 1.32 1.72 1.33 2.82 1.56 -1.76 129 1.44 Table A.1 (Continued) 258791_at AT3G04720 258919_at AT3G10525 258932_at AT3G10150 258956_at AT3G01440 259073_at AT3G02290 259104_at AT3G02170 259115_at AT3G01360 259398_at AT1G17700 259403_at AT1G17745 259441_at AT1G02300 259507_at AT1G43910 259528_at AT1G12330 259529_at AT1G12400 259531_at AT1G12460 259535_at AT1G12280 259545_at AT1G20560 259560_at AT1G21270 259561_at AT1G21250 259602_at AT1G56520 259637_at AT1G52260 259661_at AT1G55265 259664_at AT1G55330 259790_s_at AT1G29430; AT5G27780 259830_at AT1G80630 259839_at AT1G52190 259909_at AT1G60870 259954_at AT1G75130 259994_at AT1G68130 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 2.54 1.19 -1.14 -1.15 -2.36 -1.43 1.93 -1.38 g1 g1 g1 g1 g1 -1.52 130 1.70 Table A.1 (Continued) 260141_at AT1G66350 260146_at AT1G52770 260169_at AT1G71990 260327_at AT1G63840 260414_at AT1G69850 260427_at AT1G72430 260453_s_at AT1G72510; AT2G09970 260541_at AT2G43530 260592_at AT1G55850 260602_at AT1G55920 260635_at AT1G62422 260640_at AT1G53350 260734_at AT1G17600 260735_at AT1G17610 260759_at AT1G49180 260769_at AT1G49010 260770_at AT1G49200 260884_at AT1G29240 260887_at AT1G29160 260976_at AT1G53650 260999_at AT1G26580 261013_at AT1G26440 261023_at AT1G12200 261032_at AT1G17430 261081_at AT1G07350 261084_at AT1G07440 261144_s_at AT1G19660; AT1G75380 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.38 -1.34 -1.37 1.65 1.35 1.37 1.44 1.73 1.58 -1.49 131 Table A.1 (Continued) 261177_at AT1G04770 261221_at AT1G19960 261266_at AT1G26770 261280_at AT1G05860 261294_at AT1G48430 261339_at AT1G35710 261402_at AT1G79670 261407_at AT1G18810 261409_at AT1G07640 261426_at AT1G18680 261460_at AT1G07880 261485_at AT1G14360 261487_at AT1G14340 261500_at AT1G28400 261558_at AT1G01770 261570_at AT1G01120 261684_at AT1G47400 261711_at AT1G32700 261715_at AT1G18485 261758_at AT1G08250 261893_at AT1G80690 261905_at AT1G65070 261949_at AT1G64670 261982_at AT1G33780 262118_at AT1G02850 262126_at AT1G59620 262133_at AT1G78000 262137_at AT1G77920 262162_at AT1G78020 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.50 -2.79 -1.30 -2.25 -1.49 -1.23 -1.42 -1.15 1.77 -1.70 1.20 2.28 132 -1.55 Table A.1 (Continued) 262196_at AT1G77870 262232_at AT1G68600 262236_at AT1G48330 262286_at AT1G68585 262411_at AT1G34640 262533_at AT1G17090 262536_at AT1G17100 262543_at AT1G34245 262552_at AT1G31350 262598_at AT1G15260 262656_at AT1G14200 262661_s_at AT1G14230; AT1G14250 262698_at AT1G75960 262705_at AT1G16260 262736_at AT1G28570 262737_at AT1G28560 262780_at AT1G13090 262811_at AT1G11700 262850_at AT1G14920 262854_at AT1G20870 262891_at AT1G79460 262935_at AT1G79410 262986_at AT1G23390 263014_at AT1G23400 263106_at AT2G05160 263111_s_at AT1G65190; AT1G65250 263150_at AT1G54050 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.38 1.29 -2.34 1.76 -2.75 1.57 -4.99 1.18 2.36 1.22 -1.80 -1.48 g1 133 Table A.1 (Continued) 263184_at AT1G05560 263318_at AT2G24762 263369_at AT2G20480 263597_at AT2G01870 263647_at AT2G04690 263737_at AT1G60010 263776_s_at AT2G46430; AT2G46440 263947_at AT2G35820 263953_at AT2G36050 264014_at AT2G21210 264037_at AT2G03750 264091_at AT1G79110 264229_at AT1G67480 264240_at AT1G54820 264244_at AT1G60440 264306_at AT1G78890 264310_at AT1G62030 264340_at AT1G70280 264379_at AT2G25200 264408_at AT1G10240 264435_at AT1G10360 264463_at AT1G10150 264507_at AT1G09415 264517_at AT1G10120 264656_at AT1G09010 264696_at AT1G70230 264751_at AT1G23020 264769_at AT1G61350 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.19 1.35 -1.42 -1.60 1.27 -2.24 1.24 -1.45 1.34 134 1.95 1.64 1.69 Table A.1 (Continued) 264770_at AT1G23030 264826_at AT1G03410 264874_at AT1G24240 264898_at AT1G23205 264931_at AT1G60590 264947_at AT1G77020 264958_at AT1G76960 264990_at AT1G27210 265063_at AT1G61500 265265_at AT2G42900 265311_at AT2G20250 265414_at AT2G16660 265451_at AT2G46490 265452_at AT2G46510 265547_at AT2G28305 265699_at AT2G03550 265724_at AT2G32100 265767_at AT2G48110 265817_at AT2G18050 265837_at AT2G14560 265869_at AT2G01760 265871_at AT2G01680 265894_at AT2G15050 265902_at AT2G25590 265929_s_at AT2G18560; AT2G18570 265943_at AT2G19570 265985_at AT2G24220 265993_at AT2G24160 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 1.54 2.19 -1.18 2.62 -3.54 1.63 -1.43 1.14 1.14 g1 g1 g1 1.22 -1.91 135 Table A.1 (Continued) 266007_at AT2G37380 266187_at AT2G38970 266204_at AT2G02410 266257_at AT2G27820 266300_at AT2G01420 266372_at AT2G41310 266426_x_at AT2G07140; AT3G44120 266472_at no_match 266483_at AT2G47910 266552_at AT2G46330 266635_at AT2G35470 266663_at AT2G25790 266707_at AT2G03310 266869_at AT2G44660 266873_at AT2G44740 267034_at AT2G38310 267076_at AT2G41090 267135_at AT2G23430 267192_at AT2G30890 267219_at AT2G02590 267238_at AT2G44130 267265_at AT2G22980 267289_at AT2G23770 267336_at AT2G19310 267425_at AT2G34810 267462_at AT2G33735 267495_at no_match g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 g1 -1.34 -1.85 -1.48 2.70 136 Table A.1 (Continued) 267500_s_at AT2G45510; AT2G44890 267535_at AT2G41940 267569_at AT2G30790 245130_at AT2G45340 245141_at AT2G45400 245196_at AT1G67750 245277_at AT4G15550 245304_at AT4G15630 245318_at AT4G16980 245321_at AT4G15545 245479_at AT4G16140 245524_at AT4G15920 245574_at AT4G14750 245583_at AT4G14920 245626_at AT1G56700 245657_at AT1G56720 245690_at AT5G04230 245696_at AT5G04190 245759_at AT1G66900 245783_s_at AT1G35180; AT1G35170 245845_at AT1G26150 245877_at AT1G26220 245906_at AT5G11070 245984_at AT5G13090 246021_at AT5G21100 246028_at AT5G21170 246034_at AT5G08350 g1 g1 g1 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 1.74 -1.80 -1.76 -1.47 -1.12 -2.38 -1.66 -1.34 -1.22 8.78 -2.19 1.69 137 Table A.1 (Continued) 246313_at AT1G31920 246427_at AT5G17400 246462_at AT5G16940 246487_at AT5G16030 246520_at AT5G15790 246522_at AT5G15830 246540_at AT5G15600 246576_at AT1G31650 246591_at AT5G14880 246633_at AT1G29720 246681_at AT5G33280 246701_at AT5G28020 246737_at AT5G27710 246759_at AT5G27950 246768_at AT5G27400 246781_at AT5G27350 246783_at AT5G27360 246909_at AT5G25770 246932_at AT5G25190 246968_at AT5G24870 246997_at AT5G67390 247040_at AT5G67150 247162_at AT5G65730 247193_at AT5G65380 247266_at AT5G64570 247278_at AT5G64380 247284_at AT5G64410 247304_at AT5G63850 247413_at no_match g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.39 -1.96 -1.12 1.63 -1.17 -1.37 -1.52 -2.75 -1.64 -1.35 1.43 -1.39 -1.55 138 -1.50 -1.18 -1.52 Table A.1 (Continued) 247522_at AT5G61340 247696_at AT5G59780 247780_at AT5G58770 247819_at AT5G58350 247826_at AT5G58480 247848_at AT5G58120 247884_at AT5G57800 247946_at AT5G57180 247954_at AT5G56870 247977_at AT5G56850 248028_at AT5G55620 248064_at no_match 248091_at AT5G55120 248153_at AT5G54250 248179_at AT5G54380 248246_at AT5G53200 248303_at AT5G53170 248395_at AT5G52120 248460_at AT5G50915 248566_s_at AT5G49740; AT5G49730 248600_at AT5G49390 248622_at AT5G49360 248683_at AT5G48490 248719_at AT5G47910 248759_at AT5G47610 248776_at AT5G47900 248890_at AT5G46270 248912_at AT5G45670 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.50 1.13 -1.79 1.38 -1.12 3.52 139 -1.16 Table A.1 (Continued) 248924_at AT5G45960 248961_at AT5G45650 249122_at AT5G43850 249215_at AT5G42800 249355_at AT5G40500 249383_at AT5G39860 249546_at AT5G38150 249693_at AT5G35750 249694_at AT5G35790 249727_at AT5G35490 249732_at AT5G24420 249774_at AT5G24150 249775_at AT5G24160 249777_at AT5G24210 249818_at AT5G23860 249860_at AT5G22860 249862_at AT5G22920 250007_at AT5G18670 250008_at AT5G18630 250017_at AT5G18140 250079_at AT5G16650 250102_at AT5G16590 250110_at AT5G15350 250180_at AT5G14450 250217_at AT5G14120 250249_at AT5G13760 250261_at AT5G13400 250286_at AT5G13320 250304_at AT5G12110 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 2.59 -1.44 -1.22 -1.62 -1.49 -1.33 -1.31 -1.62 -1.40 1.52 140 -2.31 Table A.1 (Continued) 250478_at AT5G10250 250482_at AT5G10320 250485_at AT5G09990 250549_at AT5G07860 250598_at AT5G07690 250613_at AT5G07240 250633_at AT5G07460 250642_at AT5G07180 250669_at AT5G06870 250696_at AT5G06790 250720_at AT5G06180 250742_at AT5G05800 250880_at AT5G04070 250968_at AT5G02890 251010_at AT5G02550 251011_at AT5G02560 251028_at AT5G02230 251068_at AT5G01920 251108_at AT5G01620 251142_at AT5G01015 251160_at AT3G63240 251195_at AT3G62930 251219_at AT3G62390 251324_at AT3G61430 251360_at AT3G61210 251391_at AT3G60910 251497_at AT3G59060 251503_at AT3G59140 251519_at AT3G59400 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.24 -1.54 -1.11 1.17 -1.88 -1.48 -1.61 -1.18 -1.72 1.14 -1.18 -1.52 -1.96 141 -1.25 Table A.1 (Continued) 251524_at AT3G58990 251601_at AT3G57800 251759_at AT3G55630 251771_at AT3G56000 251773_at AT3G55960 251856_at AT3G54720 251890_at AT3G54220 251968_at AT3G53100 251982_at AT3G53190 252011_at AT3G52720 252033_at AT3G51950 252092_at AT3G51420 252143_at AT3G51150 252167_at AT3G50560 252178_at AT3G50750 252189_at AT3G50070 252317_at AT3G48720 252353_at AT3G48200 252381_s_at AT3G47760; AT3G47750 252411_at AT3G47430 252421_at AT3G47540 252433_at AT3G47560 252594_at AT3G45680 252607_at AT3G44990 252629_at AT3G44970 252648_at AT3G44630 252652_at AT3G44720 252716_at AT3G43920 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.86 -2.50 -1.64 -1.33 -1.44 -2.35 -1.86 g2 g2 g2 g2 g2 g2 g2 g2 g2 1.28 142 1.18 1.74 Table A.1 (Continued) 252827_at AT4G39950 252852_at AT4G39900 252858_at AT4G39770 252870_at AT4G39940 252911_at AT4G39510 252916_at AT4G38950 252949_at AT4G38670 253042_at AT4G37550 253048_at AT4G37560 253052_at AT4G37310 253099_s_at AT4G37530; AT4G37520 253101_at AT4G37430 253246_at AT4G34600 253270_at AT4G34160 253278_at AT4G34220 253302_at AT4G33660 253305_at AT4G33666 253332_at AT4G33420 253344_at AT4G33550 253394_at AT4G32770 253397_at AT4G32710 253412_at AT4G33000 253440_at AT4G32570 253534_at AT4G31500 253548_at AT4G30993 253579_at AT4G30610 253620_at AT4G30520 253629_at AT4G30450 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 1.15 -1.14 1.54 -1.65 -1.84 3.65 -1.27 143 -1.18 1.66 -1.76 Table A.1 (Continued) 253657_at AT4G30110 253736_at AT4G28780 253788_at AT4G28680 253806_at AT4G28270 253814_at AT4G28290 253815_at AT4G28250 253849_at AT4G28080 253856_at AT4G28100 253874_at AT4G27450 253886_at AT4G27710 253922_at AT4G26850 253940_at AT4G26950 253946_at AT4G26790 253947_at AT4G26760 253966_at AT4G26520 254016_at AT4G26150 254041_at AT4G25830 254118_at AT4G24790 254119_at AT4G24780 254132_at AT4G24660 254145_at AT4G24700 254210_at AT4G23450 254226_at AT4G23690 254280_at AT4G22756 254301_at AT4G22790 254305_at AT4G22200 254328_at AT4G22570 254332_at AT4G22730 254333_at AT4G22753 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -2.86 1.67 -1.45 -1.13 -3.13 -1.29 -2.31 -1.21 1.25 -1.40 -1.34 -1.12 144 -1.67 -1.83 Table A.1 (Continued) 254384_at AT4G21870 254505_at AT4G19985 254544_at AT4G19820 254553_at AT4G19530 254561_at AT4G19160 254564_at AT4G19170 254573_at AT4G19420 254574_at AT4G19430 254603_at AT4G19050 254649_at AT4G18570 254688_at AT4G13830 254705_at AT4G17870 254746_at AT4G12980 254773_at AT4G13410 254787_at AT4G12690 254810_at AT4G12390 254862_at AT4G12030 254938_at AT4G10770 255008_at AT4G10060 255016_at AT4G10120 255025_at AT4G09900 255026_at AT4G09900 255065_s_at AT4G08870; AT4G08900 255088_at AT4G09350 255127_at AT4G08300 255129_at AT4G08290 255150_at AT4G08160 255302_at AT4G04830 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.54 1.96 1.26 -5.20 -1.98 -1.16 -1.25 g2 g2 g2 g2 g2 -1.42 145 Table A.1 (Continued) 255310_at AT4G04955 255344_s_at AT4G04570; AT4G04540 255411_at AT4G03110 255450_at AT4G02850 255511_at AT4G02075 255561_at AT4G02050 255578_at AT4G01450 255583_at AT4G01510 255604_at AT4G01080 255617_at AT4G01330 255622_at AT4G01070 255626_at AT4G00780 255698_at AT4G00150 255753_at AT1G18570 255773_at AT1G18590 255774_at AT1G18620 255779_at AT1G18650 255793_at AT2G33250 255802_s_at AT4G10150; AT4G10160 255817_at AT2G33330 255822_at AT2G40610 255881_at AT1G67070 255895_at AT1G18020; AT1G17990 256008_s_at AT1G34040; AT1G34060 256015_at AT1G19150 g2 g2 -1.36 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.14 -2.68 1.80 1.34 -2.13 -1.28 -1.62 1.15 g2 g2 g2 g2 g2 g2 146 -1.45 Table A.1 (Continued) 256096_at AT1G13650 256186_at AT1G51680 256262_at AT3G12150 256275_at AT3G12110 256299_at AT1G69530 256321_at AT1G55020 256336_at AT1G72030 256379_at AT1G66840 256386_at AT1G66540 256603_at AT3G28270 256619_at AT3G24460 256671_at AT3G52290 256673_at AT3G52370 256674_at AT3G52360 256772_at AT3G13750 256790_at AT3G16857 256792_at AT3G22150 256873_at AT3G26310 256874_at AT3G26320 256880_at AT3G26450 256892_at AT3G19000 256894_at AT3G21870 256908_at AT3G24040 256927_at AT3G22550 257205_at AT3G16520 257209_at AT3G14920 257264_at AT3G22060 257297_at AT3G28040 257299_at AT3G28050 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.21 -2.96 -1.44 -1.18 -2.52 -1.93 1.70 -2.78 3.56 -1.18 -1.87 -1.22 -1.64 -1.27 1.93 147 Table A.1 (Continued) 257300_at AT3G28080 257314_at AT3G26590 257315_at AT3G30775 257322_at ATMG01180 257381_at AT2G37950 257506_at AT1G29440 257510_at AT1G55360 257545_at AT3G23200 257547_at AT3G13000 257556_at no_match 257567_at AT3G23930 257635_at AT3G26280 257772_at AT3G23080 257789_at AT3G27020 257793_at AT3G26960 257801_at AT3G18750 257850_at AT3G13065 257860_at AT3G13062 257880_at AT3G16910 257959_at AT3G25560 257966_at AT3G19800 257999_at AT3G27540 258003_at AT3G29030 258054_at AT3G16240 258101_at AT3G23590 258222_at AT3G15680 258262_at AT3G15770 258299_at AT3G23410 258338_at AT3G16150 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.53 1.78 1.35 1.34 -1.11 -2.38 -1.12 -1.25 -2.91 -1.96 2.56 -3.92 148 -2.59 -1.95 -1.16 Table A.1 (Continued) 258368_at AT3G14240 258375_at AT3G17470 258409_at AT3G17640 258421_at AT3G16690 258456_at AT3G22420 258460_at AT3G17330 258468_at AT3G06070 258472_at AT3G06080 258527_at AT3G06850 258651_at AT3G09920 258676_at AT3G08600 258708_at AT3G09580 258750_at AT3G05910 258764_at AT3G10720 258782_at AT3G11750 258962_at AT3G10570 258983_at AT3G08860 258993_at AT3G08940 259010_at AT3G07340 259042_at AT3G03450 259072_at AT3G11700 259234_at AT3G11620 259298_at AT3G05370 259434_at AT1G01490 259460_at AT1G44000 259476_at AT1G19000 259540_at AT1G20640 259577_at AT1G35340 259669_at AT1G52340 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.97 -1.54 -2.72 -1.45 -1.20 1.47 -1.85 -1.16 -1.12 -1.32 -1.40 149 -1.13 Table A.1 (Continued) 259731_at AT1G77460 259760_at AT1G77580 259773_at AT1G29500 259775_at AT1G29530 259786_at AT1G29660 259787_at AT1G29460 259822_at AT1G66230 259853_at AT1G72300 259856_at AT1G68440 259927_at AT1G75100 259968_at AT1G76530 260037_at AT1G68840 260041_at AT1G68780 260079_s_at AT1G35460; AT5G33210 260109_at AT1G63260 260167_at AT1G71970 260221_at AT1G74670 260266_at AT1G68520 260287_at AT1G80440 260309_at AT1G70580 260317_at AT1G63800 260363_at AT1G70550 260385_at AT1G74090 260387_at AT1G74100 260388_at AT1G74070 260395_at AT1G69780 260431_at AT1G68190 260477_at AT1G11050 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.46 -1.66 -1.97 -1.11 -1.12 -2.15 1.61 150 -2.30 Table A.1 (Continued) 260481_at AT1G10960 260603_at AT1G55960 260693_at AT1G32450 260739_at AT1G15000 260831_at AT1G06830 260914_at AT1G02640 260922_at AT1G21560 260969_at AT1G12240 260983_at AT1G53560 261016_at AT1G26560 261031_at AT1G17360 261053_at AT1G01320 261054_at AT1G01320 261060_at AT1G17340 261108_at AT1G62960 261118_at AT1G75460 261139_at AT1G19700 261308_at AT1G48480 261346_at AT1G79720 261375_at AT1G53160 261451_at AT1G21060 261488_at AT1G14345 261505_at AT1G71696 261576_at AT1G01070 261594_at AT1G33240 261609_at AT1G49740 261611_at AT1G49730 261712_at AT1G32780 261751_at AT1G76080 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.67 -2.59 -1.44 -1.13 1.65 -1.22 1.15 -1.96 -1.54 -1.12 -2.57 1.37 151 Table A.1 (Continued) 261765_at AT1G15570 261769_at AT1G76100 261782_at AT1G76110 261794_at AT1G16060 261801_at AT1G30520 261803_at AT1G30500 261826_at AT1G11580 261876_at AT1G50590 261895_at AT1G80830 261913_at AT1G65860 261914_at AT1G65870 261927_at AT1G22500 262039_at AT1G80050 262049_at AT1G80180 262134_at AT1G77990 262177_at AT1G74710 262237_at AT1G48320 262250_at AT1G48280 262290_at AT1G70985 262354_at AT1G64200 262374_s_at AT1G72910; AT1G72930 262376_at AT1G72970 262382_at AT1G72920 262563_at AT1G34210 262577_at AT1G15290 262582_at AT1G15410 262587_at AT1G15490 262608_at AT1G14120 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.15 1.22 -1.73 1.34 1.25 1.79 -1.88 -1.86 -1.74 -1.12 152 -1.94 Table A.1 (Continued) 262646_at AT1G62800 262717_s_at AT1G16410; AT1G16400 262730_at AT1G16390 262733_s_at AT1G28660; AT1G28670 262826_at AT1G13080 262830_at AT1G14700 262847_at AT1G14840 262884_at AT1G64720 263034_at AT1G24020 263073_at AT2G17500 263179_at AT1G05710 263427_at AT2G22260 263442_at AT2G28605 263443_at AT2G28630 263477_at AT2G31790 263591_at AT2G01910 263598_at AT2G01850 263606_at AT2G16280 263668_at AT1G04350 263674_at AT2G04790 263715_at AT2G20570 263718_at AT2G13570 263777_at AT2G46450 263799_at AT2G24550 263866_at AT2G36950 263918_at AT2G36590 263946_at AT2G36000 g2 g2 -1.61 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 1.27 -1.66 1.48 -1.49 -1.34 -1.42 -1.76 1.96 -1.12 -1.22 -3.39 153 -1.37 Table A.1 (Continued) 263952_s_at AT2G35830; AT2G35810 263987_at AT2G42690 264001_at AT2G22420 264022_at AT2G21185 264052_at AT2G22330 264078_at AT2G28470 264083_at AT2G31230 264191_at AT1G54730 264501_at AT1G09390 264514_at AT1G09500 264521_at AT1G10020 264582_at AT1G05230 264692_at AT1G70000 264694_at AT1G70250 264697_at AT1G70210 264738_at AT1G62250 264840_at AT1G03440 264873_at AT1G24100 264920_at AT1G60550 264930_at AT1G60800 264956_at AT1G76990 264978_at AT1G27120 264998_at AT1G67330 265053_at AT1G52000 265066_at AT1G03870 265121_at AT1G62560 265122_at AT1G62540 265149_at AT1G51400 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.13 -1.18 -1.16 -1.26 1.48 -1.26 -1.72 154 -1.82 Table A.1 (Continued) 265175_at AT1G23480 265200_s_at AT2G36800; AT2G36790 265250_at AT2G01950 265297_at AT2G14080 265342_at AT2G18300 265387_at AT2G20670 265443_at AT2G20750 265444_s_at AT2G37180; AT2G37170 265572_at no_match 265584_at AT2G20180 265609_at AT2G25420 265665_at AT2G27420 265680_at AT2G32150 265726_at AT2G32010 265873_at AT2G01630 265877_at AT2G42380 265939_at AT2G19650 265990_at AT2G24280 266001_at AT2G24150 266038_at AT2G07680 266104_at AT2G45150 266265_at AT2G29340 266277_at AT2G29310 266278_at AT2G29300 266279_at AT2G29290 266291_at AT2G29320 266324_at AT2G46710 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 1.28 -1.45 -2.75 -2.91 -1.51 1.15 -1.64 1.90 155 Table A.1 (Continued) 266326_at AT2G46650 266357_at AT2G32290 266391_at AT2G41290 266456_at AT2G22770 266460_at AT2G47930 266474_at AT2G31110 266485_at AT2G47630 266589_at AT2G46250 266591_at AT2G46225 266682_at AT2G19780 266715_at AT2G46780 266790_at AT2G28950 266899_at AT2G34620 266996_at AT2G34490 267063_at AT2G41120 267096_at AT2G38180 267112_at AT2G14750 267115_s_at AT2G32540; AT2G32530 267116_at AT2G32560 267126_s_at AT2G23600; AT2G23590 267256_s_at AT2G23000; AT2G23010 267260_at AT2G23130 267304_at AT2G30080 267344_at AT2G44230 267359_at AT2G40020 267367_at AT2G44210 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 g2 -1.46 -1.68 -1.36 -1.13 -1.15 1.65 g2 g2 g2 g2 g2 g2 g2 g2 156 Table A.1 (Continued) 267380_at AT2G26170 267470_at AT2G30490 267481_at AT2G02780 267482_s_at AT2G02770; AT3G11470 267497_at AT2G30540 267516_at AT2G30520 267517_at AT2G30520 267520_at AT2G30460 267544_at AT2G32720 245090_at AT2G40900 245119_at AT2G41640 245247_at AT4G17230 245250_at AT4G17490 245251_at AT4G17615 245276_at AT4G16780 245306_at AT4G14690 245329_at AT4G14365 245346_at AT4G17090 245397_at AT4G14560 245401_at AT4G17670 245504_at AT4G15660 245528_at AT4G15530 245558_at AT4G15430 245635_at AT1G25250 245677_at AT1G56660 245711_at AT5G04340 245731_at AT1G73500 245757_at AT1G35140 g2 g2 g2 g2 g2 g2 g2 g2 g2 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 -2.84 2.50 -1.55 -1.49 -1.31 -1.67 1.22 -1.33 2.34 1.34 1.35 157 Table A.1 (Continued) 245777_at AT1G73540 245840_at AT1G58420 245866_s_at AT1G57990; AT1G57980 245905_at AT5G11090 245925_at AT5G28770 245981_at AT5G13100 246018_at AT5G10695 246099_at AT5G20230 246108_at AT5G28630 246125_at AT5G19875 246178_s_at AT5G28430; AT3G60930 246253_at AT4G37260 246289_at AT3G56880 246293_at AT3G56710 246468_at AT5G17050 246490_at AT5G15950; AT5G15948 246495_at AT5G16200 246597_at AT5G14760 246777_at AT5G27420 246821_at AT5G26920 246949_at AT5G25140 246987_at AT5G67300 247047_at AT5G66650 247137_at AT5G66210 247177_at AT5G65300 247208_at AT5G64870 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 -1.23 2.82 1.45 2.51 -1.76 -1.46 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 -1.93 -1.88 4.24 158 Table A.1 (Continued) 247240_at AT5G64660 247279_at AT5G64310 247393_at AT5G63130 247406_at AT5G62920 247426_at AT5G62570 247431_at AT5G62520 247452_at AT5G62430 247455_at AT5G62470 247585_at AT5G60680 247655_at AT5G59820 247693_at AT5G59730 247706_at AT5G59480 247708_at AT5G59550 247925_at AT5G57560 247949_at AT5G57220 248123_at AT5G54720 248164_at AT5G54490 248190_at AT5G54130 248191_at AT5G54130 248198_at AT5G54200 248253_at AT5G53290 248327_at AT5G52750 248389_at AT5G51990 248392_at AT5G52050 248432_at AT5G51390 248502_at AT5G50450 248607_at AT5G49480 248611_at AT5G49520 248799_at AT5G47230 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 -3.47 1.23 -1.62 -1.80 2.38 2.13 1.99 -1.48 -1.13 -1.68 -1.19 159 Table A.1 (Continued) 248870_at AT5G46710 248959_at AT5G45630 248964_at AT5G45340 249191_at AT5G42760 249237_at AT5G42050 249264_s_at AT5G41750; AT5G41740 249415_at AT5G39660 249418_at AT5G39785 249423_at AT5G39785 249522_at AT5G38700 249583_at AT5G37770 249606_at AT5G37260 249622_at AT5G37550 249746_at AT5G24590 249798_at AT5G23730 249910_at AT5G22630 249918_at AT5G19240 249928_at AT5G22250 249932_at AT5G22390 250098_at AT5G17350 250099_at AT5G17300 250158_at AT5G15190 250161_at AT5G15240 250207_at AT5G13930 250246_at AT5G13700 250335_at AT5G11650 250399_at AT5G10750 250445_at AT5G10760 g3 g3 g3 g3 g3 g3 -1.52 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 1.16 -1.51 -2.40 -1.37 160 Table A.1 (Continued) 250533_at AT5G08640 250676_at AT5G06320 250690_at AT5G06530 250781_at AT5G05410 250821_at AT5G05190 250926_at AT5G03555 251281_at AT3G61640 251336_at AT3G61190 251459_at AT3G60200 251563_at AT3G57880 251642_at AT3G57520 251727_at AT3G56290 251745_at AT3G55980 251925_at AT3G54000 252009_at AT3G52800 252045_at AT3G52450 252053_at AT3G52400 252123_at AT3G51240 252131_at AT3G50930 252278_at AT3G49530 252368_at AT3G48520 252374_at AT3G48100 252470_at AT3G46930 252474_at AT3G46620 252679_at AT3G44260 253066_at AT4G37770 253104_at AT4G36010 253113_at AT4G35985 253284_at AT4G34150 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 2.77 1.44 -1.11 -1.43 1.74 -1.94 1.15 3.30 -5.97 161 Table A.1 (Continued) 253292_at AT4G33985 253323_at AT4G33920 253405_at AT4G32800 253455_at AT4G32020 253535_at AT4G31550 253608_at AT4G30290 253614_at AT4G30350 253628_at AT4G30280 253643_at AT4G29780 253794_at AT4G28720 253830_at AT4G27652 253919_at AT4G27350 254066_at AT4G25480 254074_at AT4G25490 254075_at AT4G25470 254120_at AT4G24570 254158_at AT4G24380 254159_at AT4G24240 254235_at AT4G23750 254293_at AT4G23060 254447_at AT4G20860 254520_at AT4G19960 254592_at AT4G18880 254926_at AT4G11280 255524_at AT4G02330 255568_at AT4G01250 255733_at AT1G25400 255937_at AT1G12610 256129_at AT1G18210 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 -1.72 1.25 1.16 8.25 1.54 -1.56 2.37 162 Table A.1 (Continued) 256245_at AT3G12580 256356_s_at AT5G43620; AT1G66500 256430_at AT3G11020 256522_at AT1G66160 256526_at AT1G66090 256548_at AT3G14770 256627_at AT3G19970 256633_at AT3G28340 256715_at AT2G34090 256755_at AT3G25600 256763_at AT3G16860 256799_at AT3G18560 256891_at AT3G19030 256999_at AT3G14200 257053_at AT3G15210 257057_at AT3G15310 257083_s_at AT3G20600; AT3G20590 257087_at AT3G20500 257262_at AT3G21890 257375_at AT2G38640 257654_at AT3G13310 257925_at AT3G23170 258023_at AT3G19450 258139_at AT3G24520 258167_at AT3G21560 258349_at AT3G17609 258436_at AT3G16720 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 1.93 1.22 -2.36 -1.72 -1.59 1.29 -2.34 -1.12 -4.26 6.76 163 Table A.1 (Continued) 258497_at AT3G02380 258606_at AT3G02840 258682_at AT3G08720 258752_at AT3G09520 258792_at AT3G04640 259054_at AT3G03480 259076_at AT3G02140 259129_at AT3G02150 259244_at AT3G07650 259293_at AT3G11580 259312_at AT3G05200 259364_at AT1G13260 259428_at AT1G01560 259445_at AT1G02400 259466_at AT1G19050 259479_at AT1G19020 259734_at AT1G77500 259792_at AT1G29690 259834_at AT1G69570 259879_at AT1G76650 259925_at AT1G75040 260203_at AT1G52890 260205_at AT1G70700 260227_at AT1G74450 260399_at AT1G72520 260429_at AT1G72450 260656_at AT1G19380 260744_at AT1G15010 260753_at AT1G49230 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 -2.47 -1.83 1.42 1.68 1.46 1.47 1.22 -1.34 -2.92 -3.49 1.67 164 1.55 -1.54 1.86 1.30 Table A.1 (Continued) 260774_at AT1G78290 260915_at AT1G02660 261033_at AT1G17380 261037_at AT1G17420 261150_at AT1G19640 261263_at AT1G26790 261405_at AT1G18740 261450_s_at AT1G21110; AT1G21120 261456_at AT1G21050 261470_at AT1G28370 261526_at AT1G14370 261564_at AT1G01720 261648_at AT1G27730 261754_at AT1G76130 261892_at AT1G80840 261958_at AT1G64500 262003_at AT1G64460 262028_at AT1G35560 262212_at AT1G74890 262383_at AT1G72940 262384_at AT1G72950 262526_at AT1G17050 262801_at AT1G21010 262803_at AT1G21000 262883_at AT1G64780 262940_at AT1G79520 263122_at AT1G78510 263253_at AT2G31370 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 -1.55 -1.58 -1.77 1.45 9.68 1.23 1.31 1.33 -1.84 1.13 165 -1.57 -1.35 Table A.1 (Continued) 263374_at AT2G20560 263379_at AT2G40140 263584_at AT2G17040 263613_at AT2G25250 263735_s_at AT1G60040; AT1G59810 263783_at AT2G46400 263796_at AT2G24540 263800_at AT2G24600 263845_at AT2G37040 263935_at AT2G35930 264213_at AT1G65390 264217_at AT1G60190 264232_at AT1G67470 264314_at AT1G70420 264436_at AT1G10370 264512_at AT1G09575 264617_at AT2G17660 264636_at AT1G65490 264655_at AT1G09070 264758_at AT1G61340 264852_at AT2G17480 265028_at AT1G24530 265184_at AT1G23710 265327_at AT2G18210 265359_at AT2G16720 265626_at AT2G27260 265648_at AT2G27500 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 3.12 1.15 1.18 5.72 -1.24 2.16 1.65 1.75 -1.34 166 Table A.1 (Continued) 265670_s_at AT2G32190; AT2G32210 265740_at AT2G01150 265841_at AT2G35710 265892_at AT2G15020 265999_at AT2G24100 266071_at AT2G18680; AT2G18670 266097_at AT2G37970 266316_at AT2G27080 266363_at AT2G41250 266371_at AT2G41410 266545_at AT2G35290 266908_at AT2G34650 267028_at AT2G38470 267069_at AT2G41010 267083_at AT2G41100 267293_at AT2G23810 267337_at AT2G39980 267515_at AT2G45680 267595_at AT2G32990 245152_at AT2G47490 245242_at AT1G44446 245434_at AT4G17140 245563_at AT4G14580 245619_at AT4G13990 245982_at AT5G13170 246071_at AT5G20150 246272_at AT4G37150 g3 -1.34 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g3 g4 g4 g4 g4 g4 g4 g4 g4 -2.67 1.63 -3.65 1.38 -1.28 6.23 1.17 167 Table A.1 (Continued) 246310_at AT3G51895 246523_at AT5G15850 246911_at AT5G25810 247025_at AT5G67030 247029_at AT5G67190 247055_at AT5G66740 247175_at AT5G65280 247214_at AT5G64850 247323_at AT5G64170 247346_at AT5G63770 247368_at AT5G63330 247395_at AT5G62910 247438_at AT5G62460 247477_at AT5G62340 247519_at AT5G61430 247581_at AT5G61350 247601_at AT5G60850 247707_at AT5G59450 247734_at AT5G59400 247759_at AT5G59040 247793_at AT5G58650 247903_at AT5G57340 247921_at AT5G57660 247945_at AT5G57150 248151_at AT5G54270 248160_at AT5G54470 248207_at AT5G53970 248306_at AT5G52830 248311_at AT5G52570 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 -1.81 -1.15 -2.86 2.57 -1.48 -1.78 1.69 1.88 -1.22 -2.22 168 -1.51 -1.88 Table A.1 (Continued) 248366_at AT5G52510 248537_at AT5G50100 248686_at AT5G48540 248707_at AT5G48550 248711_at AT5G48270 248777_at AT5G47920 248820_at AT5G47060 248910_at AT5G45820 248956_at AT5G45610 249011_at AT5G44670 249071_at AT5G44050 249087_at AT5G44210 249134_at AT5G43150 249148_at AT5G43260 249242_at AT5G42250 249361_at AT5G40540 249422_at AT5G39760 249527_at AT5G38710 249540_at AT5G38120 249616_s_at AT5G37750; AT5G37440 249645_at AT5G36910 249677_at AT5G35970 249754_at AT5G24530 249755_at AT5G24580 249769_at AT5G24120 249823_s_at AT5G23350; AT5G23360 250187_at AT5G14370 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 1.39 g4 g4 g4 g4 g4 g4 2.73 -1.71 -1.53 g4 169 1.59 Table A.1 (Continued) 250277_at AT5G12940 250327_at AT5G12050 250379_at AT5G11590 250413_at AT5G11160 250657_at AT5G07000 250665_at AT5G06980 250673_at AT5G07070 250680_at AT5G06570 250694_at AT5G06710 250744_at AT5G05840 250803_at AT5G04980 250956_at AT5G03210 251024_at AT5G02180 251025_at AT5G02190 251109_at AT5G01600 251171_at AT3G63220 251190_at AT3G62690 251200_at AT3G63010 251356_at AT3G61060 251372_at AT3G60520 251839_at AT3G54950 251869_at AT3G54500 251984_at AT3G53260 252037_at AT3G51920 252175_at AT3G50700 252179_at AT3G50760 252204_at AT3G50340 252210_at AT3G50410 252212_at AT3G50310 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 1.23 -1.59 -1.82 -1.63 1.76 -1.99 -1.38 1.16 170 Table A.1 (Continued) 252214_at AT3G50260 252300_at AT3G49160 252414_at AT3G47420 252415_at AT3G47340 252429_at AT3G47500 252520_at AT3G46370 252879_at AT4G39390 252917_at AT4G38960 253039_at AT4G37760 253097_at AT4G37320 253103_at AT4G36110 253125_at AT4G36040 253129_at AT4G36020 253161_at AT4G35770 253165_at AT4G35320 253173_at AT4G35110 253496_at AT4G31870 253666_at AT4G30270 253679_at AT4G29610 253717_at AT4G29440 253722_at AT4G29190 253799_at AT4G28140 253809_at AT4G28320 253824_at AT4G27940 254186_at AT4G24010 254274_at AT4G22770 254300_at AT4G22780 254432_at AT4G20830 254496_at AT4G20070 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 1.85 8.69 2.42 -2.41 -1.12 1.46 3.42 1.99 1.48 6.14 1.31 171 Table A.1 (Continued) 254508_at AT4G20170 254562_at AT4G19230 254667_at AT4G18280 254683_at AT4G13800 254685_at AT4G13790 254707_at AT4G18010 254861_at AT4G12040 254998_at AT4G09760 255255_at AT4G05070 255381_at AT4G03510 255763_at AT1G16730 255877_at AT2G40460 256013_at AT1G19270 256020_at AT1G58290 256057_at AT1G07180 256169_at AT1G51800 256266_at AT3G12320 256324_at AT1G66760 256456_at AT1G75180 256527_at AT1G66100 256577_at AT3G28220 256751_at AT3G27170 256818_at AT3G21420 256981_at AT3G13380 257076_at AT3G19680 257280_at AT3G14440 257466_at AT1G62840 257485_at AT1G63580 257643_at AT3G25730 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 1.42 1.56 1.57 -2.95 -1.40 -2.13 -1.87 -1.79 -2.75 -1.32 -2.21 -1.89 -1.11 1.38 3.14 1.82 -1.71 172 -1.62 Table A.1 (Continued) 257710_at AT3G27350 257771_at AT3G23000 257805_at AT3G18830 258019_at AT3G19470 258188_at AT3G17800 258189_at AT3G17860 258207_at AT3G14050 258239_at AT3G27690 258321_at AT3G22840 258325_at AT3G22830 258383_at AT3G15440 258402_at AT3G15450 258437_at AT3G16560 258452_at AT3G22370 258487_at AT3G02550 258723_at AT3G09600 258724_at AT3G09600 258742_at AT3G05800 258813_at AT3G04060 259001_at AT3G01960 259012_at AT3G07360 259043_at AT3G03440 259070_at AT3G11670 259185_at AT3G01550 259275_at AT3G01060 259373_at AT1G69160 259685_at AT1G63090 259751_at AT1G71030 259871_at AT1G76800 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 1.47 -1.72 -2.98 -1.23 -2.26 -1.60 1.55 1.21 1.35 -2.92 -2.00 -1.67 -1.12 1.28 173 -1.22 Table A.1 (Continued) 259887_at AT1G76360 260076_at AT1G73630 260268_at AT1G68490 260380_at AT1G73870 260474_at AT1G11090 260540_at AT2G43500 260617_at AT1G53345 260658_at AT1G19410 260676_at AT1G19450 260763_at AT1G49220 260773_at AT1G78440 260807_at AT1G78310 260956_at AT1G06040 260975_at AT1G53430 261166_s_at AT3G15750; AT1G34570 261191_at AT1G32900 261247_at AT1G20070 261569_at AT1G01060 261581_at AT1G01140 261663_at AT1G18330 261719_at AT1G18380 261766_at AT1G15580 261772_at AT1G76240 261881_at AT1G80760 261912_s_at AT1G66060; AT1G66000 261937_at AT1G22570 262113_at AT1G02820 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 -2.45 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 -2.45 -3.13 -1.53 1.83 -1.25 1.17 1.96 2.47 174 Table A.1 (Continued) 262226_at AT1G53885; AT1G53903 262259_s_at AT1G53870; AT1G53890 262448_at AT1G49450 262590_at AT1G15100 262603_at AT1G15380 262635_at AT1G06570 262677_at AT1G75860 262836_at AT1G14680 263128_at AT1G78600 263252_at AT2G31380 263320_at AT2G47180 263345_s_at AT2G05070; AT2G05100 263382_at AT2G40230 263384_at AT2G40130 263433_at AT2G22240 263452_at AT2G22190 263509_s_at ATMG00730 ;AT2G07687 263513_at AT2G12400 263549_at AT2G21650 263556_at AT2G16365 263595_at AT2G01890 263656_at AT1G04240 263739_at AT2G21320 263901_at AT2G36320 263931_at AT2G36220 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 1.63 1.99 1.13 5.13 1.38 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 -1.44 1.65 175 -1.74 -1.32 -1.15 Table A.1 (Continued) 264057_at AT2G28550 264062_at AT2G27950 264102_at AT1G79270 264153_at AT1G65390 264157_at AT1G65310 264389_at AT1G11960 264400_at AT1G61800 264467_at AT1G10140 264537_at AT1G55610 264661_at AT1G09950 264783_at AT1G08650 264788_at AT2G17880 264862_at AT1G24330 265111_at AT1G62510 265248_at AT2G43010 265450_at AT2G46620 265842_at AT2G35700 265987_at AT2G24240 266006_at AT2G37360 266072_at AT2G18700 266125_at AT2G45050 266246_at AT2G27690 266275_at AT2G29370 266572_at AT2G23840 266672_at AT2G29650 266719_at AT2G46830 266720_s_at AT2G46670; AT2G46790 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 1.13 1.13 2.52 2.97 -1.42 1.98 -1.38 1.54 -1.83 1.66 -1.15 176 -1.46 Table A.1 (Continued) 266772_s_at AT4G16540; AT2G03020 266797_at AT2G22840 266820_at AT2G44940 266875_at AT2G44800 267177_at AT2G37580 267199_at AT2G30990 267246_at AT2G30250 267411_at AT2G34930 267523_at AT2G30600 267524_at AT2G30600 267591_at AT2G39705 245038_at AT2G26560 245078_at AT2G23340 245096_at AT2G40880 245200_at AT1G67850 245237_at AT4G25520 245323_at AT4G16500 245336_at AT4G16515 245352_at AT4G15490 245427_at AT4G17550 245506_at AT4G15700 245512_at AT4G15770 245523_at AT4G15910 245533_at AT4G15130 245627_at AT1G56600 245641_at AT1G25370 245644_at AT1G25320 245699_at AT5G04250 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g4 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.78 3.15 2.66 1.49 2.74 3.39 2.57 3.65 177 1.27 Table A.1 (Continued) 245702_at AT5G04220 245734_at AT1G73480 245749_at AT1G51090 245751_s_at AT1G25682; AT1G25988 245794_at AT1G32170 245807_at AT1G46768 245821_at AT1G26270 245879_at AT5G09420 245885_at AT5G09440 245904_at AT5G11110 245998_at AT5G20830 246001_at AT5G20790 246070_at AT5G20160 246082_at AT5G20480 246088_at AT5G20600 246189_at AT5G20910 246217_at AT4G36920 246222_at AT4G36900 246282_at AT4G36580 246305_at AT3G51890 246419_at AT5G17030 246432_at AT5G17490 246435_at AT5G17460 246461_at AT5G16930 246481_s_at AT5G15960; AT5G15970 246484_at AT5G16040 246527_at AT5G15750 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 6.67 2.66 1.69 -1.29 3.55 1.52 3.82 -1.92 5.19 -1.31 g5 g5 178 Table A.1 (Continued) 246566_at AT5G14940 246703_at AT5G28080 246708_at AT5G28150 246755_at AT5G27920 246756_at AT5G27930 246779_at AT5G27520 246796_at AT5G26770 246831_at AT5G26340 246885_at AT5G26230 246922_at AT5G25110 246939_at AT5G25390 247027_at AT5G67090 247046_at AT5G66540 247095_at AT5G66400 247097_at AT5G66460 247268_at AT5G64080 247280_at AT5G64260 247287_at AT5G64230 247318_at AT5G63990 247351_at AT5G63790 247370_at AT5G63320 247378_at AT5G63120 247445_at AT5G62640 247450_at AT5G62350 247453_at AT5G62440 247474_at AT5G62280 247478_at AT5G62360 247487_at AT5G62150 247498_at AT5G61810 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 2.15 2.14 4.95 -1.12 -1.38 1.37 1.87 1.74 2.57 1.79 179 1.46 Table A.1 (Continued) 247525_at AT5G61380 247550_at AT5G61370 247593_at AT5G60790 247653_at AT5G59950 247676_at no_match 247727_at AT5G59490 247739_at AT5G59240 247774_at AT5G58660 247775_at AT5G58690 247776_at AT5G58700 247786_at AT5G58600 247794_at AT5G58670 247795_at AT5G58620 247851_at AT5G58070 247867_at AT5G57630 247937_at AT5G57110 247957_at AT5G57050 247983_at AT5G56630 247989_at AT5G56350 248000_at AT5G56190 248040_at AT5G55970 248062_at AT5G55450 248082_at AT5G55400 248100_at AT5G55180 248236_at AT5G53870 248326_at AT5G52820 248328_at AT5G52660 248337_at AT5G52310 248352_at AT5G52300 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.52 1.85 2.33 1.29 -1.69 1.39 1.53 -1.40 6.77 3.16 1.41 180 1.57 2.73 Table A.1 (Continued) 248381_at AT5G51830 248451_at AT5G51180 248466_at AT5G50720 248467_at AT5G50800 248471_at AT5G50840 248505_at AT5G50360 248581_at AT5G49900 248592_at AT5G49280 248743_at no_match 248744_at AT5G48250 248745_at no_match 248749_at AT5G47880 248762_at AT5G47455 248833_at AT5G47120 248848_at AT5G46520 248895_at AT5G46330 248911_at AT5G45830 248914_at AT5G45750 249063_at AT5G44110 249065_at AT5G44260 249091_at AT5G43860 249174_at AT5G42900 249204_at AT5G42570 249252_at AT5G42010 249303_at AT5G41460 249411_at AT5G40390 249528_at AT5G38720 249581_at AT5G37600 249741_at AT5G24470 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.38 6.76 -1.27 -1.33 -1.15 1.18 1.75 181 Table A.1 (Continued) 249750_at AT5G24570 249806_at AT5G23850 249944_at AT5G22290 249984_at AT5G18400 250053_at AT5G17850 250054_at AT5G17860 250062_at AT5G17760 250072_at AT5G17210 250151_at AT5G14570 250155_at AT5G15160 250194_at AT5G14550 250201_at AT5G14230 250213_at AT5G13820 250279_at AT5G13200 250300_at AT5G11890 250309_at AT5G12220 250316_at AT5G12140 250405_at AT5G10790 250467_at AT5G10100 250504_at AT5G09840 250522_at AT5G08500 250556_at AT5G07920 250662_at AT5G07010 250670_at AT5G06860 250711_at AT5G06110 250758_at AT5G06000 250793_at AT5G05600 250971_at AT5G02810 250974_at AT5G02820 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 2.99 1.64 2.45 1.44 3.31 4.66 2.27 2.24 1.20 1.55 2.26 182 Table A.1 (Continued) 251063_at AT5G01850 251084_at AT5G01520 251132_at AT5G01200 251225_at AT3G62660 251272_at AT3G61890 251282_at AT3G61630 251323_at AT3G61580 251346_at AT3G60980 251432_at AT3G59820 251494_at AT3G59350 251544_at AT3G58790 251603_at AT3G57760 251620_at AT3G58060 251623_at AT3G57390 251668_at AT3G57010 251735_at AT3G56090 251753_at AT3G55760 251768_at AT3G55940 251775_s_at AT2G39800; AT3G55610 251793_at AT3G55580 251800_at AT3G55510 251804_at AT3G55430 251895_at AT3G54420 251899_at AT3G54400 251910_at AT3G53810 251915_at AT3G53940 251927_at AT3G53990 251961_at AT3G53620 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.25 1.46 4.51 3.68 1.99 2.39 183 Table A.1 (Continued) 252026_at AT3G53030 252058_at AT3G52470 252076_at AT3G51660 252102_at AT3G50970 252127_at AT3G50960 252255_at AT3G49220 252321_at AT3G48510 252422_at AT3G47550 252557_at AT3G45960 252563_at AT3G45970 252573_at AT3G45260 252591_at AT3G45600 252944_at AT4G39320 252956_at AT4G38580 252986_at AT4G38380 252997_at AT4G38400 253130_at AT4G35510 253140_at AT4G35480 253167_at AT4G35310 253188_at AT4G35300 253213_at AT4G34910 253252_at AT4G34740 253253_at AT4G34750 253254_at AT4G34650 253322_at AT4G33980 253416_at AT4G33070 253423_at AT4G32280 253425_at AT4G32190 253454_at AT4G31875 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 -1.74 7.73 4.98 2.48 3.31 1.53 -1.19 3.72 2.53 1.78 1.46 1.94 1.73 1.14 -4.32 1.79 184 -2.45 -3.39 Table A.1 (Continued) 253485_at AT4G31800 253559_at AT4G31140 253595_at AT4G30830 253603_at AT4G30935 253695_at AT4G29510 253737_at AT4G28703 253813_at AT4G28150 253818_at AT4G28330 253828_at AT4G27970 253835_at AT4G27820 253841_at AT4G27830 253872_at AT4G27410 253875_at AT4G27520 253879_s_at AT4G27560; AT4G27570 253891_at AT4G27720 253892_at AT4G27620 253975_at AT4G26600 253994_at AT4G26080 254043_at AT4G25990 254073_at AT4G25500 254079_at AT4G25730 254085_at AT4G24960 254157_at AT4G24220 254178_at AT4G23880 254185_at AT4G23990 254188_at AT4G23920 254193_at AT4G23870 254204_at AT4G24160 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.57 1.12 4.76 3.46 2.25 -1.22 -1.73 1.27 4.51 1.52 1.18 -2.77 -1.66 1.46 1.62 -1.32 185 Table A.1 (Continued) 254227_at AT4G23630 254255_at AT4G23220 254268_at AT4G23040 254290_at AT4G23000 254304_at AT4G22270 254321_at AT4G22590; AT4G22592 254390_at AT4G21940 254422_at AT4G21560 254446_at AT4G20890 254563_at AT4G19120 254636_at AT4G18700 254656_at AT4G18070 254662_at AT4G18270 254670_at AT4G18390 254805_at AT4G12480 254818_at AT4G12470 254850_at AT4G12000 254858_at AT4G12070 254890_at AT4G11600 254917_at AT4G11350 254922_at AT4G11370 254952_at AT4G10955; AT4G10960 255039_at AT4G09570 255225_at AT4G05410 255236_at AT4G05520 255259_at AT4G05020 255278_at AT4G04940 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.57 -1.67 -1.19 -1.11 2.13 -5.44 4.26 2.34 -1.12 186 1.38 2.26 -1.53 Table A.1 (Continued) 255382_at AT4G03430 255452_at AT4G02880 255479_at AT4G02380 255485_at AT4G02550 255494_at AT4G02710 255501_at AT4G02400 255525_at AT4G02340 255543_at AT4G01870 255585_at AT4G01550 255625_at AT4G01120 255723_at AT3G29575 255795_at AT2G33380 255931_at AT1G12710 256014_at AT1G19200 256017_at AT1G19180 256069_at AT1G13740 256091_at AT1G20693 256114_at AT1G16850 256116_at AT1G16858; AT1G16860 256149_at AT1G55110 256235_at AT3G12490 256285_at AT3G12510 256288_at AT3G12270 256296_at AT1G69480 256310_at AT1G30360 256340_at AT1G72070 256576_at AT3G28210 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.66 -1.53 1.13 8.45 -1.17 1.53 1.79 187 Table A.1 (Continued) 256595_x_at AT3G28530; AT1G36770 256797_at AT3G18600 256833_at AT3G22910 257022_at AT3G19580 257035_at AT3G19270 257154_at AT3G27210 257188_at AT3G13150 257226_at AT3G27880 257237_at AT3G14890 257444_at AT2G12550 257484_at AT1G01650 257487_at AT1G71850 257519_at AT3G01210 257593_at AT3G24840 257610_at AT3G13810 257650_at AT3G16800 257652_at AT3G16810 257653_at AT3G13225 257708_at AT3G13330 257719_at AT3G18440 257876_at AT3G17130 258078_at AT3G25870 258092_at AT3G14595 258104_at AT3G23620 258137_at AT3G24515 258166_at AT3G21540 258180_at no_match 258202_at AT3G13940 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.31 5.19 1.56 1.76 5.45 3.59 188 Table A.1 (Continued) 258204_at AT3G13960 258209_at AT3G14060 258252_at AT3G15720 258316_at AT3G22660 258395_at AT3G15500 258505_at AT3G06530 258507_at AT3G06500 258509_at AT3G06620 258545_at AT3G07050 258562_at AT3G05980 258603_at AT3G02990 258664_at AT3G08700 258665_at AT3G08710 258683_at AT3G08760 258719_at AT3G09540 258777_at AT3G11850 258805_at AT3G04010 258809_at AT3G04070 258871_at AT3G03060 258878_at AT3G03170 258893_at AT3G05660 258965_at AT3G10530 258979_at AT3G09440 258981_at AT3G08880 259037_at AT3G09350 259077_s_at AT5G15650; AT3G02230 259105_at AT3G05500 259109_at AT3G05580 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 -1.58 -1.58 1.44 -1.47 2.68 5.66 g5 g5 189 Table A.1 (Continued) 259132_at AT3G02250 259137_at AT3G10300 259173_at AT3G03640 259175_at AT3G01560 259231_at AT3G11410 259232_at AT3G11420 259417_at AT1G02340 259426_at AT1G01470 259436_at AT1G01500 259442_at AT1G02310 259444_at AT1G02370 259516_at AT1G20450 259568_at AT1G20490 259570_at AT1G20440 259582_at AT1G28060 259588_at AT1G27930 259595_at AT1G28050 259605_at AT1G27910 259606_at AT1G27920 259611_at AT1G52280 259626_at AT1G42990 259704_at AT1G77680 259711_at AT1G77570 259789_at AT1G29395 259841_at AT1G52200 259876_at AT1G76700 259878_at AT1G76790 259971_at AT1G76580 259977_at AT1G76590 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.11 4.57 1.19 3.66 1.92 -2.92 3.30 2.34 1.66 -2.56 1.74 5.29 1.15 2.42 2.18 -1.76 1.19 190 -2.28 -1.26 1.24 -1.18 -1.55 Table A.1 (Continued) 259992_at AT1G67970 260075_at AT1G73700 260176_at AT1G71950 260209_at AT1G68552; AT1G68550 260262_at AT1G68470 260264_at AT1G68500 260276_at AT1G80450 260312_at AT1G63880 260338_at AT1G69250 260345_at AT1G69270 260352_at AT1G69295 260410_at AT1G69870 260489_at AT1G51610 260556_at AT2G43620 260628_at AT1G62320 260674_at AT1G19370 260727_at AT1G48100 260776_at AT1G14580 260804_at AT1G78410 260832_at AT1G06780 260870_at AT1G43890 260876_at AT1G21460 260921_at AT1G21540 261004_at AT1G26450 261048_at AT1G01420 261076_at AT1G07420 261077_at AT1G07430 261109_at AT1G75450 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.41 1.22 1.35 -1.31 5.36 1.28 2.22 7.38 1.39 1.82 5.26 2.22 -1.66 -1.17 1.15 2.46 1.46 1.43 3.49 191 Table A.1 (Continued) 261168_at AT1G04945 261187_at AT1G32860 261192_at AT1G32870 261225_at AT1G20100 261248_at AT1G20030 261254_at AT1G05805 261318_at AT1G53035 261356_at AT1G79660 261366_at AT1G53100 261377_at AT1G18850 261436_at AT1G07870 261453_at AT1G21130 261482_at AT1G14530 261506_at AT1G71697 261522_at AT1G71710 261566_at AT1G33230 261610_at AT1G49560 261613_at AT1G49720 261651_at AT1G27760 261655_at AT1G01940 261664_s_at AT1G18320; AT3G10110 261718_at AT1G18390 261726_at AT1G76270 261728_at AT1G76160 261745_at AT1G08500 261749_at AT1G76180 261818_at AT1G11390 261899_at AT1G80820 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 3.72 1.35 1.97 1.19 2.12 1.35 g5 g5 g5 g5 g5 g5 g5 192 1.12 Table A.1 (Continued) 262050_at AT1G80130 262061_at AT1G80110 262099_s_at AT4G37390; AT1G59500 262119_s_at AT1G02930; AT1G02920 262136_at AT1G77850 262164_at AT1G78070 262166_at AT1G74840 262219_at AT1G74750 262307_at AT1G71000 262313_at AT1G70900 262440_at AT1G47710 262452_at AT1G11210 262478_at AT1G11170 262496_at AT1G21790 262503_at AT1G21670 262584_at AT1G15440 262644_at AT1G62710 262706_at AT1G16280 262760_at AT1G10770 262843_at AT1G14687 262881_at AT1G64890 262953_at AT1G75670 263004_at AT1G54510 263082_at AT2G27200 263221_at AT1G30620 263222_at AT1G30640 263249_at AT2G31360 g5 g5 g5 3.63 1.23 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 2.95 2.42 2.19 2.64 1.11 3.60 2.17 2.16 1.17 193 Table A.1 (Continued) 263259_at AT1G10560 263330_at AT2G15320 263352_at AT2G22080 263495_at AT2G42530 263497_at AT2G42540 263517_at AT2G21620 263574_at AT2G16990 263789_at AT2G24560 263797_at AT2G24570 263823_s_at AT2G40340; AT2G40350 263963_at AT2G36080 263981_at AT2G42870 264000_at AT2G22500 264019_at AT2G21130 264024_at AT2G21180 264042_at AT2G03760 264118_at AT1G79150 264123_at AT1G02270 264131_at AT1G79150 264190_at AT1G54830 264211_at AT1G22770 264246_at AT1G60140 264261_at AT1G09240 264289_at AT1G61890 264398_at AT1G61730 264452_at AT1G10270 264458_at AT1G10410 264511_at AT1G09350 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.12 2.22 7.29 9.79 -1.19 5.45 6.85 1.94 1.46 2.34 9.15 1.69 194 2.11 1.85 Table A.1 (Continued) 264515_at AT1G10090 264516_at AT1G10090 264529_at AT1G30820 264560_at AT1G55820 264580_at AT1G05340 264624_at AT1G08930 264652_at AT1G08920 264654_s_at AT1G08890; AT1G08900 264767_at AT1G61380 264787_at AT2G17840 264818_at AT1G03530 264841_at AT1G03740 264893_at AT1G23140 264907_at AT2G17280 264948_at AT1G77050 264953_at AT1G77120 264968_at AT1G67360 264989_at AT1G27200 264999_at AT1G67310 265025_at AT1G24575 265061_at AT1G61640 265093_at AT1G03905 265119_at AT1G62570 265147_at AT1G51380 265154_at AT1G30960 265197_at AT2G36750 265214_at AT1G05000 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 2.34 1.74 1.63 1.72 2.40 2.22 2.87 1.31 1.37 4.29 -1.36 1.17 195 Table A.1 (Continued) 265244_at AT2G43018; AT2G43020 265276_at AT2G28400 265283_at AT2G20370 265290_at AT2G22590 265333_at AT2G18350 265354_at AT2G16700 265480_at AT2G15970 265634_at AT2G25530 265662_at AT2G24500 265728_at AT2G31990 265886_at AT2G25620 265913_at AT2G25625 265931_at AT2G18520 265935_at AT2G19580 265941_s_at AT2G19490; AT3G32920 266049_at AT2G40780 266100_at AT2G37980 266119_at AT2G02100 266141_at AT2G02120 266184_s_at AT3G54700; AT2G38940 266225_at AT2G28900 266229_at AT2G28840 266259_at AT2G27830 266327_at AT2G46680 266358_at AT2G32280 266510_at AT2G47990 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.75 1.57 1.66 2.41 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 1.33 3.49 3.45 -2.64 2.45 196 2.49 Table A.1 (Continued) 266514_at AT2G47890 266532_at AT2G16890 266555_at AT2G46270 266695_at AT2G19810 266702_at AT2G19860 266799_at AT2G22860 266831_at AT2G22830 266861_at AT2G26830 266934_at AT2G18900 266946_at AT2G18890 267019_at AT2G39130 267036_at AT2G38465 267081_at AT2G41210 267163_at AT2G37520 267201_at AT2G31010 267261_at AT2G23120 267266_at AT2G23150 267280_at AT2G19450 267315_at no_match 267335_s_at AT2G19440; AT1G64760 267361_at AT2G39920 267364_at AT2G40080 267429_at AT2G34850 267509_at AT2G45660 267534_at AT2G41900 267576_at AT2G30640 267631_at AT2G42150 245188_at AT1G67660 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g5 g6 2.20 1.21 1.52 2.21 3.68 1.40 1.15 1.34 2.34 1.26 1.73 -1.67 1.88 1.15 197 Table A.1 (Continued) 245319_at AT4G16146 245405_at AT4G17150 245432_at AT4G17100 245433_at AT4G17140 245437_at no_match 245505_at AT4G15690 245600_at AT4G14230 245602_at AT4G14270 245668_at AT1G28330 245694_at AT5G04170 246288_at AT1G31850 246550_at AT5G14920 246791_at AT5G27280 246829_at AT5G26570 246881_at AT5G26040 247013_at AT5G67480 247302_at AT5G63880 247348_at AT5G63810 247488_at AT5G61820 247541_at AT5G61660 247668_at AT5G60100 247723_at AT5G59220 247878_at AT5G57760 248001_at AT5G55990 248138_at AT5G54960 248410_at AT5G51570 248587_at AT5G49550 248698_at AT5G48380 248793_at AT5G47240 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 1.66 1.38 1.85 1.94 1.32 2.72 1.12 1.86 1.54 1.52 1.38 1.16 2.56 1.24 2.84 1.53 198 Table A.1 (Continued) 249015_at AT5G44730 249128_at AT5G43440 249220_at AT5G42420 249346_at AT5G40780 249456_at AT5G39410 249619_at AT5G37500 249719_at AT5G35735 249850_at AT5G23240 249917_at AT5G22460 249989_at AT5G18525 249990_at AT5G18540 250028_at AT5G18130 250096_at AT5G17190 250127_at AT5G16380 250252_at AT5G13750 250290_at AT5G13310 250412_at AT5G11150 250648_at AT5G06760 250649_at AT5G06690 250826_at AT5G05220 250858_at AT5G04760 250935_at AT5G03240 250987_at AT5G02860 251039_at AT5G02020 251221_at AT3G62550 251248_at AT3G62150 251400_at AT3G60420 251529_at AT3G58570 251725_at AT3G56260 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 3.98 2.59 1.15 1.28 5.38 1.96 2.25 1.45 1.94 1.24 1.46 1.26 1.13 1.16 -2.59 1.41 1.94 -1.42 199 1.76 1.67 1.19 1.46 1.65 Table A.1 (Continued) 251789_at AT3G55450 251791_at AT3G55500 251975_at AT3G53230 251977_at AT3G53250 252166_at AT3G50500 252355_at AT3G48250 252391_at AT3G47860 252464_at AT3G47160 252468_at AT3G46970 252475_s_at AT5G59570; AT3G46640 252880_at AT4G39730 252882_at AT4G39675 252885_at AT4G39260 252908_at AT4G39670 252927_at AT4G39090 252940_at AT4G39270 252976_s_at AT4G38550 253215_at AT4G34950 253245_at AT4G34590; AT4G34588 253293_at AT4G33905 253331_at AT4G33490 253581_at AT4G30660 253592_at AT4G30840 253622_at AT4G30560 253627_at AT4G30650 253871_at AT4G27440 253981_at AT4G26670 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 1.85 1.20 1.62 1.58 1.55 1.11 1.47 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 1.32 2.46 1.75 4.38 1.12 1.37 200 1.34 1.11 Table A.1 (Continued) 254232_at AT4G23600 254408_at AT4G21390 254490_at AT4G20320 254580_at AT4G19390 254634_at AT4G18650 254645_at AT4G18520 254646_at AT4G18530 254659_at AT4G18240 254778_at AT4G12750 254833_s_at AT4G12290; AT4G12280 255070_at AT4G09020 255221_at AT4G05150 255232_at AT4G05330 255331_at AT4G04330 255430_at AT4G03320 255477_at AT4G02370 255607_at AT4G01130 255923_at AT1G22180 255953_at AT1G22070 255980_at AT1G33970 256060_at AT1G07050 256062_at AT1G07090 256092_at AT1G20696 256221_at AT1G56300 256426_at AT1G33420 256464_at AT1G32560 256601_s_at AT3G28290; AT3G28300 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 1.21 1.24 1.57 1.72 1.50 1.62 1.68 1.78 1.82 1.74 1.24 1.11 1.98 1.86 201 1.24 1.26 3.13 1.98 1.39 Table A.1 (Continued) 256623_at AT3G19960 256676_at AT3G52180 256825_at AT3G22120 256839_at AT3G22930 257144_at AT3G27300 257147_at AT3G27270 257271_at AT3G28007 257985_at AT3G20810 258157_at AT3G18100 258347_at AT3G17520 258397_at AT3G15357 258435_at AT3G16740 258498_at AT3G02480 258735_at AT3G05880 258751_at AT3G05890 258827_at AT3G07150 258833_at AT3G07274 258887_at AT3G05630 258901_at AT3G05640 258939_at AT3G10020 258970_at AT3G10410 259080_at AT3G04910 259118_at AT3G01310 259178_at AT3G01650 259302_at AT3G05120 259705_at AT1G77450 260005_at AT1G67920 260357_at AT1G69260 260412_at AT1G69830 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 1.22 1.76 1.16 -1.38 2.19 3.50 1.47 1.89 1.48 1.28 1.59 202 Table A.1 (Continued) 260627_at AT1G62310 260688_at AT1G17665 260974_at AT1G53440 261125_at AT1G04990 261203_at AT1G12845 261272_at AT1G26665 261428_at AT1G18870 262128_at AT1G52690 262248_at AT1G48370 262296_at AT1G27630 262324_at AT1G64170 262477_at AT1G11220 262548_at AT1G31280 262607_at AT1G13990 262609_at AT1G13930 262680_at AT1G75880 262690_at AT1G62720 262703_at AT1G16510 262722_at AT1G43620 262775_at AT1G13000 262784_at AT1G10760 262892_at AT1G79440 263065_at AT2G18170 263295_at AT2G14210 263472_at AT2G31955 263493_at AT2G42520 263548_at AT2G21660 263653_at AT1G04310 263708_at AT1G09320 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 1.45 1.59 1.54 -1.13 -1.81 1.18 1.29 1.16 1.60 1.12 203 1.51 1.13 Table A.1 (Continued) 263881_at AT2G21820 263912_at AT2G36390 264045_at AT2G22450 264048_at AT2G22400 264209_at AT1G22740 264525_at AT1G10060 264888_at AT1G23070 264957_at AT1G77000 264972_at AT1G67370 264992_at AT1G67300 265082_at AT1G03830 265216_at AT1G05100 265271_at AT2G28360 265358_at AT2G16710 265478_at AT2G15890 265481_at AT2G15960 265900_at AT2G25730 266399_at AT2G38670 266462_at AT2G47770 266500_at AT2G06925 266503_at AT2G47780 266578_at AT2G23910 266839_at AT2G25930 266841_at AT2G26150 266952_at AT2G34555 267080_at AT2G41190 267084_at AT2G41180 267254_at AT2G23030 267378_at AT2G26200 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 g6 1.23 1.22 1.44 1.52 1.15 -1.63 1.55 204 Table A.1 (Continued) 267461_at AT2G33830 g6 1.14 205 2.87 1.34 REFERENCES 206 REFERENCES 1. 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