.ho-Mzbi twat...“- :Itiil. saw l“”££.b9...flw. . t 159L539:- wa .Nxaffimwuf I- :34. e. I ii}?! A H"; s . .n. . 9.3.x... ‘hr; 1“! If! 44...)... x5}... .5... .. (i... . r9...:..£.t u . . 9. FLEEMEU Tim LIBRARY 2 Do? Michigan State University This is to certify that the dissertation entitled TRANSCRIPTIONAL NETWORKS INVOLVED IN RESPONSE TO LOW TEMPERATURE STRESS IN ARABIDOPSIS THALIANA presented by Colleen J. Doherty has been accepted towards fulfillment of the requirements for the Ph.D. degree in Biochemistry and Molecular Biology 7Wr Major Professor’s Signature T/v/ V Date MSU is an afiinnative-acfion, equal-opportunity employer -.- -.-.-.— PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 5/08 K'IProleccaPres/CIRC/DaleDue.indd TRANSCRIPTIONAL NETWORKS INVOLVED IN RESPONSE TO LOW TEMPERATURE STRESS IN ARABIDOPSIS THALIANA By Colleen J. Doherty A Dissertation Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Biochemistry and Molecular Biology 2008 ABSTRACT Transcriptional Networks Involved in Response to Low Temperature Stress in Arabidopsis thaliana By Colleen J. Doherty Desirable agricultural land for crop growth is limited by extremes in temperature. Low temperatures place limits both geographically and temporally on the amount of crop land available for use. To survive in climates of low and freezing temperatures, some plants have the ability to cold acclimate, a process where plants can detect low temperature and adjust to survive freezing. Understanding how plants are capable of detecting low temperature and the signaling process involved in refocusing the transcriptome, proteome, and metabolome to levels that allow for survival of freezing temperatures will allow identification of targets for traits important to increase freezing tolerance in plants, potentially extending the growing season for some species. A complete understanding of plant cold acclimation would require creating a network of the cold response in plants as they cold acclimate. To elucidate this network two key questions need to be answered. The first, how do plants sense low temperature and secondly are there multiple mechanisms for cold acclimation and to what extent do known pathways in cold acclimation contribute? To address the first question our goal is to identify upstream components of a known pathway of cold response. The C-Repeat Binding Factor (CBF) cold response pathway has an important role in cold acclimation, the process whereby plants increase in freezing tolerance in response to low nonfreezing temperatures. In Arabidopsis, a cold acclimating plant, three AP2 domain- containing transcription factors, CBF 1, 2, and 3 (DREB 13, C, and A, respectively), are induced rapidly in response to low temperatures. Induction of CBF transcription factors is followed by expression of the CBF target genes, resulting in increased freezing tolerance. A key objective is to determine how plants sense low temperature and activate expression of the CBF genes. A cis- acting region of the CBF2 promoter that is sufficient for cold-induction of a reporter gene was identified. Here, CAMTA3, a calmodulin binding transcriptional activator, is identified as a trans-acting factor involved in the regulation of CBF2 in response to low temperature through this element, providing a potential link between calcium signaling and the CBF cold response pathway. Secondly, to identify the requirement for the CBF pathway in response to low temperature, plants expressing a dominant negative version of CBF, CBFZAC, were analyzed for their ability to cold acclimate and for changes in the cold-regulated transcriptome. This analysis showed that the expression of 40% of the cold-induced genes was affected in the CBFZAC plants. However, although not to the same extent as WT plants, CBFZAC plants were still able perceive and responds to low temperature through the process of cold acclimation. This result indicates that multiple pathways for cold acclimation exist. ACKNOWLEDGEMENTS First of all I would like to thank Dr. Michael Thomashow for the opportunity to work in the lab, for the support he has given me to try following my interests into the world of bioinforrnatic analysis, for his patience when many of the attempts did not work, and for reminding me to focus on the question. Thanks to my committee members, Dr. Arnosti, Dr. LaPres, Dr. Keegstra, and Dr. Kuo for their enthusiasm and support of my projects, their sound advice which I wish I had followed more closely, and their editorial skills to make this a readable document. I would like to thank my parents for their support. I am grateful that they were always proud of me, no matter how small the accomplishment and that they encouraged my love of “projects”. I would especially like to thank them for the interest they have shown in my work- "We found an error in your proposal, you say this is ‘interesting’.” Thanks are due to the past and present members of the Thomashow lab, especially: Heather Van Buskirk and Jonathan Vogel, who taught me molecular biology; their patience still astounds me to this day. Jonathan, thanks for showing me the real value of an autoclave. Susan Myers for her endless work, I’m not sure how many hundreds of RNA samples you tirelessly extracted, but this document would be years away from being complete without your efforts. Chin-Mei Lee, thanks for reminding me that science IS fun. Michael Mikkelson, Ryan Warner, Dona Canella, and Kanchan Pavangadkar, thanks for the science talks. Sarah Gilmour thanks for all of your help on lab projects, iv listening to talks, correcting grammar, keeping me organized, but mostly for the occasional chats on any subject imaginable. Catherine Le, Kelly McRay, Megan Sargent, Ryan Sartor, and Laura Stewart, thanks for your enthusiasm, hard work, and positive attitude. Thanks also to Marcela Carvallo, Diane Constan, Malia Dong, Bonnie St. John, Ritu Sharma, and Dan Zarka for being great lab mates. Many thanks to the biochemsitry, plant biology, and PRL students, especially Andrea Braeutigam, Lori lmboden, Joonyul Kim, Mark Linka, Janet Paper, Hiroshi Maeda, Dean Shooltz, and Dorothy Tappenden for the science discussions, encouragement, and advice. I wish I had time to do a tenth of the great ideas that were discussed. Thanks to the Biochemistry department and PRL for providing a supportive and encouraging environment for students. Dona, Andrea, and Mark, thanks for giving the journal club a go, next time, let’s meet over beer— if no one else wants to join us, we’ll still have a good time. Heather, Bonnie, Dorothy, Malia, Chin-Mei, Dean, Jessy, and Vash, without your support for the past year this would not be getting turned in now. Thanks Joe, for the stories and the whiskey. Thanks to Dunstan, Molly, and Allie, for being happy to see me no matter how late the experiments ran. Special thanks to Vash for your faith in me and for reminding me that pi is mystical, science is exciting, plants are amazing, dorkiness is acceptable, and oxygen is good! Love & Peace! PREFACE In Chapter 2, the experiments were conducted by the author of this thesis and Dr. Heather Van Buskirk. Dr. Van Buskirk was responsible for the promoter analysis and deletion experiments, the EMSA, and the construction of the CAMTA overexpression constructs. In Chapter 3, the experiments were conducted by the author of this thesis and Dr. Sarah J. Gilmour. Dr. Gilmour was responsible for the identification of the CBFZAC mutant, the creation and characterization of the CBF2AC/35s::CBF2 crosses. The metabolite analysis was a joint effort with Dr. Gilmour. In Chapter 4, Dr. Van Buskirk performed analysis of the CM2/CM1- Boszus reporter. vi TABLE OF CONTENTS LIST OF TABLES Ix LIST OF FIGURES x KEY TO ABBREVIATIONS XIII CHAPTER 1 Literature Review 1 Introduction .. .1 CBF PathWay Contributes to. the Cold AccllmatIon Ability of Arabidopsis ............................................................................... Regulation of CBFs in Response to Low Temperature” .. Regulation of CBFs in Response to Other Environmental Factors Calcium as a Potential Mechanism for the Induction of CBFs.. Evidence for CBF- Independent Pathways of Cold Acclimation . Cold Acclimation in the Absence of CBFs... . .1.1 ABA as a Potential Component of a CBF Independent Cold-AchImatIon Pathway. .12 Analysis of High Throughput Data Provides New InSIghts into Cold Acclimation .. . . ..13 Interaction between Cold and Development ..16 Developmental State Affects Low Temperature Responses ............. 17 Literature Cited. .20 cobaoibom CHAPTER 2 Roles for Arabidopsis CAMTA Transcription Factors in Cold- Regulated Gene Expression and Freezing Tolerance ...............24 Summary 24 Introduction 24 Results. 27 Discussion” 48 Materials and Methods 52 Literature Cited 58 CHAPTER 3 Dominant Negative Version of CBF2 Reveals Role of CBF Dependent and Independent Pathways in the Process of Cold Acclimation 61 Introduction 61 Results 65 Discussion ............................................................................... 93 Materials and Methods 97 Literature Cited 103 CHAPTER4 Integration of Cold Response with Other Environmental vii Responses Introduction .. Results Discussion . Materials and Methaa'g'll.‘II.'II.'II.'II.'II.'II.'IIIIIIII.‘II.‘IIIIIfIIfIIIIIIIIIIIIIIII. Literature Cited Appendix viii 107 . . 107 .108 .. 116 .119 .121 .. 123 ..166 LIST OF TABLES Table 2.1 Transcripts with a Pattern of Induction in the Cold Similar to Table 2.2 Transcripts up Early in Response to .Low Temperature ............... 40 Table 3.1 Models used for Haystack Analysis 74 Table A1 1279 Transcripts Differentially Regulated in Response to Low Temperature at 24h in Both WS and Col.. .134 ix LIST OF FIGURES Images in this dissertation are presented in color 2.1 Tetramer Containing CM2-box and CM1-box ls Able to Drive Transcription of GUS Reporter Gene in Response to Treatment at 4°C 28 2.2 CAMTAs Bind CM2-Box Region of CBF2 Promoter 30 2.3 T-DNA Insertion Lines Result in Loss of CAMTA Transcript Accumulation 31 2.4 CBF2 Levels Are Reduced in camta3 Plants 32 2.5 camta6 Growth Phenotype 33 2.6 CM2/CM1-BoszUS mRNA Levels Are Reduced in camta3 Plants ......... 34 2.7 Complementation of GUS Activity and CBF2 Transcript Levels in camta3 Plants by Ectopic Expression of CAMTA3 35 2.8 Recovery of WT Levels of CAMTA3 in camta3 x CM2/CM1-BoszUS Plants by Ectopic Expression of CAMTA3 36 2.9 CBF1, ZAT12, and CBF3 Levels in camta3 Plants 42 2.10 Galactinol synthase 3 Levels Are Reduced in camta3 Plants 43 2.11 Effect of camta3 and camta1/3 Mutations on Freezing Tolerance 45 2.12 Effect of camta1/3 Mutations on Development 46 2.13 CBF2 Levels Are Near Wild-Type Levels in camta1/3 Double Mutants .. 47 2.14 Model of CAMTA3 Regulation of CBF Expression in Response to Low Temperature . . .. . . .48 3.1 Identification of CBFZAC, a Truncated Version of CBF2 That Suppresses the 35s: C:BF2 Phenotype. . ........ ...66 3.2 CBFZAC Is a GIutamine-to-stop Codon Mutation 67 3.3 GOLS3 mRNA Levels Are Reduced in CBFZAC Plants 68 3. 4 CBF2 Levels in Plants ExpressIng 35s: :CBF2 and 35s: :CBFZAC Transgenes“ . . .. .69 3. 5 Expression of CBF Target Genes in Plants Expressmg 35s: :CBF2 and 353: :CBF2 AC Transgenes . .71 3. 6 Levels of CBFZAC mRNA Accumulate to Higher Levels than Endogenous CBF2 Levels" W. . .... . .72 3.7 Cold-Induced Transcripts 76 3.8 CBF Sufficient and Required Transcripts 78 3.9 CBF is Not Sufficient Yet Is Required for Induction of Nine Transcripts ....79 3.10 CBF is Neither Sufficient Nor Required for Induction of 370 Transcripts. 80 3.11 CBF is Sufficient But Not Required for Induction of 28 Transcripts ..... 82 3.12 CBF is Sufficient and Partially Required for the Induction of 64 CBF Integrated Transcripts“ . 84 3.13 CBF' Is Not Sufficient yet Is Partially Required for the Induction of 117 CBF Integrated Transcripts. . .85 3.14 Cold-Repressed Transcripts 86 3.15 CBF ls Sufficient But Is Not Required For the Repression of 194 Transcripts. . .88 3.16 CBF ls Neither Sufficient Nor Reqmred For the Repression of 221 Transcripts. .. . . .. .89 3.17 CBF Is Not Sufficient But Is Partially Required for the Repression of 23 CBF- -Integrated Transcripts“ . . . .90 3.18 GC-MS Metabolite Profiles of Warm and Cold Treated Plants 92 3.19 CBFZAC Survives Whole Plant Freeze Tests After Acclimation .......... 93 3. 20 Electrolyte Leakage Assays of WT and CBFZAC Plants Before and After Acclimation .94 4.1 Role for CAMTAs in Induction of CBFs by Various Stimuli 110 4.2 Histochemical staining of WT x CM2ICM1-BoszUS and camta3 x CM2ICM1-BoszUS seedlings 111 xi 4.3 Electrolyte Leakage Assay of Plants Grown in Short Day and Continuous 4.4 EL50 Values for Short Day and Constant Light Grown Plants 114 4.5 Cotyledon Phenotypes in CBF2AC Seedlings 117 4.6 Quantification of Cotyledon Phenotypes in CBF2AC Seedlings .......... 118 A1 Induction of ICICLE Transcription Factors in Response to Low Temperature Treatment and Mechanical Agitation 125 A2 Design of CBF Silencing Contruct 126 A3 CBF-Targeting RNAi Silencing Construct Produced Lines with Reduced CBF Levels .126 A4 Cold-Induced Transcript Levels in —tc T-DNA Insertion Lines 128 A5 Flower Phenotype of CBF Overexpressmg Plants Crossed to ZAT12 Overexpressing Plants” . .129 A6 COR15 Protein Levels in CBF2AC Plants 130 xii KEY TO ABBREVIATIONS ABA: Abscisic Acid ABRE: Abscisic Acid Response Element AP2: APETALAZ bHLH: Basic-Helix-Loop-Helix BP: Base Pair CAMTA: Calmodulin Binding Transcriptional Activator CAS: Cold Acclimation Specific genes CBF: C-repeat binding factor CaMV 35s: Cauliflower Mosaic Virus 35s promoter CBFZAC: CBF2 with stop codon upstream of activation domain CG-1 Element: DNA consensus sequence vCGCGb CRT: C—repeat ‘ CM-Box: Consevered Motif sequences identified in both the CBF2 and ZAT12 promoters Col-0: Columbia Ecotype of Arabidopsis thaliana COR: COId Responsive genes DRE: Dehydration Responsive Element DREB: Dehydration Responsive Element Binding factor EAR: ERF-Associated amphiphilic Repression domain ERF: Ethylene Response Factor EMS: Ethyl MethaneSulfonate EMSA: Electrophoretic Mobility Shift Assay xiii GI: Glgantea GO: Gene Ontology GOLS: GalactinOL Synthase GST: Glutathione S-Transferase GUS: beta-GlUcuronidaSe HDAC: Histone DeACetylase HOS: Hypersensitive to Osmotic Stress ICE: Inducer of CBF Expression ICEr: Inducer of CBF Expression region ICICLEs: Independent of CBF Influence Cold Late Expressed KB: KiIoBase pair MYB: MYeloBlastosis viral oncogene homolog MYC: MYeloCytomatosis viral oncogene homolog NAM: Non-Apical Meristem PCR: Polymerase Chain Reaction PKCa: Protein Kinase Cs PKD: Protein Kinase D qRT-PCR: Quantitative RT-PCR ROS: Reactive Oxygen Species RT-PCR: Reverse Transcriptase PCR SUMO: Small Ubiquitin related MOdifier T-DNA: Transfer DNA TBX: Telo-box cis-element xiv WS: Wassilewskija ecotype of Arabidopsis thaliana WT: Wild-Type ZAT: Zn transporter of Arabidopsis Thaliana ZT: ZeiTgeber XV CHAPTER ONE LITERATURE REVIEW Introduction Plants live in complex environments and'are often bombarded with signals from multiple sources at once. While cold stress is a major factor affecting the growth and development of a plant, plants do not experience cold in isolation but rather in the context of signals of nutrient stress, biotic stress, day length, light quality and quantity. These factors have an influence on the way a plant responds to low temperature. It is no surprise therefore that plants have developed a complex integrated system for handling these multiple inputs, allowing some inputs to act in synergy to give a combined response, while other times when signals conflict the plant uses these cues to inhibit its response to some signals. Understanding the response to low temperature in the midst of _ these other factors is providing new insights into how plants respond and integrate multiple input signals and how plants can survive and adapt in the complex and constantly changing world. Evaluating the cold response in the light of multiple signals can shed insights into the understanding of the mechanisms of cold acclimation. Historically, analysis of the cold response in plants has focused on isolating the cold response from all other variables. This focus on cold alone has yielded a wealth of information and provided the basis of our understanding of how plants respond to low temperature. The range of response to low temperature varies greatly between plants. Some tropical plants, such as tomato, Lycopersicon esculentum, are not able to withstand low temperatures and are considered both chilling sensitive and freezing sensitive (Zhang et al. 2004). Other plants, such as potato and some wild versions of tomato are chilling tolerant but freezing sensitive since they are able to survive low, non- freezing temperatures, but are not able to withstand temperatures that reach below freezing (Ballou et al. 2007). While no known wild-type plants are able to go directly from warm growth conditions to freezing temperatures, some temperate plants, such as wheat and Arabidopsis are able to cold acclimate after exposure to low, non-freezing temperatures. During this process of cold acclimation, large changes in transcription, metabolism, membrane composition and accumulation of cryoprotectant molecules allow plants to prepare for and survive freezing temperatures (Thomashow 1999; Viswanathan and Zhu 2002). Even among the freezing tolerant plants there is a range of ability for cold acclimation, with Arabidopsis being able to make changes to survive down to - 5°C while rye after acclimation can survive temperatures as low as -40°C (Jaglo-Ottosen et al. 1998; Thomashow 1999). CBF Pathway Contributes to the Cold Acclimation Ability of Arabidopsis One important pathway that contributes to the process of cold acclimation is the CBF cold-responsive pathway. CBFs are a family of AP2 domain containing DNA binding transcription factors defined by their signature sequences flanking the AP2 DNA binding domain. In the model plant, Arabidopsis, there are 6 members of the CBF family, three of which are cold- induced (CBF 1, 2, and 3). CBFs bind the CRT/DRE (C-Repeat Element! Drought Response Element) element in the promoter of their target genes and induce transcription of these genes in response to low temperature. CBFs are important components of the cold acclimation process; overexpression of the three cold-inducible CBFs leads to induction of the CBF target genes at warm temperatures and results in the ability of the plants to be freezing tolerant without a requirement for a period of cold acclimation (Thomashow 2001). It is clear that expression of CBFs is sufficient for cold acclimation in Arabidopsis, however, two important questions remain: How are CBFs themselves induced in response to low temperature? And, how much do CBFs contribute to the ability of plants to cold acclimate? Regulation of CBFs in Response to Low Temperature The accumulation of CBF 1,2, and 3 mRNA in response to low temperature occurs rapidly, within 15 minutes after exposure to 4°C (Zarka et al. 2003). The CBF transcripts peak at around 2h and then are reduced after continued time in the cold to a level slightly higher than their expression in warm grown plants. Upon return to warm temperature the CBF transcripts are rapidly degraded (Zarka, Vogel et al. 2003). ICE1 (Inducer of CBF expression1), a MYC-like bHLH protein that binds to MYC recognition sites, was shown to be involved in the induction of CBFs (Chinnusamy et al. 2003). A point mutation in this bHLH transcription factor has a dramatic reduction in CBF3 expression in response to low temperature. ICE1 is a target for ubiquitination by HOS1, an E3 Iigase and negative regulator of cold acclimation (Dong et al. 2006). SIZ1, a SUMO-E3 Iigase, prevents ubiquitination of ICE1, leading to an increase in accumulation of CBFs in response to low temperature (Chinnusamy et al. 2007). Although the promoters of CBF1 and 2 have MYC recognition sequences, potential binding elements for ICE1, there is little effect on the expression level of CBF1 and 2 in the ice1 mutant, indicating that there may be independent regulation of the three CBF transcription factors in response to low temperature. In contrast to the ice1 point mutation, a complete knock out of this transcription factor does not have any significant effect on expression levels of CBF1, 2, or 3, indicating that there may be redundant factors that allow for regulation of CBF3 in the absence of ICE1. Overexpression of ICE1 increases the accumulation of CBF2 and CBF3 mRNA in response to low temperature. However, ICE1 overexpression does not induce any of the three CBFs in the warm, indicating that expression of ICE1 alone is not sufficient for induction of CBFs. Perhaps there are repressive factors that ICE1 can not overcome,—there are modifications of ICE1 or other regulators that occur only at low temperature, or there is a change in stability of CBF mRNA that only allows accumulation of CBF mRNA at low temperature. Cycloheximide treatment also induces the accumulation of CBF mRNA, one interpretation of this result is translation of a potential negative regulatory element is important for the warm-repression of CBFs (Zarka, Vogel et al. 2003). MYB15 and ZAT12 were identified as negative regulators of CBFs. Overexpression of either MYB15 or ZAT12 reduces the cold-induced accumulation of CBFs (AganNal et al. 2006, Vogel et al. 2005). Loss of MYB15 results in increased accumulation of these potential factors in response to low temperatures (Agarwal et al. 2006). However, the effect seen in the knockout plants of either MYB15 or ZAT12 on the CBFs is slight, perhaps due either to redundancy among the large MYB and Zinc finger families in Arabidopsis or to the function of other potential negative regulatory elements on CBF regulation. Regulation of CBFs in Response to Other Environmental Factors As we start to look at the overlapping responses of plants to different environmental signals, a complex regulation of CBFs in response to multiple environmental conditions is revealed. Circadian rhythms have an effect on CBF induction. CBF3 mRNA cycles in the warm due to circadian rhythms, peaking early in the morning (Harmer et al. 2000). lmportantly, the circadian clock has an effect on the cold-induction of CBF, with the clock gating the induction. When plants are shifted to low temperature at 2T4 (dawn), which coincides with the peak of the CBF3 circadian expression, the induction of CBFs in the cold is higher than when shifted to cold at the trough of CBF expression (Fowler et al. 2005). This indicates that the clock works together with cold signals to ensure induction of CBFs and the cold response at the appropriate times. In a similar manner, light quality has an effect on cold-induction of CBFs and their target transcripts. A reporter gene composed of four copies of the CRT fused to GUS was shown to require light for cold induction. A 10min pulse of red light was sufficient for this response; however, this response was eliminated when the pulse of red light was followed by a pulse of far-red light (Kim et al. 2002) . This reversal of the red light response when followed by treatment with far red light is indicative of regulation by phytochrome signaling. Kim et al. demonstrated that this is a phytochrome B dependent response. The involvement of light in regulating CRT elements suggests that there is either a regulation of the accumulation of the CBFs themselves in response to light or that light signals are integrated at a downstream checkpoint between CBF mRNA accumulation and CBF target gene regulation. Light quality signals have been shown to regulate the induction of CBF transcripts at 16°C. A low ratio of Red/Far Red light causes an increase in the circadian based induction of CBF at 16°C. Additionally, this light treatment of low red/far red can be used to increase freezing tolerance of seedlings grown at 16°C, but not at 22°C (Franklin and Whitelam 2007). Thus integration of light signals is important for both CBF induction and activity. Additionally, it has been shown that CBFs are also transcriptionally induced by another environmental factor, mechanical agitation. In response to touch or agitation of Arabidopsis plants, CBFs 1, 2, and 3 are rapidly induced to levels equal or greater than that of their cold level of induction (Gilmour et al. 1998). Integrating the information from these multiple methods of CBF regulation may provide clues to the mechanisms of CBF induction in response to low temperature. Calcium as a Potential Mechanism for the Induction of CBFs One common thread among all of these environmental regulators of CBF is a spike in cytosolic calcium. A transient increase in calcium levels is observed in response to a shift to low temperature, mechanical agitation, or treatment with red light. In addition, cytosolic calcium has a circadian regulation with a peak around dawn, coincident with circadian induction of CBFs (Knight et al. 1996, Knight et al. 1992, Shacklock et al. 1992, Johnson et al. 1995) While calcium signals appear to be ubiqUitous in most environmental responses, it is interesting to speculate that the calcium signature maybe a common thread in these conditions to which CBF responds. Perhaps calcium may provide a mechanism for the induction of CBF in response to these treatments. One CBF-target transcript has been shown to be up-regulated in response to treatment with calcium ionophores in the warm and inhibited from cold-induction by treatment with calcium chelators and calcium channel inhibitors (Knight et al. 1996). Similarly, cold-regulated transcripts were shown to be inducible at warm temperatures by treatment with calcium ionophores in alfalfa (Monroy and Dhindsa 1995). There are no reports of the regulation of CBFs by calcium. However, in light of the role of circadian rhythms and light quality in control of CBF induction, experiments to determine the effect of calcium on CBF induction will need to take into consideration the time of day and light quality when CBF induction is analyzed. This involvement of calcium in cold induction is suggested to be through the CRTs. Treatment with okadaio acid inhibited the cold induction of a 4xCRT reporter construct (Kim et al. 2002). Okadaic acid has been shown to inhibit sharp peaks in calcium accumulation while having little effect on gradual calcium accumulation, reducing calcium influx through inhibition of protein phosphatase activity (Hescheler et al. 1988; Kuo et al. 1996). The concentration required for inhibition of the 4xCRT reporter was specific to the inhibition of type 20 protein phosphatases (Kim et al. 2002). Two type 2C protein phosphatases, ABI1 and AB|2 are known components of ABA signaling upstream of calcium influx in Arabidopsis guard cells (Allen et al. 1999). The specific effect of okadaic acid on cold-induced calcium levels is unknown; however, this experiment raises the possibility that the calcium signal activates the CBF target genes through the CRT element. The role of calcium in cold signaling is not straightforward in that overexpression of a calmodulin results in a reduced cold-induction of a CBF target gene, Kin1 (Townley and Knight 2002). This surprising result suggests perhaps that the response to the calcium signal is tightly regulated to a specific concentration threshold or signature of calcium and its downstream signals. Perhaps adjusting the levels of any portion of the calcium signal away from this . critical threshold can result in feedback inhibition of the response pathway. Reactive oxygen species (ROS) mediated signaling pathways may also have significant involvement in the induction of CBFs. F R01 is a NADH dehydrogenase subunit of mitochondrial respiratory chain complex I, and the fro1 mutant shows constitutive accumulation of ROS (Lee et al. 2002). The fro1 mutant is sensitive to freezing, have reduced induction of cold-responsive transcripts, COR153, COR47, Kin1, and R029a. This reduction leads to a decrease in freezing tolerance of fro1 plants. It is interesting to note that the only change seen in CBF induction in response to low temperatures was seen at 12h, where CBF levels when increased in fro1 mutants compared with WT levels. Perhaps this indicates that ROS signals are involved in the circadian or light-base trough of CBF expression. It would be interesting to examine the circadian and R/FR regulation of CBFs in the fro1 mutant background. Extensive crosstalk exists between ROS signaling and calcium signaling. For example, ROS signaling can activate Ca2+ permeable ion channels in the plasma membranes while Ca2+ regulates ROS scavenging mechanisms(Mori and Schroeder 2004; Yan et al. 2006). The evidence for involvement of calcium signaling in the response to regulation of transcripts at low temperature is clear. However, it remains unknown if this involvement is upstream of the CBF factors. Involvement of calcium in the induction of CBFs could be a key link in understanding the response of CBF to these multiple environmental signals. Evidence for CBF-Independent Pathways of Cold—Acclimation While it is clear that CBFs are important in the cold-regulation and that their induction is complex and involves integration of many environmental cues, it remains unknown if there are other pathways that can independently lead to an increase in freezing tolerance. Interestingly, many freezing sensitive plants, such as tomato contain cold-inducible and functional CBF proteins. CBF1 in tomato is cold-inducible and is functional in Arabidopsis, leading to constitutive freezing tolerance of Arabidopsis when overexpressed (Zhang, Fowler et al. 2004). However, even with this functional and cold-inducible CBF protein, tomato is not freezing tolerant. This suggests that either there is a required component downstream of CBF that is missing in tomato or that there are other pathways that are required for freezing tolerance present in Arabidopsis that are missing from tomato. Mutational analysis has revealed mechanisms of affecting freezing tolerance independent of the CBF pathway. The esk1 mutant is constitutively freezing tolerant, however it does not show a significant increase in CBFs or many of the CBF target genes (Xin and Browse 1998; Xin et al. 2007). esk1 mutants accumulate high-levels of proline, a cryoprotectant, suggesting a possible mechanism for the increase in freezing tolerance. However, ESK1 encodes a novel protein with a domain of unknown function, so the mechanisms of the increase in proline levels and this increase in freezing tolerance remain unclear. A mutation in a MYB transcription factor, hos10, which has an effect on the regulation of ABA biosynthetic gene NCED3, . reduces freezing tolerance yet has an increase in expression of CBF regulon genes, COR15a, R029a, ADH, and KIN1 (Zhu et al. 2005). This reduction in freezing tolerance in the presence of a functional CBF pathway indicates that CBF alone may not be sufficient to provide full wild-type levels of freezing tolerance. Analyzing how the cold-response pathway is affected by mutations in other environmental and developmental responses may help to reveal the importance of the CBF pathway in contributing to cold acclimation and help to identify potential CBF-independent pathways. Glgantea (GI), a protein shown to be involved in developmental regulation of flowering in response to day- 10 length and circadian clock, may play a role in CBF-independent cold acclimation. GI itself is cold induced and loss of GI in gi1-3 plants resulted in increased sensitivity to freezing without and effect on CBFs or their target genes, COR15a, KIN1, and Rd29a (Cao et al. 2005). Additionally, the delay in flowering caused by the gi1-3 mutation is enhanced by intermittent cold treatment, indicating a potential link to developmental regulation by cold through GI. Additional evidence for CBF-independent pathways comes from microarray data which indicates that the transcriptional regulation of a majority of cold-regulated genes is not affected by overexpression of CBFs in warm grown plants, even though constitutive expression of CBF is sufficient for freezing tolerance (Fowler and Thomashow 2002; Vogel et al. 2005). These transcripts may be entirely CBF independent or they may require CBF and an , additional factor present only at low temperatures. Additionally, CBF2 overexpressing plants still have a significant and dramatic increase in freezing tolerance after a period of cold acclimation over their warm-grown levels. This additional increase in freezing tolerance may be due to CBF independent pathways that contribute to freezing tolerance or a quantitative effect due to the increase in the total CBF mRNA levels in the cold due to the additional accumulation of the endogenous CBF mRNA in response to their induction at low temperature. 11 Cold Acclimation in the Absence of CBFs An ideal way to address the contribution of CBF independent pathways to the process of cold acclimation would be to knock out CBF function and assess the remaining ability of these mutants to cold acclimate. However, the three cold-induced CBF genes lie in tandem, making recombination of single T- DNA insertion lines impractical. No triple, CBF1, 2, and 3 knock-out plants have been generated to examine the ability of Arabidopsis to acclimate in the absence of CBFs. Knock-out and miRNA studies of individual CBFs revealed that they have distinct roles in regulation of the target genes, not evident from overexpression (Novillo et al. 2004; Novillo et al. 2007). These distinct roles are in agreement with their tissue specific expression. Promoter GUS fusion reporters demonstrated that during development warm-grown tissue expresses CBF2 highly in the shoots and not in the roots, while CBFs 1 and 3 accumulate in the roots, but not the shoots. It is unknown if these genes are preferentially expressed in different tissue during low temperature induction. Mature plants showed little staining for the reporter genes in the warm, and the cold-induction of the three CBFs was similar in response to low temperatures in shoot tissue. However, root tissue of mature plants was not examined for differences in cold- induced expression of CBFs1, 2, and 3. Double miRNA constructs targeting CBF1 and CBF 3 show a dramatic reduction in freezing tolerance of acclimated plants (Novillo, Medina et al. 2007). However, it is not clear if this remaining freezing tolerance is due to CBF2 activity or CBF-independent pathways. It is 12 still unknown if Arabidopsis plants lacking all three cold-inducible CBFs are still able to cold acclimate. ABA as a Potential Component of a CBF-Independent Cold-Acclimation Pathway Cross-talk between cold and other stresses may provide clues to potential CBF—independent mechanisms of achieving freezing tolerance. Levels of ABA, a plant growth hormone, important in signaling in response to drought stress, also increase in response to low temperature in Arabidopsis and pre- treatment with ABA increases freezing tolerance (Lang et al. 1994). Many cold regulated transcripts are also regulated in response to drought conditions, in an ABA dependent manner. It is not clear if the effects of ABA on cold-responsive transcripts and freezing tolerance are due to direct involvement of ABA in the cold-response or are secondary to the overlap in response between drought f and cold. Support for the latter argument comes from the fact that drought stressed plants also have an increase in freezing tolerance similar to the levels seen by treatment with ABA (Mantyla et al. 1995). Analysis of High-Throughput Data Provides New Insights into Cold Acclimation It appears that there is substantial integration of multiple signals which converge in regulating the process of cold-acclimation both through the CBF pathway and possible CBF-independent pathways. One method of analyzing the networks that compose the cold response is to perturb the system one variable at a time, changing either the functional components of known 13 pathways of cold response, or altering the environmental signals which may be involved in regulating this response. Quantitative analysis of the effects each perturbation has on the system of cold response will be important for building accurate networks of signal transduction as qualitative changes maybe masked by redundancy. Fortunately, methods exits for sensitive analysis of such changes on a global scale. The primary focus here is on global transcriptional changes, however advances in the analysis of proteins, metabolites, ions, and other cellular components on a global scale is also improving and will add significantly to our understanding of the cold response. The analysis of the effects of different growth conditions in publically available cold-response studies is already yielding interesting results. Comparison of the cold-induced transcripts from several labs revealed an enhancement of circadian-regulated genes in those considered to be cold- induced. Further analysis of these transcripts revealed that the large change in these cold and circadian transcripts was likely due to changes in output from the circadian clock. Therefore many transcripts were considered to be cold regulated because the phase of their circadian regulation shifted in response to low temperature. However, this was not a universal effect on all clock-regulated transcripts as both the phase and amplitude of some transcripts were maintained in response to low temperature (Blasing et al. 2005). Also by comparing the cold-treatment conditions of various labs and linking this to changes in cold-regulated gene expression Blasing et al. found that sugar 14 signaling was also an important component of regulation of cold-responsive transcripts. Analysis of the representation of previously described promoter elements and transcription factor binding sites in the promoters of cold-regulated transcripts indicates that there is extensive combinatorial regulation by trans- acting factors on cold-responsive genes. Each transcription factor family is predicted to interact with multiple other transcription factors, each interaction resulting in the regulation of a subset of cold-responsive target genes (Chawade et al. 2007). However, many cold-responsive transcripts are also regulated in response to other stresses. Therefore, the presence of multiple motifs on a single promoter could be a mechanism of regulation by Independent transcription factors as downstream signals originating from distinct stresses and may not necessarily represent combinatorial control of the transcript in response to cold stress. Analysis of the response of these transcripts across multiple stresses using bi-clustering methods, which allow for the representation of a transcript into multiple clusters, will further divide cold regulated transcripts into patterns of regulation across multiple stresses. Analysis of the overrepresented promoter elements in these refined classes may provide additional insights into the role of these promoter elements in regulation of these transcripts to the various environmental stimuli to which they respond. Analysis of transcriptional or metabolite data on a genome-wide scale in response to perturbations in the genome or environment during cold acclimation has the potential to provide a wealth of information on networks involved in the 15 cold-response. Changes in gene expression can be correlated with different environmental factors or regulatory genes. However, for this information to be translatable into an improvement in ability of plants to survive low temperature, it will be important to establish a link between changes in the networks and the phenotype of freezing tolerance. Current methods of measuring freezing tolerance are incomplete, as they lack the capacity for quantitative assessment of the complex phenotype of freezing tolerance in a high-throughput manner. Methods such as electrolyte leakage analysis or thermal imaging, while quantitative, measure only one aspect of a complex response. In some cases the results of the measurements of these specific components of freezing tolerance are at odds with the overall phenotype. For example, Zat12 overexpressing plants are more freezing tolerant when measured by whole plant freeze tests, yet have a lower electrolyte leakage measure (Vogel et al. 2005, Vogel, unpublished). Whole plant freeze tests are not an ideal answer either because, as a qualitative measure, they are not sensitive enough to pick up changes in phenotype which maybe subtle due to compensation by multiple pathways of cold-acclimation. Since wild-type plants are able to survive after cold acclimation, it is difficult to quantify an improvement above complete survival. Similarly, as basal acclimation is not sufficient for survival of wild-type plants, non-acclimated wild-type plants do not survive freezing. Therefore, it is not possible to measure a decrease in basal acclimation ability, as mutations resulting in a decrease in basal freezing tolerance will show the same phenotype after freezing tolerance as wild-type plants, non-survival. To fully 16 take advantage of the powerful tools of high-throughput analysis of the transcriptome and metabolome which are becoming available in plants, it will be necessary to develop a quantitative, high-throughput method for measuring freezing tolerance, possibly by incorporating measurements of several components of freezing tolerance. Interaction between Cold and Development Low temperature treatment plays a significant role in the regulation of many other processes. Cold-entrainment of the circadian clock is an important factor in the regulation of many circadian transcripts (Michael et al. 2008). This effect has physiological significance; plants grown in warm nights and cold days have decreased growth rates (T hingnaes et al. 2003). Many cold-regulated transcripts are also circadian regulated and their low-temperature regulation involves changes in the amplitude of the circadian clock in response to cold (Bieniawska et al. 2008). Further understanding of how cold contributes to these changes in the circadian regulation of target genes can help identify CBF- lndependent mechanisms for regulation of cold-responsive transcripts. Low-temperature treatment also has an effect on development; short term cold treatment causes a delay in flowering. However, long-term cold treatment promotes flowering by activating the vernalization pathway. In this process, exposure to low temperatures for extended periods, results in repression of FLC, a negative regulator of flowering. This repression of FLC is maintained through epigenetic modifications to the FLC locus (Sung and Amasino 2005). 17 Integration of multiple signals, including cold, is also important for germination. Environmental cues of light, temperature, and water availability must all be analyzed in the decision to germinate. It will be interesting to see if the mechanisms of integration of these environmental signals at this stage of development are similar to the mechanisms of integration during cold acclimation. Developmental State Affects Low Temperature Responses The range to which an individual species can adapt to freezing depends on other environmental factors and the developmental state of the plant. Some environmental factors that have been shown to affect the process of cold acclimation include altitude, exposure to growth hormones, light quality and light quantity (Zarter et al. 2006; Franklin and Whitelam 2007; Soitamo et al. 2008). Development as well plays a role in cold acclimation levels. During the switch . to a reproductive state from a vegetative state by induction of the vernalization pathway, rye and wheat have a reduction in their ability to induce cold-regulated transcripts and a loss in their ability to cold acclimate (Fowler et al. 1996). Other developmental factors including sex in dimorphous plant species has an effect on freezing tolerance (Li et al. 2005). Environmental cues have an effect on freezing tolerance. Arabidopsis, a model plant examined for freezing tolerance, has a dramatic increase in freezing tolerance when grown under short days compared to continuous light (Doherty, unpublished). The mechanisms that result in this increase in freezing tolerance remain unknown. One mechanism may be the gating of the cold response by day-length signals 18 ensuring that the full cold response occurs only during short day conditions. Another possible mechanism for this increase in freezing tolerance seen in short day grown plants may be due to differences in developmental stage between short day and continuous light grown plants, if the continuous light grown plants have already made the switch to reproductive phase, their ability to cold acclimate maybe reduced. The roles of CAMTAs, calmodulin regulated transcriptional activators, in regulating CBFs are discussed in this thesis. CAMTA regulation of CBF could serve as the integration point of calcium into the cold-response pathway. The second question of the role of CBF in the cold-acclimation process is addressed using a dominant negative version of CBF. Identifying a potential integration point for calcium signaling in the cold response and understanding the requirement for the CBF based pathway in the ability of Arabidopsis to cold acclimate will serve as important contributions to our knowledge of how plants interact with their environment. Additionally, these contributions may lay the groundwork for understanding how plants are able to integrate multiple signals in order to adapt to the complex environments in which they grow. With increased environmental changes on the near horizon due to increased atmospheric COZ concentrations, it is of vital importance to understand how plants integrate complex environmental signals and the limits of the incredible plasticity plants demonstrate in responding to their ever-changing environment. 19 Literature Cited Agarwal, M., Y. Hao, et al. (2006). "A R2R3 Type MYB Transcription Factor Is Involved in the Cold Regulation of CBF Genes and in Acquired Freezing Tolerance." J. Biol. Chem. 281(49): 37636-37645. Allen, G. J ., K. Kuchitsu, et al. (1999). "Arabidopsis abil-l and abi2-1 Phosphatase Mutations Reduce Abscisic Acid—Induced Cytoplasmic Calcium Rises in Guard Cells." Plant Cell 11(9): 1785-1798. Ballou, S. M., K. Y. Yun, et a1. (2007). 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Plant Physiology 104(4): 1341. 21 Lee, B.-h., H. Lee, et a1. (2002). "A Mitochondrial Complex I Defect Impairs Cold- Regulated Nuclear Gene Expression." Plant Cell 14(6): 1235-1251. Li, C., Y. Yang, et al. (2005). "Sexual differences in cold acclimation and freezing tolerance development in sea buckthom (Hippophae rhamnoides L.) ecotypes." Plant Science 168(5): 1365-1370. Mantyla, E., V. Lang, et al. (1995). "Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LTI78 and RAB l 8 proteins in Arabidopsis thaliana." Plant Physiol 107: 141-148. Michael, T. P., T. C. Mockler, et al. (2008). "Network Discovery Pipeline Elucidates Conserved Time-of-Daya€“Specific cis-Regulatory Modules." PLoS Genetics 4(2): e14. Monroy, A. F. and R. S. Dhindsa (1995). "Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25 degrees C." Plant Cell 7(3): 321-31. Mori, I. C. and J. I. Schroeder (2004). "Reactive Oxygen Species Activation of Plant Ca2+ Channels. A Signaling Mechanism in Polar Growth, Hormone Transduction, Stress Signaling, and Hypothetically Mechanotransduction." flan; Physiol. 135(2): 702-708. Novillo, F., J. M. Alonso, et al. (2004). "CBF2/DREB1C Is a Negative Regulator of CBF1/DREB1B and CBF3/DREB1A Expression and Plays a Central Role in Stress Tolerance in Arabidopsis." Proceedings of the Nag'gnal Academy of Sciences of the United States of America 101(11): 3985-3990. Novillo, F., J. Medina, et al. (2007). "Arabidopsis CBF1 and CBF3 have a different function than CBF 2 in cold acclimation and define different gene classes in the CBF regulon." Proceedings of the National Academy of Sciences 104(52): 21002. Soitamo, A., M. Piippo, et al. (2008). "Light has a specific role in modulating Arabidopsis gene expression at low temperature." BMC Plant Biology 8(1): 13. Sung, S. and R. M. Amasino (2005). "REMEMBERING WINTER: Toward a Molecular Understanding of Vemalization." Annual Review of Plant Biology 56(1): 491-508. Thingnaes, E., S. Torre, et al. (2003). "Day and Night Temperature Responses in Arabidopsis: Effects on Gibberellin and Auxin Content, Cell Size, Morphology and Flowering Time." Ann Bot 92(4): 601-612. 22 Thomashow, M. F. (1999). "PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms." Annual Review of Plant Physiology and Plant Molecular Biology 50(1): 571-599. Thomashow, M. F. (2001). "So What's New in the Field of Plant Cold Acclimation? Lots!" Plant Physiol. 125(1): 89-93. Townley, H. E. and M. R. Knight (2002). "Calmodulin as a Potential Negative Regulator of Arabidopsis COR Gene Expression." Plant Physiol. 128(4): 1169- 1172. Viswanathan, C. and J. K. Zhu (2002). "Molecular genetic analysis of cold-regulated gene transcription." PhilosophicflTraliaactiona of the Royal Society B: Biological Sciences 357(1423): 877. Vogel, J. T., D. G. Zarka, et al. (2005). "Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis." m Plant Journal 41(2): 195-211. Xin, Z. and J. Browse (1998). "eskimol mutants of Arabidopsis are constitutively freezing-tolerant." Proceedings of the National Academy of Sciences 95(13): 7799-7804. Xin, Z., A. Mandaokar, et al. (2007). "Arabidopsis ESK1 encodes a novel regulator of freezing tolerance." The Plant Journ_al 49(5): 786-799. Yan, Y., C.-l. Wei, et a1. (2006). "Cross-talk between calcium and reactive oxygen species signaling." Acta Pharmacologica Sinica 27(7): 821-826. Zarka, D. G., J. T. Vogel, et al. (2003). "Cold induction of Arabidopsis CBF genes involves multiple ICE (inducer of CBF expression) promoter elements and a cold-regulatory circuit that is desensitized by low temperature." Plant Physiol 133: 910 - 918. Zarter, C. R., W. W. Adams, et al. (2006). "Winter acclimation of PsbS and related proteins in the evergreen Arctostaphylos uva-ursi as influenced by altitude and light environment." Plant, Cell & Environment 29(5): 869-878. Zhang, X., S. G. Fowler, et al. (2004). "Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing- tolerant Arabidopsis." The Plant Journal 39(6): 905-919. Zhu, J ., P. E. Verslues, et al. (2005). "HOSIO encodes an R2R3-type MYB transcription factor essential for cold acclimation in plants." Proceedings of the National Academy of Sciences 102(28): 9966-9971. 23 CHAPTER TWO Roles for Arabidopsis CAMTA Transcription Factors in Cold- Regulated Gene Expression and Freezing Tolerance SUMMARY The ability of some plants to cold-acclimate is an important survival trait that increases both the seasonal and geographic range of plant growth. Understanding the process by which plants are able to cold acclimate will allow us to extend the growing season and available land for crop growth. The CBF cold-response pathway is an important component of the cold acclimation process. To understand how plants are able to turn on the CBF pathway in response to low temperature we analyzed the CBF2 promoter in Arabidopsis. This analysis led to the identification of a 27 bp region of the CBF2 promoter. the CM1/CM2-Box that is sufficient for cold responsiveness. We identified CAMTA3 as a positive trans-acting regulator of CBF1 and CBF2 in response to low temperature. Loss of CAMTA 3 results in a reduced cold-induction of CBF1 and CBF2. Loss of both CAMTA1 and CAMTA3 results in reduced ability of Arabidopsis to cold acclimate. These results indicate that the CAMTA 1 and CAMTA3 transcription factors are components of the cold acclimation response in Arabidopsis. INTRODUCTION Many plants from temperate regions increase in freezing tolerance upon exposure to low non-freezing temperatures through a process known as cold acclimation. One major pathway involved in cold acclimation is the CBF cold- 24 response pathway. In Arabidopsis, the pathway includes three genes, CBF 1, CBF2 and CBF3, also known as DREB1b, DEB 1c and DREB1a, respectively, whose transcript levels increase within 15 minutes of exposure to low temperature (Gilmour et al. 1998). The CBF genes encode three closely related members of the AP2/ERF domain family of transcription factors. CBF proteins regulate the transcription of many downstream target genes, known as the CBF regulon, by binding to CRT/DRE (C-repeat/dehydration responsive element) DNA regulatory element found in the promoters of these genes (Stockinger et al. 1997; Liu et al. 1998). Expression of the CBF regulon, which includes COR and other cold-responsive genes, results in an increase in freezing tolerance (Jaglo—Ottosen et al. 1998). This increase in freezing tolerance involves a variety of biochemical and physiological changes that are mediated by changes in expression of CBF regulon genes, including the ‘ accumulation of compatible solutes such as raffinose, sucrose and proline and the production of cryoprotective polypeptides such as COR15a (Gilmour, Zarka et al. 1998). While the CBF pathway has been demonstrated to have a major role in plant cold acclimation, relatively little is known about the mechanism(s) that induces CBF1-3 gene expression in response to low temperature. Zarka et al. (Zarka et al. 2003) identified a 155-bp region of the CBF2 promoter that is capable of driving cold-induced transcription and found that within this promoter fragment that there are two short sequences, lCEr1 and lCErZ (Inducer of CBF expression region 1 and 2), that contribute to cold-induction. Zhu and 25 colleagues have identified two transcription factors involved in CBF gene expression (Chinnusamy et al. 2003; Agarwal et al. 2006). One of these, MYB15, appears to act as a negative regulator of CBF1-3 expression; loss of MYB15 function does not affect CBF1-3 expresSion at warm temperature, but results in a detectible increase in CBF1-3 expression at low temperature. The other transcription factor, ICE1, is a MYC-like bHLH protein that binds to MYC recognition sites in the CBF3 promoter. A dominant-negative mutation of ICE1 was isolated, ice1, that nearly eliminates cold-induction CBF3, indicating the protein acts as a positive regulator CBF3 expression. The ice1 mutation, however, has little effect on cold-induced expression of CBF1 and CBF2 indicating that there are differences in the specific transcription factors that regulate expression of CBF1-3. Upon exposure to low temperature there is a transient increase in cytosolic calcium levels. A vacuolar release of calcium in response to low temperature has been demonstrated in Arabidopsis through use of aequorin, a bioluminescent calcium reporter (Knight et al. 1996). This rapid influx of calcium into the cytosol is required for proper regulation of KIN1, a target of CBF, in response to low temperature. Similarly, in alfalfa, regulation of two cold acclimation specific (cas) genes, cas15 and cas18 was shown to involve calcium signaling (Monroy and Dhindsa 1995). These results point to the importance of calcium in the response of plants to low temperature. However, the molecular mechanism connecting the calcium increase and cold-regulated gene expression is not known. 26 Here the goal was to further an understanding of the cis-acting DNA regulatory elements and trans-acting factors involved in regulation of the CBF cold response pathway. A cold-regulatory element overlapping the lCEr2 region was identified in the CBF2 promoter that is sufficient for cold-induction of a reporter construct. Additionally, it is shown here that members of the CAMTA (calmodulin-binding transcription factors) family of transcription factors can bind to this region of the CBF2 promoter; CAMTA3 is a positive regulator of CBF2 expression; and that the increase in freezing tolerance that occurs with cold acclimation involves concerted action of CAMTA1 and CAMTA3. These results identify new cis-acting cold-regulatory elements, establish a role for CAMTA proteins in cold-acclimation, and provide a possible point of integrating calcium into the cold response pathway. RESULTS A 27 bp Sequence from the CBF2 Promoter is Sufficient to lmpart Cold- lnduced Transcription We recently identified seven DNA motifs, 6 to 9 nt in length, that are present in both the 155 bp cold-responsive promoter fragment of CBF2 and a 224 bp region of the ZAT12 promoter that is sufficient to impart cold-induced transcription (Van Buskirk, in preparation). One of these sequences, the CM2- box, overlaps the ICEr2 element. To determine whether this region of the CBF2 promoter was sufficient to impart cold-induced gene expression, a 27 bp region of the CBF2 promoter (Fig 2.1A) that included the lCEr2 sequence, as well as 27 the overlapping CM2-box and the CM1-box (another of the seven conserved DNA motifs), was fused as a tetramer to the GUS reporter gene and the construct tested for expression in Arabidopsis. The results indicated that the 27 bp fragment could indeed impart cold-induced gene expression (Fig 218.0). A .— wCTCCGCGTTCGACCCCAcm'm- 3 'cl GUS 3' -cnremcccmecreeccrmnr-sv WI :2— ATA GUS §'-GmcraatathGACCCCACAMTA- ' E— CM2-box mutant 3'-crrrcntcatacecrccccrcrrrnr-5' F ‘l L “ ”I ATA B WT CM2-box mutant WCWCWCWCWCWCWCWCWCWC GUS uni-ageing; .l j ' ' '. - Oi '- 9 o o g oIF4a a. '1 7“?““flfivw «nu wash e 93% c WT CM2-box mutant hoursat4'C o 1 2 4 8240 1 2 4 824 GUS Mflfihww CBF2 fig. 3. vu- ' elF4a “‘9’ Figure 2.1 Tetramer Containing CMZ-box and CM1-box Is Able to Drive Trans- cription of GUS Reporter Gene in Response to Treatment at 4°C. (A) Sequence of conserved region defining CM2-box (clear) and CM1-box (shaded) and mutated version of CM2-box. ICER2 sequence is indicated by bar. (B) Northern blot analysis of plants expressing either WT version of the CM2/CM1-box tetramer or plants expressing a reporter gene with a mutated CM2-box sequence. Plants were harvested after growth in the (W) warm or (C) after treatment at 2h4°C. Five independent transgenic lines of the reporter construct are shown for WT and CM2-box mutant. (C) Induction of GUS reporter and CBF2 in response to treatment at 4°C of one line shown in B. 28 In addition, it was found that nucleotide substitutions that resulted in the elimination of the CM2-box resulted in a DNA fragment that could not impart cold-inducible gene expression (Fig. 2.1B,C). These results indicated that the 27 bp sequence can impart cold-inducible tranScription and that this requires the CM2-box sequence. CAMTA Proteins Bind the CMZ-box Sequence The CAMTA family of calmodulin-binding transcription factors comprises six members in Arabidopsis (Bouche et al. 2002; Yang and Poovaiah 2002). Each protein includes a DNA binding domain referred to as the CG-domain which binds to a core consensus sequence, vCGCGb, referred to as the CG-l element (de Costa e Silva 1994). This sequence matches the CMZ-box sequence, ACGCGG, and overlaps the ICEr1 element (Figure 2.2A). Thus, the possibility raised was that one or more of the CAMTA proteins have a role regulating CBF2 expression. As a first test of this possibility, we asked whether the 06-1 DNA binding domain of the CAMTA proteins could bind to the CM2-box sequence (Figure 2.23). Specifically, the CG-1 DNA-binding domains of CAMTA1, 2, 3 and 5 (CAMTA4 and 6 were not tested) were fused to GST, expressed in E. coli, and soluble protein extracts were assayed for DNA binding using the electro- mobility shift assay (EMSA). No shift was detected when lysates from E. coli lacking the expression vector or expressing a recombinant protein consisting of GST fused to a disrupted version of the CAMTA5 CG-1 domain with a premature early stop codon. In contrast, a band shift occurred with proteins 29 comprised of the CAMTA1, 2, 3 or 5 CG-1 domains fused to GST. The binding could be competed off with unlabeled DNA containing the wild-type CM2-box sequence, but not with unlabeled DNA containing a mutation in the CM2-box sequence indicating specificity of protein-DNA binding. A vCGCGb GT'I‘TC‘I'I‘ATCCACGTGGCA'I'I‘CACAGAGACAGAAACTCCGCGTTCGACCCCACAAATATCCAAATATC'I‘TCCGGC _ _ _ CAAAGAATAGGTGCACCGTAAGTGTCI‘CTGTCTTTGAGGCGCAAGCTGGGGTGT'I'I‘ATAGGT'I'I‘ATAGAAGGCCG '35 1: ICE r2 2 23. B : g protein; - 8 C1 02 03 C5 05‘ competitor: — - —wm—wm-wm—wm- U U U Dawn“. free probe [W 0-. Figure 2.2 CAMTA5 Bind CMZ-Box Region of CBF2 Promoter (A) Overlap of CMZ-Box (open box), ICEr2 region, and putative CG-1 DNA recognition sequence. (B) EMSA of CM2/CM1-Box by CAMTA CG-1 DNA binding domain. Probe is the CM2/CM1-Box sequence as in Figure 1A. Lanes are lysate from non-vector containing E.coli (lysate), E.coli expressing CG-1 DNA binding domains from CAMTA1 (C1), CAMTA2(C2), CAMTA3(C3), and CAMTA5(05), and a scrambled version of the CAMTA5 CG-1 DNA binding domain(C5*). Competition with WT CM2/CM1-Box (w) and with CM2-Box mutant (m) sequence as in Figure 1A CAMTA3 has a Role in Cold-Induced Expression of CBF2 30 To further test the possibility that one or more of the CAMTA proteins have a role in CBF2 expression, we identified homozygous T-DNA insertion lines for each CAMTA gene that resulted in undetectable transcripts for each corresponding gene (Figure 2.3). Each camta mutant plant was tested for differences in CBF2 expression from wild-type plants after 2h0°C treatment. Initial northern analysis suggested that the camta3 mutation resulted in a reduction of cold-induced accumulation of CBF2 transcripts (Figure 2.4A). This was confirmed by quantitative RT-PCR analysis; CBF2 transcript levels in cold- treated camta3 plants were reduced to about 50% of the level found in cold- treated wild-type plants (Figure 2.48). No statistically significant reduction in CBF2 expression was observed in camta1, 2, 4, 5 or 6 at a p-value <0.05. [.— C! E r. a a r. e e ‘2’ CAMTA1 . - . . .; .. CAMTAZ a a; .. .3 rev“;- . CAMTA3[- - o .- .. . J CAMTA4|.. .. .. . . - ] CAMTAel- .. .. .. - .. j ACTIN3[.!----- I E a Figure 2.3 T-DNA Insertion Lines Result in Loss of CAMTA Transcript Accumulation. RT-PCR analysis of CAMTA T-DNA insertion lines camta1(c1), camta2(02), camta3(c3), camta5(c5), camta6(06) shows a loss of transcript in each T-DNA insertion line compared to WT (Col-0) 31 2h 0° A E z a a a e s CBF2[.,q--- nun-j 18srRNA|7 J B 1.20 a; 1.00- E a co .1 0.80 O S g 'a 0.60- % 8 — - 0.40- (D o: 3 L“ 0.20- 0.00- WT Warm WT 2h0° c3 2h0° Figure 2.4 CBF2 Levels Are Reduced in camta3 Plants (A) RNA gel blot analysis for cold-induced CBF2 expression. Transcript levels of CBF2 gene were examined by gel blot analysis using total RNA prepared from seedlings of WT and all camta T-DNA insertion lines grown for 2h at 0°C. The same blot probed for 18s rRNA is shown for a loading control. (B) QPCR analysis. Relative expression level of CBF2 transcript in terms of WT 2h0°C. Error bars indicate SE. The averages for WT warm and camta3 2h0°C are significantly different than WT 2h0°C (p-value <0.0001) (n=6 for WT warm, n=12 for WT 2h0°C. n=12 for camta3 2h0°C). In growing the plants for these experiments, it was noted that those carrying either camta1, 2, 3 ,4 or 5 mutations showed no obvious abnormalities in growth and development when their life cycle was carried out at either warm (22°C) or cold (4°C) temperatures. The camtaG mutant plants displayed yellowing of the veins and midrib at warm temperature, which was suppressed in new leaves produced at low temperatures (Figure 2.5). We have not yet tried to suppress this phenotype by overexpressing CAMTA6, therefore we cannot 32 rule out the possibility that this phenotype was due to a separate mutation unlinked to camta6. Figure 2.5 camtaG Growth Phenotype (A) camta6 T-DNA insertion line showed a yellowing of the veins when grown at 22°C. (B) WT Col-0 plants grown at 22°C (C) camta6 germinated and grown for 40d at 4°C did not show this phenotype (D) WT Col-0 plants germinated and grown for 40d at 4°C The CM2ICM1-box Sequence is a Site of CAMTA3 Action In Plants The results presented above indicated that the 27 bp fragment of the CBF2 promoter that includes both the CM2-box and CM1-box imparts cold- induced gene expression, and that this required the CM2-box sequence to which the CAMTA3 protein can bind. If the CAMTA3 protein contributes to CBF2 cold-induction through binding to the CM2-box in planta, then it would be anticipated that the cath mutation would affect cold-induction of the GUS reporter gene driven by the 27 bp promoter fragment. This was the case. 33 Northern analysis indicated that whereas the camta1 mutation had no detectable effect on cold-induction of the reporter gene, the camta3 mutation appeared to eliminate it (Figure 2.6A,B). Further analysis by qRT-PCR confirmed that the camta3 mutation eliminated cold-induced expression of the 27 bp::GUS reporter gene (Figure 2.6C). 1.20 A B C 1.00- 0.80 - 0.60 - 0.40 - C3 x CM2/CM1 GUS WT x CM2/CM1:GUS C3 x CM2/CM1iGUS WT x CM2/CM1iGUS C1 x CM2/CM1zGUS WI" x CM2/CM12GUS C1 x CM2/CM1iGUS 1 WT x CM2/CM1:GUS 0.20 - Gusl I -IGusl I: l 185 18 s 0.00 4 Warm 2h0°C Warm 2h0°C Relative GUS Expression Level . WT x WT x c3 x CMZ/CM1:GUS CM2/CM1:GLB CMZ/CIIM :GUS w arm 2hO'C 2h0‘C Figure 2.6 CM2/CM1-BoszUS mRNA Levels Are Reduced in camta3 Plants (A) Northern analysis of camta3T-DNA insertion lines crossed to the CM2/CM1- BoszUS reporter construct lines (C3 x CM2/CM1:GUS)revealed a reduction in cold-induced GUS mRNA levels compared to WT plants crossed to the CM2/CM1-BoszUS lines (WT x CM2/CM12GUS). (B) Northern analysis of camta1 T-DNA insertion lines crossed to the CM2/CM1-BoszUS reporter construct lines (C1 x CM2/CM1zGUS) showed no reduction in cold-induced GUS mRNA levels. (C) QPCR analysis. Relative expression level of GUS transcript in terms of WT 2h0°C. Error bars indicate SE. The average Gus level in the camta3 background at 2h0°C are significantly different than WT 2h0°C (p-value <0.0001) (n=2 for WT warm, n=6 for WT 2h0°C, n=6 for camta3 2h0°C). Histochemical staining of lines carrying the 27bp::GUS reporter indicated that GUS activity was dramatically reduced in plants carrying the camta3 mutation whether they were grown at warm or cold-temperature (Figure 2.7A). The small amount of GUS staining in the camta3 plants was limited to the roots 34 of the plants, indicating that CAMTA3 may only be responsible for regulation of the CM2/CM1-Box in shoot tissue. The staining of the wild-type plants grown under warm conditions was presumably due to the low level expression of the reporter gene observed at warm temperature (Figure 2.1B,C). Transformation of these plants with the wild-type CAMTA3 gene under control of the 358 CaMV promoter resulted in recovery of staining confirming that the camta3 mutation was responsible for the lack of reporter gene expression (Figure 2.7A). Recovery of CBF2 expression levels was also observed in the camta3 plants transformed with the wild-type CAMTA3 gene (Figure 278). Finally, the levels of expression of the transgenic CAMTA3 gene were near equal to or less that the level of the endogenous CAMTA3 gene in wild-type plants (Figure 2.8). 03 x CM2/CM1:GUS / B A WTx c3x CM2/CM1zGUS CM2/CM1:GUS 355;;c3 413 c'\ Q WARM f "a if. :9, 9. 48h0°C {g‘gik’ll 9d0°C 650‘s Mi I yr: p a: Relative CBF2 Expression Level . WT 210°C (:3 210°C c3l 3582C3 003‘"? 2I‘0°C Figure 2.7 Complementation of GUS Activity and CBF2 Transcript Levels in camta3 Plants by Ectopic Expression of CAMTA3. (A) Histochemical analysis of CM2/CM1-Box:Gus reporter activity. Staining of seedlings of WT plants (WT x CM2/CM1zGUS), camta3 plants crossed to CM2/CM1-Box:GUS (03 x CM2/CM1zGUS), and camta3 plants crossed to CM2/CM1-Box:GUS transformed with 35s:CAMTA3 (03 x CM2/CM1:GUS / 35$:C3) after growth in the warm, treatment for 48h at 0°C or 9d at 0°C. (B) QPCR analysis. Relative expression of CBF2 transcript levels to WT 2h0°C (n=3). Error bars indicate SE. 35 1.8 . 1.6 . 1.4 4 1.2— 0.8 - 0.6 - 0.4 ,. 0.2 4 O - 1 _ 1 WT 2h0°C 03 xXIZ:GUS 2h0°C c3 xX/ZZGUS B55303 Relative CAMTA3 Expression Levels Figure 2.8 Recovery of WT Levels of CAMTA3 in camta3 x CM2/CM1-BostUS Plants by Ectopic Expression of CAMTA3. QPCR analysis of relative expression levels of CAMTA3 in terms of WT level of expression in camtaB plants crossed to CM2/CM1-BoszUS (03 x CM2/CM1:GUS) and camta3 plants crossed to CM2/CM1-BoszUS transformed with 35$:CAMTA3 (03 x CM2/CM1:GUS / 35s:C3) after treatment for 2h0°C. Error bars indicate SE. CG-1 Element/CAMTA Binding Sites are Enriched in the Promoter Regions of Early Cold-Responsive Genes The results presented above indicated that CAMTA3 has a role in cold- regulated expression of a single gene, thus raising the question of whether CAMTA3 has a more general role in cold regulation. Computational analysis indicated that the CAMTA binding sites are enriched in the promoters of cold- regulated genes. A search of publicly available microarray data (Vogel et al. 2005; Kilian et al. 2007) identified 46 genes having an expression pattern that highly correlated with that of CBF2, with a minimum Pearson’s correlation of 0.85 (Table 2.1). These 46 genes were analyzed for the presence of the 06-1 binding site using Promomer (T oufighi et al. 2005). Nineteen of these genes had at least one vCGCGb sequence present in the upstream 500 bp sequence 36 (TAIR7). Hypergeometric testing, a statistical method to determine if an event is overrepresentation in a subset of a population, was used to determine if the CG-1 element was overrepresented. Analysis of the presence of the 06-1 element in this set of cold-regulated transcriptscompared to its presence in the 500 bp upstream sequences of all sequences indicated that the CG-1 element was highly enriched in the promoters of these cold-responsive genes (p-value, <10 '5). To test if the 06-1 sequence was overrepresented in early cold-induced genes, transcripts were selected that are up-regulated early in response to low temperature, regardless of their overall correlation with CBF2 expression and were tested for enrichment of the 06-1 sequence. Publicly available microarray data were analyzed to create a list of genes that responded rapidly to low temperature. Thirty genes (Table 2.2) were selected that were up- regulated two-fold in wild-type plants in Vogel et al. and were also up 2 fold at 1 h and 3h in ATGenExpress cold-treated samples (Vogel et al. 2005; Kilian et al. 2007). Of these 30 genes, 12 contained at least on CG-1 element in the promoter region 500 bp upstream of the start site (TAIR7). Hypergeometric testing showed the occurrence of the element in these genes was enriched as compared to the rest of the genome (p<0.001). Together, these results indicate that the 06-1 sequence is overrepresented in early cold-responsive transcripts and those with a pattern of expression at low temperature similar to CBF2. 37 Table 2.1 Transcripts with a Pattern of Induction in the Cold Similar to CBF2 Forty-six transcripts that have a pattern of induction similar to that of CBF2 in response to treatment at 4°C. Those in bold contain the sequence vCGCGb in the region 500bp upstream of the 5’UTR (TAIR7). 38 Transcripts with pattern of induction similar to CBF2 in cold microarray experiments AGI At1909070 Soybean ILegulated by Cold SRC2 At1g13260 RAV1 At1g21010 Unknown Protein At1927730 ZAT10 At1L61340 F-box family protein At1965390 ATPP2-A5 At1g69570 Dof-type zinc finger domain-containing protein At1g70420 Unknown Protein At1 g14450 Unknown Protein AtLg75020 LPAT4 At1g75180 Unknown Protein At2g27260 Similar to Hydroxyproline-Rich Glycoprotein Family Protein AtZQBZZO Unknown Protein At2937970 SOUL-1 At2g38790 Unknown Protein At3gO4640 Glycine-Rich Protein At3923170 similar to AtBET12 At3928340 GATL10 At3g48520 CYP94B3 At3g51920 CAM9 At3gS2800 Zinc finger (AN1-like) family protein At4g16780 ATHB-2 At-tg18280 Glycine-Rich Cell Wall Protein At4925470 CBF2 At4g25480 CBF3 At4g25490 CBF1 Attg29190 Zinc Finger (CCCH-type) Family Protein At4929610 Putative Cytidine Deaminase At4g32020 Unknown Protein At4g3_3920 Protein Phosphatase 2C Family Protein At4936500 Putative Protein At4$37260 MYB73 AtSMMO CZF2 At5fl0695 Unknown Protein At5g20230 Blue Copper BindiggProteln At5g39785 Unknown Protein At5g46710 Zinc Binding Family Protein At5gfl230 ATERF5 At5954470 Zinc Finger (B-boxype) Family Protein At5g58650 PSY1 At5959730 A member of EXO7O gene family At5g59820 Zat12 At5960680 Unknown Protein At§g62460 Zinc Finger (C3HC4-type RING finger) Family Protein At5963130 OcticosapeptidelPhox/Bem1 p (PB1) Domain-Containing Protein At5g66070 Zinc Figer (C3HC4-type RING finger) Family Protein Table 2.1 Transcripts with a Pattern of Induction in the Cold Similar to CBF2 39 Transcripts up early in response to low temperature AGI Description At1g19050 ARR7 AtLq21050 Unknown Protein At1gZ1910 Encodes a Member of the DREB subfamily A-5 of ERF/AP2 transcription factor family. At1gz7730 ZAT10 At1968840 RAV2 At1g74890 ARR15 At1g76600 Unknown Protein At1fl440 Kelch Repeat-containing F-box Family Protein At1g80840 WRKY40 1112125900 CTH At2g26530 Unknown Protein At2938470 WRKY33 At3g15450 Unknown Protein At3g48100 ARR5 At3g48360 BT2 Atagsseao Zinc Firngr (CCCH-type) Family Protein At4gO1250 WRKY22 At4g24570 Mitochondrial Substrate Carrier Family Protein; At4g25470 CBF2 At4g25480 CBF3 At4g29780 Unknown Protein At4g34150 C2 Domain-containiryg Protein At4936040 DNAJ heat shock N-terminal domain-containing protein At4g§7610 BT5 Atagzozso Blue Copper Binding Protein At5l28770 bZlP 63 At5L37260 C|R1 At5g57560 Cell wall-modifying enzyme At5959820 Zat12 Atsgg920 ARR6 Table 2.2 Transcripts up Early in Response to Low Temperature Thirty transcripts up-regulated at 1h in response to treatment at 4°C. Those in bold contain the sequence vCGCGb in the region 500bp upstream of the 5’UTR (TAIR7). 40 A Role for CAMTA3 in Cold-Induction of CBF1, ZAT12 and GOLS3 Like CBF2, the promoter regions (1kb region upstream of start codon) of CBF1 and ZAT12 include a CG-1 sequence whereas that of CBF3 does not. It was therefore of interest to determine whether any of the camta mutations affected cold-induced expression of these genes. The results indicated that the camta3 mutation reduced CBF1 transcript levels by about 40% (Figure 2.9A) and reduced ZAT12 transcript levels by about 50% (Figure 2.98). Although a slight decrease in CBF3 expression was suggested, there was great variability over repeated experiments indicating that this was not statistically significant at a p-value <0.05 (Figure 2.90). One downstream target of CBF, GOLS3, was reduced about 40% in the camta3 plants (Figure 2.10). The effect on GOLS3 levels indicates that the reduction in CBF1 and CBF2 levels seen in camta3 plants may have an effect on downstream cold responses. 41 > w . 1.20 , 1.20 E3 1.00 e T; 1.00« in 3 I- a) 0 0.80 < .1 0.80- c N c 3.9 0.60 0 .9 -0.60« -,—. m .2 m g 3 0.40 5 g 0.40- I! 3 0-20 c? 5% 0.20- ul LIJ 0,00 0.00- WTwarmWT2h0°C c32hO°C WTwarm WT2h0°C c32h0°C C 1.20 m: 1.004 u_> 003 0.801 o: as 0.60 “In aficio- (xx ""' 0.201 0.00- WTwarm WT2h0°C c32h0°C Figure 2.9 CBF1, ZAT12, and CBF3 Levels in camta3 Plants (A)QPCR analysis shows relative expression of CBF1 transcript levels in terms of WT2h0°C. Error bars indicate SE. The averages for WT warm and camta3 2h0°C are significantly different than WT 2h0°C (Student's t-test p-value <0.01) (n=10 for WT warm, n=12 for WT and camta3 2h0°C ) (B)Relative expression of Zat12 transcript levels in terms of WT2h0°C. Error bars indicate SE. The averages forWT warm and camta3 2h0°C are significantly different than WT 2h0°C (Student’s t-test p-value <0.01) (n=10 for WT warm, n=12 for WT and camta3 2h0°C ) (C)Relative expression of CBF3 transcript levels in terms of WT2h0°C. Error bars indicate SE. The average for camta3 2h0°C is not significantly different than WT 2h0°C (Student's t-test cutoff p-value <0.05) (n=2 for WT warm, n=4 for WT and camta3 2h0°C ) 42 1.20 ...: 1.00 ‘3 2 03 0.80 0.60 0.40 :3 0.20 0.00 Relative G pressm Figure 2.10 Galactinol synthase 3 Levels Are Reduced in camta3 Plants QPCR analysis shows relative expression levels of galactinol synthase3 are significantly reduced in 24h0°C camta3 plants compared to WT at 24h0°C (Student’s t-test p-value <0.01, n=4). Concerted Action of both CAMTA1 and CAMTA3 Are Required to Attain Full Levels of Freezing Tolerance The results presented above establish that the camta3 mutation caused about a 40-50% decrease in cold-induced accumulation of transcripts for CBF1, CBF2, ZA T12, and one of the CBF targets, GOLS3. It was therefore of interest to determine whether the camta3 mutation had an effect on freezing tolerance. The results of both electrolyte leakage (Figure 2.11A) and whole plant freeze tests (not shown) did not reveal any difference in freezing tolerance wild-type and camta3 mutant plants grown at either warm temperature or cold-acclimated for 7d4°C. Additional testing indicated that the camta1, 2, 4, 5, and 6 mutants were also not affected in freezing tolerance (not shown). 43 The CAMTA1, 2 and 3 proteins have similar protein structure and thus might have overlapping functions (ref; Figure 2.43). To determine if redundancy between CAMTAs 1 and 3 might contribute to the lack of freezing tolerance seen in the camta3 mutant plants, camta1 and camta3 mutants were crossed to obtain a camta1/3 double mutant which was then tested for freezing tolerance (Figure 2.113). There was no significant difference in freezing tolerance between the camta1/3 and WT plants when they were grown at 22°C, but there was a considerable difference after cold acclimation. A 7 day period of cold acclimation at 4°C resulted in about a 5°C increase in freezing tolerance in WT plants, but the camta1/3 plants only increased about 2°C in freezing tolerance. Thus, for Arabidopsis to attain full levels of freezing tolerance, it needs the concerted action of both the CAMTA1 and CAMTA3 genes. The basis for this requirement is not known, but the camta1/3 plants displayed outward phenotypic differences that might have accounted, at least in part, for the differences in freezing tolerance. The camta1/3 plants were smaller than WT plants when grown at 22°C and showed an increase in chlorosis compared to WT plants (Figure 2.12A). Moreover, both of these phenotypes were more pronounced when grown at 4°C (Figure 2.128). The CBF2 levels in response to 2h0°C treatment were near WT levels in camta1/camta3 double mutants (Figure 2.13 A,B). 44 l l l r. __ 3» 4 87° :80~£%- .na‘: -- - N n 360-----,-~---77 ~»----l =2 40 " _¢_ WTWarm_j 1“,...- --o--c3Warm l 20 +wr7d4°c7 o , , , , ,i—o—c37d4°c_J o -2 4 43 -8 -1o .12 -14 Tem p (C) % Leakage Temp (C) Figure 2.11 Effect of camta3 and camta1/3 Mutations on Freezing Tolerance (A) Electrolyte leakage assay of non-acclimated WT (dashed lines, open triangles), non—acclimated camta3(c3) (dotted lines, open circle), WT acclimated for 7d4°C (solid line, filled triangles) and camta3(c3) acclimated for 7d4°C(solid line, filled circles). (B) Electrolyte leakage assay of non-acclimated WT (dashed lines, open triangles), non-acclimated camta1/camta3 (c1/c3) (dotted lines, open squares), WT acclimated for 7d4°C (solid line, filled triangles), and camta1/camta3 (c1/c3) acclimated for 7d4°C (solid line, filled squares) 45 Figure 2.12 Effect of camta1/3 Mutations on Development (A) Warm grown Columbia-0 (Col) and camta1/3 (C1/C3) double mutants. Bar indicates 18mm. (B) Colombia—0 (Col) and camta1/3(c1/c3) double mutants grown at 4°C DISCUSSION CAMTA3 Regulates CBF2 Expression in Response to Low Temperature Through the CM2-Box Region of the CBF2 Promoter. This study took an in depth look at the CBF2 promoter and identified multiple elements present in the promoter that are involved in regulation of CBF2 transcription. Conserved regions in the similarly regulated ZAT12 promoter were identified and through mutational analysis we determined that a 27bp sequence, the CM2ICM1-box is necessary for cold-induced regulation of a reporter construct. This result is consistent with the overlap between the CM2- box region and the previously identified ICEr2 region (Zarka et al. 2003). Additionally, four copies of the CM2-box. in combination with the region downstream, the CM1-box, was sufficient to induce cold-transcription of a reporter gene in a manner dependent on an intact CM2-box sequence. We showed that several members of the CAMTA family of transcription factors are able to bind specifically to this region of the CBF2 promoter. A 1.20 1.00 - 0.80 4 0.60 . 0.40 . 0.20 - Relative CBF2 Expression Level 0.00 r . WT Warm WT 2h0°C c1lc3 2h0°C 03 1.40 1.20 - 1.00 - 0.80 - 0.60 4 0.40 - 0.20 - 0.00 - Relative CBF2 Expression Level . WT 2h0°C c1 2h0°C c3 2h0°C c1/c3 2h0°C Figure 2.13 CBF2 Levels Are Near Wild-Type Levels in camta1/3 Double Mutants (A) QPCR analysis shows relative expression levels of CBF2 in camta1/ camta3 double mutants (c1/c3) are similar to WT levels in plants treated with 2h0°C (B) QPCR analysis shows that relative expression levels of CBF2 are significantly reduced in camta3 mutants(c3) compared to WT, but not camta1 or camta1/3 double mutants (C1/c3) 47 Analysis of T-DNA insertion lines in each of the CAMTA family members revealed a functional role for CAMTA3 in the regulation of CBF2 in response to low temperature. Loss of CAMTA3 reduced CBF2 levels in response to low temperature. This was shown to be through the CM2/CM1-box region of the CBF2 promoter (Figure 2.14). Complementation of the camta3 T-DNA insertion line recovered CBF2 expression. This effect was not limited to the CBF2 cold- response, the CAMTA transcription factor family has been shown to bind DNA containing a sequence vCGCGb (Bouche et al. 2002; Yang and Poovaiah 2002). We have shown that this binding plays an important role in regulation of cold-responsive transcripts, particularly CBF1, CBF2 and Zat12. . ‘ .‘é ‘i;' _ ..l' ."f‘II'. " d'rZTLIi‘ ""1"” ....ri | -‘ m‘l'f “34171-21 q ,i....,._._ub, _*:§...74,, LEVI. $93459: nus-f- 1.‘ ""T':o.'1,i4.;'."§i::h. Figure 2.14 Model of CAMTA3 Regulation of CBF Expression in Response to Low Temperature The effect on CBF3 mRNA levels was not statistically significant across repeated experiments. CBF3 lacks a CAMTA DNA binding site in the 1KB upstream promoter region and thus may not be regulated by CAMTAs. The slight effect seen in the CBF3 transcript could be due to the proximity of the three cold-induced CBFs. Since these three genes are located in tandem on chromosome 4, the effects seen in CBF1 and CBF2 may perpetuate along the 48 chromosome extending their effects to CBF3 perhaps through changes in chromatin modification. The differences in regulation of CBFs 1, 2, and 3 are consistent with results from studies of the ice1 mutant (Chinnusamy et al. 2003). The distinct regulation mechanisms for these key cold response regulators CBF2 and CBF3 may have functional importance to cold response, providing a redundant mechanism for the induction of the CBF cold response pathway in response to low temperature stress. CAMTAs Are Potential Regulator of Cold Acclimation in Arabidopsis Three lines of evidence support a model that CAMTA proteins, in particular, CAMTA3, may be important regulators of the response to cold stress in Arabidopsis. First CAMTA3 appears to be a positive regulator of the induction of CBF1, CBF2, ZAT12, and GOLS3 in response to low temperature. Secondly, camta1/3 double mutants have a significant reduction in freezing tolerance compared to wild-type plants. Finally, CAMTA binding sites are over- represented in the promoters of early cold responsive genes. The effect of camta3 on CBF1, CBF2, and known CBF2 target gene, GOLS3 indicates that at least part of this response is through the CBF cold-acclimation pathway. The significant reduction in freezing tolerance seen in the camta1/3 double mutants while CBF2 levels are unaffected in the camta1 mutant suggests that CAMTAs may also play a role in CBF independent cold- acclimation pathways. It will be interesting to examine the global effects of the loss of CAMTA3 and CAMTA1/3 on the cold-regulated transcriptome. 49 The recovery of the CBF2 levels in the camta1/3 double mutants suggests that interaction between the CAMTA proteins may be complex. In Drosophila, which contains only one copy of a CAMTA transcription factor, the dimerization domain is required for nuclear IoCalization and proper function of the CAMTA protein (Gong et al. 2007). This dimerization domain is conserved in the plant CAMTA transcription factors. The possibility exists that in Arabidopsis this family can form heterodimers or heteroligomers and that the loss of CAMTA3 impairs this multimerization, thus explaining the significant effects seen in camta3 plants. Analysis of CAMTA proteins in Arabidopsis and other organisms indicates that CAMTAs can act both positively and negatively on targets and through two distinct mechanisms (Choi et al. 2005). In Drosophila dCAMTA regulates transcription of a gene containing an F box motif through direct activation through a CGCG box region of the promoter (Han et al. 2006). However, in mammals CAMTA2 is brought to the promoter through interaction with another DNA binding protein (Song et al. 2006). Transcriptional activity of CAMTA2 in mammals is inhibited by interaction with HDACs. This inhibition is relieved by protein kinase C e (PKCe) and protein kinase D (PKD) (Song, Backs et al. 2006). This study provides evidence that CAMTA3 interacts with the CBF2 promoter in a mechanism similar to that seen in the Drosophila CAMTA functions. It will be interesting to see if this is the predominant mode of action of CAMTAs in Arabidopsis or if there are some promoter/ CAMTA protein 50 combinations that act in concert with additional transcription factors as seen in mammals. Potential Role of CAMTAs in Early Responses to Abiotic and Biotic Stresses Transcripts containing the putative CG-1 DNA binding site within 500 bp upstream of their ATG are overrepresented in early cold-responsive genes and in transcripts that show a pattern of induction similar to CBF2. This suggests that perhaps CAMTA transcription factors may have a larger role in early responses to low temperature. Recently, Walley et al. identified a novel cis- element overrepresented in the promoters of wound-induced genes and postulated that this could be an early signaling response to many biotic and abiotic stresses (Walley et al. 2007). This element contains the recognition sequence for CAMTA binding. Therefore, it will be interesting to see if the CAMTAs bind to this overrepresented early stress response element and if they are primary regulators of the signaling response for multiple abiotic and biotic stresses. A recent report identified CAMTA3 as a suppressor of defense responses (Galon et al. 2008). My work contributes to the potential role of CAMTAs as early components of abiotic stress responses, showing the role for CAMTA3 as a positive regulator of early cold responses. Consistent with the role of CAMTAS in the stress responsive regulation of hypertrophic cardiac growth in mammals and the recently demonstrated role for CAMTA3 in response to biotic stress, my study provides evidence that 51 CAMTAs are important signaling components of cold-stress response in Arabidopsis. The high conservation of CAMTA proteins and their target binding sites across distantly related species, Arabidopsis, rice, Drosophila, & mammals suggests that this family of transcription factors may be and ancient tool for response to environmental stresses on an organism. MATERIALS AND METHODS Plant Material and Growth Conditions All plants were stratified for 3-5 days in the dark at 4°C then transferred to constant illumination at 24°C for 10 days prior to treatment. Cold treatment consisted of moving the plates to a 4°C or 0°C chamber with constant light at a reduced level (approximately 35 p mol m'zsec"). For experiments with the CAMTA mutants and complimented lines, plants were grown on Gamborg’s 85 nutrients (Caisson Laboratories, www.caissonlabs.com) and 0.8% phytagar (Caisson Laboratories, www.caissonlabs.com) without sucrose. Experiments on the reporter constructs from CBF2 and 28112 were grown on the same medium with the addition of 0.2% sucrose. The experiments testing for the complementation of CAMTA3 by overexpression in the camta3 background were performed on shoot tissue, all other experiments were done on whole seedfings. T-DNA insertion mutants were identified using SIGnAL database (Alonso et al. 2003) and obtained from ABRC for camta1(Salk_008187), camtaZ(Salk_007027), camta3(Salk_001152), camta4(Salk_O13723), and 52 camt86(Salk_O78900). camta5 was obtained from GABI-Kat Line ID 815808 (Rosso et al. 2003). The CM2/CM1-boszus plasmids were transformed in Arabidopsis ecotype WS using standard procedures. SeleCted lines of homozygous CM2/CM1-box:Gus plants were crossed into camta mutant lines. Seven independent T4 lines of camta3-l- CM2/CM1-boszUS +/+ and four lines of camta1 -/- CM2/CM1-boszUS +/+ were analyzed for Gus expression level by northern analysis (two representative lines shown in manuscript). Two lines of camta3-l- CM2/CM1-boszUS were transformed with 353::CAMTA3. T2 lines were analyzed by histochemical staining for GUS and selected T3 homozygous lines were analyzed for GUS, CBF2, and CAMTA3 mRNA levels by qRT-PCR. EMSA EMSA analysis was performed by Dr. Heather Van Buskirk. The protein coding regions of the DNA binding domain for CAMTAs 1, 2, 3, and 5 were expressed in E. coli. and lysate was harvested. CM2/CM1-box and mutated CM2/CM1-box sequences were generated and labeled with [d-32P]dCTP. Probe was incubated with protein and unlabeled competitor, if indicated and then resolved on a 5% polyacrylimide gel. The gel was dried and exposed to a phosphorimager screen. RNA isolation and Analysis Total RNA was extracted from plant material with the use of RNeasy Plant Mini kits (Qiagen, Valencia, CA) with modifications as described (Zarka, 53 Vogel et al. 2003). Northern transfers were prepared and hybridized as described (Hajela et al. 1990) and washed with high-stringency conditions (Stockinger, Gilmour et al. 1997). For RT-PCR cDNA sythesis was performed using Promega Reverse Transcription system according to the manufacuturer's directions using random primers with the following modifications. Total reaction volumes were doubled and starting RNA was adjusted. Starting RNA was 0.01ug for warm and 2h0°C samples for analysis of CBF 1,2,3, and ZAT12 mRNA and 0.1ug for all 24h0°C samples and for warm and 2h0°C samples for analysis of GUS and CAMTA3 mRNA. cDNA was diluted five-fold with water and 3uL was used as a template for quantitative real-time PCR (qRT-PCR). qRT-PCR using SYBR Green was performed using the Applied Biosystems 7500 real-time PCR system in Standard Mode with SYBR Green PCR Core Reagents Mix (Applied Biosystems). qRT-PCR was performed according to manufacturers protocols with the following modification. Reactions were performed in a 30pL volume. For CBF2 mRNA analysis, the annealing/extension temperature was 62°C. Serial dilutions of 2h0°C WT samples were performed to determine the efficiencies of the primers for CBF1,2, 3, ZAT12, GUS, and ACTIN3(ACT3). Serial dilutions of 24h0°C samples were performed to determine the efficiencies of GOLS3 and ACT3 primers. Reactions were performed in triplicate and products checked by melting curve analysis. The abundance of transcripts was analyzed with the relative standard curve method normalizing to the reference transcript, ACT3 (AT3GS3750). The primers used for amplification were CBF1 54 (CGACTATCGAATATTAGTAACTCCAAAGCGACACG-3' and 5’GGAGACAATGTTTGGGATGC-3’), CBF2 (5’-GGA TGCTCATGGTCTTGACAT-3’ and 5’-TCTTCATCCATATAAAACGCATCTTG- 3’), CBF3 (5’-CAACAAACTCGGCATCTCAA-3’ and 5’- GGCGTTTCAGGATGAGATGT-3’), ZAT12 (5’-CCTTAGGAGGTCACCGTGC-3’ and 5’- CAAGCCACTCTCTTCCCACT-B’), ACT3 (5’- GGTCGTACTACTGGTATTGTGCT-3’ and 5’-TGACAATTTCACGCTCAGCT- 3’), GOLS3 (5’-GGAGTGGTTGGTCTGGCTAA-3’ and 5’- TTGGTTATCCGGTGGGTAAA-3’), CAMTA1 (5’- CTGTCAGAAGCCCAACACAG-3’ and 5’-CCTTGAGCTTCTCATGAGCTTCTC- 3’), CAMTA2 (5’-GGCAAGGAGCACATGAAAAT-3’ and 5’- TAAGATCCTCGGGGCCTAAT-3’), CAMTA3 (5’- CAACGACATCCAAGAAAGCA-3’ and 5'-TGAGGACATAGGCAACATCAA-3’), CAMTA4 (5’-‘ITTGGAAAGGGCAGGAACTA-3’ and 5’- . TTTGGTAACCTCGCACATGA-3’), CAMTA5 (5’- ATCGCGAGACACATGAGGTT-3’ and 5’- GACTGTTGCTCCGCACTGTA—3’), CAMTA6 (5’-TTGTCTTCAGGGACGGTCTT-3’ and 5’- TGGAGTCTACCGTTGCATCA-3’). Statistical Analysis Experiments consisting of three or more conditions were tested for statistical significance using two way ANOVA followed by a protected t-test. Experiments with only two comparisons were tested for significance using Student’s t-test. 55 Staining for GUS activity Gus activity was analyzed as described by Zarka et al. (2003). Overrepresentation of Motif Analysis CEL files were obtained from TAIR for Warm and cold treated shoot samples by Kilian et al. (2007) and warm and cold treated samples by Vogel et al. (2005). RMA normalization was performed using AF FY package for Bioconductor (Irizarry et al. 2003, www.bioconductor.org). Resulting data was analyzed through BAR expression Angler (Toufighi et al.2005) and selected for genes with a minimum Pearson’s correlation coefficient to CBF2 of 0.85. The 500 kb region upstream of the transcriptional start site (TAIR7) was analyzed for the presence of vCGCGb using Promomer (Toufighi et al. 2005). The number of genes that contained this motif was compared to the number in the entire genome (3794). A list of transcripts that responded rapidly to low temperature was identified as those that were differentially regulated at one or three hours at 4°C (Kilian et al. 2007) or one hour in Vogel et al. (2005) by ANOVA with a multiple testing correction of 0.05 FDR and were two fold or more induced in these conditions. These were analyzed for the presence of the vCGCGb sequence in their promoter using Promomer and compared to the promoters of the entire genome. Whole Plant Freeze Test Whole plant freeze tests were performed essentially as previously described (Vogel et. al. 2005). 56 Electrolyte Leakage Freeze Test Electrolyte leakage freeze tests were performed essentially as described (Gilmour et al. 2000) with minor modifications. The SAS system was not used to aid in randomization Accession Numbers The Arabidopsis Genome Initiative locus identifiers for the CAMTAs are as follows: CAMTA1 AT5G09410, CAMTA2 AT5GG4220, CAMTA3 AT2622300, CAMTA4 At1667310, CAMTA5 ATSG16150, and CAMTA6 At3G16940. 57 Literature Cited Agarwal, M., Y. Hao, et al. (2006). "A R2R3 Type MYB Transcription Factor Is Involved in the Cold Regulation of CBF Genes and in Acquired Freezing Tolerance." J. Biol. Chem. 281(49): 37636-37645. Alonso, J. M., A. N. Stepanova, et al. (2003). "Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana." Science 301(5633): 653-657. Bouche, N., A. Scharlat, et al. (2002). "A Novel Family of Calmodulin-binding Transcription Activators in Multicellular Organisms." J. Biol. Chem. 277(24): 21851-21861. Chinnusamy, V., M. Ohta, et al. (2003). "ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis." Genes Dev. 17(8): 1043- 1054. Choi, M. S., M. C. Kim, et al. (2005). 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Chem. 282(29): 21253-21258. Hajela, R. K., D. P. Horvath, et al. (1990). "Molecular Cloning and Expression of cor (Cold-Regulated) Genes in Arabidopsis thaliana." Plant Physiol. 93(3): 1246- 1252. Han, J ., P. Gong, et al. (2006). "The Fly CAMTA Transcription Factor Potentiates Deactivation of Rhodopsin, a G Protein-Coupled Light Receptor." Cill 127(4): 847-858. 58 Jaglo-Ottosen, K. R., S. J. Gilmour, et al. (1998). "Arabidopsis CBF1 Overexpression Induces COR Genes and Enhances Freezing Tolerance." Science 280(5360): 104-106. Kilian, J ., D. Whitehead, et al. (2007). "The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses." The Plant Journal 50(2): 347-363. Knight, H., A. J. Trewavas, et a1. (1996). "Cold Calcium Signaling in Arabidopsis Involves Two Cellular Pools and a Change in Calcium Signature after Acclimation." Plant Cell 8(3): 489-503. Liu, Q., M. Kasuga, et al. (1998). "Two Transcription Factors, DREBl and DREB2, with an EREBP/APZ DNA Binding Domain Separate Two Cellular Signal Transduction Pathways in Drought- and Low-Temperature-Responsive Gene Expression, Respectively, in Arabidopsis." Plant Cell 10(8): 1391-1406. Monroy, A. F. and R. S. Dhindsa (1995). "Low-Temperature Signal Transduction: Induction of Cold Acclimation-Specific Genes of Alfalfa by Calcium at 25[deg]C." Plant Cell 7(3): 321-331. Rosso, M. G., Y. Li, et al. (2003). "An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics." Plant Molecular Biology 53(1): 247-259. Song, K., J. Backs, et al. (2006). "The Transcriptional Coactivator CAMTA2 Stimulates Cardiac Growth by Opposing Class II Histone Deacetylases." Qe_ll 125(3): 453- 466. Stockinger, Eric J ., Sarah J. Gilmour, et al. (1997). "Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C- repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit." Proceedings of the National Academy of Sciences 94(3): 1035-1040. Toufighi, K., S. M. Brady, et al. (2005). "The Botany Array Resource: e-Northems, Expression Angling, and promoter analyses." The Plant Journal 43(1): 153-163. Vogel, J. T., D. G. Zarka, et al. (2005). "Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis." IE Plant Journal 41(2): 195-211. Walley, J. W., S. Coughlan, et al. (2007). "Mechanical Stress Induces Biotic and Abiotic Stress Responses via a Novel cis-Element." PLoS Genetics 3(10): e172. 59 Yang, T. and B. W. Poovaiah (2002). "A calmodulin-binding/CGCG Box DNA-binding protein family involved in multiple signaling pathways in plants." J. Biol. Chem: M207941200. Zarka, D. G., J. T. Vogel, et al. (2003). "Cold Induction of Arabidopsis CBF Genes Involves Multiple ICE (Inducer of CBF Expression) Promoter Elements and a Cold-Regulatory Circuit That Is Desensitized by Low Temperature." Plant Physiol. 133(2): 910-918. 60 CHAPTER THREE Role of CBF in the Regulation of Cold-Responsive Transcripts Introduction Large changes in gene expression occur In response to low temperature treatment, and many of these changes in transcription are controlled by the CBF family of transcription factors, however, the extent to which CBFs are required for the process of cold acclimation is not known. Clearly, these transcription factors do play an important role because constitutive expression of CBFs 1, 2, and 3, which are cold-inducible themselves, is sufficient to confer freezing tolerance to Arabidopsis plants without the period of cold-acclimation required by WT plants. Previous work by Vogel et al identified CBF sufficient transcripts, those which were up-regulated by both cold-treated wild type plants and warm-grown plants overexpressing CBF2 (Vogel et al. 2005). This analysis identified a CBF regulon of 85 up- and eight down- regulated transcripts. Over 80% of the identified cold-regulated transcriptome was not regulated by CBF2 overexpression (Vogel, Zarka et al. 2005). It is of interest to know if these transcripts that are not up-regulated by CBF overexpression contribute to the ability of Arabidopsis to cold-acclimate and if so, what controls their regulation in response to low temperature. The regulation of these additional transcripts may be due to many possible sources. One possibility is that CBF1 or CBF3 are responsible for the regulation of these transcripts. Novillo et al. recently demonstrated that CBF1 61 and 3 are expressed in different tissues than CBF2 and that the loss of either CBF1 or CBF3 individually or in combination through miRNA targeting showed specificity in their effects on certain cold-induced transcripts (Novillo et al. 2007). This information refines the CBF expression network and identifies that each CBF has a specific role in the regulation of different CBF-regulon transcripts. Analysis of CBF1, 2 and 3 overexpression lines revealed extensive overlap in the transcripts regulated by these three CBFs (Fowler and Thomashow 2002). This supports the idea that the specificity of the CBF cold- induction pathway likely comes from controlled expression of the three cold- induced CBF transcripts with respect to tissue and timing. However, since the effects seen in plants constitutively expressing CBF1 or CBF3 were very similar to the effect of constitutive expression of CBF2, including the transcripts differentially regulated by the overexpression of each CBF, the specificity of the CBFs for their targets is likely involved in the complex regulation of the CBF regulon genes and does not contribute much of an effect on the CBF- independent transcripts. Therefore, while the specificity of the three cold- inducible CBFs is important for establishing the network cascade of regulation of CBF-regulon transcripts, CBF1 and CBF3 are not likely to be major contributors to the regulation of the CBF-independent transcripts. Another possible explanation for the regulation of these CBF- independent transcripts is that their differential expression is due to disruption of the circadian clock at low temperature (Bieniawska et al. 2008). Comparative analysis by Bieniawska et al. showed that many of the transcripts identified as 62 differentially regulated in response to low temperature in several studies appear to be expressed at different levels from warm due to a shift in the circadian clock in response to low temperature. Many output genes from the clock show a dampening or loss of their cyclical expression in low temperature. It is possible that the CBF-independent transcripts are comprised mostly of transcripts regulated by the clock, whose change in response to low temperature is due to disruption of cycling. However, in experiments where plants are grown in constant light and shifted to low temperature in constant light, there are still a large number of CBF-independent transcripts that are cold- regulated. These results indicate that while the effects of the cold on the clock may explain the cold-regulation on some CBF-independent transcripts, it does not account for the regulation of all CBF-independent transcripts. Two additional explanations for the regulation of these CBF-independent transcripts are that either they are truly CBF-independent and are induced in response to low temperature by a parallel pathway to CBF or perhaps these transcripts are regulated by CBF but require some additional factor for their regulation that is not present in warm grown CBF overexpressing plants. Identifying if these transcripts are indeed CBF-independent will help to establish the role of CBFs in response to low temperature. Additionally, while it is known that CBF is sufficient for the regulation of a set of cold-regulated genes, it is not known if CBF is required. In order to establish which transcripts, if any, are CBF-independent and to identify the extent to which CBFs are required for cold acclimation in Arabidopsis, we attempted to identify plants that were incapable 63 of inducing the CBF regulon in response to low temperature. Elimination of CBFs 1, 2, and 3 was attempted initially by using an RNAi construct or the addition of an EAR domain to the CBF protein, turning CBF from a transcriptional activator to a repressor (Hiratsu et al. 2003). However, a dominant negative version of the CBF protein was identified that was successful in inhibiting the response of the CBF regulon to low temperature. A dominant negative version of the CBF protein, CBF2AC, was identified that affects the regulation of many CBF regulon transcripts in response to low temperature. Use of this mutated version of CBF2 enabled us to identify 398 up-regulated transcripts (60% of the cold induced transcriptome) that are CBF independent. These transcripts are not enriched for circadian regulation and the promoters of these transcripts are overrepresented for the TBX (telo—box) element, suggesting a novel method of cold-induction of these genes. Several transcripts which were induced in the warm by CBF2 overexpression and were identified as CBF sufficient were still able to be induced in CBFZAC plants in response to low temperature indicating that there are multiple mechanisms for inducing these transcripts in response to low temperature. The induction of 40% of the cold-induced transcripts was affected in CBF2AC plants, indicating a larger role for CBF in the cold-acclimation pathway than previously thought. The transcripts down-regulated in response to low temperature were largely unaffected in the CBF2AC plants. Ninety percent of these down-regulated transcripts were still repressed in CBFZAC plants, indicating that either down- regulation of these transcripts in response to low temperature is independent of 64 CBF or that an intact activation domain of CBF is not required for their down- regulation. CBF2AC plants showed a decrease in the freezing tolerance as well as a reduction of the basal freezing tolerance. However, CBF2AC plants were still able to cold acclimate suggesting an important role for both the CBF- independent and CBF co-regulated transcripts in the process of cold acclimation. 32% Identification of a Dominant Negative Version of CBF2 Overexpression of CBF1,2, or 3 results in a dwarf phenotype (Gilmour et al. 2004). A population of CBF2 overexpressing plants was mutagenized using EMS. Offspring were selected that were larger than CBF2 overexpressing plants, indicating a suppression of the CBF size phenotype. One identified mutant showed a recovery of WT size (Figure 3.1A). This mutant was tested for expression of CBF2 target gene COR15. Northern analysis of these genes indicated that the expression levels of both COR15 and COR78 mRNA levels were reduced in both warm grown plants and plants grown for 7d4°C (Figure 3.18). Sequencing of the CBF2 transgene in these plants revealed a point mutation converting glutamine 136 to a stop codon positioned just past the AP2 DNA binding domain and prior to the activation domain (Figure 3.10, Figure 3.2). This truncated version of CBF2 was called CBF2AC. 65 A C \‘l 1 32 44 47 106 213 ‘21 «t ‘_6 NLS AP2 Domain Acidic Region Ws-2 35s::CBF2 AC CBFZAC B COR78 COR15 """'"'~-~ CBF2 Figure 3.1 Identification of CBF2AC, a Truncated Version of CBF2 That Suppresses the 35s::CBF2 Phenotype (A) CBF2AC (AC) plants show a recovery of WT (Ws-2) phenotype compared to their 35s::CBF2 parents (8) Northern analysis of COR78 and COR15 levels in WT(WS), 35s::CBF2, CBFZAC (AC) plants and two other mutants (1-2) and (2-4) after growth in warm (w) and treatment for 24h4°C (C) (C) Sequencing of the CBF2 transgene in CBF2AC plants revealed a stop codon just past the DNA binding domain CBF2AC Effectively Inhibits Induction of GOLS3, a CBF Regulon Transcript. We hypothesized that CBF2AC might be acting as a dominant negative version of the CBF proteins. The CBF2AC protein, with its intact DNA binding domain, may recognize and bind the C-repeat (CRT) cis-element, RCCGAC, in the promoters of the CBF target genes. However, with the stop codon upstream of the activation domain, CBF2AC would not be able to activate transcription of these genes. Thus, the presence of the CBF2AC transgene may block access of the endogenous CBFs to the CRT elements in the CBF 66 target genes. If CBFZAC is functioning as a dominant negative version of CBF2, this construct could serve as a tool to improve our understanding of the role of CBF in low temperature responses. CBFZAC Mutation 1 MNSFSAFSEMFGSDYESPVSSGGDYSPKLATSCPKKPAGRKKFRETRHP1 51 AP2 domain YRGVRQRNSGKWVCELREPNKKTRIWLGTFQTAEMAARAHDVAAIALRQR 101 SACLN FADSAWRLRIPESTCAKEIQKAAAEAALNFQDEMCHMTTDAHGLD 151 MEETLVEAIYTPEQSQDAFYMDEEAMLGMSSLLDNMAEGMLLPSPSVQWN 201 YNFDVEGDDDVSLWSY Figure 3.2 CBFZAC Is a Glutamine-to-stop Codon Mutation Sequence of CBF2 highlighting the glutamine that is converted to a stop codon in CBFZAC. To confirm that CBF2AC was functioning as a dominant negative protein we cloned CBF2AC and transformed it into wild type plants under control of the 35s CaMV promoter. These CBFZAC-containing plants were then tested for the expression of Galactinol Synthase 3 (GOLS3), one of the most highly induced transcripts in plants constitutively expressing CBF2 (Vogel et al. 2005). There was an 80% reduction in the cold-induced accumulation of GOLS3 transcript in the CBFZAC plants after 24h4°C treatment (Figure 3.3). The reduction seen in the expression level of GOLS3, a CBF regulon transcript, indicates that the presence of the CBFZAC construct is responsible for the inhibition of the induction of CBF target genes in response to low temperature. 67 100004 UWarm 7, 9000 I24h 4°C > 3 8000 4 S ,7, 7000- E a 6000 - X “J 5000- 53 _r 4000 - o ‘9 3000 ll) .2 E 2000 . l I: 1000 - _ 0' r ‘1 WT 355::CBF2 AC Figure 3.3 GOLS3 mRNA Levels Are Reduced in CBF2AC Plants QPCR analysis of exprssion of GOLSS levels in WT, 35s::CBF2, and CBF2AC (AC) lines relative to WT warm levels in either warm grown (grey) or 24h4°C treatment (black). CBF2AC Functions as a Dominant Negative Protein The effect of the CBF2AC construct in preventing the large fold increase of GOLS3 mRNA accumulation in response to low temperature and the increase in plant size seen in the original mutant suggest that CBF2AC is acting as a dominant negative protein and preventing the access of wild-typeONT) CBF proteins to their targets. To test the ability of CBF2AC to block WT-CBF2 proteins we transformed plants containing 35s::CBF2 with the 355::CBF2AC construct. If CBF2AC functions as a dominant negative protein, when mRNA levels of CBF2AC and WT—CBF2 accumulate to equal levels, CBF2AC should inhibit the induction of CBF target genes by the WT-CBF2 construct in warm grown plants, where the expression and activity of the endogenous CBFs should be minimal compared to the WT-CBF2 transgene. Primers were designed which could amplify transcripts originating from the vector expressing 68 CBF2, but do not amplify CBF2AC transcripts. Three lines were identified which did not show silencing of the CBF2 transcript (2AC 6-4, 8-3, and 9-4). These lines expressed transcripts of the WT-CBF2 construct at levels similar to the parent plant (Figure 3.4A). A CBF2 expressed from PGA643 vector Warm FuaflwCEZTrangane BrpreesionLevel O O h or 1 Ws 35s: C2 6-4 B CBF2 Total Warm 2.5 - 1.5 - WTMCEZ Ws 358:02 84 8-3 9-4 Figure 3.4 CBF2 Levels in Plants Expressing 35s::CBF2 and 35s::CBF2AC Transgenes (A) QPCR analysis of CBF2 transgene levels in WT, 35s::CBF2 (35s:CZ), and 35s::CBF2 x 35s::CBF2AC (6-4, 8-3, and 9-4) plants. Expression is relative to levels in 35s::CBF2 plants (35s:C2). (B) QPCR analysis of total CBF2 levels in WT, 353::CBF2 (35$:C2), and 353::CBF2 x 35s::CBF2AC (6-4, 8-3, and 94) plants. Expression is relative to levels in 35s::CBF2 plants (35s:CZ). 69 Analysis of total CBF expression levels with primers that would amplify either CBF2 or CBF2AC indicated that, after subtraction of WT-CBF2 levels from total CBF levels, CBF2AC levels were expressed at approximately equivalent levels as WT-CBF2 (Figure 3.48). After identifying these three lines which showed approximately equal expression of WT-CBF2 and CBF2AC transcripts, we examined the expression level of two CBF regulon genes, COR15 and GOLS3. Analysis of two CBF target genes COR15a and GOLSS indicated that the CBF2AC construct was able to greatly reduce the expression of these transcripts by WT-CBF2 although the CBF2AC mRNA accumulates to only equal levels as the WT-CBF2 transgene mRNA levels (Figure 3.5 A, B). This is consistent with the possibility that CBF2AC is acting as a dominant negative protein and that when CBF2AC is expressed at levels similar to WT-CBF2 it is able to inhibit expression of two CBF regulon genes. When expressed in WT background, the CBF2AC transcript accumulates to even higher levels, accumulating to over 15 times the level of the endogenous CBF2 transcripts at 2h4°C (Figure 3.6). 70 > 1.27 0.8 - 0.6 2 Relative COR15a Expression Level . 0.2 — Ws 35s:CZ AC 6-4 8-3 9-4 1.2] 0.8 J 0.6 - 0.4 - Relative GOLS3 Expression Level . 0.2 — Ws 35$:C AC 6-4 8-3 9-4 Figure 3.5 Expression of CBF Target Genes in Plants Expressing 35s::CBF2 and 353::CBF2 AC Transgenes (A) QPCR analysis of COR15 levels in WT, 353::CBF2 (353:02), 35s::CBF2AC (AC), and 353::CBF2 x 355::CBF2AC (6-4, 8-3, and 9-4) plants. Expression is relative to levels in 35s::CBF2 plants (35SZC2). (B) QPCR analysis GOLS3 levels in WT, 35$:2CBF2 (35s:C2), 353::CBF2AC (AC), and 35s::CBF2 x 35s::CBF2AC (6—4, 8-3, and 9-4) plants. Expression is relative to levels in 353::CBF2 plants (353:02). Since CBF2AC is able to inhibit the targets of constitutively expressed WT-CBF2 when both transgenes are expressed at the same level, it would be expected that the effects of CBF2AC in wild-type plants will be even more profound since CBF2AC mRNA levels accumulate to a higher level than the endogenous CBF mRNA levels. For GOLS3 transcripts, the level of induction by WT-CBF2 transgene is reduced about 50% in plants containing both the WT- CBF2 transgene and the CBF2AC transgene. In agreement with the dosage effect predicted on the endogenous transcripts, cold-induction of GOLS3 mRNA at 24h is reduced about 80% in the CBF2AC plants compared to WT plants (Figure 3.5 B). The ability of CBF2AC to block the access of the endogenous CBFs to their targets in its role as a dominant negative form of CBF makes it a potential tool to assess the requirement and involvement of CBFs in the process of cold acclimation. Total CBF2 Warm and 2h Cold .3 00 4 16 1 Iwarm I 2 hr cold r-r ........ ‘ I“: H. . .3 .3 .3 on O N A 1 r r 1 Relative Expression Level . Ws 355202 ACp89 Figure 3.6 Levels of CBF2AC mRNA Accumulate to Higher Levels than Endogenous CBF2 Levels QPCR analysis of total CBF2 levels in WT, 35s::CBF2 (35$:C2), and 35s::CBF2AC (AC) plants grown in warm (grey) or cold-treated, 2h4°C (black). Expression is relative to WT cold levels. 72 Analysis of cold regulated transcripts in CBF2AC These results indicate that CBF2AC construct is effectively acting as a dominant negative protein, blocking the function of the endogenous CBFs. This suggests that in the presence of the CBF2AC construct, these plants are not able to mount a functional CBF pathway-based response to low temperature. To determine the full effects of an inhibited CBF pathway on the cold-regulated transcriptome, Affymetrix expression arrays were performed on WT and CBF2AC plants cold treated for 24h4°C. Analysis was focused on a robust cold-regulated set of transcripts in order to minimize the effects of growth conditions and to minimize the selection of circadian-regulated transcripts identified as differentially regulated due to cold-induced changes in the clock (Ramos et al. 2005; Bieniawska, Espinoza et al. 2008). RankProd, a Rank Product method was used to identify transcripts that were cold-regulated at 24h across three different experiments in two different growth conditions, 12/12 day/night cycles and constant light (Breitling et al. 2004; Vogel, Zarka et al. 2005; Hong et al. 2006; Kilian et al. 2007). This analysis identified 2368 probesets that were robustly cold regulated. To further refine this list we selected those that were cold regulated in both WS and Col-0 ecotypes at 24h4°C, resulting in 1279 probesets regulated by low temperature treatment in two cold-acclimating ecotypes (Appendix Table A1). 73 ' .1 ~55 I..”..yiiiir“ 3‘35 ‘. if? Titer-g ."~'__‘:._.. Model 1 0 0 1 1 0 0 Model 2 0 0 1 1 0 0 0.1 0.1 0.1 0.1 Model 3 0 0 1 1 0 0 0.5 0.5 0.5 0.5 Model 4 0 0 1 1 0 0 1 1 1 1 Model 5 0 0 1 1 0.5 0 5 0 0 0 0 Model 6 0 0 1 1 0.5 0.5 0.1 0.1 0.1 0.1 Model 7 0 0 1 1 0.5 0.5 0.5 0.5 0.5 0.5 Model_8 0 0 1 1 0.5 0.5 1 1 1 1 Model_9 0 0 1 1 1 1 0 0 0 0 Model_10 0 0 1 1 1 1 0.1 0.1 0.1 0.1 Model_11 0 0 1 1 1 1 0.5 0.5 0.5 0.5 Model_12 0 0 1 1 1 1 1 1 1 1 Model_13 1 1 0 0 0 0 0 0 0 0 Model_14 1 1 0 0 0 0 0.1 0.1 0.1 0.1 Model_15 1 1 0 0 0 0 0.5 0.5 0.5 0.5 Model_16 1 1 0 0 0 0 1 1 1 1 Model_17 1 1 0 0 0.5 0.5 0 0 0 0 Model__18 1 1 0 0 0.5 0.5 0.1 0.1 0.1 0.1 Model_19 1 1 0 0 0.5 0.5 0.5 0.5 0.5 0.5 Model_20 1 1 0 0 0.5 0.5 1 1 1 1 Model_21 1 1 0 0 1 1 0 0 0 0 Model_22 1 1 0 0 1 1 0.1 0.1 0.1 0.1 Model_23 1 1 0 0 1 1 0.5 0.5 0.5 0.5 Model_24 1 1 0 0 1 1 1 1 1 1 Table 3.1 Predetermined models of expression used for Haystack analysis of cold- regulated transcripts. Samples compared are WS_0h and 24h, 355::CBF2AC (AC) Oh and 24h, 353::CBF 2 (CBF2) 0h. Each row represents a potential model of expression for a transcript. Transcripts whose relative pattern of expression across all samples closely has a Pierson’s correlation of >080 to a particular model were considered matches of that model. To identify groups of genes with patterns of expression across the various genotypes Haystack analysis was employed (Mockler et al. 2007). Haystack uses a pattern matching algorithm that compares the expression pattern of each transcript across treatments to user-defined models of expression. Analysis of the expression pattern of these 1279 probesets was compared to 24 predefined models of expression (Table 3.1) across the following samples: WT 0h, WT24h, 355::CBF2 0h, 35s::CBF2AC Oh and 74 35s::CBF2AC 24h. Of these 1279 probesets 1129 had a correlation coefficient greater than 0.80 to one of 24 predefined models of expression, 1086 of these are predicted to bind to a unique transcript. These included 642 transcripts up- regulated in WT at 24h4°C and 444 down-regulated transcripts. Effect of 353::CBF2AC on cold-induced transcripts Ten percent (63) of the up-regulated transcripts matched a pattern where the expression at 24h4°C in 35s::CBF2AC plants was similar to wild type warm plants, corresponding to models 1,2,5,6,9, or 10 (Table 3.1, Figure 3.7A). These genes were classified as requiring CBF. Over 60 percent, 398, of cold- induced transcripts in WT plants were classified as CBF independent; these transcripts were still induced to their WT levels in 35s::CBF2AC plants, corresponding to models 4,8, or 12 (Table 3.1, Figure 3.78). The final group of 181 transcripts were induced in both WT and 35s::CBF2AC plants, however, their level in 35s::CBF2AC was significantly reduced from the WT level of expression at 24h4°C, corresponding to models 3,7, or 11 (Table 3.1, Figure 3.70). 75 A 20000 - E 18000 - __ .1, 16000 g 8 140001 3 c 12000 c .3 10000 ,9 g 8000 g a 6000 9 III 4000 4 a. ‘ 2000 - LU 0 _ 1 . WTOh WT24h4°C CBF2AC ‘ 24h4°C WT 0h WT 24h4°C CBF2AC 24h4°C C_ 14000 - D . 3 120001 642 Up-Regulated Transcripts 0 c -1 '9 8000 28% ,4 $8,, DRequif-ed 3 6000 1 . 3‘ ,5; , 1:4, 8 4000 - 133113511; m“. Ilndependent 5. 2000 4 . lEllntegrated 0 — ,. , wron WT24h4°C came , 24h4°C 62 A Figure 3.7 Cold-Induced Transcripts (A) Expression level of 63 transcripts that are upregulated in WT plants, but are not significantly upregulated at 24h4°C in CBF2AC plants. (8) Expression level of 398 transcripts that are upregulated in WT plants, and are expressed to a level equal to that of WT in CBF2AC plants at 24h4°C . (C) Expression level of 181 transcripts that are still induced in CBF2 AC plants, but are not induced to their full WT level. (D) Pie-chart showing the distribution of each category of transcripts based on their requirement for CBF, required (A), independent (8), integrated(C) Some transcripts previously identified as CBF-regulated due to their induction by CBF2 overexpression in warm-grown plants were still found to be induced to their full wild-type level in CBF2AC plants, suggesting that there are multiple mechanisms for inducing these transcripts in response to low-temperature. Additionally, this analysis indicates that there is a larger role for CBF than previously identified by analysis of warm grown CBF overexpressing plants suggesting that CBF maybe involved in the regulation of up to 40% of the cold responsive transcripts. 76 Constitutive Expression of CBF2 is also Sufficient for Most CBF Dependent Transcripts Of the 63 transcripts identified as requiring CBF for their cold-induction, 54 were induced by constitutive expression of CBF2 in warm-grown plants, matching models 9 or 10. This indicates that CBF expression is both sufficient and required for the expression of these transcripts (Figure 3.8). As would be expected for a group of genes that are regulated by CBF, the presence of the CRT element to which CBF binds, RCCGAC is overrepresented in the promoters region, with 44 of these 54 genes containing at least one CRT element in the 1K8 upstream promoter region, p-value<10"°. All but eight of these transcripts contained the core element of the CRT, CCGAC. The remaining 8 transcripts lack a known CBF binding site in the 1K8 upstream region and maybe regulated by CBF through an indirect mechanism. A subgroup of the genes whose induction was inhibited by the presence of CBF2AC was not induced by overexpression of CBF2 in the warm. These 9 transcripts matched haystack model 2, where their induction in response to low temperature was inhibited in the presence of CBF2AC; however, expression of CBF2 was not sufficient for their expression in the warm. For this subgroup perhaps there is a requirement for an additional factor, present only at low temperatures, to induce their transcription (Figure 3.9). Promoters of these transcripts are overrepresented for the presence of the CG-1 binding element (ATSR1) vCGCGb, p-value <10'3 (Yang and Poovaiah 2002). This element was shown to be important for the induction of CBF2 in response to low temperature 77 and is overrepresented in early-cold responsive transcripts (Doherty, unpublished). A B Cold > 46 Transcripts 0 15000 .C F CBF WTOh WT24h 35s::CBF CBF2AC Raul”, 2 Warm 24h 8 Transcripts C . 6000 AT1609350 GolS3 T) 5000 5 .1 4000 '5 3000 g 2000 a 1000 J} 0 WT 0h WT24h4°C 355::CBF2 CBF2AC W a r m 24h 4°C Figure 3.8 CBF Sufficient and Required Transcripts (A) Model of regulation for 54 transcripts identified as CBF sufficient and required (8) Expression level of 54 transcripts in WT warm, WT 24h4°C, 35s:CBF2 warm and 35s:CBF2AC 24h4°C (C) Representative transcript expression level for one transcript GOLSS for samples in (B) 78 aooo - Cold 7; 250° , 3 2000- C 0, ‘ ,9 1500- : 3 3 1000 s : e '1 . V : E4" soo~ ? = L“ o. V \‘ wron WT24h4°C 35s::CBF2 CBF2AC §$ VCGCGb ? , , Warm 24h4°C Regulon C 9 Transcripts AT1 (375390 ATBZIP44 1000 ~ 800 '1 BOO -1 400 4 200 -‘ mt.“ WTOh WT24h4°C 35s::CBF2 CBF2AC Warm 24h4°C Figure 3.9 CBF is Not Sufficient Yet Is Required for Induction of Nine Transcripts (A) Model of regulation for 9 transcripts identified as CBF required but not sufficient (8) Expression level of 9 transcripts in WT warm, WT 24h4°C, 35$:CBF2 warm and 35$:CBF2AC 24h4°C (C) Representative transcript expression level for one transcript, ATBZIP44 for samples in (B) ’ TBX Element is Present in Promoters of many CBF-Independent Transcripts The induction of 398 transcripts in CBF2AC plants at 24h4°C was similar to the level of induction in wild-type plants in response to low temperature (Figure 3.7 8) indicating that a functional CBF pathway is not required for their induction. The majority of these probesets, 370, matched model 4, in that they were not up-regulated in response to overexpression of CBF2 in the warm, suggesting that they are entirely independent of CBF (Figure 3.10). 79 Cold _. 370 Transcripts [51:82:12. 8 c AT4G19120 ERD3 T) T 0 71 2000 -' B c E’ 1500 « 'g '3 1000 4 (D l «g B. I l “>5 x o . UJ ‘ . wron WT24h4°C 35s::CBF2 CBF2AC WT 01) WT 24h4°C CBF2AC 24h4°C Warm 240‘ C Figure 3.10 CBF is Neither Sufficient Nor Required for Induction of 370 Transcripts (A) Model of regulation for 370 transcripts where CBF is neither sufficient nor required (8) Expression level of 370 transcripts in WT warm, WT 24h4°C, and 35$:CBF2AC 24h4°C (C) Representative transcript expression level for one transcript, ERD3 for samples in WT warm, WT 24h4°C, 35$:CBF2 warm and 35$:CBF2AC 24h4°C The 1K8 upstream promoter region of these CBF independent transcripts was enriched for the Telo-box binding element, AAACCCTAA, p- value <10'1°. The telo-box element is a cis-element known to be involved in the regulation of gene expression in root meristems, eEF 1A gene expression, and rp40 expression (Manevski et al. 2000; Tremousaygue et al. 2003). The core teIo-box sequence, ACCCTA, was present in the 1kb upstream promoter region of 144 of these transcripts. This element was previously identified as being enriched in a midnight specific module of circadian regulated transcripts (Michael et al. 2008). However, the set of transcripts with the TBX element 80 present in the 1K8 upstream region of their promoter was enriched for those not matching any circadian pattern in the 5 experiments analyzed by Michael et al., p-value <0.01. The overrepresentation of the TBX element in the promoters in these non-circadian regulated transcripts suggeSts that there may be an additional role for the TBX in regulating response to low temperature, in addition to circadian rhythms. A subset of these transcripts that were induced to near their full wild-type level in CBF2AC plants corresponded to models 8 or 12, indicating that they were up-regulated in response to CBF2 overexpression. Overexpression of CBF2 is sufficient for induction of these 28 transcripts, yet they are unaffected by the CBF2AC transgene (Figure 3.11). Promoters of 21 of these 28 transcripts contain the core CRT, p-value <10‘. The overrepresentation of the CBF binding site in the promoters of these transcripts supports their induction by CBF2 overexpression in warm-grown plants. However, no other known elements are overrepresented in this set of transcripts to explain their induction in the presence of CBF2AC. This could indicate that either the truncated version of CBF2 is enough for induction of these transcripts or that there are independent pathways that are sufficient for their induction. 81 a,“ 310000 3 8000~ IE CRT ~ 1 3 4000,- “3’ 2000- Ly l . car on d} 0- “9”“ , WTOh WT24h4°C 35s::CBF2 camc 28 Transcripts wan“ 24h4°C AT4G12470 Protease Inhibitor 8000 l 7000 1 6000 r 5000 ~ 4000 - 3000 1 2000 ~ 1000 1 ression Level Exp WTOh WT24h4°C 358:2CBF2 CBF2AC Warm 24h4°C Figure 3.11 CBF is Sufficient But Not Required for Induction of 28 Transcripts (A) Model of regulation for 28 transcripts identified as CBF sufficient but not required (8) Expression level of 28 transcripts in WT warm, WT 24h4°C, 35s:CBF2, and 35s:CBF2AC 24h4°C . (C) Representative transcript expression level for one transcript, AT4G12470 for samples in (B) CBF-Integrated Transcripts are Induced in 35s::CBF2AC Plants, but not to Their WT Levels A large number of transcripts, 181, were still induced in response to low temperature in the CBF2AC expressing plants; however, their expression level was reduced when compared to WT plants. This suggests that while CBF is not required for the low-temperature induction of these transcripts, CBF is required for them to reach their full level of induction after 24h of cold treatment (Figure 3.12). Constitutive expression of CBF2 was sufficient for induction of 64 of these transcripts, matching haystack models 7 and 11. As would be 82 expected for transcripts that can be induced by overexpression of CBF, the CRT element was overrepresented in the 1K8 upstream region of these transcripts, p-value 10"° with 54 of these transcripts containing the core CRT element. Additionally, the promoters of these transcripts were also overrepresented, p-value 10”, for the presence of an ABRE-like element BACGTGKM, with 31 of the 64 promoters containing this element (Shinozaki and Yamaguchi-Shinozaki 2000). The pattern of regulation of these transcripts and overrepresentation of both of these elements suggest a model where CBF is sufficient for induction of these transcripts through the CRT element, however, in the CBF2AC plants, these transcripts can be activated via another mechanism, although not to the same level as they reach in WT plants. The overrepresentation of both the CRT element and the ABRE-like element in the promoters of these transcripts indicates that perhaps ABA might play a role in the induction of these transcripts in the CBF2AC plants. In support of this model, GO annotation of these transcripts shows that they are enriched for genes which respond to ABA, p-value 10'7. Precedence for such a cooperative effect between CBF and an ABRE-like binding protein has been demonstrated with the RDZQa promoter where CRT elements and ABRE elements are interdependent for expression in response to ABA treatment (Narusaka et al. 2003). 83 Cold [:j‘cer CRT -—-—~ 64 Transcripts - OF OR LYjABRE car AND v l'ke cgulo B C 14000 “I .. ._—. -.- — -— ....__ ——-—-——~ ATZG42540 COR153 §12000< ‘ 8 310000 3 mac 1 g 0000 . l 3 6000 .4 To r: 5000 r g 6°°°‘ ] «3 4000 - 2' 40001 3 3000 - a 2000 - w 20°01 5} 1000 - 0‘ o . wron wr24n4'c 35mm cerzac wron WT24h4°C 35s::CBF2 CBF2AC Warm 24140 Warm 24h4°C Figure 3.12 CBF Is Sufficient and Partially Required For the Induction of 64 CBF Integrated Transcripts (A) Model of regulation for 64 transcripts identified as CBF-Integrated, sufficient and partially required. (8) Expression level of 64 CBF-Integrated transcripts in WT warm, WT 24h4°C, 35s:CBF2, and 35$:CBF2AC 24h4°C (C) Representative transcript expression level for one transcript, COR15A for samples in (B) The majority of these transcripts, 117, matched haystack model 3 and were not induced in response to constitutive expression of CBF2. For these transcripts, while CBF is required for their full, wild-type level of induction, CBF2 alone is not sufficient for induction of these transcripts in the warm (Figure 3.13). The CRT element was overrepresented in the promoters of these transcripts, p-value 104°, perhaps contributing to the quantitative effect seen in the CBF2AC plants. The ABRE-like element, was also overrepresented (p- value, 10”), perhaps indicating a role for ABA in the induction of these transcripts in response to low temperature in the absence of CBF. 84 Cold i ~— 117 Transcripts y J ABRE” BFAND ulon like "‘9 B C 8000.. . , . ,_ s -_-, .- _ AT1G1717O ATGSTU24 E 7000‘ 3000 36°°°‘ . '6 ' c 5000 ‘ 3 2500 ~ ~§ 4ooo~ El 2000 ‘1 9 3000+ 3, 1500 4 32000‘ 3 1000 . 100% s- 500 ‘ o~ o _ WTOh WW‘C 35sz 23% WW?! wr24n4°c 35s::CBF2 CBF2AC Warm 24M'C Figure 3.13 CBF Is Not Sufficient Yet ls Partially Required For the Induction of 117 CBF Integrated Transcripts (A) Model of regulation for 117 transcripts identified as CBF-Integrated, not sufficient and partially required. (B) Expression level of 117 CBF- Integrated transcripts in WT warm, WT 24h4°C, 35s: CBF2, and 35s: CBF2AC 24h4°C (C) Representative transcript expression level for one transcript, ATGSTU24 for samples in (B) No Down-Regulated Transcripts Are Completely Dependent on CBF None of the 444 transcripts down-regulated in WT plants in response to low temperature completely lost their repression in response to low temperature in 35s::CBF2AC plants. However 29 of these transcripts were not repressed to the same extent in 35s::CBF2AC plants as they were in WT plants (Figure 3.14 A). 85 > B — 1400 > 1200 —l 1000 800 600 400 200 0 WTOh Wl’ 24h4°C C BFZAC ....-s- E. W.,, . "H,_ , a, 24h4°C wron WT24h4“C CBF2AC 24n4°c ee Expression Level Ex 0 res 5 io n O 444 Down-Regulated Transcripts 0% 7% Cl Required I Independent [:3 Integrated 93% Figure 3.14 Cold-Repressed Transcripts (A) Expression level of 415 transcripts that are repressed in WT plants and are also reduced to a level equal to that of WT in CBF2AC plants at 24h4°C (B) Expression level of 29 transcripts that are still repressed in CBF2 AC plants, but are not reduced to their full WT level. (C) Pie-chart showing the distribution of each category of transcripts based on their requirement for CBF, independent (A) and integrated (B). The repression level of the remaining 415 transcripts was unaffected by the presence of the 355::CBF2AC construct (Figure 3.14 B). This suggests that either CBF is not required for the down-regulation of transcripts or that the DNA binding domain, still intact in the CBF2AC constructs is sufficient for the repressive function of CBF. Of the 415 transcripts whose repression in response to low temperature was not affected by the presence of the CBF2AC construct, 194 were repressed in warm grown plants constitutively expressing CBF2, indicating that 86 while wild-type CBF is sufficient for the down-regulation of these transcripts, a full-length CBF protein is not required for their repression (Figure 3.15). The CBF binding sequence, the CRT, was not overrepresented in the promoters of these transcripts; only 34 of the 194 contained this CRT in the 1KB upstream region, p-value 0.66. Therefore, either direct binding of CBF through the CRT element is not a likely mechanism of repression for these transcripts or the regulatory regions for these genes is not in the upstream 1KB region. A possible regulatory mechanism is through the l-Box, which is present in the promoters of 108 of these 194 transcripts, p-value 10'”. In tomato a MYB transcription factor was identified as a potential binding factor to the I-Box (Rose et al. 1999).The l-Box motif, GATAAG, is also enriched in the promoters of light-regulated genes (Giuliano et al. 1988). Mutation of this sequence in the context of the ribulose-1,5-bisphosphate carboxylase small subunit promoter sequence causes reduced expression of a reporter gene (Donald and Cashmore 1990). In agreement with this group of I-Box enriched transcripts being involved in light regulation, these transcripts were also enriched for the GO cellular component ontology term, chloroplast (10”). In addition to the l- Box, a second cis-element, a MYC recognition sequence, CACATG present in the promoter of RD22, was also overrepresented in these promoters, with 92 of these 194 containing at least one copy of this element, p-value 10'7. Cooperation between MYC and MYB binding elements has previously been demonstrated for the RD29a stress responsive promoter (Abe et al. 1997). Perhaps the overrepresentation of the MYC element in this set of genes 87 indicates cooperative regulation of the MYC elementand the potential MYB binding site in the l-Box. 5”; ..... ,* I-BOX . CBF OR 194 Transcripts l """" “4 MYC ‘ LI Regulon B C 8000 AT1G52190 ECS1 no Expression Level wron WT24h4'C 35s::CBF2 CBF2AC WTOh W724?! 35s:CBF2 CBF2AC 24h4'C w: 24h Figure 3.15 CBF ls Sufficient But Is Not Required For the Repression of 194 Transcripts (A) Model of regulation for 194 transcripts identified as CBF sufficient but not required. (B) Expression level of these 194 transcripts in WT warm, WT 24h4°C, 3552CBF2, and 35s:CBF2AC 24h4°C (C) Representative transcript expression level for one transcript, ECS1 for samples in (B) Both the l-Box and the same MYC recognition sequence were overrepresented in the CBF independent down-regulated transcripts, p-value 104° and 10'7 respectively. These 221 transcripts were reduced in CBF2AC plants to the same level as WT plants after 24h4°C, and were not reduced in 355::CBF2 plants in the warm indicating that they are entirely CBF independent (Figure 3.16). As would be expected for CBF independent transcripts, the CBF 88 recognition sequence, CRT, is not overrepresented in the promoters of these transcripts A Cold rim-l I..:BQX . Z 221TranscrIpts “‘TT‘T‘J M I Regulon YC B C m, AT1G49480 RTV1 E m. — 3 sooo< 2 50° ‘ c: “I 3 400 'l '5 3°°°‘ 8 3 2000« 'a ‘5‘: 1000« 3 Lu 0‘ g o .. UJ WW“ W724“ C 355~C3F2 CBFZf‘C wron WT24h 4°C 353::CBF2 CBF2AC Warm 24M C Warm 24h4°C Figure 3.16 CBF ls Neither Sufficient Nor Required For the Repression of 221 Transcripts (A) Model of regulation for 221 transcripts identified as CBF Independent. (B) Expression level of these 221 transcripts in WT warm, WT 24h4°C, 35s:CBF2, and 35s:CBF2AC 24h4°C (C) Representative transcript expression level for one transcript, RTV1 for samples in (B) The 29 transcripts that are quantitatively affected in their level of down- regulation in CBF2AC expressing plants at low temperature suggest a mechanism of regulation where CBF is not required for their repression; yet, achieving complete reduction to the level seen in WI' plants is dependent on CBF. Most of these transcripts, 23, that are no longer repressed to their WT level, are also not repressed in 35s::CBF2 plants in the warm, indicating that CBF is not sufficient for their repression, but is required for achieving the full, WT level of repression (Figure 3.17). No known motifs showed any 89 overrepresentation in the promoters of these transcripts. Six transcripts that were qualitatively affected were repressed in 35s::CBF2 plants in the warm, demonstrating that CBF is sufficient for their repression, and is required for their being reduced to their WT level (Figure 3.18). There was no overrepresentation for any known cis-elements in this group, however, this could be due to the small sample size, for example, the I-Box, which is present in 5 of the 6 promoters, is not statistically overrepresented in this set of transcripts, p-value 0.01. A Cold "r“? 23 Transcripts D I ; BFAND ..__, I Regulon B c . AT1G68520 COL6 3 5 _l 800 g 600 338 I g. o W WT24h4'C 3552:0sz CBF2AC ul WTOh WT24h 4°C 358::CBF2 CBF2AC Warm 24M‘C Warm 24h4'C Figure 3.17 CBF Is Not Sufficient But Is Partially Required for the Repression of 23 CBF-Integrated Transcripts (A) Model of regulation for 23 CBF-Integrated Transcripts. (B) Expression level of these 23 transcripts in WT warm, WT 24h4°C, 35$:CBF2, and 35$:CBF2AC 24h4°C (C) Representative transcript expression level for one transcript, COL6 for samples in (B) 90 Effects on Transcript Levels Are Reflected in Metabolite Profiles Transcript levels are not always reflective of the protein levels, enzyme activity, or phenotypic activity of the organism. To determine if the transcript changes seen in the 35s::CBF2AC plants was reflective of downstream effects we analyzed changes in metabolites in response to low temperature in both WT and 35s::CBF2AC plants. In previous work Cook et al. showed that the metabolite profile of plants overexpressing CBF2 at warm temperature resembled that of cold-treated plants(Cook et al. 2004). With the changes seen in the transcript of CBF2AC plants we would expect that there would be an effect on metabolite levels, particularly of raffinose. GOLS3 is one of the most highly up-regulated transcripts in CBF2 overexpression and its level of induction is drastically reduced in 35s::CBF2AC plants (Figure 3.3, 3.8 C). GOLS3 is one member of the six gene galactinol synthase family and is a component of the raffinose metabolism pathway. Therefore, if this change in transcript level is reflective of a change in the metabolism of the plant, we would expect there to be a dramatic reduction in raffinose levels. When metabolite levels of WT warm and cold plants were compared to cold-treated 35s::CBF2AC plants, there was in fact a dramatic reduction of raffinose levels in the cold-treated 35s::CBF2AC plants compared to WT indicating that the changes in the transcript level in this case are reflective of changes in the metabolic profile of the plant (Figure 3.18). 91 Ws warm 1 ‘ Rafflnose , 1 AAA“ ‘ l _.-_-——' L- J; L A A A— v - - v r 1 - . v v—v v fi v - j Ws 7d cold l 1 ; M ‘ L AA+ 1;. 424—. CBF2 A Cp93 7d cold -A.AIJAAL 4. A‘ _A. Relative Abundace Relative Abundace Relative Abundace ALLL A L _ A A L t _. Figure 3.18 GC-MS Metabolite Profiles of Warm and Cold Treated Plants. Arrow indicates raffinose peak. X-axis indicates retention time in two minute increments beginning at 20.00 min and continuing through 48.00 min. (A) WT warm-grown plants, (B) WT plants after treatment at 4°C for 7d (C) CBF2AC plants after 7d4°C treatment. CBF2AC Plants Cold Acclimate, but at a Drastically Reduced Level Compared to WT Plants The results presented here indicate that CBF2AC construct is effectively acting as a dominant negative gene blocking the function of the endogenous CBFs. The presence of the CBF2AC construct drastically reshapes the cold- regulated transcriptome at both a quantitative and qualitative level. To determine if these plants with an inhibited CBF response are still able to cold acclimate, three lines of CBF2AC plants were tested for their ability to cold 92 acclimate by whole plant freeze tests and electrolyte leakage assays. The CBF2AC plants were able to cold acclimate as analyzed by a whole plant freeze test (Figure 3.19). Acclimation: None Acclimation: 7Days at 4°C CBF2AC#89V~ WT CBF2AC#89 WT CBF2AC#93 358::CBF2 CBF2AC#93 CBF2AC#96 Figure 3.19 CBF2AC Survives Whole Plant Freeze Tests After Acclimation Plants were treated for 1h at -10°C either without acclimation or after acclimation of 7d at 4°C. However, when measured by electrolyte leakage assay, CBF2AC were not able to acclimate to the same degree as M plants (Figure 3.20). EL50 values for WT plants were -10.5°C alter cold acclimation while EL50 of CBF2AC plants only reaches -8.5°C after acclimation. Interestingly, CBF2AC plants also had a lower basal level of freezing tolerance than WT plants. DISCUSSION Previous work suggested that CBF was not sufficient for regulation of all cold-responsive transcn'pts, and that CBF independent pathways for cold acclimation may exist (Vogel et al. 2005). However, it was not known if CBF 93 was required for the induction of these transcripts, and to what extent Arabidopsis can cold-acclimate without a functional CBF pathway. 120 § 1:: —EI -ws 7d Acc : 60 WS Non-Acc 3 40 —A -CBF2AC 7d Acc 3line Avg 3 20 +CBF2AC Non-Acc 3Iine Avg Temp (C) Figure 3.20 Electrolyte Leakage Assays of WT and CBF2AC Plants Before and After Acclimation. Percent electrolyte leakage of non-acclimated WT (solid squares,solid lines), non-acclimated CBF2AC (solid triangles,solid lines), acclimated WT (open squares, dashed lines), and acclimated CBF2AC (open triangles, dashed lines). Cold acclimation was for 7d 4°. Data for CBF2AC plants is an average of three independent transgenic lines. This work has further refined the CBF regulon, adding two new categories of CBF-regulated transcripts: those where CBF is required but not sufficient for their regulation and those where CBF is not required for their response to low temperature, but is required for their reaching their wild-type level of induction or repression. Comparison of warm-grown and 24h4°C WT plants with 35s::CBF2AC warm-grown and 24h4°C and with warm-grown 35s::CBF2 plants revealed several complicated models of regulation for cold- induced transcripts. The two groups of transcripts, those where CBF is sufficient, but not required and those where CBF2AC results in a quantitative, but not absolute loss of induction comprise one third of the cold-induced transcripts. The evidence for combinatorial regulation seen here for these modules is in agreement with predictions of large combinatorial regulation in 94 response to low temperature based on motif analysis of cold-regulated genes (Chawade et al. 2007). The further refinement of the CBF-independent transcripts through the removal from this group of those transcripts where CBF is not sufficient, but is required, should provide a more cohesive group for regulatory element analysis. In fact, analysis of the promoters of these more narrowly defined CBF- independent transcripts identified the telo-box (TBX) as a highly overrepresented element in this group of transcripts. This element has been previously identified as a “midnight element” overrepresented in transcripts peaking at midnight in the circadian cycle (Michael et al. 2008). One possible interpretation of the strong presence of the TBX could be that the effect of cold on the circadian clock has caused this group of transcripts to be shifted from their normal expression pattern (Bieniawska et al. 2008). However, preliminary analysis of this CBF-independent group showed that these transcripts were not over-represented for circadian peak at midnight (or any time point) and in fact the transcripts containing the TBX element were overrepresented for those that did not match any tested model of circadian induction in five different circadian analysis conditions (Michael et al. 2008). One possible interpretation of this result is that the TBX represents a cold-responsive element that has been adapted for cold-entrainment of the circadian clock in a subset of transcripts. This could be one mechanism for the described effect on many clock output transcripts by low temperature (Bieniawska et al. 2008). 95 Down—regulation of transcripts in response to abiotic stress in general, and in particular in response to cold- treatment, has not been examined as thoroughly as regulation of induced transcripts. Like induced transcripts, there is evidence for cooperativity in the repression of these transcripts (Chawade et al. 2007). The quantitative effect seen in CBF2AC plants supports this idea of cooperativity in the down-regulation of cold-responsive genes. While no repressed transcripts were entirely dependent on CBF for their repression, a large number did not reach their wild-type level of repression in CBF2AC plants, suggesting that CBF and another factor or factors may contribute cooperatively to their repression. Additionally, by refining the cold-repressed transcripts into those that are repressed in both WS and Col and those that are independent of CBF in their repression, a potentially important cis-element for cold-responsive repression, the l-Box, was identified. The I-Box is highly overrepresented in transcripts that are down-regulated in response to low temperature and are CBF not affected by the presence of the CBF2AC construct. The large number of transcripts that were still reduced to their wild-type levels in CBF2AC plants could indicate that either CBF is not involved in the repression of these transcripts in response to low temperature or that the CBF DNA binding domain is sufficient to initiate repression in these transcripts. In support of the latter idea, the CBF DNA binding domain was shown to interact with the co-activator protein ADA2a (Mao et al. 2006). However, the CRT element was not present in the promoters of many of these genes indicating 96 that if the CBF DNA binding domain is sufficient for this repression, it is likely recruited to these genes through a novel mechanism. lmportantly, this work identified the importance of the CBF pathway for the ability of Arabidopsis to cold-acclimate. Without a fully-functional CBF pathway, plants containing the CBF2AC transcript, while still able to acclimate, are not able to reach the freezing tolerance capabilities of their WT counterparts. Interestingly, even the basal freezing tolerance was affected in CBF2AC plants. The expression of CBFs in response to circadian rhythms may play some role in the basal freezing tolerance (Harmer et al. 2000). Further combination of CBF2AC with other factors known or identified as important in freezing tolerance will narrow down the set of transcripts that are responsible for contributing to freezing tolerance and distinguish those that are actively contributing to increase freezing tolerance and those that are responding to the change in temperature. The CBF-independent transcripts identified here, which may contribute to this remaining ability to cold acclimate, can now be used as a tool to work upstream and identify potential cold sensors and regulators, providing additional targets for improving cold tolerance in plants. MATERIALS AND METHODS EMS Mutagenesis EMS Mutagenesis of 353::CBF2 overexpressing plants, E2, was performed essentially as described in Kim et al. (2005). Mutants of interest were selected 97 from M2 populations based on size; gene expression analysis was performed on M3 plants. Plant Material and Growth Conditions All plants were stratified for 3-5 days in the dark at 4°C then transferred to constant illumination at 24°C for 10 days prior to treatment. Cold treatment consisted of moving the plates to a 4°C chamber with constant light at a reduced level (approximately 35 u mol m'zsec'1). Plants were grown on Gamborg's 85 nutrients (Caisson Laboratories, www.caissonlabs.com) and 0.8% phytagar (Caisson Laboratories, www.caissonlabs.com) without sucrose. All experiments were performed on tissue from whole seedlings. 35s::CBF2 plants were as described in Gilmour et al. (2004). The plasmid containing 35s::CBF2AC in the PGA643 vector was transformed into Arabidopsis ecotype WS using standard procedures (Gilmour et. al. 2004). Three transforrnant lines (89, 93, and x96) were selected based on their level of CBF2AC expression in warm grown conditions and were taken to homozygosity. RNA isolation and Analysis Total RNA was extracted from plant material with the use of RNeasy Plant Mini kits (Qiagen, Valencia, CA) with modifications as described (Zarka et al. 2003). 98 Northern transfers were prepared and hybridized as described (Hajela et al. 1990) probes prepared, and membranes washed with high-stringency conditions (Stockinger et al. 1997). For RT-PCR cDNA synthesis was perforrned using Promega Reverse Transcription system according to the manufacturer’s directions using random primers with the following modifications. Total reaction volumes were doubled and starting RNA was adjusted. Starting RNA was 0.01ug for warm and 2h0°C samples for analysis of CBF1, 2, 3, and Zat12 mRNA and 0.1ug for all 24h0°C samples. cDNA was diluted five-fold with water and 3uL was used as a template for quantitative real-time PCR (qRT-PCR). qRT-PCR using SYBR Green was performed using the Applied Biosystems 7500 real-time PCR system in Standard Mode with SYBRGreen PCR Core Reagents Mix (Applied Biosystems). qRT-PCR was performed according to manufacturer’s protocols with the following modification. Reactions were performed in a 30uL volume. For CBF2 mRNA analysis, the annealing/extension temperature was 62°C. Serial dilutions of 2h0°C WT samples were performed to determine the efficiencies of the primers for CBF2, transgeneCBFZ, and Actin3. Serial dilutions of 24h0°C samples were performed to determine the efficiencies of COR15, GOLS3, and Actin3 primers. Reactions were performed in triplicate and products checked by melting curve analysis. The abundance of transcripts was analyzed with the relative standard curve method normalizing to the reference transcript, Actin3 (AT3G53750). The primers used for amplification were CBF2 (5’-GGA TGCTCATGGTCTTGACAT-S’ and 5’- 99 TCTTCATCCATATAAAACGCATCTTG-3’), transgeneCBF2 (5'- GCTCGTTAACGGTACCATCG-3’ and 5’-GTAATCACCGCCTGAGGAAA—3’), ACTIN3 (5’-GGTCGTACTACTGGTATTGTGCT-IB’ and 5’- TGACAATTTCACGCTCAGCT-3’), GOL83 (5’-GGAGTGGTTGGTCTGGCTAA- 3’ and 5’- TTGGTTATCCGGTGGGTAAA-S’), COR15 (5’- ATGGCTTCTTCT'ITCCACAGC-3’ and 5’-GAAGCTTTCT'I'TGTGGCCTC-3’), Affymetrix GeneChip Hybridization and Data Collection Biotinylated target RNA was prepared from 15ug of total RNA equally pooled from three plates. Labeling was performed using Affymetrix IVT labeling kit according to manufacturer’s directions (Affymextrix, Santa Clara, CA, USA). The samples were hybridized to the Affymetrix Arabidopsis ATH1 GeneChip. Two biological replicates were analyzed for CBF2AC warm, WS 24h, Col 24h, and four biological replicates were analyzed for CBF2AC 24h. Affymetrix GeneChip Data Analysis Publically available microarray data available from ATGE and Vogel et al was used in addition to new chips (Vogel, Zarka et al. 2005; Kilian, Whitehead et al. 2007). Each experiment was maintained as an individual unit and normalized using RMA in the AFFY package for Bioconductor (Irizarry et al. 2003; Vincent et al. 2004). RankProduct analysis was performed with the RankProd package (Hong, Breitling et al. 2006). Cold-regulated transcripts were selected as those with a fold change greater than 2 and significance cutoff of p value <0.01 (FDR corrected) in any of the three selected time points (1h, 24h, or 7d). Differentially expressed transcripts between WS and Col at 24h4° 100 and 24h0° were determined using limma with a 2 fold cutoff and FDR corrected p-value of 0.05 (Smyth 2004). These transcripts were removed from the analysis of cold-regulated genes. CBF2 was removed from all ensuing analysis. Overrepresentation of Motif Analysis Prior to promoter analysis probe sets known to hybridize to multiple genes were removed. Probesets that do hybridize multiple genes remain in the total numbers of each category and are listed in tables as “multiple”. Analysis for overrepresentation of known elements was performed using Athena (O'Connor et al. 2005). Frequency of occurrence for modifications of known elements was performed using Promomer and hypergeometric testing performed using R (Toufighi et al. 2005; Team 2008). Overrepresentation of GO Terms Each subgroup of genes was analyzed for enrichment of GO terms using GOStats and Athena (O'Connor, Dyreson et al. 2005; Falcon and Gentleman 2007). Extraction of Polar Metabolites 100mg (warm) or 25mg (cold treated) of powdered tissue was extracted in 700uL cold methanol with 10uM ribitol. 130 uL of methanol/water phase was dried under vacuum. Derivatization and Analysis 50uL of methoxyamine hydrochloride (20 mg/mL) was added to extraction and incubated for 120 min at 37°C while shaking. 80pL MSTFA was 101 added and incubated 30 min at 37°C while shaking. Incubate 120 min at 22°C. 100uL was transferred to GC/MS vial. 50pL of derivitized metabolites were injected on a 30m HP5 ramping from 80°C to 325°C at 25°C / min, then 3 min at 325°C. The injection was split for cold samples. Peaks were identified by retention time referenced to a standard mix. Quantification was analyzed based on the ribitol internal standard. Whole Plant Freeze Test Whole plant freeze tests were performed essentially as previously described (Vogel et. al. 2005). 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"Bioconductor: Open software development for computational biology and bioinformatics." Genome Biology 5: R80. Vogel, J. T., D. G. Zarka, et al. (2005). "Roles of the CBF 2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis." lh_e_ Plant Journal 41(2): 195-211. Yang, T. and B. W. Poovaiah (2002). "A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants." J Biol Chem 277: 45049 - 45058. 105 Zarka, D. G., J. T. Vogel, et al. (2003). "Cold Induction of Arabidopsis CBF Genes Involves Multiple ICE (Inducer of CBF Expression) Promoter Elements and a Cold-Regulatory Circuit That Is Desensitized by Low Temperature." Plant Physiol. 133(2): 910-918. 106 CHAPTER FOUR Integration of Cold Response with Other Environmental Responses Introduction The overall goal of this project is to build a descriptive network of the regulation of the response to low temperature and cold acclimation in Arabidopsis. The creation of such a network would allow for identification of key regulatory steps in the response to low temperature. These key regulatory steps are potential hubs of cold-response and would then be excellent targets for further study to improve tolerance of plants to low temperatures. This work has provided additional information in two aspects of this network. First, CAMTA3 has been identified as an upstream regulator of the CBF cold responsive pathway. Secondly, transcriptome analysis of a dominant negative version of CBF2, CBF2AC, provided a refined classification of cold-regulated transcripts and demonstrated the importance of CBF on both basal and acclimated freezing tolerance levels. The identification of CAMTA3 as a positive regulator of CBF2 induction in response to low temperature provides insight into a possible link between calcium signaling and the cold-response. Additional experiments have shown that CAMTAs are also involved in the regulation of CBF2 in response to mechanical stimulus and cycloheximide treatment. These results indicate that perhaps CAMTAs are integration points for multiple environmental signals. As potential hubs for integration points of multiple environmental inputs, further 107 understanding of the role of CAMTAs in stress response may provide mechanisms for improving stress tolerance in plants. Networks describing responses in biological systems often reveal the extensive overlap of the many pathways of an organism with each other and the interaction of many pathways previously considered independent. The cold regulation pathway is no exception to this interconnectedness. The role of development and environmental conditions on the response of Arabidopsis to low temperature is only beginning to be analyzed, but there is a clear role for involvement of the circadian clock and light quality signaling (Fowler et al. 2005; Ramos et al. 2005; Franklin and Whitelam 2007; Bieniawska et al. 2008; Michael et al. 2008). In the course of this analysis additional evidence for the complex integration of environmental signals and development with the cold- response were identified. It was discovered that Arabidopsis has improved cold-acclimation ability when grown under short days, implicating a possible link between day-length signals and the cold response. Additionally, developmental phenotypes observed in the CBF2AC plants indicate that CBF may have roles in normal growth and development in addition to its role in cold acclimation. ML! CAMTA3 is a Positive Regulator of CBF Expression in Response to Mechanical Agitation and Cycloheximide Treatment CBF2 is induced in response to low-temperature, mechanical agitation, and cycloheximide treatment. One common link between these three stimuli is a rapid influx of cytosolic calcium (Knight et al. 1992; Knight et al. 1996). 108 CAMTA3 was identified as a positive regulator of low-temperature induction of CBF2. The binding of calmodulin by CAMTA proteins suggests a possible link to calcium signaling. It is therefore of interest to determine if CAMTAs are involved in the regulation of CBF2 in response 'to these other stimuli. In support of this possibility, the CM2/CM1-Boszus reporter, which Is regulated by CAMTA3 in response to low temperature, was shown by Van Buskirk to be induced in response to cycloheximide and mechanical treatment (Figure 4.1A, Unpublished Data). To test the potential role for the CAMTAs in the regulation of CBF2 in response to other environmental inputs, mRNA accumulation was measured in camta mutants after treatment with cycloheximide. As was seen in response to low temperature, loss of CAMTA3 resulted in lower induction of CBF2 in response to cycloheximide treatment (Figure 4.1 B). There also seems to be a role for CAMTA2 in the regulation of CBF2 to cycloheximide as CBF2 mRNA does not accumulate as high in the camta2 mutant as in WT. In response to mechanical agitation, there is little effect on CBF2 induction in the camta3 mutant (Figure 4.1C). Indicating that induction of CBF2 in response to mechanical agitation may occur through a distinct pathway than that of cold- induction. However, loss of CAMTA3, did have an effect on CBF1 induction in respon to mechanical agitation (Figure 4.1 D). This result suggests that CAMTAs may contribute to specificity in the response to various external signals. 109 1 «mm W c M r ‘ GUS -I cm H 75551 mi: e ...-m Iii-1m- — B Control 15 min Mechanical C 8‘; 8 E ‘2; 8 ‘3, '8 B Cycloheximide ZAT12 e e e a o e e CBF1 o .I ' i” " ' _, CBF1 ERm ~ ' W .. W .. W 18 rRNA 1BsrRNAt__. M] s CBF2 = c a o 6 Ii 0 I3 CBF2 TUB8 w W 18srRNA M Figure 4.1 Role for CAMTAs in Induction of CBFs by Various Stimuli (A)CM2/CM1-Box is sufficient for induction in response to mechanical agitation (B)Northern blot analysis of plants treated for 15 min with mechanical agitation then harvested after 10 min. Control is untreated plants. mRNA levels of ZAT12, CBF1, and ERD7 were analyzed in Columbia (Col), camta1(c1), camtaZ(02), camta3(c3), camta4(c4), camta5(c5), camta6(06) plants and 185 rRNA was used as a loading control. CBF2 mRNA levels were analyzed with TUB8 as a loading control. (C)Northern blots of plants treated with 10 pg ml'1 cycloheximide Columbia (Col), camta1(c1), camtaZ(c2), camta3(c3), camta6(c6) analyzed for CBF2 mRNA levels 18s rRNA was used as a loading control. CAMTAs as Potential Hubs in Response to Stress in Arabidopsis The ability for a similar calcium signature to precede a multitude of environmental responses indicates the importance of downstream signals in the calcium cascade for interpretation of the calcium signal and initiation of an appropriate response. The CAMTA proteins could be one potential method of interpreting this signal and deriving the specificity of the response. For example, loss of CAMTA3 showed a loss of GUS staining in the shoots of Arabidopsis expressing the CM2/CM1zGus reporter, but not in the roots, indicating a 110 possible role for CAMTA 3 in providing tissue specificity in response to low temperature signals (Figure 4.2) . The observation that CAMTAs are involved in the regulation of different stimuli to induce CBF expression indicates that CAMTA proteins are a potential hub for interaction of multiple signals and initiation of the desired response. If CAMT As are a point of regulation for the calcium response they will be interesting targets for improving stress tolerance in plants. 1 ' . WT x CM2ICM1-BoszUS camta3 x cm1-Boxzcus . ' Figure 4.2 Histochemical staining of WT x CM2ICM1-BoszUS and camta3 x CM2/CM1-BoszUS seedlings Arabidopsis Has an Increased Capacity for Cold Acclimation When Grown in Short-Days There have been multiple environmental inputs demonstrated to affect the cold-acclimation pathway in Arabidopsis; induction of CBFs and other early cold-responsive genes is gated by the circadian clock, light-quality affects induction of downstream targets of CBFs, and pre-treatment with ABA or 111 drought increases freezing tolerance (Mantyla et al. 1995; Fowler et al. 2005; Franklin and Whitelam 2007). Results from this study indicated that there is a potential role for the integration of photoperiod signals into the cold acclimation ability in Arabidopsis. Electrolyte leakage assays of plants grown in short-day photoperiods (8h light, 16h dark) showed a dramatic increase in the EL50 values of cold acclimated plants over plants grown in constant light (Figure 4.3, 4.4). The increased freezing tolerance after acclimation was statistically significant (Student's t-test, p-value <0.01). Interestingly, there was not a dramatic difference seen in basal levels of freezing tolerance. This suggests that the change in freezing tolerance may be due to an increase in the capacity of a plant to cold acclimate rather than a difference in the composition of the plant itself when grown under sh0It days. 112 A Short Day 120 , . . ..--.--__..___. . .— _, ___,__________.._----..____-.-.~- +WS 7d Aoc —E] -WS Non-Ace 100 -——— --~¢-e- ~r~ —— ’m— "E‘ ‘ .9 so -’ r? gww 4- I 3 4o~- :2 20 o -2oi — - Temp (C) B Continuous Light 120 ~- -A -WTNonacc +WI’ 7d acc 100 - Id: 80 --.___ j’§_fiA—‘§A 1+ I 60 T v .\° 40 - 20 ’ 0 I f I I I 7 T 0 -2 -4 -6 -8 -10 -12 -14 Temp (C) F igure4.3 Electrolyte Leakage Assay of Plants Grown in Short Day and Continuous Light (A) Electrolyte leakage assay of non-acclimated WS (open squares, dashed lines) and WS acclimated for 7d4°C (closed squares, solid line) of plants grown in 8h Iight/ 16h dark (B) Electrolyte leakage assay of non-acclimated WS (open triangles, dashed lines) and WS acclimated for 7d4°C of plants grown in continuous light 113 Light Regimen Non-Acclimated EL5°(°C) Acclimated EL5°(°C) Change in EL50(°C) Constant -3 -7 4 Constant —4.5 -6.5 2 Constant -4 -5.5 1.5 8h -6 -10 4 8h -5.5 -10.5 5 8h -4 -9.5 5.5 B 12 -10 § 3 u] E s .E 3 4 -2 Constant Liqht Short Days Figure 4.4 EL50 Values for Short Day and Constant Light Grown Plants (A) Table of ELsoValues for plants grown in constant light and short days. (B) Graph of acclimated EL50 values, (p-value <0.01) The mechanisms for this increase in freezing tolerance are not clear. One possibility is that when grown in continuous light, plants make the switch from vegetative to reproductive state very early and that switch may be delayed I in short day grown plants. In rye and wheat, after switching from vegetative to reproductive growth, there is a reduction in the ability of these plants to cold acclimate (Fowler et al. 1996). Another possibility Is that the plant uses day- length clues to gate the response to low temperature. Gating of the response to low temperature to day-length would ensure that a full response to low temperature in preparation for the potential threat of freezing occurs only when days get shorter. 114 Potential Role for CBFs in Development Many developmental changes occur in response to growth at low temperature. Exposure to short periods of low temperature causes a delay in flowering, while extended periods at low temperature are responsible for vernalization, which induces flowering (Sung and Amasino 2005; Franklin and Whitelam 2007). Inversion of thennocycles, warm nights and cold days, results in reduced growth (Thingnaes et al. 2003). Therefore, when constructing a network of cold-response signals, the integration of developmental and other environmental cues must be considered. Interestingly, the loss of CBF results in a decrease in basal freezing tolerance of non-acclimated plants. This decrease in basal freezing tolerance suggests a potential role for CBF in normal growth and development in Arabidopsis. This role may simply be in maintaining a prepared state for cold acclimation. However, functional CBFs are present in many species of plants, including those which are chilling and freezing sensitive(Zhang et al. 2004; Ballou et al. 2007). The maintenance of a functional CBF in a diverse array of plant species, including those that are not chilling tolerant suggests that there is some pressure on preservation of a functional CBF, independent of its function in cold acclimation. One would expect that if there were a role in development for CBF, CBF overexpressing plants and CBF2AC plants may show some developmental phenotypes. CBF overexpressing plants have a dramatic dwarf, late-flowering phenotype that appears to correlate with CBF function (Canella and Gilmour, 115 unpublished data)(GiImour et al. 2004). Comparison of plants under normal growth conditions revealed no obvious phenotype differences in adult plants between WT and CBF2AC. However, CBF2AC plants have a consistent developmental phenotype; approximately 10% of offspring have single, fused, or tri-cotyledons (Figure 4.5, 4.6). This result is consistent from generation to generation, regardless of the parent plant’s cotyledon phenotype. NAM (non- apical meristem) transcription factors have a similar cotyledon phenotype to the one seen in CBF2AC plants. Comparison of the expression level of the NAM genes in Arabidopsis revealed that two NAM genes, AT5922290 and AT5g39610 were two-fold or more higher in expression in CBF2AC than in WT plants with a p-value <0.05. Perhaps this indicates a role for CBF in the development of Arabidopsis, but that redundancy in the developmental network, like that of the cold network, provides the plant with plasticity to overcome the loss of CBF. DISCUSSION As would be expected from a living organism that has to survive in the environment where it landed as a seed, with only the information provided in its genome, there is a great deal of plasticity and redundancy in the response of Arabidopsis to environmental stresses. In this work, this plasticity is evident in the response of Arabidopsis to low temperature. Loss of the CBF pathway, which is sufficient for cold acclimation, does cause a reduction in freezing tolerance. However, in the absence of the CBF pathway Arabidopsis can still sense low temperature and make changes necessary to increase its ability to 116 survive freezing, even without the ability to accumulate raffinose, an important cryoprotectant molecule. This redundancy implies a complicated network, complete with multiple back-ups, alternative routes, and fail-safe mechanisms to ensure that the plant has the best chance 0f survival no matter what environment it finds itself in. Figure 4. 5 Cotyledon Phenotypes in CBF2AC Seedlings CBF2AC seedlings with wild-type (W), tri—cotyledons (T), or single-cotyledon (S) phenotypes. Cotyledons are indicated by C and leaves are indicated by. 117 16 5X96 n=191 I 14 — G89 n=404 — 12 ——~ I 93 n=328 e 10 fl DWS n=211 . % of Plants with Phenotype \ «0‘0 N '5 Figure 4.6 Quantification of Cotyledon Phenotypes in CBF2AC Seedlings Percent of total plants with abnormal cotyledon phenotype from WS (white) and three lines of CBF2AC plants, 93 (solid black), 89 (grey), x96 (checkered). Total number of plants analyzed (n) is presented in the legend. An important component in a preparedness system is to be able to rapidly and accurately predict changes in the environment and translate this into an appropriate response. It is easy to imagine the evolutionary pressure on plants to incorporate all possible environmental cues to anticipate changes in the environment and prepare for them when necessary, while not wasting resources preparing needlessly. In this study, the CAMTAs are identified as important regulators of the response to low temperature. Their possible role as integration points for processing and responding to multiple environmental signals makes them interesting targets for further study and potential targets for improving stress responses in crop plants. 118 This study also describes the effect of the loss of the CBF pathway on the ability of plants to cold acclimate. While CBF2AC plants are still able to cold acclimate, one level of redundancy in response to low temperature has been removed in these plants. The use of CBF2AC'as a background for future studies will facilitate the identification of other pathways for cold acclimation. While plants have had millions of years to fine tune their perception and response to environmental stimuli, the recent rapid climate changes may disrupt the ability of plants to accurately predict and prepare for fluctuations in the environment. It is encouraging to see the incredible plasticity of plants in their response to the stress of low temperature. However, it is important to remember that survival of Arabidopsis in the face of an impaired cold acclimation pathway does not directly translate into a sustained yield of crop plants in response to low temperature. Recognizing and investigating the ability of plants to incorporate these subtle signals into their response will allow us to describe more accurately the network of responses to low temperature and other stresses that plants encounter. A thorough understanding of the plant stress response network will allow us to target areas for improvement in plant species we depend on for survival. MATERIALS AND METHODS Plant Material and Growth Conditions All plants were stratified for 3-5 days in the dark at 4°C then transferred to constant illumination at 24°C for 10 days prior to treatment. Mechanical 119 treatment consisted of tapping the plates on the bench for 15 minutes prior to harvesting the tissue. Cycloheximide treatment consisted of floating seedlings grown on a filter paper in 10ug ml’1 cylcoheximide. Plants were grown on a filter placed on Gamborg’s 85 nutrients (Caiss0n Laboratories, www.caissonlabs.com) and 0.8% phytagar (Caisson Laboratories, www.caissonlabs.com) without sucrose. All experiments were performed on tissue from whole seedlings. RNA isolation and Analysis Total RNA was extracted from plant material with the use of RNeasy Plant Mini kits (Qiagen, Valencia, CA) with modifications as described (Zarka et al. 2003). Northern transfers were prepared and hybridized as described (Hajela et al. 1990) probes prepared, and membranes washed with high- stringency conditions (Stockinger et al. 1997). Electrolyte Leakage Freeze Test Electrolyte leakage freeze tests were performed essentially as described (Gilmour et al. 2000) with minor modifications. The SAS system was not used to aid in randomization. Staining for GUS Activity Gus activity was analyzed as described by Zarka et al. (2003). 120 LITERATURE CITED Ballou, S. M., K. Y. Yun, et al. (2007). "Cold Sensitivity Gradient in Tuber-Bearing Solanum Based on Physiological and Transcript Profiles." Crop Science 47(5): 2027. Bieniawska, Z., C. Espinoza, et al. (2008). "Disruption of the Arabidopsis Circadian Clock Is Responsible for Extensive Variation in the Cold-Responsive Transcriptome." Plant Physiol. 147(1): 263-279. Fowler, D. B., L. P. Chauvin, et al. (1996). "The regulatory role of vernalization in the expression of low-temperature. induced genes in wheat and rye." Theor Appl Genet 93: 554-559. Fowler, S. G., D. Cook, et al. (2005). "Low temperature induction of Arabidopsis CBF1, 2, and 3 is gated by the circadian clock." Plant Physiol 137: 961 - 968. Fowler, S. G., D. Cook, et al. (2005). "Low Temperature Induction of Arabidopsis CBF1, 2, and 3 Is Gated by the Circadian Clock." Plant Physiol 137(3): 961 - 968. Franklin, K. A. and G. C. Whitelam (2007). "Light-quality regulation of freezing tolerance in Arabidopsis thaliana." Nat Genet 39(1 1): 1410-1413. Gilmour, S. J., S. G. Fowler, et al. (2004). "Arabidopsis Transcriptional Activators CBF1, CBF 2 and CBF3 have Matching Functional Activities. " Plant Molecular Biology 54(5). 767- 781. Gilmour, S. J ., A. M. Sebolt, et al. (2000). "Overexpression of the Arabidopsis CBF3 Transcriptional Activator Mimics Multiple Biochemical Changes Associated with Cold Acclimation." Plant Physiol. 124(4): 1854-1865. Hajela, R. K., D. P. Horvath, et al. (1990). "Molecular Cloning and Expression of cor (Cold-Regulated) Genes in Arabidopsis thaliana." Plant Physiol. 93(3): 1246- 1252. Knight, H., A. J. Trewavas, et al. (1996). "Cold Calcium Signaling in Arabidopsis Involves Two Cellular Pools and a Change in Calcium Signature after Acclimation." Plant Cell 8(3): 489—503. Knight, M. R., S. M. Smith, et al. (1992). "Wind-induced plant motion immediately increases cytosolic calcium." Proceediangs of the National Academy of Sciences of the United gates of America 89(11): 4967-4971. 121 Mantyla, E., V. Lang, et al. (1995). "Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LTI78 and RABIS proteins in Arabidopsis thaliana." Plant Physiol 107: 141-148. Michael, T. P., T. C. Mockler, et al. (2008). "Network Discovery Pipeline Elucidates Conserved Time-of-Daya€“Specific cis-Regulatory Modules." PLoS Genetics 4(2): e14. Ramos, A., E. Perez-Solis, et al. (2005). "From the Cover: Winter disruption of the circadian clock in chestnut." Proceedings of the National Acafieray of Sciences 102(19): 703 7-7042. Stockinger, Eric J ., Sarah J. Gilmour, et al. (1997). "Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C- repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit." Proceedmasof the National Academy of Sciences 94(3): 1035-1040. Sung, S. and R. M. Amasino (2005). "REMEMBERING WINTER: Toward a Molecular Understanding of Vemalization." Annual Review of Plant Biolpgy 56(1): 491-508. Thingnaes, E., S. Torre, et al. (2003). "Day and Night Temperature Responses in Arabidopsis: Effects on Gibberellin and Auxin Content, Cell Size, Morphology and Flowering Time." Ann Bot 92(4): 601-612. Zarka, D. G., J. T. Vogel, et al. (2003). "Cold Induction of Arabidopsis CBF Genes Involves Multiple ICE (Inducer of CBF Expression) Promoter Elements and a Cold-Regulatory Circuit That Is Desensitized by Low Temperature." Plant Physiol. 133(2): 910-918. Zhang, X., S. G. Fowler, et al. (2004). "Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing- tolerant Arabidopsis." The Plant Joumg 39(6): 905-919. 122 APPENDIX 123 EAR Repression Attempts were made to reduce CBF function using an EAR repressive domain (Hiratsu et al. 2003). CBF full length and two versions of the CBF DNA binding domain were fused to the EAR repressive domain. No viable colonies of the CBF full length/EAR fusion construct were obtained from cloning attempts by either Sarah Gilmour or me. Both versions of the CBF DNA binding domain fused to EAR were obtained and plants were transformed. Plants were tested for a reduction in COR15 and COR78 using northern blot analysis. lines were found with reduced COR15 and COR78 expression. However, the level of reduction was not as great as that seen in the CBF2AC lines. ICICLES In an interest to identify other factors that maybe involve in regulation of cold-responsive transcripts a reverse genetics approach was taken. Seven transcription factors that were induced in response to low temperature were studied for their role in cold acclimation. The group of transcription factors analyzed was classified as ICICLEs, Independent of CBF Influence and Cold Late Expressed, that is analysis of Affymetrix experiments by Jonathan Vogel indicated that they were induced after long-term exposure to low temperature and were not up-regulated by overexpression of CBF in the warm. The expression pattern of these transcripts was confirmed by northern blot analysis (Figure A1). Homozygous knockouts were obtained and tested for changes in freezing tolerance. No changes were seen in freezing tolerance in any of the 124 homozygous knockout mutants either by electrolyte leakage assay or whole plant freeze tests. In contrast to the transcription factors induced early in response to low temperature which were induced rapidly after mechanical agitation, these transcription factors either did not have a detectable response to mechanical agitation, At2955580 and At3902990, or were induced an hour after treatment (Figure A1 ). AGI Cold 4°C Mechanical Description 0‘) C 1h 2h 4h 24h 7d 0.25 0.5 1 Altg49720 _. “W e ‘1‘. ABA response element Transcription Factor Ilke At1069570 . 4 Dofzinc finger protein Al3902990 ,1" ,I V ”IL I l l 1.2:, r: "f7" . C3HC4- ezincfin er roteln fami gm: , ft“ m *- data "" 9 p " Angsssao $334 . l” is". _ Regulator of Chromatin Condensation like; UVRB like Arsgozaw m "N w... ..." . Pseudo-response regulator, APRR7 (APRR1fI' OCI family) P45954470 CONSTANS B-box zinc finger family protein, A5957660 .... __ .- ..I. -- CONSTANS 8- box zinc finger family protein Figure A1 Induction of ICICLE Transcription Factors in Response to Low Temperature Treatment and Mechanical Agitation Northern blot analysis for induction of each transcript for control (C), after treatment at 4°C for time specified, and after 15 min mechanical agitation and recovery for time specificed. Silencing of CBF A hairpin RNAi construct was designed that would target CBF 1, 2, and 3 for silencing. This construct was based on CBF2 sequence homology. The construct was a 537bp sense construct fused to a reverse oriented 338bp antisense construct (Figure A2). The sense construct started 31bp upstream of the CBF2 translation start site. Both the sense and the anti-sense construct 125 ended at bp 509. Thus the overlapping region targeted the 3' end of the DNA binding domain and the CBF specific sequence, PKKPAGER. Initial results from the T2 generation of plants transformed with this construct showed a reduced binding to CBF1 full-length probe indicating a reduction in CBF 1, 2, and 3 levels (Figure A3). However, when homozygous offspring from the lines which showed the lowest levels were analyzed for CBF mRNA levels there was a distribution of reduction similar to that seen in the original lines indicating that this reduction was not stable. CBF Silencing Design Construct / ni- ense ense '\ \ A t s | s / Figure A2 Design of CBF Silencing Contruct. 2hrs at 4°C WI" P1 P2 P3 P4 P5 P6 P7 P3 P9 p10 p11 CBF1 Full Length - w “= 3" w m. w .... y. .. _~ a. Figure A3 CBF-Targeting RNAi Silencing Construct Produced Lines with Reduced CBF Levels. Northern blot analysis of CBF levels after treatment for 2h at 4°C in T2 plants containing the CBF silencing construct. Wild-type (WT) plants were used as a control for comparisons of lines P1 through P11. rRNA was probed as a loading control. TRP Channels Members of the Transient Receptor Potential (TRP) family of calcium ion channel proteins have been shown to be involved in sensing cold and heat in 126 mammals ((McKemy et al. 2002). There are four homologs of these channels in Arabidopsis, TC1-4. Dr. Richard Amasino provided homozygous T-DNA insertion lines for each of these TC channels for analysis of CBF induction in these channels in response to low temperature treatment. There was no significant difference in the early cold responsive transcripts levels of CBF or ZAT12 in these lines in response to low temperature compared with wild type plants. However, there was a reduction in two downstream cold-responsive genes, both COR15 and COR78 mRNA did not reach the cold-induced level of wild type plants in the -tc1 and -t02 T-DNA insertion lines (Figure A4). Not all late-cold-induced transcript levels were affected in -tc1 and -th plants, however. Phenyl ammonia lyase (PAL2) was present at levels comparable to wild type in both -tc1 and -t02 plants (Figure A4). The analysis on CBF mRNA levels was performed using a full length CBF probe which measures levels of CBF 1, 2 and 3. Analysis with probes specific for each CBF might provide additional information since we now know that there are differences in regulation of each of these three cold-responsive CBFs. 127 cm(wn) TC1 TC2 TC3 TC4 Timeat0°C(h) 0 2 4 824168 0 2 4 824168 0 2 4 824168 0 2 4 824168 0 2 4 824168 CBF1 F .... a... 00¢ 0.0. ...». COR15 " “‘ " '- COR 78 ' -- rRNA ZAT1 2 PAL2 rRNA Figure A4 Cold-induced Transcript Levels in —tc T-DNA Insertion Lines Northern blot analysis of mRNA levels for cold-responsive transcripts in Columbia (Col), -tc1 (TC1), -th (TCZ), -tc3 (TC3), -tc4 (TC4) plants. Probes were for CBF1 full length (CBF1 F.L.), COR15, COR78, ZAT12, PAL2. rRNA was probed as a loading control. Phenotypes of CBF Overexpressing Plant Crossed to CBF2AC CBF2 overexpressing plants crossed to CBF2AC plants produced T3 offspring that were near WT size however showed a thickening of the leaves and stem compared to WT. Plants that contained both constructs showed an earlier flowering time and recovery of WT size compared to CBF2 overexpressing plants. However, since both constructs were in the same vector and the sequences only differed by one base pair, it was difficult to identify offspring homozygous for both CBF2 and CBF2AC constructs. 128 Phenotypes of CBF Overexpressing Plants Crossed to ZAT12 Overexpressing Plants CBF overexpressing plants crossed to ZAT12 overexpressing plants showed an interesting phenotype. While both CBF overexpressors and ZAT12 overexpressors show a dwarf phenotype plants containing at least one copy of each construct showed a recovery of WT size. The plants also had a phenotype in flower development with downtumed buds (Figure A5). This phenotype was consistent across crosses with several different ZAT12 overexpressing lines. \ ‘. ",1 ‘i‘j‘; ‘3552CBF2/ ‘1‘ 1 WT 353:ZAT12 Q) l 91;» Figure A5 Flower Phenotype of CBF Overexpressing Plants Crossed to ZAT12 Overexpressing Plants. Plants containing at least one copy of both the CBF overexpression construct (35s:CBF2) and one copy of the ZAT12 overexpression construct (35$:ZAT12) showed a downtumed flower phenotype indicated by arrow. Siliques remain downtumed as indicated by dashed arrow. 129 Other CAMTA Phenotypes CAMTA3 overexpressing plants are early flowering. camta1/3 double mutants are chlorotic, have lower FV/FM in both warm and low temperature and show early senescence of leaves. COR Protein Levels in CBF2AC Plants The protein levels of two CBF target genes COR15 and COR78 were analyzed in CBF2AC expressing plants. These plants showed a dramatic decrease in both COR15 levels (Figure A6) and COR78 levels. a a A m B I— CD (”molt—N EmmOFN LO LONCOCO Emfifig §§m### Cor15 — Figure A6 COR15 Protein Levels in CBF2AC Plants. (A) Western blot analysis of COR15 in plants treated for 7d at 4°C. Multiple bands in 355:CBF2 are possibly due to cross-reactivity with other COR genes or breakdown products of COR15. (B) Comassie stain of samples in (A) for loading comparison 130 Analysis of CBF2AC Transcript Categories for Novel Cis-Elements The categories of transcripts based on CBF dependence identified in the analysis of CBF2AC plants (Chapter 3) were tested for the overrepresentation of novel cis-elements in the promoters of each group of transcripts. The analysis was performed with Motif Sampler and Weeder. Overrepresentation of the known elements, CRT, CG-1, TBX, and ABRE was identified by both programs. However, no novel element was consistently picked in any category of transcripts by both programs. Kinetic Analysis of Known Cis-Elements Identified in CBF2AC Plants The known cis-elements identified as being overrepresented in each category of transcripts based on CBF dependence (Chapter 3) were analyzed for their potential role in the different kinetic responses to low temperature of the cold-regulated transcripts. Each category was analyzed separately. The expression levels of the after treatment with low temperature for different periods of time were obtained from publically available microarray data. Each category was then divided into clusters based on the. kinetics of the induction of its transcripts in response to low temperature. Two methods of clustering were used, K-means and the pattern-based method of Haystack. These sub-clusters were then analyzed for the overrepresentation of known motifs. For the motifs analyzed, ABRE-like, CG-1, CRT, and TBX only CG-1 showed significant enrichment in any kinetic category. As discussed in Chapter 2, 06-1 was enriched in those transcripts with an early response to low temperature. 131 LC-MS Analysis of CBF2AC Plants Initial attempts at characterizing the affects of the CBF2AC construct on the metabolite profile in response to low temperature involved using LC-MS analysis of metabolites. This analysis identified Some metabolites that were significantly difference between CBF2AC and wild-type (WT) samples after treatment at low temperature. However, the majority of metabolites did not show a significant difference between WT and CBF2AC. There was also no detectable difference in the majority of metabolites between CBF2 overexpressing plants and WT plants in the warm as was previously seen by Cook et al. (Cook et al. 2004). This discrepancy is most likely due to the fact that many of the metabolite differences detected were in polar metabolites, detectable by the GC-MS method used by Cook et al. These polar metabolites are possibly excluded by the LC/MS method employed. Therefore, I switched to GC/MS analysis and identification of these metabolites was not pursued. Identification of CBF2 T-DNA Insertion Lines with Reduced Expression of CBF2 mRNA Isolating knock-out plants for each of the three cold-inducible CBFs would provide a tool for understanding the function of each CBF and the possible redundancy and specificity of the three proteins. In collaboration with Ryan Sartor, we attempted to identify T-DNA insertion lines affecting the CBF 1, 2, or 3 genes. However, the position of the T-DNA insertion in all publically available lines in the CBF locus is limited to the promoter regions of the three CBFS. 132 Therefore, to determine if plants that were homozygous for the T-DNA insertion had significantly reduced mRNA levels we extracted RNA and performed semi- quantitative RT PCR with primers specific for each CBF. The two T-DNA insertion lines in the promoters of CBF2, CBF2c P7 and CBF2a P23, showed almost no accumulation of CBF2 mRNA in response to low temperature. These were the only two lines with any affect on the expression levels of CBF1, 2 or 3. The two CBF2 knock-down lines were analyzed for their affect on the other CBF transcripts and downstream target genes. No dramatic differences, as detectable by semi-quantitative RT PCR, were seen. However, quantitative differences could not be determined by this method. CM2ICM1 Mutant Screen In collaboration with Megan Sargent a population of plants containing the CM2ICM1:GUS reporter construct were mutagenized with ethyl methane- sulphonate (EMS). The plants were screened for mutants that no longer stained for GUS after exposure to low temperatUre. Megan identified two lines with reduced staining, L-3 and S-14. Line S-14 still showed GUS staining in the warm although at a lower level than the parental plants and showed almost no GUS staining after treatment at low temperature. L-3 had normal staining in the warm, and after low temperature treatment had a blotchy staining pattern. 133 Table A] 1279 Transcripts Differentially Regulated in Response to Low Temperature at 24h in Both WS and C01. 134 Probe Set 261 572_at 261 048_at 261049_at 259426_at 259436_at 259431_at 261558_at 261655_at 2641 23__at 259442_at 259444__at 260903_at 260914_at 262113_at 2621 12_at 2631 18_at 2631 14_at 264357_at 264843_at 264818_at 265066_at 265093_at 263656_at 263668_at 261130_at 261168_at 264583_at 263231_at 263179_at 260955_at 259392_at 260824_at 260832_at 256065_at 261081_at 261077_at 261084_at 264803_at 264806_at 264652_at AGI AT1601170 AT1601420 AT1601430 AT1601470 AT1GO1500 AT1601620 AT1601770 AT1GO1940 AT1602270 AT1602310 AT1602370 AT1602460 AT1 602640 AT1602820 AT1602870 AT1603090 AT1603130 AT1603360 AT1603400 AT1603530 AT1603870 AT1603905 AT1 604240 AT1 604350 AT1604870 AT1604945 AT1 6051 70 AT1 605680 AT1 60571 0 AT1606000 AT1606380 AT1606720 AT1 606780 AT1607070 AT1607350 AT1607430 AT1607440 AT1608580 AT1 60861 0 AT1608920 Description ozone-responsive stress-related protein UDP-glucoronosyllUDP-glucosyl transferase family protein similar to unknown protein Encodes late-embryogenesis abundant protein similar to unknown protein a member of the plasma membrane intrinsic protein subfamily PIP1. similar to Protein of unknown function DUF1446 peptidyI-prolyl sis-trans isomerase cyclophilin-type family protein endonucleaselexonuclease/phosphatase family protein glycosyl hydrolase family protein 5 I cellulase family protein pentatricopeptide (PPR) repeat-containing protein glycoside hydrolase family 28 protein l polygalacturonase (pectinase) family protein encodes a protein similar to a beta-xylosidase located in the extracellular matrix. late embryogenesis abundant 3 family protein I LEA3 family roteln Similar to hypothetical protein MtrDRAFT_AC16103299v1 MCCA is the biotinylated subunit of the dimer MCCase Encodes a protein predicted by sequence similarity with spinach PsaD ATRRP4; exonuclease; similar to Os0490520000 A single copy gene that encodes a protein with sequence similarity to tomato E8 similar to unknown protein fasciclin-Iike arabinogalactan-protein 9 (F Ia9) ABC transporter family protein; Identical to Non-intrinsic ABC protein 4 SHYZ/IAA3 regulates multiple auxin responses in roots. encodes a protein whose sequence is similar to 2—oxoglutarate- dependent dioxygenase protein arginine N-methyltransferase family protein similar to 050190962500 similar to galactosyltransferase family protein UDP-glucoronosyI/UDP-glucosyl transferase family protein ethylene-responsive protein encodes a flavonol-7-O-rhamnosyltransferase ribosomal protein-related similar to unknown protein Encodes a protein with putative galacturonosyltransferase activity. 608 ribosomal protein L35a (RPL35aA); Identical to 608 ribosomal protein L35a-1 transformer serinelarginine—rich ribonucleoprotein protein phosphatase 20 tropinone reductase similar to Pm52 pentatricopeptide (PPR) repeat-containing protein sugar transporter 135 264656_at 264261_at 264511_at 264501__at 264672_at 264668_at 26451 5_at 264452_at 264435_at 264436_at 264458_at 262784_at 260481_at 262452_at 261818_at 260969_at 259537_at 261211_at 262777_at 262766_at 259364_at 256096_at 256098_at 26261 1_at 261485_at 262830_at 262840_at 262544_at 262584_at 259500_at 261845_at 262706_at 262703_at 255764_at 256114_at 262518_at 262517_at 262539_at 261060_at 259398_at 261661_at AT1609010 AT1609240 AT1609350 AT1609390 AT1609750 AT1609780 AT1610090 AT1610270 AT1610360 AT1610370 AT1610410 AT1610760 AT1610960 AT1611210 AT1611390 AT1612240 AT1612370 AT1612780 AT1613030 AT1613160 AT1613260 AT1613650 AT1613700 AT1614060 AT1614360 AT1614700 AT1614900 AT1615420 AT1615440 AT1615740 AT1615960 AT1616280 AT1616510 AT1616720 AT1616850 AT1617170 AT1617180 AT1617200 AT1617340 AT1617700 AT1618360 glycoside hydrolase family 2 protein nicotianamine synthase ATGOLS3 (ARABIDOPSIS THALIANA GALACTINOL SYNTHASE 3) GDSL-motif lipase/hydrolase family protein chloroplast nucleoid DNA-binding protein-related 2,3-biphosphoglycerate-independent phosphoglycerate mutase, putative similar to RXW8 GRP23 (GLUTAMINE-RICH PROTEIN23) Encodes glutathione transferase belonging to the tau class of GSTs. ATGSTU17/ER09/GST30/GST3OB (EARLY-RESPONSIVE TO DEHYDRATION 9 similar to CW14 SEX1 ATFD1 (FERREDOXIN 1); electron carrier! iron ion binding similar to unknown protein ABC1 family protein; similar to ABC1 family protein ATBETAFRUCT4NAC-INV (VACUOLAR INVERTASE) significant homology to the recently characterized type II photolyases Encodes a UDP-glucose epimerase sphere organelles protein-related; similar to unknown protein SDA1 family protein; similar to SDA1 family protein Encodes an AP2/B3 domain transcription factor similar to 18$ pre-ribosomal assembly protein gar2-related glucosamine/galactosamine-6-phosphate isomerase family protein similar to unknown protein ATUTR3/UTR3 (UDP-GALACTOSE TRANSPORTER 3) ATPAP3/PAP3 (purple acid phosphatase 3) Encodes a protein belonging to the subgroup of HMGA (high mobility group A) proteins similar to transducin family protein I WD-40 repeat family protein transducin family protein lWD-40 repeat family protein leucine-rich repeat family protein; similar to leucine-rich repeat family protein member of Nramp2 family DEAD/DEAH box helicase auxin-responsive family protein oxidoreductase/ transcriptional repressor; similar to transcriptional repressor similar to unknown protein Encodes glutathione transferase belonging to the tau class of GSTs. Encodes glutathione transferase belonging to the tau class of GSTs. integral membrane family protein; similar to integral membrane family protein phosphoinositide phosphatase family protein prenylated rab acceptor (PRA1) family protein hydrolase 136 255774_at 255779_at 261377_at 261428_at 259461_at 259476_at 25601 5_at 25601 7_at 260674_at 260668_at 255786_at 261 143_at 261248_at 259570_at 259516_at 256091_at 259560_at 260876_at 260877_at 260921_at 262503_at 262496_at 255968_at 255962_at 264211_at 264774_at 264751_at 264895_at 264893_at 262986_at 263014_at 263016_at 265175_at 265188_at 263035_at 263019_at 264864_at 245637_at 245639_at 245845_at 26101 1_at AT1618620 AT1618650 AT1618850 AT1618870 AT1618900 AT1619000 AT1619150 AT1619180 AT1619370 AT1619530 AT1619670 AT1 61 9770 AT1 620030 AT1 620440 AT1 620450 AT1 620693 AT1621270 AT1621460 AT1621500 AT1621540 AT1621670 AT1621790 AT1622270 AT1622335 AT1622770 AT1622890 AT1623020 AT1623100 AT1623140 AT1623390 AT1623400 AT1623410 AT1623480 AT1623800 AT1623860 AT1623870 AT1624310 AT1625230 AT1625260 AT1626150 AT1626340 similar to unknown protein glycosyl hydrolase family protein 17; similar to glycosyl hydrolase family protein 17 similar to 050190112100 Encodes a protein with isochorismate synthase activity pentatricopeptide (PPR) repeat-containing protein myb family transcription factor; similar to myb family transcription factor PSI type II chlorophyll aIb-binding protein (Lhca2‘1) mRNA similar to unknown protein similar to unknown protein unknown protein Chlorophyllase is the first enzyme involved in chlorophyll degradation. Member of a family of proteins related to PUP1 pathogenesis-related thaumatin family protein Belongs to the dehydrin protein family Encodes a gene induced by low temperature and dehydration. Encodes a protein belonging to the subgroup of HMGB (high mobility group B) proteins cytoplasmic serinelthreonine protein kinase nodulin MtN3 family protein; similar to nodulin MtN3 family protein similar to conserved hypothetical protein AMP-binding protein similar to unknown protein similar to 050190869600 Identical to TRM112-like protein At1922270 similar to RNA recognition motif (RRM)-containing protein GI . similar to unknown protein Encodes a ferric chelate reductase whose transcription is regulated by FIT1. 10 kDa chaperonin C2 domain-containing protein; similar to C2 domain-containing protein kelch repeat-containing F-box family protein; similar to F-box family protein ATCAF2/CAF2 (ARABIDOPSIS THALIANA HOMOLOG 0F MAIZE CAF2) ubiquitin extension protein encodes a gene similar to cellulose synthase Encodes a mitochondrial aldehyde dehydrogenase Encodes a 9G8-Iike serine-arginine rich (SR) protein that interacts in vivo with U1-70K Encodes an enzyme putatively involved in trehalose biosynthesis. similar to transporter purple acid phosphatase family protein acidic ribosomal protein PO-related; similar to 60$ acidic ribosomal protein P0 (RPPOC) protein kinase; similar to protein kinase family protein member of Cytochromes b5 137 261276_at 261263_at 264989_at 264990_at 264445_at 262296_at 261648_at 261651_at 259588_at 259582_at 245668_at 261500_at 259789_at 259773_at 260049_at 245770_at 261801_at 263221_at 256497_at 246575_at 260653_at 261711_at 261187_at 261192_at 261566_at 261594_at 261981_at 259577_at 260869_at 260868_at 260870_at 245803_at 262440_at 260727_at 262248_at 261294_at 261 308_at 261 301_at 246623_at 260769_at AT1 626670 AT1 626790 AT1627200 AT1 627210 AT1 627290 AT1 627630 AT1 627730 AT1 627760 AT1 627930 AT1 628060 AT1 628330 AT1 628400 AT1 629395 AT1629500 AT1 629940 AT1 630240 AT1 630520 AT1 630620 AT1631580 AT1631660 AT1632440 AT1632700 AT1632860 AT1632870 AT1633230 AT1633240 AT1 63381 1 AT1635340 AT1643800 AT1 643860 AT1 643890 AT1 647128 AT1647710 AT1 6481 00 AT1 648370 AT1 648430 AT1 648480 AT1 648570 AT1 648920 AT1 649010 member of VTI1 Gene Family. Ies. ‘Dof-type zinc finger domain-containing protein similar to zinc finger (C3HC4-type RING finger) family protein binding; similar to binding [Arabidopsis thaliana] (TAIR:AT1GS9850.1) similar to conserved hypothetical protein cyclin family protein; similar to cyclin family protein Related to CysZ/His2-type zinc-finger proteins found in higher Iants. interferon-related developmental regulator family protein similar to unknown protein small nuclear ribonucleoprotein family protein I snRNP family protein dormancy-associated protein (DRM1) similar to unknown protein encodes a protein similar to the cold acclimation protein WCOR413 in wheat. auxin-responsive protein Encodes a subunit of RNA polymerase 1 (aka RNA polymerase A). binding; similar to unnamed protein product acyl-activating enzyme 14 (AAE14) encodes a type-ll membrane protein that catalyzes 4- epimerization of UDP-D-Xylose to UDP-L-Arabinose in vitro Encodes cell wall protein. ECS1 is not a ch750 resistance ene gimilar to 050990352400 pyruvate kinase zinc-binding family protein; similar to zinc-binding family protein glycosyl hydrolase family 17 protein; Identical to Putative glucan endo-1 ‘ ANAC013 (Arabidopsis NAC domain containing protein 13) similar to unknown protein GTL1 GDSL-motif lipase/hydrolase family protein ATP-dependent protease La (LON) domain-containing protein acyl-(acyI-carrier-protein) desaturase transcription factor; similar to Shwachman-Bodian-Diamond syndrome ras-related small GTPase cysteine proteinase (RD21A) lthiol protease serpin glycoside hydrolase family 28 protein I polygalacturonase (pectinase) family protein Arabidopsis thaliana metal-nicotianamine transporter YSL4 dihydroxyacetone kinase family protein; similar to dihydroxyacetone kinase family protein Arabidopsis thaliana receptor-like protein kinase (RKL1) gene zinc finger (Ran-binding) family protein; similar to zinc finger (Ran-binding) family protein The predominant form of the two nucleolin genes in Arabidopsis. myb family transcription factor 138 260770_at 262448_at 262400_at 261610_at 261613_at 261611_at 261638_at 245749_at 265142_at 265147_at 265149_at 260489_at 259839_at 262159_at 260203_at 2601 57_at 261 31 8_at 260614_at 260983_at 260984_at 262956_at 262964_at 264186_at 264191_at 264238_at 264240_at 256353__at 259659_at 259666_at 259664_at 257510_at 265078_at 264535_at 260592_at 260596_at 260603_at 262094_at 262098_at 256225_at AT1 649200 AT1 649450 AT1 649480 AT 1 649560 AT1649720 AT1649730 AT1649975 AT1651090 AT1651360 AT1651380 AT1651400 AT1651610 AT1652190 AT1652720 AT1652890 AT1652930 AT1653035 AT1 653390 AT1653560 AT1653645 AT1654270 AT1654380 AT1654570 AT1654730 AT1654740 AT1654820 AT1655000 AT1655170 AT1655310 AT1655330 AT1655360 AT1655500 AT1 655690 AT1655850 AT1655900 AT1655960 AT1656110 AT1656170 AT1656220 zinc finger (03HC4-type RING finger) family protein; Identical to ATL1I (ATL1 I) transducin family protein / WD-40 repeat family protein RTV1 (RELATED TO VERNALIZATION1 1) myb family transcription factor; similar to myb family transcription factor Identified as a protein that binds to abscisic acid response elements. ' protein kinase family protein; similar to protein kinase family protein similar to expressed protein heavy-metaI-associated domain-containing protein; similar to metal ion binding similar to unknown protein eukaryotic translation initiation factor 4A photosystem II 5 kD protein cation efflux family protein I metal tolerance protein proton-dependent oligopeptide transport (POT) family protein similar to unknown protein encodes a NAC transcription factor whose expression is induced by drought brix domain-containing protein; similar to brix domain-containing protein similar to unknown protein ATPase similar to unknown protein hydroxyproline-rich glycoprotein family protein; similar to actin binding member of elF4A - eukaryotic initiation factor 4A spliceosome protein-related; similar to spliceosome protein- related ‘ esterase/Iipase/thioesterase family protein sugar transporter similar to unknown protein protein kinase family protein; similar to protein kinase family protein peptidoglycan-binding LysM domain-containing protein similar to unknown protein Encodes 3 SR spliceosome protein that is localized to nuclear specks Encodes a putative arabinogalactan-protein (AGP21). similar to unknown protein similar to ECT2 SEC14 cytosolic factor family protein / phosphoglyceride transfer family protein encodes a protein similar to cellulose synthase component of a translocase in the mitochondrial inner membrane similar to unknown protein NOP56-like protein Encodes a protein with similarity to a subunit of the CCAAT promoter motif binding complex donnancylauxin associated family protein 139 256221_at 245628_at 245868_at 246396_at 256022_at 262076_at 263679_at 264217_at 264940_at 264920_at 264963_at 259908_at 259721_at 265061_at 264398_at 264400_at 264289_at 264736_at 2651 19_at 262644_at 262691_at 259690_at 2601 09_at 260323_at 260317_at 260316_at 262354_at 261956_at 261972_at 261949_at 262884_at 262881_at 263142_at 262930_at 260140_at 255852_at 255857_at 264471_at 264992_at 264968_at 264229_at 245196_at 245200_at AT1 656300 AT1 656650 AT1 658030 AT1658180 AT1658360 AT1659580 AT1659990 AT1 660190 AT1 660470 AT1660550 AT1660600 AT1660850 AT1660890 AT1 661640 AT1661 730 AT1661 800 AT1661890 AT1662200 AT1662570 AT1 662710 AT1 662740 AT1 663160 AT1 663260 AT1 663780 AT1 663800 AT1 66381 0 AT1 664200 AT1 664590 AT1 664600 AT 1 664670 AT 1 664720 AT1 664890 AT1 665230 AT1 665690 AT1 666390 AT1 666970 AT1 667080 AT1 667120 AT1 667300 AT1 667360 AT1 667480 AT1 667750 AT1667850 DNAJ heat shock N-terrninal domain-containing protein Encodes a putative MYB domain containing transcription factor Arabidopsis thaliana amino acid permease family protein (A11958030) carbonic anhydrase family protein I carbonate dehydratase family protein neutral amino acid transporter expressed in seeds encodes a mitogen-activated kinase involved in innate immunity DEAD/DEAH box helicase armadillo/beta-catenin repeat family protein I U-box domain- containing protein ATGOLS4 (ARABIDOPSIS THALIANA GALACTINOL SYNTHASE 4) naphthoate synthase Encodes a protein similar to 1 ATRPAC42 (Arabidopsis thaliana RNA polymerase I subunit 42) phosphatidylinositol-4-phosphate 5-kinase family protein ABC1 family protein DNA-binding storekeeper protein-related; similar to transcription regulator gIucose6-Phosphatelphosphate transporter 2 MATE efflux family protein; similar to MATE efflux family protein proton-dependent oligopeptide transport (POT) family protein flavin-containing monooxygenase family protein I FMO family protein Encodes a vacuolar processing enzyme stress-inducible protein replication factor C 40 kDa Member of TETRASPANIN family Small nucleolar ribonucleoprotein protein involved in ribosomal RNA processing. UBCS (ubiquitin-conjugating enzyme 37); ubiquitin-protein Iigase similar to Nrap protein VHA-E3 (VACUOLAR H+-ATPASE SUBUNIT E ISOFORM 3) short-chain dehydrogenase/reductase (SDR) family protein similar to unknown protein BDG1 membrane related protein 0P5 integral membrane transporter family protein; similar to hypothetical protein MtrDRAFT_AC14817192v1 NDR1/HIN1-LIKE 25 production of anthocyanin pigment 2 protein (PAP2) glycerophosphoryl diester phosphodiesterase family protein Involved in the photoprotection of PSII. midasin-related; similar to unknown protein hexose transporter rubber elongation factor (REF) family protein kelch repeat-containing F-box family protein pectate lyase family protein; Identical to Probable pectate lyase 5 precursor similar to unknown protein 140 259996_at 260014_at 260431_at 260262_at 260264_at 260266_at 262281_at 262286_at 260037_at 259672_at 260338_at 260357_at 260360_at 256299_at 260410_at 264700_at 264697_at 264339_at 262288_at 262313_at 261516_at 257487_at 260167_at 259803_at 260398_at 260427_at 260425_at 259891_at 245734_at 260076_at 260075_at 260070_at 260388_at 260221_at 259953_at 259925_at 259927_at 259954_at 256458_at 2611 16_at 261 1 14_at 261 1 1 8_at 262953_at AT1667910 AT1668010 AT1668190 AT1 668470 AT1 668500 AT1 668520 AT1668570 AT1668585 AT1668840 AT1668990 AT1 669250 AT1669260 AT1669370 AT1 669530 AT1 669870 AT16701 00 AT1 67021 0 AT1 670290 AT1670760 AT1670900 AT1671750 AT1671850 AT1671970 AT1672150 AT1672320 AT1672430 AT1672440 AT1672730 AT1 673480 AT1 673630 AT1673700 AT1 673830 AT1 674070 AT1674670 AT1674810 AT1675040 AT1675100 AT1 6751 30 AT1 675220 AT1 675370 AT1 675390 AT1 675460 AT1 675670 similar to unknown protein Encodes hydroxypyruvate reductase. zinc finger (B-box type) family protein; Identical to Putative zinc finger protein A11968190 exostosin family protein; similar to MUR3 (MURUS 3) unknown protein zinc finger (B-box type) family protein; Identical to CONSTANS- LIKE 6 (COL6) proton-dependent oligopeptide transport (POT) family protein metal ion binding; contains lnterPro domain Heavy metal transport/detoxification protein RAV2 DNA-directed RNA polymerase nuclear transport factor 2 (NTFZ) family protein similar to unknown protein Encodes chorismate mutase 3 (0M3). Member of Alpha-Expansin Gene Family. proton-dependent oligopeptide transport (POT) family protein similar to unknown protein Encodes a D-type cyclin that physically interacts with CDCZA. Encodes an enzyme putatively involved in trehalose biosynthesis. inorganic carbon transport protein-related; similar to NADH dehydrogenase subunit similar to unknown protein phosphoribosyltransferase family protein similar to unknown protein similar to unknown protein novel cell-plate—associated protein APUM23 (ARABIDOPSIS PUMILIO 2.3) auxin-responsive protein-related EDA25 (embryo sac development arrest 25); similar to unknown protein eukaryotic translation initiation factor 4A hydrolase calcium-binding protein MATE efflux family protein; similar to MATE efflux family protein BEE3 (BR ENHANCED EXPRESSION 3) peptidyI-prolyl cis-trans isomerase cyclophilin-type family protein gibberellin-responsive protein anion exchange family protein; Identical to Putative boron transporter-like protein 3 Thaumatin-Iike protein involved in response to pathogens. Contains a J-domain at the C-terminus which is similar to the J- domain of auxilin member of CYP721A integral membrane protein SEC14 cytosolic factor bZIP transcription factor family protein ATP-dependent protease La (LON) domain-containing protein similar to RNA polymerase Rpa43 subunit-like 141 262677_at 262682_at 261751_at 261769_at 261782_at 261726_at 259971_at 259977_at 256332_at 264953_at 264482_at 259708_at 25971 1_at 25971 3_at 2621 36_at 262164_at 263127_at 2641 18_at 264131_at 262892_at 262943_at 262941_at 262039_at 262061_at 262050_at 262049_at 260331_at 260287_at 261911_at 261 887_at 257474_at 261 901_at 266300_at 266348_at 2661 19_at 266141_at 266707_at 265699_at 264027__at 264037_at 264042_at 263334_at AT1675860 AT1675900 AT1676080 AT1676100 AT16761 10 AT1676270 AT1676580 AT1676590 AT1 676890 AT1 677120 AT1677210 AT1677420 AT1677570 AT1677610 AT1677850 AT1678070 AT1678610 AT1679150 AT1679150 AT1679440 AT1679470 AT1679490 AT1680050 AT1 6801 1 0 AT16801 30 AT1680180 AT1680270 AT1680440 AT1680750 AT1680780 AT1680850 AT1680920 AT2601420 AT2601450 AT2602100 AT2602120 AT260331 0 AT2603550 AT2603670 AT2603750 AT2603760 AT2603820 similar to unknown protein family II extracellular lipase 3 (EXL3); similar to family ll extracellular lipase 1 (EXL1) ATCDSP32/CDSP32 plastocyanin high mobility group (HMG1I2) family protein similar to unknown protein, transcription factor; Identical to (SPL1-related protein 3) (SPL16) zinc-binding family protein; similar to zinc-binding family protein encodes a plant trihelix DNA-binding protein Catalyzes the reduction of acetaldehyde using NADH as reductant. sugar transporter hydrolase DNA binding I transcription factor; similar to AT-HSFA5 glucose-6-phosphatelphosphate translocator-related Posttranscriptionally regulated by miR160 and is essential for proper development. WD-40 repeat family protein; similar to WD-40 repeat family protein mechanosensitive ion channel domain-containing protein binding; similar to unknown protein binding; similar to unknown protein Encodes a mitochondrial succinic semialdehyde dehydrogenase (SSADH). inosine-5'-monophosphate dehydrogenase EM82217 (EMBRYO DEFECTIVE 2217) Encodes an adenosine phosphoribosyl transferase(E.C:2.4.2.7) ATPP2-B1 1 binding; similar to unknown protein similar to unknown protein DNA-binding protein kelch repeat-containing F-box family protein 606 ribosomal protein L7 (RPL7A); Identical to 60$ ribosomal protein L7-1 (RPL7A) CCR4-NOT transcription complex protein methyladenine glycosylase family protein J8 mRNA Encodes a putative auxin efflux carrier member of MAP Kinase LCR69IPDF2.2 (Low-molecular-weight cysteine—rich 69); protease inhibitor;+C494 LCR70/PDF2.1 (Low-molecular-weight cysteine-rich 70); protease inhibitor unknown protein similar to unknown protein CD048 - like protein AAA-type ATPase sulfotransferase family protein; similar to sulfotransferase family protein High similarity to flavonol sulfotransferases (FSTs). nonsense-mediated mRNA decay NMD3 family protein 142 263811_at 263647_at 263674_at 265511_at 263391_at 257444_at 266614_at 265481_at 265480_at 263239_at 265354_at 266532_at 263574_at 26341 5_at 264906_at 264907__at 264591_at 264590_at 264787_at 265326_at 265342_at 265931_at 266072_at 266934_at w 267436_at 267336_at 267309_at 267280_at 265947_at 265935_at 266693_at 266695_at 265584_at 263712_at 265394_at 264019_at 264024_at 264014_at 263739_at 263544_at AT2604350 AT2604690 AT2604790 AT2605540 AT261 1 81 0 AT2612550 AT2614910 AT261 5960 AT261 5970 AT2616570 AT2616700 AT2616890 AT2616990 AT2617250 AT2617270 AT261 7280 AT2617670 AT261 7710 AT2617840 AT2618220 AT261 8300 AT261 8520 AT261 8700 AT2618900 AT26191 90 AT261931 0 AT2619385 AT2619450 AT2619540 AT2619580 AT2619800 AT261 981 0 AT2620180 AT2620585 AT2620725 AT2621 130 AT2621 180 AT2621210 AT2621 320 AT2621590 Iong-chain-fatty-acid-CoA Iigase family protein cellular repressor of E1A-stimulated genes (CREG) family similar to conserved hypothetical protein glycine-rich protein; similar to glycine-rich protein MGDG synthase type C ubiquitin-associated (UBA)fTS-N domain-containing protein; similar to H0413E07.5 , similar to unknown protein unknown protein encodes an alpha form of a protein similar to WCOR413 in wheat. Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) Encodes actin depolymerizing factor 5 (ADF5). UDP-glucoronosyllUDP-glucosyl transferase family protein; similar to transferase tetracycline transporter; similar to unknown protein similar to 050490585300 mitochondrial substrate carrier family protein; similar to mitochondrial phosphate transporter phosphogcherate/bisphosphoglycerate mutase family protein pentatricopeptide (PPR) repeat-containing protein similar to 050490560700 Identified as drought-inducible gene by differential hybridization. Identical to Nucleolar complex protein 2 homolog (Protein N002 homolog) basic helix-loop-helix (bHLH) family protein pentatricopeptide (PPR) repeat-containing protein Encodes an enzyme putatively involved in trehalose biosynthesis. transducin family protein IWD-40 repeat family protein Receptor-like protein kinase. Involved in early defense signaling. similar to HSP18.2 (HEAT SHOCK PROTEIN 18.2) nucleic acid binding Izinc ion binding Encodes Acyl-CoA:diacylglycerol acyltransferase (DGAT) transducin family protein IWD-40 repeat family protein Member of TETRASPANIN family Encodes a myo-inositol oxygenase family gene. zinc finger (CCCH-type) family protein; similar to zinc finger (CCCH-type) family protein Member of the basic helix-Ioop-helix (bHLH) family of transcription factors. NFD6 (NUCLEAR FUSION DEFECTIVE 6); similar to unknown protein CAAX amino terminal protease family protein peptidyl—prolyl cis-trans isomerase I cyclophilin (CYP2) I rotamase similar to unknown protein Putative auxin-regulated protein whose expression is downregulated in response to chitin oligomers. zinc finger (B-box type) family protein; similar to zinc finger (8- box type) family protein Encodes the large subunit of ADP-glucose pyrophosphorylase 143 263517_at 263875_at 263352_at 263452_at 264052_at 264001_at 264000_at 265290_at 267265_at 267262_at 267261_at 267266_at 245078_at 266578_at 266567_at 265999_at 266001_at 265990_at 265662_at 263799_at 257435_at 264379_at 265614_at 265634_at 265886_at 265913_at 266656_at 267377_at 263082_at 266209_at 266253_at 263435_at 263443_at 263412_at 266225_at 266790_at 266778_at 266279_at 2662 77_at 266265_at 266237_at 26661 7_at 266668_at 255866_at AT2621620 AT2621970 AT2622080 AT26221 90 AT2622330 AT2622420 AT2622500 AT2622590 AT2622980 AT2622990 AT26231 20 AT26231 50 AT2623340 AT2623910 AT2624050 AT26241 00 AT26241 50 AT2624280 AT2624500 AT2624550 AT2624590 AT2625200 AT2625355 AT2625530 AT2625620 AT2625625 AT2625900 AT2626250 AT2627200 AT2627550 AT2627840 AT2628600 AT2628630 AT2628720 AT2628900 AT2628950 AT2629090 AT2629290 AT262931 0 AT2629340 AT2629540 AT2629670 AT2629760 AT2630350 Encodes gene that is induced in response to dessieation stress enhanced protein 2 (SEP2) chlorophyll aIb-binding protein similar to zinc finger protein-related trehalose-phosphatase; similar to trehalose-6-phosphate phosphatase Encodes a cytochrome P450. peroxidase 17 (PER17) (P17) mitochondrial substrate carrier family protein glycosyltransferase family protein SCPL13; serine carboxypeptidase; similar to SCPL7 sinapoylglucosezmalate sinapoyltransferase. similar to unknown protein Encodes a member of the Nramp2 metal transporter family encodes a member of the DREB subfamily A-5 of ERF/AP2 transcription factor family. cinnamoyl-CoA reductase-related; similar to cinnamoyI-CoA reductase-related MIF4G domain-containing protein I MA3 domain-containing protein similar to unknown protein heptahelical transmembrane protein HHP3 serine carboxypeptidase S28 family protein Encodes a 02H2 zinc finger protein FZF. similar to unknown protein splicing factor similar to unknown protein exonuclease—related; similar to unknown protein AFG1-like ATPase family protein; similar to ATPase protein phosphatase 2C similar to 050590575000 putative Cys3His zinc finger protein (ATCTH) mRNA epidermis-specific GTP-binding family protein encodes a protein similar to TF L1 Belongs to the plant specific HD2 type proteins ATP binding IATP-dependent helicase] nucleic acid binding beta-ketoacyl-CoA synthase family protein histone H28 Encodes At0EP16 Encodes an expansin. Naming convention from the Expansin Working Group Encodes a protein with ABA 8'-hydroxyla5e activity tropinone reductase tropinone reductase short-chain dehydrogenase/reductase (SDR) family protein RNA polymerase I(A) and lIl(C) 14 kDa subunit binding; similar to binding [Arabidopsis thaliana] (TAIR:AT2G29670.1) pentatricopeptide (PPR) repeat-containing protein endo/excinuclease amino terminal domain-containing protein; similar to 050790230500 144 267516_at 267517_at 267576_at 266471_at 266474_at 263249_at 263252_at 263474__at 263467_at 265680_at 265730_at 2671 16_at 245164_at 255793_at 25581 7_at 255795_at 267454_at 267004_at 266996_at 266903_at 266899_at 267406_at 267429_at 267432_at . 265795_at 263951_at 263946_at 263919_at 265197_at 263867_at 263866_at 265471_at 265953_at 265952_at 267163_at 267177_at 267168_at 266093_at 267034_at 267036_at 26641 1_at 266979_at AT2630520 AT2630520 AT2630640 AT2631060 AT2631 1 10 AT2631360 AT2631380 AT2631725 AT2631730 AT2632150 AT2632220 AT2632560 AT2633210 AT2633250 AT2633330 AT2633380 AT2633730 AT2634260 AT2634490 AT2634570 AT2634620 AT2634780 AT2634850 AT2635020 AT2635780 AT2635960 AT2636000 AT2636470 AT2636750 AT2636830 AT2636950 AT26371 30 AT2637480 AT2637480 AT2637520 AT2637580 AT2637770 AT2637990 AT2638310 AT2638465 AT2638730 AT2639470 light inducible root phototropism 2 light inducible root phototropism 2 Mutator-like transposase family elongation factor family protein; similar to elongation factor family protein similar to unknown protein homologous to delta 9 acyl-lipid desaturases of cyanobacteria a B-box zinc finger protein that interacts with COP1. similar to unknown protein ethylene-responsive protein haloacid dehalogenase-like hydrolase family protein 608 ribosomal protein L27 (RPL27A); Identical to 60S ribosomal protein L27-1 (RPL27A) F-box family protein; similar to F-box family protein chaperonin similar to unknown protein 33 kDa secretory protein-related; similar to 33 kDa secretory protein-related Encodes a calcium binding protein whose mRNA is induced upon treatment with NaCl DEAD box RNA helicase transducin family protein I WD-40 repeat family protein Encodes a protein with C22-sterol desaturase activity. MEE21 (maternal effect embryo arrest 21); similar to Protein of unknown function DUF652 mitochondrial transcription termination factor-related I mTERF- related EMB1611/MEE22 (EMBRYO DEFECTIVE 1611 MEE25 (maternal effect embryo arrest 25); catalytic UTPuglucose-1-phosphate uridylyltransferase family protein SCPL26 (serine carboxypeptidase-like 26) Encodes a protein whose sequence is similar to tobacco hairpin-induced gene (HIN1) mitochondrial transcription termination factor-related I mTERF- related similar to unknown protein UGT7201 (UDP-glucosyl transferase 72C1) encodes a tonoplast intrinsic protein heavy-metal-associated domain-containing protein peroxidase 21 (PER21) (P21) (PRXR5) similar to unknown protein similar to unknown protein PHD finger family protein; similar to PHD finger transcription factor zinc finger (C3HC4-type RING finger) family protein aldo/keto reductase family protein; similar to aldo/keto reductase family protein ribosome biogenesis regulatory protein (RRS1) family protein similar to unknown protein unknown protein peptidyl-prolyl sis-trans isomerase photosystem II reaction center PsbP family protein 145 266965_at 266984__at 267592_at 245063_at 267356_at 267359_at 263824_at 263802_at 255877_at 255822_at 266076_at 266049_at 267080_at 263495_at 263497_at 263987_at 265265_at 260540_at 260556_at 260585_at 267367_at 267344__at 267371_at 266873_at 266876_at 266822_at 266820_at 2661 06__at 267509_at 266925__at 266927_at 266604_at 266583_at 266591_at 266552_at 266327_at 266324_at 26671 9_at 263320_at 266503_at 266514_at AT2639510 AT2639570 AT26 3971 0 AT2639795 AT2639930 AT2640020 AT2640360 AT2640430 AT2640460 AT264061 0 AT2640700 AT2640780 AT2641 1 90 AT2642530 AT2642540 AT2642690 AT2642900 AT2643500 AT2643620 AT2643650 AT264421 0 AT2644230 AT264451 0 AT2644740 AT2644820 AT2644860 AT2644940 AT26451 70 AT2645660 AT2645740 AT2645960 AT2646030 AT2646220 AT2646225 AT2646330 AT2646680 AT264671 0 AT2646830 AT26471 80 AT2647780 AT2647890 nodulin MtN21 family protein; similar to nodulin MtN21 family protein ACT domain-containing protein; similar to ACT domain- containing protein Encodes a Cysteine-rich peptide (CRP) family protein mitochondrial glycoprotein family protein I MAM33 family protein Encodes an isoamylase—type debranching enzyme. similar to unknown protein transducin family protein I WD-40 repeat family protein Identical to Protein At2940430 proton-dependent oligopeptide transport (POT) family protein member of Alpha-Expansin Gene Family DEAD/DEAH box helicase RNA binding I translation initiation factor amino acid transporter family protein; similar to amino acid transporter family protein cold-responsive protein I cold-regulated protein (cor15b) A cold-regulated gene whose product is targeted to the chloroplast Hpase similar to 050590582000 RWP-RK domain-containing protein chitinase Sas10/U3 ribonucleoprotein (Utp) family protein; similar to unknown protein similar to unknown protein similar to unknown protein p21Cip1-binding protein-related; Identical to Protein BCCIP homolog CYCP4;1 (cyclin p4;1); cyclin-dependent protein kinase; similar to CYCP4 similar to 050190224500 60$ ribosomal protein L24 encodes a member of the DREB subfamily A—4 of ERFIAP2 transcription factor family. Involved in autophagy. Under nutrient starvation the protein localizes to autophagosomes. AGL20 member of the peroxin11 (PEX11) gene family a member of the plasma membrane intrinsic protein subfamily PIP1. Ubiquitin conjugating enzyme E2 similar to unknown protein Encodes a subunit of the WAVE complex. Encodes arabinogalactan protein (AGP16). encodes a putative transcription factor rac GTPase activating protein CCA1 ATGOLS1 (ARABIDOPSIS THALIANA GALACTINOL SYNTHASE1) rubber elongation factor (REF) protein-related zinc finger (B-box type) family protein; Identical to CONSTANS- LIKE 13 (COL13) 146 266483_at 266460_at 266510_at 259278_at 258949_at 259181_at 258996_at 258998_at 258859_at 259104_at 259131_at 259132_at 259075_at 258502_at 258487_at 258474_at 258618_at 258603_at 258871_at 258849_at 259173_at 259347_at 258805_at 258537_at 258800_at 259311_at 259105_at 258893_at 258742_at 258562_at 258468_at 258472_at 256401_at 258882_at 258505_at 258535_at 258527_at 258552_at 258545_at 258834_at 259017_at AT2647910 AT2647930 AT2647990 AT3601 160 AT3601370 AT3601690 AT3601 800 AT3601820 AT3602120 AT3602170 AT3602180 AT3602250 AT3602320 AT3602490 AT3602550 AT3602650 AT3602885 AT3602990 AT3603060 AT3603250 AT3603640 AT3603920 AT3604010 AT3604210 AT3604550 AT3605060 AT3605500 AT3605660 AT3605800 AT3605980 AT3606070 AT3606080 AT3606200 AT3606330 AT3606530 AT3606750 AT3606850 AT360701 0 AT3607050 AT3607270 AT3607310 CRR6 (CHLORORESPIRATORY REDUCTION 6); similar to conserved hypothetical protein AGP26/ATAGP26 (ARABINOGALACTAN PROTEINS 26) Encodes a transducin family nucleolar protein with six WD40 similar to unknown protein similar to unknown protein similar to unknown protein, ribosome recycling factor family protein I ribosome releasing factor family protein adenylate kinase family protein; similar to ADKIATPADK1 (ADENOSINE KINASE) ' hydroxyproline-rich glycoprotein family protein Encodes LONGIFOLIA2 (LNG2). SPIRAL1-LIKE3 belongs to a six-member gene family in Arabidopsis+C949 similar to unknown protein RNA binding I tRNA (guanine—N2-)-methyltransferase pentatricopeptide (PPR) repeat-containing protein LOB domain protein 41 I lateral organ boundaries domain protein 41 (LBD41) pentatricopeptide (PPR) repeat-containing protein; similar to bindin GASAQS (GAST1 PROTEIN HOMOLOG 5); similar to gibberellin- regulated family protein member of Heat Stress Transcription Factor (Hsf) family ATPase; similar to AAA-type ATPase family protein ls thought to encode a cytosolic UDP-glucose pyrophosphorylase Encodes beta-glucosidase (GLUC). Gar1 RNA-binding region family protein glycosyl hydrolase family 17 protein; similar to glycosyl hydrolase family 17 protein disease resistance protein (TIR-NBS class) similar to unknown protein SAR DNA-binding protein rubber elongation factor (REF) family protein kinase/ protein binding; similar to disease resistance family protein transcription factor; similar to transcription factor similar to unknown protein similar to unknown protein similar to unknown protein guanylate kinase zinc finger (C3HC4-type RING finger) family protein BAP28-related; Identical to Hypothetical protein At3906530 hydroxyproline-rich glycoprotein family protein dihydrolipoamide branched chain acyltransferase pectate lyase family protein; Identical to Probable pectate lyase 8 precursor GTP-binding family protein; similar to GTP-binding family protein GTP cyclohydrolase 1; similar to GTP cyclohydrolase l similar to unknown protein 147 259018_at 259068_at 259227_at 258692_at 258993_at 258987_at 259207_at 259037_at 258979_at 258719_at 258708_at 258723_at 258928_at 258965_at 258774_at 256427_at 258727_at 256661_at 256262_at 256288_at 256266_at 256230_at 256235_at 256245_at 256237_at 257702_at 257694_at 257860_at 257188_at 257193_at 257658_at 257708_at 256649_at 256772_at 256779_at 258202_at 257280_at 258094_at 257237_at 257211_at AT3607390 AT3607560 AT3607750 AT3608640 AT3608940 AT3608950 AT3609050 AT3609350 AT3609440 AT3609540 AT3609580 AT3609600 AT3610070 AT3610530 AT3610740 AT361 1090 AT361 1 930 AT361 1 964 AT36121 50 AT3612270 AT3612320 AT3612340 AT3612490 AT3612580 AT361261 0 AT3612670 AT3612860 AT361 3062 AT361 31 50 AT361 3160 AT361 3230 AT3613330 AT3613570 AT3613750 AT3613784 AT361 3940 AT3614440 AT3614690 AT3614890 AT361 5080 isolated from differential screening of a cDNA library from auxin- treated root culture. Encodes peroxin 13 (PEX13) involved in protein transport into peroxrsomes. 3' exoribonuclease family domain 1-containing protein alphavirus core protein family; similar to unknown protein Lhcb4.2 protein (Lhcb4.2 electron transport SCO1ISenC family protein similar to 050190853000 armadillo/beta-catenin repeat family protein heat shock cognate 70 kDa protein 3 (HSC70-3) (HSP70-3) pectate lyase family protein; similar to pectate lyase family protein amine oxidase family protein; similar to HEMGZIMEE61 (maternal effect embryo arrest 61) myb family transcription factor; similar to DNA binding I transcription factor putative TBP-associated 58 kDa subunit protein (TAFII58) transducin family protein IWD-40 repeat family protein Encodes a bifunctional alpha-I-arabinofuranosidase/beta-d- xylosidase LOB domain family protein / lateral organ boundaries domain family protein (LBDZ1) universal stress protein (USP) family protein S1 RNA-binding domain-containing protein similar to unnamed protein product methyltransferase; similar to protein arginine N- methyltransferase similar to unknown protein F K506 binding I peptidyl-prolyl cis-trans isomerase; similar to immunophilin-related ' cysteine protease inhibitor HSP70 (heat shock protein 70); ATP binding Plays role in DNA-damage repair/toleration. EMBZ742 (EMBRYO DEFECTIVE 2742); CTP synthase; similar to CTP synthase nucleolar protein Nop56 similar to unknown protein pentatricopeptide (PPR) repeat—containing protein pentatricopeptide (PPR) repeat-containing protein nucleic acid binding; similar to lsopenicillin N synthetase; KH binding; similar to proteasome activator subunit 4-Iike encodes an SC35—like splicing factor of 30 kD that is localized to the nuclear specks. beta-galactosidase ATCWINV5 (ARABIDOPSIS THALIANA CELL WALL INVERTASE 5) DNA binding / DNA-directed RNA polymerase; similar to 051190615100 Encodes 9-cis-epoxycarotenoid dioxygenase putative cytochrome P450 phosphoesterase; similar to poly (ADP-ribose) polymerase exonuclease family protein; similar to exonuclease 148 257058_at 258397_at 258402_at 258396_at 258225_at 258270_at 258054_at 258055_at 259329_at 259375_at 257206_at 258434_at 257650_at 257652_at 257648_at 256790_at 257880_at 257876_at 258375_at 258214_at 258215_at 258218_at 258156_at 258155_at 256797_at 257801_at 257803_at 257035_at 258015_at 258024_at 258006_at 258009_at 257966_at 257964_at 257131_at 258034_at 258166_at 258167_at 257262_at 256796_at 258316_at AT361 5352 AT361 5357 AT3615450 AT361 5460 AT361 5630 AT3615650 AT3616240 AT3616250 AT3616360 AT3616370 AT3616530 AT3616770 AT3616800 AT3616810 AT3616840 AT3616857 AT3616910 AT3617130 AT3617470 AT3617970 AT3617970 AT3618000 AT3618050 AT361 81 30 AT3618600 AT361 8750 AT3618790 AT3619270 AT3619340 AT3619360 AT3619400 AT3619440 AT3619800 AT3619850 AT3620240 AT3621300 AT3621 540 AT3621560 AT3621 890 AT3622210 AT3622660 Encodes protein similar to yeast COX17 similar to zinc finger protein-related similar to unknown protein brix domain-containing protein; similar to brix domain-containing pnnefii similar to unknown protein phospholipase/carboxylesterase family protein Delta tonoplast intrinsic protein ferredoxin-related; similar to ferredoxin-related Encodes AH P4 GDSL-motif lipase/hydrolase family protein; similar to GDSL- rnofiffipase Lectin like protein whose expression is induced upon treatment with chitin oligomers. RAP2.3 protein phosphatase 2C APUM24 (ARABIDOPSIS PUMILIO 24) ATP-dependent helicase Encodes an Arabidopsis response regulator (ARR) protein Encodes a peroxisomal protein with acetyI-CoA synthetase acfivfl invertzselpectin methylesterase inhibitor family protein RelA/SpoT domain-containing protein I calcium-binding EF- hand family protein chloroplast outer membrane translocon subunit chloroplast outer membrane translocon subunit Arabidopsis thaliana N-methyltransferase-Iike protein mRNA. similar to unknown protein RACK1 DEAD/DEAH box helicase Encodes a member of the WNK family (9 members in all) of protein kinases similar to 2 coiled coil domains of eukaryotic ori (GB:BAD19345.1) Encodes a protein with ABA 8'-hydroxyla5e activity sodiumzdicarboxylate symporter; similar to unknown protein zinc finger (CCCH-type) family protein; similar to zinc finger (CCCH-type) family protein cysteine proteinase pseudouridine synthase family protein similar to conserved hypothetical protein phototropic-responsive NPH3 family protein mitochondrial substrate carrier family protein RNA methyltransferase family protein; similar to zinc finger (CCCH-type) family protein transducin family protein IWD-40 repeat family protein Encodes a protein with sinapic acid:UDP-glucose glucosyltransferase activity. zinc finger (B-box type) family protein; similar to DNA binding I zinc ion binding similar to fortune-1 rRNA processing protein-related 149 258317_at 258321_at 258101_at 258104_at 257203_at 257171_at 257173_at 256890_at 256914_at 256905_at 257253_at 257868_at 256754_at 257643_at 258078_at 258079_at 256881_at 256880_at 25761 5_at 25731 3_at 25761 1_at 257832_at 258254_at 258256_at 256980_at 257789_at 257154_at 257149_at 257970_at 257226_at 257271_at 257299_at 257300_at 257071_at 256577_at 257745_at 256924_at 245228_at 252661_at 252639_at 252625_at 252615_at 252591_at 252548_at AT3622670 AT3622840 AT3623590 AT3623620 AT3623730 AT3623760 AT362381 0 AT3623830 AT3623880 AT3623990 AT3624190 AT3625070 AT3625690 AT3625730 AT3625870 AT3625940 AT3626410 AT3626450 AT362651 0 AT3626520 AT3626580 AT3626740 AT3626782 AT3626890 AT3626932 AT3627020 AT362721 0 AT3627280 AT3627570 AT3627880 AT3628007 AT3628050 AT3628080 AT36281 80 AT3628220 AT3629240 AT3629590 AT362981 0 AT3644450 AT3644550 AT3644750 AT3645230 AT3645600 AT3645850 pentatricopeptide (PPR) repeat-containing protein; similar to bflufln Encodges an early light-inducible protein. similar to structural constituent of ribosome brix domain-containing protein; Identical to Brix domain- containing protein 1 homolog xyloglucanzxyloglucosyl transferase similar to transferase ' SAHH2 (S-ADENOSYL-L-HOMOCYSTEINE (SAH) HYDROLASE 2) encodes a glycine-rich RNA binding protein. F-box family protein; similar to F-box family protein mitochondrial chaperonin HSP ABC1 family protein; similar to ABC1 family protein RIN4 actin binding protein required for normal chloroplast positioning AP2 domain-containing transcription factor similar to unknown protein transcription factor S-Il (TFIIS) domain—containing protein methyltransferase] nucleic acid binding; similar to RNA methylase-like protein major latex protein-related I MLP-related; similar to major latex protein-related / MLP-related octicosapeptide/Phox/Bem1p (PB1) domain-containing protein gamma tonoplast intrinsic protein 2 (T 1P2) binding; similar to hypothetical protein CCL binding; similar to pentatricopeptide (PPR) repeat-containing protein similar to unknown protein . DRB3 (DSRNA-BINDING PROTEIN 3) Arabidopsis thaliana metal-nicotianamine transporter YSL6 similar to unknown protein ATPHB4 (PROHIBITIN 4) similar to unknown protein similar to unknown protein nodulin MtN3 family protein nodulin MtN21 family protein nodulin MtN21 family protein encodes a gene similar to cellulose synthase meprin and TRAF homology domain-containing protein similar to unknown protein At3929590 (At5MAT) encodes a malonyI-CoA phytochelatin synthetase family protein I COBRA cell expansion protein COBL2 similar to unknown protein oxidoreductase Encodes a histone deacetylase. , hydroxyproline-rich glycoprotein family protein; similar to unknown protein Member of TETRASPANIN family kinesin motor protein-related 150 252534_at 252508_at 252529_at 252467_at 252429_at 252433_at 252374_at 252353_at 252355_at 252367_at 252321_at 252316_at 252317_at 252252_at 252305_at 252281_at 252250_at 252239_at 252214_at 252199_at 252168_at 252167_at 252178_at 252179_at 2521 54_at 2521 27_at 2521 02_at 2521 23_at 252092__at 252117_at 252076_at 246307_at 246304_at 246310_at 252034_at 252040_at 256676_at 256677_at 256671_at 256674_at 25201 1_at 251973_at 251975_at AT36461 30 AT364621 0 AT3646490 AT3647080 AT3647500 AT3647560 AT36481 00 AT3648200 AT3648250 AT3648360 AT364851 0 AT3648700 AT3648720 AT36491 80 AT3649240 AT3649320 AT3649790 AT3649990 AT3650260 AT3650270 AT3650440 AT3650560 AT3650750 AT3650760 AT3650880 AT3650960 AT3650970 AT3651 240 AT3651420 AT3651430 AT3651660 AT3651800 AT3651840 AT3651895 AT3652040 AT3652060 AT3652180 AT3652190 AT3652290 AT3652360 AT3652720 AT3653180 AT3653230 Encodes a putative transcription factor (MYB48). 3' exoribonuclease family domain 1-containing protein oxidoreductase binding; similar to binding [Arabidopsis thaliana] (TAI R:AT3G47080. 1 ) Def-type zinc finger domain-containing protein esterase/lipaselthioesterase family protein Encodes a transcription repressor similar to 050590594500 pentatricopeptide (PPR) repeat-containing protein BT2 similar to unknown protein similar to unknown protein transferase family protein transducin family protein I WD-40 repeat family protein EMB1796 (EMBRYO DEFECTIVE 1796) similar to unknown protein similar to ATPP2-A10 (Phloem protein 2-A10) similar to 050390372700 encodes a member of the DREB subfamily A-5 of ERFIAP2 transcription factor family. transferase family protein hydrolase; similar to esterase short-chain dehydrogenase/reductase (SDR) family protein brassinosteroid signalling positive regulator-related; Identical to BEH1 Encodes a protein with putative 9alacturonosyltransferase activity. HhH-GPD base excision DNA repair family protein similar to unknown protein Belongs to the dehydrin protein family Encodes flavanone 3-hydroxylase strictosidine synthase family protein; similar to YLS2 (yellow- leaf-specific gene 2) strictosidine synthase-like protein macrophage migration inhibitory factor family protein I MIF family protein putative nuclear DNA-binding protein G2p (AtG2) mRNA Encodes a short-chain acyl-CoA oxidase Encodes a sulfate transporter. similar to unknown similar to unknown protein Encodes a plant-specific protein phosphatase Encodes a plant specific protein structurally related to the SEC12 IQD3 (IQ-domain 3); calmodulin binding; similar to IQDZ (IQ- domain 2) similar to unknown protein carbonic anhydrase family protein; similar to carbonic anhydrase family protein glutamate-ammonia Iigase; similar to glutamine synthetase cell division cycle protein 48 151 251984_at 251987_at 251941_at 251931_at 251927_at 251886_at 251899_at 251827_at 251800_at 251793_at 251759_at 251753_at 251768_at 251740_at 251725_at 251657_at 251668_at 251667_at 251629_at 251641_at 251638_at 251593_at 251620_at 251575_at 251529_at 251538_at 251506_at 251476_at 251432_at 251427_at 251371_at 251372_at 251391_at 251 346_at 251 356_at 251 336_at 251360_at 251324_at 251323_at 251296_at 251265_at 251267_at AT3653260 AT3653280 AT3653470 AT3653950 AT3653990 AT3654260 AT3654400 AT3655120 AT3655510 AT3655580 AT3655630 AT3655760 AT3655940 AT3656070 AT3656260 AT3657000 AT3657010 AT36571 50 AT365741 0 AT3657470 AT3657490 AT3657660 AT3658060 AT36581 20 AT3658570 AT3658660 AT3659090 AT3659670 AT3659820 AT36601 30 AT3660360 AT3660520 AT3660910 AT3660980 AT3661 060 AT3661 1 90 AT3661210 AT3661430 AT3661580 AT3662010 AT3662310 AT3662330 Encodes phenylalanine lyase. cytochrome P450 monooxygenase similar to 050390285100 glyoxal oxidase—related; similar to glyoxal oxidase-related universal stress protein (USP) family protein similar to unknown protein aspartyl protease family protein; similar to pepsin A Catalyzes the conversion of chalcones into flavanones. similar to unknown protein regulator of chromosome condensation (RCC1) family protein ATDF D (A. THALIANA DHFS—FPGS HOMOLOG 0) similar to unknown protein phosphoinositide-specific phospholipase C rotamase cyclophilin 2 (R002) similar to unknown protein nucleolar essential protein-related; similar to 050290290400 strictosidine synthase family protein; similar to strictosidine synthase family protein Encodes a putative pseudouridine synthase (NAP57). Encodes a protein with high homology to animal villin. peptidase M16 family protein I insulinase family protein 408 ribosomal protein 82 (RPSZD); Identical to 40S ribosomal protein 824 (RPS2D) Encodes a subunit of RNA polymerase I (aka RNA polymerase A). cation efflux family protein I metal tolerance protein bZIP transcription factor family protein DEAD box RNA helicase 608 ribosomal protein-related similar to TOM1 (TOBAMOVIRUS MULTIPLICATION 1) similar to unknown protein calcium-binding mitochondrial protein-related glycosyl hydrolase family 1 protein I beta-glucosidase EDA14IUTP11 (U3 SMALL NUCLEOLAR RNA-ASSOCIATED PROTEIN 11 zinc ion binding; similar to unknown protein catalytic; similar to catalytic [Arabidopsis thaliana] (TAIR:AT3G17365. 1) pentatricopeptide (PPR) repeat-containing protein+C612 ATPP2-A13; similar to ATPP2-A12 (Phloem protein 2-A12) Encodes a protein with a 02 domain that binds to BON1 in yeast two hybrid analyses. embryo-abundant protein-related; similar to em bryo-abundant protein-related a member of the plasma membrane intrinsic protein subfamily PIP1. delta-8 sphingolipid desaturase (SLD1); similar to delta-8 sphingolipid desaturase metal ion binding I oxidoreductase; similar to 050390586700 RNA helicase zinc knuckle (CCHC-type) family protein; similar to unknown protein 152 251218_at 251227_at 251229_at 251235_at 251200_at 251205_at 251169_at 255700_at 255637_at 255626_at 255645_at 255617_at 255579_at 255543_at 255561_at 255517_at 255524_at 255501_at 255452_at 255411_at 255436_at 255434_at 255365_at 255278_at 255310_at 255259_at 255225_at 255070_at 255032_at 255011_at 255008_at 255807_at 254991_at 254938_at 254919_at 254931_at 254890_at 254848_at 254850_at 25481 5_at AT366241 0 AT3662700 AT3662740 AT3662860 AT366301 0 AT3663080 AT366321 0 AT4600200 AT4600750 AT4600780 AT4600880 AT4601 330 AT4601460 AT4601 870 AT4602050 AT4602290 AT4602330 AT4602400 AT4602880 AT46031 1 0 AT46031 50 AT46031 80 AT4604040 AT4604940 AT4604955 AT4605020 AT460541 0 AT4609020 AT4609500 AT461 0040 AT461 0060 AT461 0270 AT461 0620 AT461 0770 AT461 1 360 AT461 1460 AT461 1600 AT461 1 960 AT4612000 AT4612420 CP12-2 encodes a small peptide found in the chloroplast stroma. member of MRP subfamily glycosyl hydrolase family 1 protein; similar to glycosyl hydrolase family 1 protein esterase/lipaselthioesterase family protein Encodes a gibberellin (GA) receptor ortholog of the rice GA receptor gene (05GID1). ' Encodes glutathione peroxidase. encodes a novel zinc-finger protein with a proline-rich N- terminus DNA binding; similar to DNA-binding family protein dehydration-responsive family protein; similar to dehydration- responsive family protein meprin and TRAF homology domain-containing protein I MATH domain-containing protein auxin-responsive family protein protein kinase family protein; similar to protein kinase family protein basic helix-loop-helix (bHLH) family protein toIB protein-related; similar to unknown protein sugarfiansponer glycosyl hydrolase family 9 protein; Identical to Endoglucanase 17 precursor (EC 3.2.1.4) pectinesterase family protein; similar to pectinesterase family protein similar to U3 ribonucleoprotein (Utp) family protein similar to unknown protein RNA-binding protein similar to 051290534100 similar to hypothetical protein DDBDRAFT_0185878 MEE51 (maternal effect embryo arrest 51) transducin family protein I WD-40 repeat family protein Encodes an allantoinase which is involved in allantoin degradation and assimilation. NDBZ (NAD(P)H DEHYDROGENASE B2); disulfide oxidoreductase transducin family protein I WD-40 repeat family protein Encodes an isoamylase-Iike protein. glycosyltransferase family protein Encodes cytochrome c similar to unknown protein wound-responsive family protein similar to GTP binding oligopeptide transporter Encodes a putative RING-H2 finger protein RHA1b. protein kinase family protein; similar to protein kinase family protein Encodes glutathione peroxidase. similar to unknown protein similar to unknown protein encodes a protein of unknown function involved in directed root tip growth. 153 254806_at 254818_at 254805_at 254832_at 254819_at 254747_at 254688_at 245325_at 245605_at 245265_at 245592_at 245563_at 245306_at 245533_at 245352_at 245321_at 245281_at 245512_at 245523_at 245319_at 245391_at 245318_at 245266_at 245346_at 245264_at 245399_at 245362_at 245308_at 245426_at 245427_at 245401_at 254656_at 254662_at 254667_at 254636_at 254609_at 254563_at 254561_at 254564_at 254562_at 254580_at 254573_at AT461 2430 AT461 2470 AT461 2480 AT461 2490 AT461 2500 AT461 3020 AT461 3830 AT46141 30 AT4614300 AT4614400 AT4G14540 AT461 4580 AT4614690 AT461 51 30 AT461 5490 AT461 5545 AT461 5560 AT461 5770 AT461 5910 AT4616146 AT4616520 AT461 6980 AT461 7070 AT461 7090 AT461 7245 AT461 7340 AT461 7460 AT461 7486 AT461 7540 AT461 7550 AT461 7670 AT461 8070 AT461 8270 AT4618280 AT4618700 AT4618970 AT461 9120 AT461 9160 AT461 91 70 AT4619230 AT4619390 AT4619420 trehalose-6-phosphate phosphatase protease inhibitor/seed storage/lipid transfer protein (LTP) family protein a putative lipid transfer protein protease inhibitor/seed storage/lipid transfer protein (LTP) family protein protease inhibitor/seed storage/lipid transfer protein (LTP) family protein ' Encodes a member of the cd62+ family of protein kinases MHK. DnaJ-Iike protein (J20); nuclear gene xyloglucan endotransglycosylase-related protein (XTR7) heterogeneous nuclear ribonucleoprotein encodes a novel protein with putative ankyrin and transmembrane regions. CCAAT-box binding transcription factor subunit 8 (NF-YB) (HAP3 ) (AHAP3) family CBL-interacting protein kinase Encodes an early light-induced protein. cholinephosphate cytidylyltransferase Encodes a protein that might have sinapic acid2UDP—glucose glucosyltransferase activity. similar to unknown protein Encodes a protein with 1-deoxyxylulose 5-phosphate synthase acfiv' 60$ lrtiybosome subunit biogenesis protein DI21 similar to unknown protein ATG8F (AUTOPHAGY 8F); microtubule binding; similar to AtATG8e (AUTOPHAGY 8E) arabinogalactan-protein family similar to 050390100300 Encodes a beta-amylase targeted to the chloroplast. zinc finger (C3HC4-type RING finger) family protein DELTA-TIPZI'I'IP2;2 (tonoplast intrinsic protein 2;2) Encodes homeobox protein HAT1. Identical to UPF0326 protein At4g17486 similar to 051090563400 transporter-related; similar to glycerol-3-phosphate transporter senescence-associated protein-related similar to unknown protein Encodes protein similar to similar to bacterial translocase I (mra Eyeine-rich cell wall protein-related Encodes CBL-interacting protein kinase 12 (CIPK12). GDSL-motif lipase/hydrolase family protein ERD3 (EARLY-RESPONSIVE T0 DEHYDRATION 3) binding; similar to similar to Uncharacterized conserved protein similar to nine-cis-epoxycarotenoid dioxygenase Encodes a protein with ABA 8'-hydroxylase activity similar to structural constituent of ribosome pectinacetylesterase family protein; similar to pectinacetylesterase 154 254574_at 254553_at 254543_at 254547_at 254505_at 254446_at 254452_at 254455_at 254378_at 254387_at 254384_at 254305_at 254304_at 254355__at 254314_at 254321_at 254333_at 254256_at 254239_at 25421 0_at 254232_at 254227_at 2541 88_at 254185_at 2541 57_at 254145_at 2541 19_at 2541 33_at 254085_at 254105_at 2541 10_at 254054_at 254076_at 254075_at 254066__at 254073_at 254080_at 254077_at 254079_at 254032_at 254043_at AT461 9430 AT461 9530 AT461981 0 AT461 9860 AT4619985 AT4620890 AT4621'100 AT4621 140 AT4621 81 0 AT4621850 AT4621 870 AT4622200 AT4622270 AT4622380 AT4622470 AT4622590 AT4622753 AT46231 80 AT4623400 AT4623450 AT4623600 AT4623630 AT4623920 AT4623990 AT4624220 AT4624700 AT4624780 AT462481 0 AT4624960 AT4625080 AT4625260 AT4625320 AT4625340 AT4625470 AT4625480 AT4625500 AT4625630 AT4625640 AT4625730 AT4625940 AT4625990 unknown protein disease resistance protein (TlR-N BS—LRR class) glycosyl hydrolase family 18 protein; similar to glycosyl hydrolase family 18 protein lecithin2cholesterol acyltransferase family protein I LACT family roteln gCNS-related N-acetyltransferase (GNAT) family protein tubulin 9 ' DDB1 similar to unknown protein Der1 -like family protein Idegradation in the ER-Iike family protein methionine sulfoxide reductase domain-containing protein I SelR domain-containing protein 26.5 kDa class P-related heat shock protein (HSP26.5-P) Encodes a photosynthate- and light-dependent inward rectifying potassium channel similar to unknown protein ribosomal protein L7AeIL30e/S12elGadd45 family protein protease inhibitor/seed storage/lipid transfer protein (LTP) family protein trehalose-6-phosphate phosphatase Encodes a member of the SM01 family of sterol 4alpha-methyl oxidases. Encodes a receptor-like protein kinase. PIP1 zinc finger (C3HC4—type RING finger) family protein Encodes cystine lyase which is expected to be involved in amino acid metabolism BTI1 (VIRBZ-INTERACTING PROTEIN 1) Encodes a protein with UDP-D-glucose 4-epimerase activity. encodes a protein similar to cellulose synthase encodes a novel protein containing mammalian death domain similar to 050290595200 pectate lyase family protein similar to ABC1 family protein Homologous to a eukaryote specific ABA- and stress-inducible ene Encodes a protein with methyltransferase invertase/pectin methylesterase inhibitor family protein DNA-binding protein-related; similar to DNA—binding protein- related immunophilin-related I F KBP-type peptidyl-prolyl cis-trans isomerase-related CBF2 CBF3 encodes an arginine/serine-rich splicing factor. encodes a fibrillarin MATE efflux family protein; similar to MATE efflux family protein FtsJ-like methyltransferase family protein epsin N-terminal homology (ENTH) domain-containing protein chloroplast import apparatus CIA2-like. 155 253994_at 253971_at 253973_at 253975_at 253949_at 253917_at 253872_at 253874_at 253875_at 253886_at 253835_at 253841_at 253824_at 253828_at 253849_at 253803_at 253815_at 253806_at 253777_at 253722_at 253709_at 253695_at 253684_at 253608_at 253627_at 253597_at 253595_at 253548_at 253559_at 253534_at 253490_at 253425_at 253440_at 25341 1_at 253412_at 253416_at 253302_at 253305_at 253293_at 253322_at 253282_at AT4626080 AT4626530 AT4626555 AT4626600 AT4626780 AT4627380 AT462741 0 AT4627450 AT4627520 AT4627710 AT4627820 AT4627830 AT4627940 AT4627970 AT4628080 AT4628200 AT4628250 AT4628270 AT4628450 AT4629190 AT4629220 AT462951 0 AT4629690 AT4630290 AT4630650 AT4630690 AT4630830 AT4630993 AT4631 140 AT4631500 AT4631 790 AT46321 90 AT4632570 AT4632980 AT4633000 AT4633070 AT4633660 AT4633666 AT4633905 AT4633980 AT46341 20 Involved in abscisic acid (ABA) signal transduction. fructose-bisphosphate aldolase immunophilin I FKBP-type peptidyI-prolyl cis-trans isomerase family protein nucleolar protein unknown function similar to hypothetical protein MtrDRAFT_AC12601395v1 Encodes a NAC transcription factor induced in response to dessieation. similar to unknown protein plastocyanin-like domain-containing protein member of CYP7098 glycosyl hydrolase family 1 protein; similar to hydrolase glycosyl hydrolase family 1 protein; similar to glycosyl hydrolase family 1 protein mitochondrial substrate carrier family protein C4-dicarboxylate transporter/malic acid transport family protein binding; similar to binding [Arabidopsis thaliana] (TAIR:AT4G28080.1) similar to Ribosomal protein L29 putative beta-expansinlallergen protein. zinc finger (C3HC4-type RING finger) family protein; similar to RMA1 transducin family protein I WD-40 repeat family protein zinc finger (CCCH-type) family protein; similar to zinc finger (CCCH-type) family protein phosphofructokinase family protein protein arginine N-methyltransferase type I phosphodiesterase/nucleotide pyrophosphatase family rotein gutative xyloglucan endotransglycosylaselhydrolase hydrophobic protein translation initiation factor 3 (IF-3) family protein similar to unknown protein similar to unknown protein glycosyl hydrolase family 17 protein; Identical to Putative glucan endo-1 Encodes an oxime-metabolizing enzyme in the biosynthetic pathway of glucosinolates. diphthine synthase centromeric protein-related; similar to protein transport protein- related contains lnterPro domain ZIM; (lnterProzlPR010399) Encodes transcription factor involved in photomorphogenesis. Encodes a member of the calcineurin B-like calcium sensor gene family. pyruvate decarboxylase similar to proline-rich family protein unknown protein peroxisomal membrane protein 22 kDa similar to unknown protein LEJ1 (LOSS OF THE TIMING 0F ET AND JA BIOSYNTHESIS 1) 156 253277_at 253254_at 253203_at 253252_at 253255_at 253219_at 253141_at 253130_at 253163_at 253161_at 253104_at 253129_at 246275_at 246282_at 246249_at 2461 96_at 253049__at 253097_at 253061_at 253040_at 253021_at 253027_at 252986_at 252997_at 252990_at 252956_at 252950_at 252972_at 252965_at 252968_at 252888_at 252882_at 252880_at 252863_at 252871_at 251 142_at 251 136__at 251090_at AT4634230 AT4634650 AT463471 0 AT4634740 AT4634760 AT4634990 AT4635440 AT463551 0 AT4635750 AT4635770 AT463601 0 AT4636020 AT4636540 AT4636580 AT4636680 AT4637090 AT4637300 AT4637320 AT4637610 AT4637800 AT4638050 AT46381 50 AT4638380 AT4638400 AT4638440 AT4638580 AT4638690 AT4638840 AT4638860 AT4638890 AT4639210 AT4639675 AT4639730 AT4639800 AT4640000 AT5601015 AT5601290 AT5601340 Encodes a catalytically active cinnamyl alcohol dehydrogenase squalene synthase encodes a arginine decarboxylase (ADC) Encodes glutamine 5-phosphoribosylpyrophosphate amidotransferase. auxin-responsive family protein Member of the R2R3 factor gene family. CLC-e chloride‘channel protein similar to unknown protein Rho-GTPase-activating protein-related; similar to unknown protein Senescence—associated gene that is strongly induced by phosphate starvation. pathogenesis-related thaumatin family protein; CSDP1 (COLD SHOCK DOMAIN PROTEIN 1) BEE2 (BR ENHANCED EXPRESSION 2); DNA binding I transcription factor AAA-type ATPase family protein; similar to AAA-type ATPase family protein pentatricopeptide (PPR) repeat-containing protein similar to 050290186700 MEE59 (maternal effect embryo arrest 59); similar to 050590451300 member of CYP81 D BT5 (BTB and TAZ domain protein 5); protein binding I transcription regulator xyloglucanzxyloglucosyl transferase permease; Identical to Nucleobase-ascorbate transporter 11 (AtNAT11) pentatricopeptide (PPR) repeat-containing protein MATE efflux protein-related; similar to MATE efflux family protein member of EXPANSIN-LIKE. Naming convention from the Expansin Working Group similar to 050690574400 putative farnesylated protein (At4g38580) mRNA 1-phosphatidylinositol phosphodiesterase-related auxin-responsive protein auxin-responsive protein dihydrouridine synthase family protein; similar to FAD binding I oxidoreductase Encodes the large subunit of ADP-Glucose Pyrophosphorylase which catalyzes the first unknown protein lipid-associated family protein; similar to lipid-associated family protein Columbia myo-inositol-1-phosphate synthase mRNA NOL1INOP2I5un family protein; similar to NOL1INOP2I5un family protein similar to unknown protein mRNA guanylyltransferase; similar to mRNA capping enzyme family protein mitochondrial substrate carrier family protein 157 251084_at 251063_at 251066_at 251068_at 251029_at 250994_at 251005_at 251017_at 250974_at 250987_at 245701_at 245699_at 245714_at 245711_at 250842_at 250891_at 250856_at 250817_at 250825_at 250777_at 250779_at 250752_at 250742_at 250758_at 250711_at 250729_at 250679_at 250648_at 250669_at 250665_at 250582_at 250558_at 250546_at 246011_at 250529_at 250538_at 250533_at 245879_at 250502_at 250504_at 250452_at 245906_at AT5601 520 AT5601850 AT5601880 AT5601 920 AT5602050 AT5602490 AT5602590 AT5602760 AT5602820 AT5602860 AT56041 40 AT5604250 AT5604280 AT5604340 AT5604490 AT5604530 AT5604810 AT5604940 AT5605210 AT5605440 AT5605470 AT5605690 AT5605800 AT5606000 AT5606110 AT5606460 AT5606550 AT5606760 AT5606870 AT5606980 AT5607580 AT5607990 AT56081 80 AT5608330 AT560861 0 AT5608620 AT5608640 AT5609420 AT5609590 AT5609840 AT5610630 AT561 1 070 zinc finger (C3HC4—type RING finger) family protein protein kinase zinc finger (C3HC4-type RING finger) family protein; Identical to ATL5A (ATL5A) STN8 mitochondrial glycoprotein family protein I MAM33 family protein heat shock cognate 70 kDa protein 2 (HSC70—2) (HSP702) chloroplast lumen common family protein protein phosphatase 2C family protein I PP2C family protein Involved in the patterning and shape of leaf trichomes. pentatricopeptide (PPR) repeat-containing protein Encodes a gene whose sequence is similar to Fd-GOGAT OTU-like cysteine protease family protein glycine-rich RNA-binding protein; similar to glycine-rich RNA- binding protein putative c2h2 zinc finger transcription factor mRNA Encodes a protein with phytol kinase activity involved in tocopherol biosynthesis. beta-ketoacyI-CoA synthase family protein pentatricopeptide (PPR) repeat-containing protein Encodes a SU(VAR)3-9 homolog nucleolar matrix protein-related; similar to nucleolar matrix protein-related similar to unknown protein protein synthesis initiation factor elF2 alpha Encodes a member of the CP90A family similar to unknown protein One of the 2 genes that code for the G subunit of eukaryotic initiation factor 3 (EIF3). DNAJ heat shock N-terminal domain-containing protein I cell division protein-related Encodes a ubiquitin-activating enzyme (E1) similar to transcription factor jumonji (jij) domain-containing rotein lpate embryogenesis abundant group 1 domain-containing protein polygalacturonase inhibiting protein 2 (PGIP2) mRNA similar to unknown protein encodes a member of the ERF subfamily B-3 of ERFIAP2 transcription factor family. Required for flavonoid 3' hydroxylase activity. ribosomal protein L7AeIL30e/S12e/Gadd45 family protein TCP family transcription factor DEAD box RNA helicase (RH26) DEAD box RNA helicase (RH25) Encodes a flavonol synthase that catalyzes formation of flavonols from dihydroflavonols. chloroplast outer membrane translocon subunit heat shock protein 70 (Hsc70—5); nuclear similar to unknown protein elongation factor 1-alpha similar to unknown protein 158 245904_at 250418_at 250318_at 250309_at 245984_at 250279_at 250252_at 250203_at 250222_at 250217_at 250180_at 250192_at 250194_at 250196_at 246596_at 246597_at 246566_at 250152_at 250158_at 250110_at 246559_at 246528_at 246527_at 246487__at 246484_at 246457_at 246461_at 24641 9_at 246467_at 246468_at 250072_at 250083_at 246435_at 250062_at 250053_at 250066_at 250012_x_at 250016_at 250017_at 249984_at 250009_at AT5611110 AT561 1240 AT5612200 AT561 2220 AT561 3090 AT561 3200 AT5613750 AT5613980 AT5614050 AT5614120 AT5614450 AT5614520 AT5614550 AT5614580 AT5614740 AT5614760 AT5614940 AT561 5120 AT561 5190 AT561 5350 AT5615550 AT561 5640 AT5615750 AT5616030 AT5616040 AT5616750 AT5616930 AT5617030 AT5617040 AT5617050 AT561 721 0 AT5617220 AT561 7460 AT561 7760 AT561 7850 AT5617930 AT5618060 AT5618100 AT561 8140 AT5618400 AT5618440 Encodes a protein with putative sucrose-phosphate synthase acfivi. transgucin family protein I WD-40 repeat family protein dihydropyrimidinase I DH Pase I dihydropyrimidine amidohydrolase I hydantoinase (PYDZ) las1-like family protein; similar to Las1-like similar to unknown protein GRAM domain-containing protein IABA-responsive protein- related ZIFL1 (ZINC INDUCED FACILITATOR-LIKE 1) glycosyl hydrolase family 38 protein transducin family protein l WD-40 repeat family protein nodulin family protein; similar to nodulin family protein GDSL-motif lipase/hydrolase family protein pescadiIIo-related; similar to BRCT; Pescadillo similar to unknown protein polyribonucleotide nucleotidyltransferase Encodes a beta carbonic anhydrase likely to be localized in the cytoplasm. At5914760 encodes for L-aspartate oxidase involved in the early steps of NAD biosynthesis proton-dependent oligopeptide transport (POT) family protein similar to unknown protein unknown protein plastocyanin-like domain-containing protein transducin family protein / WD-40 repeat family protein mitochondrial substrate carrier family protein RNA-binding S4 domain-containing protein; similar to hypothetical protein similar to unknown protein _ regulator of chromosome condensation (RCC1) family protein mutant has Female gametophyte; WD-40 Repeat Protein AAA-type ATPase family protein; similar to AAA-type ATPase family protein UDP-glucoronosyllUDP-glucosyl transferase family protein UDP-glucoronosleUDP-glucosyl transferase family protein The At5917050 encodes a anthocyanidin 3-0- glucosyltransferase similar to unknown protein Encodes glutathione transferase belonging to the phi class of GSTs. similar to conserved hypothetical protein AAA-type ATPase family protein; similar to AAA-type ATPase family protein cation exchanger RNA binding; similar to MIF4G domain-containing protein I MA3 domain-containing protein auxin-responsive protein A putative peroxisomal CuZnSOD inducible by a high-light puke. DNAJ heat shock N-terrninal domain-containing protein similar to Protein of unknown function DUF689 similar to 050190814000 159 249996_at 250008_at 250000_at 250007_at 249977_at 249923_at 245957_at 245913_at 246125_at 246149_at 246070_at 246073_at 246099_at 246114_at 246088_at 246001_at 245998_at 246189_at 246021_at 246028_at 249941_at 249886_at 249938_at 249862_at 249848_at 249830_at 249806_at 249818_at 249774_at 249775_at 249777_at 249785_at 249732_at 249741_at 249742_at 246965_at 246922_at 246932_at 246917_at 246901_at 246909_at 246831_at 246796_at 246781_at AT561 8600 AT561 8630 AT5618650 AT5618670 AT5618820 AT5619120 AT5619590 AT5619860 AT5619875 AT5619890 AT5620160 AT5620180 AT5620230 AT5620250 AT5620600 AT5620790 AT5620830 AT5620910 AT5621 1 00 AT5621 1 70 AT5622270 AT5622320 AT5622330 AT5622920 AT5623220 AT5623300 AT5623850 AT5623860 AT56241 50 AT56241 60 AT562421 0 AT5624300 AT5624420 AT5624470 AT5624490 AT5624840 AT56251 10 AT5625190 AT5625280 AT5625630 AT5625770 AT5626340 AT5626770 AT5627350 glutaredoxin family protein; Identical to Monothiol glutaredoxin- S2 (AterSZ) (GRXSZ) lipase class 3 family protein; similar to lipase class 3 family protein zinc finger (C3HC4-type RING finger) family protein putative beta-amylase BMY3 (BMY3) EM83007 (EMBRYO DEFECTIVE 3007); ATP binding / protein bindin ' pepsing A; similar to extracellular dermal glycoprotein similar to unknown protein similar to unknown protein similar to oxidoreductase/ transition metal ion binding peroxidase ribosomal protein L7AeIL30eIS12e/Gadd45 family protein ribosomal protein L36 family protein; similar to 050390811800 Al-stress-induced gene encodes a member of glycosyl hydrolase family 36. similar to unknown similar to unknown protein Encodes a protein with sucrose synthase activity (SUS1). zinc finger (C3HC4-type RING finger) family protein L-ascorbate oxidase 5'-AMP-activated protein kinase beta-2 subunit similar to unknown protein leucine-rich repeat family protein; similar to Ieucine—rich repeat family protein ATTIP49AIRIN1 zinc finger (C3HC4-type RING finger) family protein isochorismatase hydrolase family protein; similar to isochorismatase hydrolase family protein dihydroorotate dehydrogenase similar to unknown protein beta-tubulin squalene monooxygenase gene homolog squalene monooxygenase 1 lipase class 3 family protein; similar to triacylglycerol lipase SSI glucosaminelgalactosamine—6-phosphate isomerase-related APRR5 30$ ribosomal protein methyltransferase; Identical to Probable tRNA (guanine-N(7)-)- methyltransferase member of AtClPKs encodes a member of the ERF subfamily B-6 of ERF/AP2 transcription factor family. serine-rich protein-related pentatricopeptide (PPR) repeat-containing protein similar to 050690163200 Encodes a protein with high affinity similar to unknown protein Encodes a sugar-porter family protein that is induced during leaf senescence. 160 246783_at 246759_at 246701_at 246708_at 245925_at 246651_at 249694_at 249645_at 249622_at 249542_at 249528_at 249475_at 249494_at 249493_at 249426_at 249410_at 249411_at 249378_at 249355_at 249327_at 249337_at 249315_at 249303_at 249266_at 249265_at 249233_at 249204_at 249190_at 249174_at 249138_at 249134_at 249148_at 249091_at 249073_at 249011_at 249008_at 248912_at 248879_at 248839_at 248870_at 248868_at 248820_at 248795_at 248786_at AT5627360 AT5627950 AT5628020 AT56281 50 AT5628770 AT5635170 AT5635790 AT5636910 AT5637550 AT5638140 AT5638720 AT5638890 AT5639050 AT5639080 AT5639840 AT5640380 AT5640390 AT5640450 AT5640500 AT5640890 AT5641 080 AT5641 1 90 AT5641460 AT5641 670 AT5641 700 AT56421 50 AT5642570 AT5642750 AT5642900 AT5643070 AT56431 50 AT5643260 AT5643860 AT5644020 AT5644670 AT5644680 AT5645670 AT5646180 AT5646690 AT5646710 AT5646780 AT5647060 AT5647390 AT5647410 Encodes a sugar-porter family protein that unlike the closely related gene ' kinesin motor protein-related; similar to kinesin motor protein- related Encodes cysteine synthase AtcysDZ. similar to unknown protein bZIP protein BZOZH3 mRNA adenylate kinase family protein; similar to adenylate kinase Encodes a plastidic glucose-6-phosphate dehydrogenase Encodes a thionin similar to unknown protein histone—like transcription factor (CBF/NF—Y) family protein similar to unknown protein exoribonuclease-related; similar to exoribonuclease-like transferase family protein transferase family protein ATP-dependent RNA helicase protein kinase family protein; similar to protein kinase family protein Encodes a protein which might be involved in the formation of verbascose. similar to unknown protein similar to 050490482900 Encodes a member of the voltage—dependent chloride channel. glycerophosphoryl diester phosphodiesterase family protein similar to unnamed protein product fringe-related protein; similar to fringe-related protein 6-phosphogluconate dehydrogenase family protein One of the polypeptides that constitute the ubiquitin-conjugating enzyme E2 » electron carrierl protein disulfide oxidoreductase similar to unknown protein _ Encodes a plasma-membrane associated phosphoprotein similar to unknown protein WPP1 (VVPP domain protein 1) similar to hypothetical protein MtrDRAFT_AC14110994v1 chaperone protein dnaJ-related; similar to drought-induced protein 1 Encodes a Chlorophyllase acid phosphatase class 6 family protein; similar to acid phosphatase class B family protein similar to unknown protein methyladenine glycosylase family protein GDSL-motif lipase/hydrolase family protein ornithine delta-aminotransferase BHLI-l071 (BETA HLH PROTEIN 71) zinc-binding family protein; similar to zinc-binding family protein VQ motif-containing protein senescence-associated protein-related myb family transcription factor; similar to myb family transcription factor similar to hypothetical protein 25.t00068 161 248790_at 248762_at 248753__at 248749_at 248732_at 248744_at 248683_at 248686_at 248625_at 248596_at 248622_at 248607_at 248614_at 248510_at ‘248466_at 248467_at 248471_at 248463_at 248451_at 248410_at 248375_at 248381_at 248398_at 248337_at 248357_at 248329_at 248326_at 248303_at 248252_at 248243_at 248236_at 248186_at 248207_at 248185_at 248160_at 248136_at 248140_at 248100_at 248101_at 248082_at 248036_at 248045_at AT5647450 AT5647455 AT5647630 AT5647880 AT5648070 AT5648250 AT5648490 AT5648540 AT5648880 AT5649330 AT5649360 AT5649480 AT5649560 AT56 5031 5 AT5650720 AT5650800 AT5650840 AT5651 130 AT5651 180 AT5651570 AT5651 710 AT5651830 AT5651970 AT5652310 AT5652380 AT5652780 AT5652820 AT5653170 AT5653250 AT5653590 AT5653870 AT5653880 AT5653970 AT5654060 AT5654470 AT5654910 AT5654980 AT56551 80 AT5655200 AT5655400 AT5655920 AT5656030 Tonoplast intrinsic protein similar to unknown protein acyl carrier family protein IACP family protein; similar to mtACP-1 Encodes a eukaryotic release factor 1 homolog. putative xyloglucan endotransglycosylase/hydrolase zinc finger (B-box type) family protein; Identical to CONSTANS- LIKE 10 (COL10) protease inhibitor/seed storage/lipid transfer protein (LTP) family protein 33 kDa secretory protein-related; similar to receptor protein kinase-related Encodes a peroxisomal 3-keto-acyI-CoA thiolase 2 precursor. Member of the R2R3 factor gene family. encodes a beta-xylosidase located in the extracellular matrix. AtCP1 encodes a novel Ca2+-binding protein similar to unknown protein Mutator—like transposase family . Encodes one of five HVA22 homologs in Arabidopsis. nodulin MtN3 family protein; similar to nodulin MtN3 family protein similar to nuclear matrix constituent protein-related similar to 050890540500 similar to unknown protein bam7mmemmm member of Putative potassium proton antiporter family pflrB-type carbohydrate kinase family protein sorbitol dehydrogenase cold regulated gene zinc knuckle (CCHC-type) family protein; similar to zinc knuckle (CCHC-type) family protein similar to unknown protein WD—40 repeat family protein I notchless protein encodes an FtsH protease that is localized to the chloroplast andthernfiochondnon AGP22IATAGP22 (ARABINOGALACTAN PROTEINS 22) auxin-responsive family protein plastocyanin-like domain-containing protein unknown protein encodes tyrosine aminotransferase UF3GT (UDP—GLUCOSEzFLAVONOID 3-0- GLUCOSYLTRANSFERASE) zinc finger (B-box type) family protein; similar to zinc finger (B- box type) family protein DEAD/DEAH box helicase integral membrane family protein; similar to integral membrane family protein glycosyl hydrolase family 17 protein; similar to hydrolase co-chaperone grpE protein fimbrin-Iike protein nucleolar protein a member of heat shock protein 90 (HSP90) gene family. 162 248007_at 247989_at 247983_at 247977_at 247954_at 247957_at 247937_at 247942_at 247926_at 247914_at 247921_at 247880_at 247882_at 247838_at 247851_at 247816_at 247819_at 247786_at 247775_at 247776_at 247754_at 247763_at 247739_at 247655_at 247650_at 247649_at 247638_at 247593_at 247608_at 247575_at 247550_at 247524_at 247540_at 247497_at 247498_at 247488_at 247487_at 247463_at 247478_at 247454_at 247453_at AT5656260 AT5656350 AT5656630 AT5656850 AT5656870 AT5657050 AT56571 1 0 AT5657120 AT5657280 AT5657540 AT5657660 AT5657780 AT5657785 AT5657990 AT5658070 AT5658260 AT5658350 AT5658600 AT5658690 AT5658700 AT5659080 AT5659180 AT5659240 AT5659820 AT5659960 AT5660030 AT5660490 AT5660790 AT5660990 AT5661 030 AT5661 370 AT5661440 AT5661 590 AT5661770 AT5661 81 0 AT5661820 AT56621 50 AT5662210 AT5662360 AT5662440 AT5662440 Regulator of ribonuclease-like protein 3 pyruvate kinase phosphofructokinase family protein similar to unknown protein beta-galactosidase Encodes a protein phosphatase 20 Arabidopsis-autoinhibited Ca2+ -ATPase similar to unknown protein similar to SAM (and some other nucleotide) binding motif xyloglucanzxyloglucosyl transferase zinc finger (B-box type) family protein; Identical to CONSTANS- LIKE 5 (COL5) similar to transcription factor contains lnterPro domain Helix-Ioop-helix DNA-binding; (lnterProzlPR01 1598) Encodes a ubiquitin-specific protease. Iipocalin Encodes subunit NDH-N of NAD(P)H:plastoquinone dehydrogenase complex (Ndh complex) Encodes a member of the WNK family (9 members in all) of protein kinases Belongs to a large family of plant-specific genes of unknown function. phosphoinositide-specific phospholipase C family protein phosphoinositide-specific phospholipase C family protein similar to unknown protein DNA-directed RNA polymerase II; Identical to DNA-directed RNA polymerase II 406 ribosomal protein 68 (RPSBB); Identical to 40$ ribosomal protein $8-2 (RPS8B) ZAT12 similar to 050190151600 similar to unknown protein F LA12 (fasciclin-like arabinogalactan-protein 12) member of GCN subfamily DEAD/DEAH box helicase encodes a glycine-rich RNA binding protein. pentatricopeptide (PPR) repeat-containing protein thioredoxin family protein; Identical to Thioredoxin-like 3 encodes a member of the ERF subfamily B-3 of ERFIAP2 transcription factor family. brix domain-containing protein; Identical to Peter Pan-like protein (PPAN) mitochondrial substrate carrier family protein similar to hypothetical protein peptidoglycan-binding LysM domain-containing protein embryo-specific protein-related; similar to ATS3 (ARABIDOPSIS THALIANA SEED GENE 3) invertase/pectin methylesterase inhibitor family protein similar to defective chloroplasts and leaves protein-related I DCL protein-related similar to defective chloroplasts and leaves protein-related I DCL protein-related 163 247396_at 247378_at 247377_at 247374_at 247361_at 247356_at 24731 8_at 247268_at 247323_at 247287_at 247282_at 247278_at 247284_at 247277_at 247241_at 2471 75_at 247162_at 247168_at 247119_at 247072_at 247043_at 246985_at 246996_at 247013_at 264270_at 251230_at 265572_at 248743_at 254963_at 267315_at 245866_s_at 254331_s_at 248566_s_at 249658_s_at 258977_s_at 265444_s_at 263048_s_at 245877_at 255908_s_at 247388_s_at 2571 75_s_at 245783_s_at 247266_at AT5662930 AT5663120 AT5663180 AT56631 90 AT5663480 AT5663800 AT5663990 AT5664080 AT56641 70 AT5664230 AT5664240 AT5664380 AT566441 0 AT5664420 AT5664680 AT5665280 AT5665730 AT5665860 AT5665900 AT5666490 AT5666880 AT5667290 AT5667420 AT5667480 AT1 G60260 AT3G62750 AT2G2821 0 AT5G48240 AT4G1 1 060 AT2G34720 multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple GDSL-motif lipase/hydrolase family protein; similar to carboxylic ester hydrolase ethylene-responsive DEAD box RNA helicase pectate lyase family protein; Identical to Probable pectate lyase 22 precursor MA3 domain-containing protein similar to OSJNBa0053B21.8 member of Glycoside Hydrolase Family 35 3(2) protease inhibitor/seed storage/lipid transfer protein (LTP) family protein dentin sialophosphoprotein-related; similar to unknown protein similar to unknown protein latex-abundant family protein (AMC3) I caspase family protein fructose-1 oligopeptide transporter DNA polymerase V family; similar to zinc finger protein-related similar to 050190259900 lanthionine synthetase C-Iike family protein; similar to catalytic xyloglucanzxyloglucosyl transferase ankyrin repeat family protein DEAD/DEAH box helicase similar to unknown protein encodes a member of SNF1-related protein kinases (SnRK2) FAD-dependent oxidoreductase family protein; similar to 0507901 55100 LOB domain protein 37 I lateral organ boundaries domain protein 37 (LBD37) BT4 (BTB AND TAZ DOMAIN PROTEIN 4) Unknown Protein Unknown Protein Unknown Protein Unknown Protein Unknown Protein Unknown Protein purine permease-related; similar to ATPUP18 (Arabidopsis thaliana purine permease 18) member of CYP706A Encodes a putative ferric chelate reductase. phosphoglycolate phosphatase encodes a monofunctional aspartate kinase a member of the plasma membrane intrinsic protein subfamily PIP2. similar to unknown protein Potential natural antisense gene similar to unknown protein Encodes a protein with similarity to a subunit of the CCAAT promoter motif binding complex cyclopropane-fatty-acyI-phospholipid synthase similar to unknown protein Encodes a beta-d-xylosidase that belongs to family 3 of glycoside hydrolases. 164 261 144_s_at 257634_5_at 255895_at 256337_at 264022_at 267126_s_at 262733_s_at 250670_at 264654_s_at 253879_s_at 266932_s_at 250151_at 261664_s_at 265941_s_at 266720_s_at 258449_s_at 2671 62_s_at 251 775_s_at 2661 84_s_at 255685_s_at 249888_s_at 263823_s_at 2461 73_s_at 265670_5_at 259077_s_at 254740_s_at 256376_s_at 262054_s_at 262306_s_at 267335_s_at 256595_x_at 254952_at 246481_s_at multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple multiple wound-responsive family protein; similar to wound-responsive protein-related putative cytochrome P450 12-oxophytodienoate reductase serine-type endopeptidase inhibitor; similar to 050390734300 unknown protein hydrolase fipase polygalacturonase inhibiting protein 1 (PGIP1) mRNA sugar transporter family protein glycosyltransferase family protein DEAD box RNA helicase Encodes a microRNA. mitochondrial import inner membrane translocase subunit Tim17fl'im22fl'im23 family protein recA family protein pseudo-response regulator DEAD box RNA helicase phosphoribosylaminoimidazole carboxylase family protein IAIR carboxylase family protein encodes a delta1-pyrroline-5-carboxylate synthase phosphate transporter (AtPT2) tetrahydrofolate dehydrogenase/cyclohydrolase zinc finger (ZPR1-type) family protein encodes a member of the DREB subfamily A-2 of ERFIAP2 transcription factor family pentatricopeptide (PPR) repeat-containing protein similar to unknown protein reversibly glycosylated polypeptide possibly involved in plant cell wall synthesis . SHM5 (SERINE HYDROXYMETHYLTRANSFERASE 5) S-adenosyl-L-methioninezcarboxyl methyltransferase family protein heat shock protein 70 SYNC3 glycosyl hydrolase family 17 protein gypsy-like retrotransposon family (Athila) lipase class 3 family protein cold and ABA inducible protein kin1 165 Literature Cited Cook, D., S. Fowler, et al. (2004). "From The Cover: A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis." Proceedings of the National Academy of Sciences 101(42): 15243. Hiratsu, K., K. Matsui, et al. (2003). "Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis." The Plant Journ_a_l 34(5): 733-739. McKemy, D. D., W. M. Neuhausser, et al. (2002). "Identification of a cold receptor reveals a general role for TRP channels in thermosensation." Nature 416(6876): 52-58. 166 BIO . . m .111 4 R 1 m1. 7 . . m“ 6 . . . Sll 1| 5 . . W111 w . WM. 0 . . El . . . . vAI 3 . . . 9 PM“ 9 1 , N" 2 . . . m 1 . . W ml . . . M . . . . . . . : . . . . . . he..un.rln_ .