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D . degree in Genetics f/EM;//I/% é /7 Major professor Michael Thomashow Date March 3, 1993 M5 U is an Affirmative Action ’Equal Opportunity lnsn’xuu'on 0-12771 '_ _~__—._—.~_ -._-4- 4- __.—. _ 'm-—‘~ LIBRARY Michigan State Unlverslty r PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE % ll fl MSU Is An Affirmative Action/Equal Opportunity Institution encircMuna-ej Ch Cloning, characterization and regulation of four cold-induced genes from Arabidopsis thaliana By David P. Horvath A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirments for the degree of DOCTOR OF PHILOSOPHY Program in genetics 1993 Plants to is known resPonse Changes ECnes fr fOur dis 10W ten COl’ gm 3316.5 1 the ER C0r78‘ IEVel, Stum- demO impu Pail“ Abstract Cloning, characterization and regulation of four cold-induced genes in Arabidopsis thaliana BY David P. Horvath Considerable work has been done to characterize the physiological responses of plants to low temperatures and during the process of cold-acclimation. However, little is known about how plants sense cold and alter their pattern of gene expression in response to low temperatures. In order to gain insight into the mechanisms involved in changes in gene expression during cold acclimation, it was decided that cold-regulated genes from Arabidopsis thaliana should be isolated and studied. cDNAs representing four distinct cam-regulated (cor) genes were cloned. The car genes are all induced by low temperatures, drought stress, and by exogenous application of abscisic acid. The cor genes are not induced, (or are poorly induced) by heat shock and wounding. These genes demonstrate a threshold of induction at 10°C and are expressed throughout all of the growing stages of the plant. Work focusing on one of these cor genes (designated cor78) has demonstrated that this gene is regulated primarily at the transcriptional level, and it is expressed throughout most of the plant in response to low temperatures. Studies on the kinetics of the induction and inactivation of the cor78 promoter has demonstrated that this gene has a half-life of approximately 35 minutes. Finally, the implications of all of these results concerning the possible signal transduction pathway(s) that may function in the regulation of this gene are discussed. ii l W throughout t Ravindra Ha Wilhelm, N; (SCicntific ar efforts and p manuscript 1 given to Dr. Short notice ; Committee, during the 10 quesm. Fina mammal, a! ACKNOWLEDGMENTS I would like to thank Dr. Mike Thomashow for his support, help, and ideas throughout this project. I would also like to thank my co-workers Sarah Gilmour, Ravindra Hajela, Tim Lynch, Brett McLamey, Tod Cotter, Stokes Baker, Kathy Wilhelm, Nancy Artus and Deane Lehmann for their encouragement and discussions (scientific and otherwise). Special thanks goes to Sarah Gilmour for her tremendous efforts and patience in the critical reviews of this dissertation and every other manuscript I have written since I have known her. Also, special thanks should be given to Dr. Lee Maclntosh and Dr. Rebecca Grumet for joining my committee on short notice and to Dr. Jerry Dodgson for his help as the lone original member of my committee. I am also grateful to my wife and son for their patience and understanding during the long periods of time that I have spent away from them during my scientific quests. Finally, I would like to acknowledge Dr. Terry Mathews, Dr. Mark Levanthal, and Dr. Jeff Bennetzen for the inspiration and encouragement that I received from them early in my scientific career. iii LIST 0? I‘ll LIST OF TA CWUWER 1: Intrt Cold Post Trai Lit CHAPTER Sn] In Me CHAPT TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES CHAPTER 1: Literature Review Introduction 1 Cold Stress Physiology 1 Post-transcriptional Regulation 7 Transcriptional Regulation 9 Literature Cited 17 CHAPTER 2: Cloning and Expression of Cold-Regulated Genes in Arabidopsis thaliana Summary 23 Introduction 24 Materials and Methods 26 Results 29 Discussion 53 Acknowledgments 57 Literature Cited 58 CHAPTER 3: Regulation of the Arabidopsis cor78 gene Summary 61 Introduction 62 Materials and Methods 64 Results 70 Discussion 110 Literature Cited 120 iv SUMMARY A Lite APPENDIX Corr indt Kim COD' Lit SUMMARY AND CONCLUSIONS Literature Cited APPENDIX Correlation of freezing tolerance and cor gene induction. Kinetics of expression of the cor78/GUS gene constructs. Literature Cited 123 127 129 139 148 Table 2. Table 2 Table 3 Table 2.1 Table 2.3 Table 3.1 LIST OF TABLES Size of cDNA clones, mRNAs and Proteins. Nuclear runon results compiled from three separate experiments. List of conserved promoter sequences found in all of the known cor gene. vi Page 30 36 113 Figure Figure Figure Figure Figure Figure Figure FigUre ; Figure Figure Figure Figure Figure FiQUre Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 2.3 2.4 2.7 LIST OF FIGURES Page cDNA clones or cor genes are induced by low temperature. 31 Temperature threshold of cor genes. 33 Representative data from runon transcription assays. 37 Cor genes are induced by drought. 39 Cor genes are induced by salt stress. 42 Cor genes are not induced by heat shock. 44 Cor genes are not induced by wounding. 46 Tissue specificity of cor gene expression. 49 Ability of plants to express cor genes in response to low temperatures at several stages throughout their life cycle. Sequence of the cor78 gene. Determination of the transcriptional start site via primer extension. In vitro transcription and translation of the near-full-length cDNA clone of cor78. Hydrophilicity plot of the predicted COR78 protein. Cor78 promoter/GUS fusion constructs. Cor78 promoter/GUS constructs are induced by cold, drought, and ABA. 78P/gus and 78PI/gus are cold induced, but 355/gus is.not. vii 51 71 75 78 80 83 85 87 Figure 3.4 Figure 3. Figure 3 . Figure 3 Figure 3 Fiqure Figure PigUre Fiqu, Figur Fight Figu Fig, Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Rch/GUS/cor78 3' constructs. BSS/cor78 transcribed sequences construct. Northern analysis on the rch/GUS/cor78 3' constructs in transgenic plants. RNase protection assays on the 3SS/cor78 transcribed construct in transgenic plants. Histochemical staining for GUS activity in plants carrying the cor78 promoter/gus constructs. Histochemical staining for GUS activity in plants carrying the cor78 promoter/gus constructs, and thin sections of specific tissues. Kinetics of cor78 induction and loss after a three hour cold treatment. Kinetics of cor78 inactivation after a three hour cold treatment. of cor78 inactivation overnight cold treatment. Kinetics after an Freezing tolerance in salt stressed plants. tolerance in drought plants. Freezing stressed Freezing tolerance in plants placed at 10°C for two days. Freezing tolerance in plants acclimated and deacclimated for various lengths of time. Kinetics of cor78 induction and loss after a 3 hour cold treatment in plants carrying the cor78 promoter/GUS constructs. viii page 90 92 94 96 99 101 104 106 108 131 133 135 137 140 Figure Figure Figure Figure A.6 Figure A.7 Figure A.8 Kinetics of GUS induction and loss after a 3 hour cold treatment in plants carrying the cor78 promoter/GUS constructs. Kinetics of cor78 inactivation after an overnight cold treatment in plants carrying the cor78 promoter/GUS constructs. Kinetics of GUS inactivation after an overnight cold treatment in plants carrying the cor78 promoter/GUS constructs. ix page 142 144 146 ABA: ab oC: degn CaMV: c cDNA: c CIM: cal DNA: de DS: dout DTT: dit EtOH; e1 GBF: (3-] GUS: bet Lt502 letl NEB; Ne List of abbreviations ABA: abscisic acid oC: degrees centigrade CaMV: cauliflower mosaic virus cDNA: copy deoxyribonucleic acid CIM: callus inducing media DNA: deoxyribonucleic acid DS: double stranded D'l'l‘: dithiothreitol EtOH: ethanol GBF: G-box binding factor GUS: beta glucuronidase Lt50: lethal temperature 50% NEB: New Engalnd Biolabs Nos: nopalin synthase PCI: phenol/chloroform/iso-amyl alcohol PCR: polymerase chain reaction RNA: ribonucleic acid SDS-PAGE: sodium dodecyl sulfate- poly acrylamide gel electrophoresis SIM: shoot inducing media URE: upstream regulatory element UTP: uridine 5 '-triphosphate UTR: untranslated region Chapter 1 LITERATURE REVIEW I. INTRODUCTION Plants that are indigenous to temperate regions of the earth are often exposed to extremes in low temperature. Unlike their animal counterparts, plants are unable use avoidance as a means of countering this type of stress. Thus, it is not surprising that some plants have evolved means by which they can sense temperature drops and alter their biochemistry in order to prevent or minimize systemic damage caused by freezing. In fact it is known that many plants, if first subjected to low but non-freezing temperatures, are able to become more tolerant to freezing stress (see Levitt, 1980; Sakai and Larcher, 1987; Thomashow, 1990). This process is called cold acclimation. II. COLD STRESS PHYSIOLOGY Much is known about the changes in plant biochemistry that occur during the cold acclimation process. Many plants show a build up of small soluble molecules such as proline and sugars (Sakai and Larcher, 1987). Often, changes in the composition of membrane lipids also accompany the plant's response to the cold (Steponkus and Lynch, 1989). Finally, it has been shown that there are specific changes in the protein profile of plants that have undergone cold acclimation (Levitt, 1980; Thomashow, 1990; Guy, 1990), and there is evidence to suggest that protein synthesis is necessary for the cold acclimation process to occur (Weiser, 1970). Although it is known that all 2 these changes occur, with the exception of a few cases (such as changes in the phospholipid composition of the plasma membrane (Steponkus 1989)), it has yet to be demonstrated that any are directly responsible for bringing about a greater tolerance to freezing temperatures in the plant. Arabidopsis thaliana was chosen as a model system for studying cold acclimation due to its many advantages for molecular study (Meyerowitz, 1989), and because earlier work on this plant has demonstrated that it is capable of acclimating to cold temperatures (Gilmour er al., 1988). Studies using electrolyte leakage to determine the relative cellular damage caused by a freeze-thaw cycle demonstrated that leaf tissue from acclimated Arabidopsis plants leaked less than 50% of their electrolytes at a temperature of -8°C. When compared to non-acclimated tissue, which reached the same level of leakage at a temperature of -3°C, this represented a significant change in the ability of the acclimated tissue to survive freezing. However, since the leakage of electrolytes can be the result of relatively minor or non-permanent damage to the cell membrane, these electrolyte leakage experiments may not provide an accurate representation of the true survivability of an Arabidopsis plant to a freeze-thaw cycle. Acclimation studies using growth after a freeze-thaw cycle as an indication of incurred damage have shown that both non-acclimated and acclimated plants are capable of - withstanding lower temperatures (-5°C and -20°C, respectively) than one would expect given the results of electrolyte leakage experiments (Singh, unpublished results). To better understand what a plant must do in order to acclimate, one needs to understand the physical nature of the damage caused by a freeze-thaw cycle. As the temperature drops below 0°C, water in the extracellular spaces begin to freeze. This has the effect of lowering the water potential outside the cell, and thus water moves into the extracellular spaces with the lower potential and the cell becomes dehydrated. As the cell thaws, water moves back into the cell at a rate that is dependent on the 3 rapidity of the temperature increase (Gordan-Kamm and Steponkus, 1984). The stress caused by this freezing induced dehydration and subsequent rehydration is a major cause of damage to plant cells that are exposed to temperatures. between 0°C and -10°C (Steponkus, 1984). Studies using isolated protoplasts from rye as a model system, have shown that nonacclimated cells form intracellular vesicles in response to the decrease in cell volume caused by freezing induced dehydration, and that when the cell rehydrates upon thawing these vesicles are not reincorporated back into the membrane fast enough and the cell bursts. In acclimated cells, the cell membrane forms extracellular extrusions that are readily reincorporated into the membrane upon thawing (Gordon-Kamm and Steponkus, 1984). Moreover, there is evidence that the ability to form these extracellular extrusions is the result of specific changes in the phospholipid composition of the acclimated cell's membrane (Steponkus and Lynch, 1989). Thus, the acclimating cell may increase the production or activity of enzymes that can effect changes in the phospholipid composition of the membrane. Freeze-thaw induced cell lysis is not the only mechanism by which a cell can be damaged during freezing. Many proteins may also be susceptible to the dehydration effects experienced during freezing (Brandts, 1967), as interactions with water are required to maintain their three dimensional form. When water in the cell becomes limiting, these proteins are likely to become denatured and form aggregates which precipitate out of solution. Therefore, chaperonin-like proteins (Gatenby, 1992) may be induced in cells acclimating to the cold in order to protect exposed hydrophobic surfaces. Alternatively, some highly charged proteins can order the water molecules around membranes and other proteins (Reviewed in Carpenter and Crowe, 1988). This may have the effect of decreasing the energy potential of the water molecules in their immediate area (Reviewed in Carpenter and Crowe, 1988). Since water will move 4 from areas of higher water potential to areas with lower water potential, the stabilizing effect of these charged proteins may help maintain a layer of water around other proteins and membranes (Reviewed in Carpenter and Crowe, 1988). Since freezing stress often involves severe dehydration, it is not surprising that a number of parallels to drought stress have been documented. In both stresses, the levels of the plant hormone abscisic acid (ABA) have been shown to become elevated (albeit only transiently in some cold-treated plants) (Daie and Campbell, 1981; Chen et al., 1983 ). It has been shown that in some plants, both drought stress and the exogenous application of ABA can increase freezing tolerance (Chen and Gusta, 1983; Gusta, 1983; Lang er al., 1989). Finally, many of the gold-regulated (cor) genes which have been cloned, are also induced by both drought and ABA (Mohapatra et al., 1988; Hejela er al., 1990). Such observations have led to the development of a model where some environmental signal for drought stress or for cold stress leads to the accumulation of ABA which then triggers a common signal transduction pathway involved in the changes in gene expression common to both conditions. However, the signal transduction pathways involved in the induction of these genes in response to ABA, cold, and drought appears to be more complicated. Recent experiments involving the temporal expression of one particular copy of a cold-regulated gene (cor78) in response to environmental signals other than low temperatures have suggested this gene responds to drought stress in a manner that is not observed during cold stress (Kazuko Y. and Kazuko S., 1992). Drought stress induces this gene very rapidly, (within 20 minutes) but accumulation of the message drops off after 40 minutes and remains at near control levels up to 3 hours after the initiation of the drought stress, whereupon it again begins to accumulate to significant levels. The transcript begins to accumulate in response to cold between 60 and 90 minutes after the plant has reached inducing temperatures and continues to accumulate steadily for 7 to 5 10 hours after which the message level appears to remain constant for as long as the plant is left in the cold (Yamaguchi—Shinozaki and Shinozaki, 1992). ABA treatment induces this gene with kinetics very similar to cold, with the mRNA accumulating within 60-90 minutes after treatment. These data suggest that this cor78 responds differently to drought than it does to either cold or ABA, and that there are at least two different pathways that are capable of inducing it. Interestingly though, a second closely linked copy of cor78 responds to drought, cold, and ABA with nearly identical kinetics (Yamaguchi-Shinozaki and Shinozaki, 1992). A number of other studies have focused on the expression and regulation of several cor genes in Arabidopsis plants with altered ABA responses. In these studies, . Arabidopsis plants with a mutation that renders them insensitive to exogenous applications of ABA (designated abiI for ABA insensitive 1), were either cold treated, drought stressed, or sprayed with ABA, and the induction of the cor genes was monitored. The results showed that the cor genes were still capable of responding to drought and cold stress, but were not induced by exogenous application of ABA (Nordin er al., 1991; Gilmour and Thomashow, 1991). Since initial analysis of the abiI mutation has shown that the exogenously applied ABA does accumulate in such plants (suggesting that these plants are not deficient in ABA uptake), these studies appear to indicate that the induction of the cor genes in response to both cold and drought is not dependent on the plant's ability to perceive increases in internal ABA levels (Finkelstein and Sommerville, 1990). Such results point to the possibility that the ABA response operates via a different (or partially different) signal transduction pathway than does the drought or cold responses. It is noted however, that since the nature of the abil mutation is not known, it is possible that endogenous ABA may still be functioning as an intermediate, and that some part of the pathway involved with the 6 transmission of signal from exogenously applied ABA may be interrupted in the abil mutants. Finally, it has been demonstrated recently that there are at least two copies of one of the cold-regulated genes in Arabidopsis (designated Kin] and Kin2, and analogous to cor6.6). Both genes are induced by cold and by ABA, but Kin] is only induced by drought stress (Kurkela and Borg-Franck, 1992). This suggests that both the cold and the ABA response in the induction of this gene may be separable from the drought response. This also suggests that the signal transduction pathway involved in the drought response does not operate through ABA (at least for KinI). Work on two distinct copies of the corIS gene from Arabidopsis appears to give similar results (Wilhelm, manuscript in preparation). 1 For future manipulation of the expression of the cor genes it is of interest to study how plants sense cold and the signal transduction pathway(s) that may be involved in altering gene expression in response to particular environmental stimuli. Traditionally, studies of this sort have relied upon the isolation and characterization genes that are regulated by the environmental stimulus, and the cis and trans acting factors controlling them. A considerable amount of success has been obtained in animal systems using this approach. The signal transduction pathway of post- transcriptionally regulated genes such as c-fos (Shyru er al. , 1989) and the iron transferrin genes (Leibold and Munro, 1988) as well as many transcriptionally regulated genes (Gronemeyer, 1992; Hunter and Karin, 1992; Karin and Smeal, 1992;) have been, or are being characterized through studies facilitated by the identification of key cis and trans acting factors from genes responsive to these stimuli. III. POST-TRANSCRIPTION AL REGULATION Factors which bind specific sequences in RNA and alter its stability have been found in animals (Koeller et al. , 1989, Ross, 1989), and are likely to be found in plants (Elliott et al. , 1989). There are two fairly well characterized systems involving changes in RNA stability in animal cells that have been studied. These are the regulation of c-myc and iron transferrin mRNA stability. In both systems, specific sequences in the 3' untranslated region (UTR) of the gene have been shown to be necessary for appropriate regulation. In the case of c—myc mRNA, the sequences of interest are a series of AUUUA repeats in it's 3' UTR. Repeats of this sequence when placed in the 3'UTR of a reporter gene can dramatically reduce the stability of its RNA (Shaw and Kamen, 1986). Specific proteins have been shown to bind to this AUUUA motif in viva (Myer er al. , 1992) and site specific changes in this sequence that prevents binding of the AUUUA binding factor also result in a more stable RNA (Malter, 1989). With the iron transferrin mRNA, the 3'UTR was shown to impart an iron dependent decrease in the stability of RNA from a reporter gene. A hypothetical hairpin loop structure within the 'U' rich region of the 3' UTR was shown to bind a protein factor, and specific changes in these sequences that disrupted the hairpin formation also prevented the protein binding and resulted in a more stable RNA (Mullner and Kuhn, 1988). In plants, the ferridoxin I gene has been shown to be primarily regulated at the level of RNA stability (Elliott er al., 1989). In this system, the sequences that regulate stability appear to be located in the 5' UTR, and reporter genes engineered with the ferridoxin 5' UTR become less stable in response to far red light. This system however, has not been as well characterized, and the action of any specific RNA binding factors has not been demonstrated. In the above cases, it is 8 generally assumed that the binding of these specific sites in the 5' or 3' end of these messages protects these sites from endonucleases with specificities for the same or nearby sequences (Malter, 1989; Mullner er al., 1989). Other mechanisms which may be functioning to bring about changes in RNA stability are those which specifically alter the affinity of poly A binding proteins which are involved in protecting the 3' end of specific RNAs (Bernstein and Ross, 1989). It has been observed that generally mRNAs with longer poly A tails are usually more stable than mRNAs with shorter poly A tails (Shapiro et al., 1987). The reason for this is thought to be due to the presence of poly A binding proteins (PABPs) that may be involved in protecting the 3' end of the RNA from 3'- >5' exonucleases (Ross, 1989). There is some evidence to support the proposal that the AU rich regions which have been correlated with reduced RNA stability (including the AUUUA sequences in the c-myc gene) may compete for PABPs with the poly A tail, and thus leave the 3' end more open to degradation by exonucleases (Bernstein er al. , 1989). Along with RNA stability, RNA splicing mechanisms offer points of control for gene regulation. It has been found that the presence of certain introns within a number of different structural genes can increase the expression level of these genes (Luehrsen and Walbot, 1991). The mechanisms of how the introns enhance the accumulation of these messages is not well understood. However, the data suggest that the introns, or the proteins acting on, or bound to the intron containing RNAs may protect these RNAs from nucleases present within the nucleus. This protection may be in the form of steric hindrance or competition for nuclease binding sites, and/or of increased efficiency of transport from the nucleus. IV. TRANSCRIPTIONAL REGULATION Although a number of post-transcriptionally regulated genes have been characterized, in most cases experiments have focused on genes that are regulated primarily at the transcriptional level and thus much has been learned about the functional components of the eucaryotic promoter. Most eucaryotic promoters have several common (but not ubiquitous) features that appear to be conserved. These are the TATA box, and upstream regulatory elements (UREs) (sometimes referred to as upstream activating sequences in yeast) (Dynan and Tjian, 1985) The TATA box serves as the binding site for a general transcription factor (TFIID) (Greenblatt, 1991). Other general transcription factors necessary for basal level transcription appear to bind to or in someway interact with TFIID (Sawadogo and Sentenac, 1990; Sharp, 1992). Also, there appear to be factors that interact specifically with TFIID and facilitate the formation of the transcriptional initiation complex (HOrikoshi et al., 1988). The subsequent complex probably serves to recruit RNA polymerase II (Treisman and Maniatis, 1985). It is also thought that the TATA box complex is necessary for placement of the RNA polymerase in order to ensure that transcription is initiated at the appropriate site (Greenblatt, 1991). However, there are a number of promoters that do not appear to contain a functional TATA box, although they still support active transcription with a defined transcriptional initiation site (Pugh and Tjian, 1991). In these cases, however, it has been demonstrated that specific sequences around the start of transcription are required for appropriate initiation (Roy er al., 1991). 10 UREs are short sequences in the promoter that act as binding sites for proteins that can interact either directly or indirectly with TFIID or other proteins in the transcription complex in order to facilitate complex formation and/or RNA polymerase II binding (Klein-Hitpass er al. , 1990). UREs can affect transcription even when they are positioned some distance from the transcription start site (up to several thousand bases away) (Struhl, 1987). However, it has been found that when such sequences were placed at varying distances from the start site, full activity was only observed when the distance to the TATA box was altered in multiples of ten to eleven bases (Takahashi et al., 1986). However, as the distance from the start site was increased, the requirement for this periodicity for full activity was relaxed. This suggests that the URE binding proteins must be present in a specific orientation for enhanced RNA polymerase activity, and that this requirement may be fulfilled if there is sufficient distance between the URE and the site of complex formation. A likely model for how these URE binding factors work at variable distances from the transcription start is that they interact with or recruit other components of the transcription complex by a looping out of the intervening DNA. Therefore, if the distance between the site of URE factor binding and the assembly of the basal transcription complex is sufficient to allow enough flexibility in the DNA for the URE binding protein to be appropriately presented, then activation is possible. In fact, in vitro studies have shown, that if the concentration of bound and active URE binding proteins is high enough, full activation of a promoter may be achieved in vitro, even if the URE is present on a completely separate piece but linked piece of DNA (Muller er al. , 1989). Much work has been focused on determining the exact mode of action of the trans acting factors that bind to the UREs and how they bring about enhanced transcription. There appear to be several classes of such factors with differing modes of action. One common class contains a characteristic acid activating domain, whose 11 role was discovered by creating fusion proteins containing various portions of a known activating factor connected to a DNA binding domain specific for a sequence engineered into a reporter gene. What this acid activating domain specifically interacts with, and how it brings about increased transcription has not yet been elucidated. Early studies have indicated that such domains may interact directly with TFIID or with polymerase directly (Ingles er al., 1991; Cullen, 1990). Alternatively, they may be involved in specifically interacting with nucleosomes and thus open regions of the promoter for TFIID binding (Taylor et al. , 1991). However, there are also indications that there may be other factors which are required for such acidic domains to be functional, and that these factors may act as adapters between the URE binding proteins and other components of the basal transcriptional complex (specifically TFIIA) (Wang et al., 1992). Another interesting and little understood aspect of URE binding factors is how such proteins recognize their appropriate binding sites. Since many of these factors recognize and bind to fairly short DNA sequences (4-10 bps), it is not clear what prevents them from binding inappropriately to similar sequences that are likely to be present throughout the genome. Currently, there are several common DNA binding motifs in URE binding factors that have been characterized. These include zinc fingers, helix turn helix, leucine zippers, and c—myb like binding factors. Although the gross interaction of the amino acids within these domains and the DNA itself are reasonably well understood (Johnson and McKnight, 1989), there are a number of interesting ambiguities concerning the specificity of the binding factors and the recognition of their appropriate binding sites. There are a number of cases in which the site to which a given factor binds is significantly degenerate (Payvar er al. , 1983; deVargas er al., 1988; Weintraub er al. , 1991). How these factors recognize such different sequences, and what prevents them from binding inappropriately to other 12 similar sequences found randomly in other promoters throughout the genome is not well understood (Johnson and McKnight, 1989). Also, there are a number of cases where there appears to be a family of very similar but distinct factors which all recognize the same sequence, but which appear to regulate different genes in different ways (Weintraub et al. , 1991). In their review, Johnson and McKnight used the OCT family of transcription factors to serve as an illustration of how this type of system may operate. In this case, there are at least two distinct factors which both recognize the sequence TAATGARAT albeit with slightly different affinities, and which demonstrate virtually identical gene activating properties in virro. OCT] is ubiquitous in mammalian cells, but OCT2 is present only in lymphoid cells and plays a role in transcription from the Ig heavy chain promoter. Since OCTl is ubiquitous, and binds to the same sequence, there is question as to why the Ig heavy chain promoter is not activated in non-lymphoid cells. Assuming the Ig heavy chain promoter is open, there appears to be nothing that prevents the binding and subsequent activation by OCTl. Although there are no significant differences in the DNA binding capacities of these two factors, there do appear to be some differences in their ability to interact with other proteins. For instance, OCTl can interact with and form part of the ternary complex with the herpes virus activator protein, VP16, whereas OCT2 cannot. Also, OCT2 has a leucine zipper-like motif outside its DNA binding domain that OCTl does not have. It has been suggested that this zipper domain may allow the OCT2 protein to interact with factors that do not recognize OCTl , and that these separate proteinzprotein interactions are responsible for the binding and/or activating differences observed in vivo. Another family of seemingly promiscuous binding factors may be particularly relevant to the study of our cold-regulated genes. Of interest is a family of leucine zipper type binding factors called GBFs (G-Box binding factors), which recognize 13 variations of the core sequence CACGTG (G-Box) (Schindler et al., 1992; Williams et al., 1992). These G-Box sequences were first characterized as protein binding sites that played some role in the expression of light regulated genes such as the small subunit of Rubisco (Guiltinan et al., 1988). Also, very similar sequences were found to be important (if not sufficient) for the expression of a number of ABA-regulated genes (Guiltinan er al., 1990; Mundy et al., 1990). Subsequently, similar sequences have been found in the promoters of a number of very differently regulated genes from a large number of different species. As examples, in A. thaliana alone, the HSP70-I, adh, chs, rch-IA, and all of the known cor genes (which have also been shown to be ABA-regulated) contain similar sequences in their promoters (Williams et al., 1992). Whether the binding of factors to these sites is sufficient for the induction of any of these genes, or if binding simply functions to enhance or allow the transcription of already activated genes is not known. There is, however, some evidence that points to the former. It has been shown that a tetramer of a 21 bp piece of DNA containing the sequence GGACGCGTGGC was able to confer ABA and desiccation induced regulation to the both the -46 and -90 minimal CaMV promoter fragments (Lam and Chua, 1991). The sequence within this tetramer is very similar to the sequence GGACACGTGGC which has been shown to bind a similar factor and has been shown to play an important role in the induction of Eml, an ABA-regulated gene from wheat (Guiltinan er al., 1990). It should be noted, however, that a tetramer of the sequence TACGTGGC, which is implicated in ABA regulation from several rice and cotton genes, was incapable of imparting ABA regulation in tobacco, even when specific binding of the endogenous GBF (in this case the GBF homologue from tobacco named TAFl) was demonstrated (Oeda er al., 1991). Much recent work has been directed at determining how these G-box like sequences can control such a diverse set of genes. In Arabidopsis, at least three distinct 14 binding factors which recognize G-Box sequences have been cloned and characterized (Schindler et al., 1992). At least two such binding activities have also been isolated from nuclear extracts of leaves from parsley, cauliflower, wheat, rice, and tobacco (Armstrong er al., 1992; Williams et al. , 1992; Guiltinan er al., 1990; Mundy er al. , 1990; Katagiri er al. , 1989), and the genes for a number of these binding factors have also been cloned (Armstrong er al., 1992; Guiltinan er al., 1990; Oeda er al. , 1991). As was the case for OCT 1 and OCT2, it appears that these binding factors are able to differentiate between several genes with G-box binding sites within the same nucleus (Williams et al., 1992). The fact that there appear to be several differentially regulated genes all bound by GBFs within a given cell suggests a number of possible explanations for how this family of binding factors operate. It is possible that the promoters for these different genes are bound by other factors which prevent binding of the GBFs when the genes are inactive. In this scenario, when the appropriate environmental signal is perceived, these blocking factors are moved away, and the promoters become "open" for G-Box binding and subsequent gene activation. Another possibility is that there are a large number of GBFs that are all ”activated" by specific environmental signals m that all recognize subtly different flanking sequences around their respective G-Boxes. In this case, when the appropriate environmental signal is perceived and the GBF is activated, it then binds only to the promoters that contain G-Boxes in the context of specific flanking sequences. Alternatively, there may only be a limited number of GBFs, but each type could be specifically altered in different ways by various environmental signals. Such alterations could cause the factors to recognize subtle differences in the G-Box sequences or in the various other proteins that are likely to be present on the promoters of their respective genes. Such alterations may be in the form of specific phosphorylations, acetylations, or methylations, or they may be the result of other 15 protein:protein interactions (as was suggested for OCTl and OCT2 specificities). Finally, the reality of how these factors bring about the specific regulation of the many different genes may well be the result of a combination or subtle derivation of any of the possibilities just discussed. A number of significant observations have been made in investigating how the GBFs operate, which have lent support to the various models just discussed. These are: 1) G-box binding factors, like other known luecine zipper DNA binding factors (see Ziff, 1990 for a review), are capable of forming heterodimers (Schindler et al., 1992). This means that with a limited number of GBF monomers many novel GBF dimers could be formed. If it is shown that different GBF combinations have slightly different binding specificities and sensitivities to different environmental signals, then the specificity requirement will be met. 2) GBFs can be modified via phosphorylation, and this phosphorylation affects the GBFs' gene activating abilities (Klimczak er al., 1992). It will be interesting to see if any specific modifications occur in response to a particular environmental signal, and if so, whether any of these modifications alter the ability of the GBF to bind subtly different G-Box sequences or form specific GBF heterodimers. 3) In vitro binding studies with several different GBFs isolated from a single nuclear preparation have demonstrated distinct binding activities in a sequence dependent manner (Williams et al. , 1992; Schindler et al. , 1992). Also, GBFs from parsley interact with other nuclear factors and that these interactions alter their binding specificities (Armstrong er al. , 1992). Thus, it appears that given GBFs can be distinguished by their ability to recognize and bind subtly different G-Box sequences. If it is demonstrated that there are indwd many different GBFs (or heterodimer combinations), then again the specificity requirements will be met. 4) Arabidopsis GBF3 and tobacco TAFl both appear to be expressed primarily in the roots, while Arabidopsis GBFl and GBF2 are expressed primarily in green tissue (Schindler et al, 16 1992; Odea et al., 1991). This means that within the GBF gene family, certain members can be expressed in a tissue specific manner. Therefore, at least a portion of the specificity requirements may be met by limiting the expression of certain GBF forms to specific tissues. Expanding on this idea, it is also possible that the presence of other additional factors might inhibit the expression of genes that would otherwise be activated by GBFs in specific tissues (i.e. tissue specific silencers). Finding and characterizing UREs and the proteins which bind them is only the initial step in elucidating any given signal transduction pathway. One must next determine the factors which influence the presence of, the activation of, and the binding of these proteins to the promoters. Such factors include the spatial and temporal regulation of the expression of the genes which encode these DNA binding proteins, and also the proteins involved in the phosphorylation or other post-translational modifications of these proteins which may alter their ability to bind to their specific sequences or alter their ability to interact appropriately with the transcriptional apparatus. For example, the chromatin structure in and around the promoter has been demonstrated to alter the binding pattern of the essential components of the transcriptional apparatus (Layboum and Kadonaga (1991); see Eissenberg and Elgin, (1991) for a review). Finally, the spatial orientation and packing of the chromatin within the nucleus and the channels through it may even play a role by determining which mRNAs are allowed to leave the nucleus and which are physically blocked by - the maze of proteins and DNA (Blobel, 1985; Chang and Sharp 1990). Much general knowledge has been learned about how eucaryotic genes may be regulated. However, at the time this dissertation wasbegun, little was known about how plants perceived environmental signals or the signal transduction pathways that existed to bring about alter gene expression in response to changes in the environment. There has also been much learned concerning the changes in physiology and 17 biochemistry that occur in plants that are placed at low temperature. However, few conclusive studies have been done to illuminate the affects of any of the given changes that are known to occur. In order to learn more about both environmental signal perception, and to obtain the tools to study the molecular aspects of cold stress physiology, the work in this dissertation was undertaken. Specifically, the goal of this work was to clone cold-regulated genes from Arabidopsis, and to study the regulation of those genes to gain insight into how Arabidopsis senses low temperatures and brings about changes in gene expression. Literature Cited Armstrong GA, Weisshaar B, Hahlbrock (1992) Homodimeric and heterodimeric leucine zipper proteins and nuclear factors from Parsley recognize diverse promoter elements with ACGT cores. The Plant Cell 4, 525-537 Berstein P, Ross J (1989) Poly(A)-poly(A) binding protein and the regulation of mRNA stability. TIBS 14, 373-377 Bernstein P, Peltz S, Ross J (1989) The poly(A)-poly(A) binding protein complex is a major determinant of mRNA stability in virro. Mol. Cell. Biol. 9, 659-670 Blobel G (1985) Gene gating: A hypothesis. PNAS 83, 8527-8529 Brandts EJ ( 1967) Thermobiology. Academic Press, New York, pp25-73 Carpenter JF, Crowe JH (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25, 244-255 Chang DD, Sharp PA (1990) Messenger RNA transport and HIV rev regulation. Science 249, 614-615 Chen THH, Gusta LV (1983) Abscisic acid-induced freezing resistance in cultured plant cells. Plant Physiol. 73, 71-75 Chen H, Li PH, Brenner ML (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71, 362-365 18 Cullen BR (1990) The HIV-1 Tat protein: An RNA sequence specific processivity factor. Cell 63, 655-657 Dale J, Campbell WF (1981) Response of tomato plants to stressful temperatures. Plant Physiol. 67, 26-69 DeVargas L, Pargellis C, Hasan N, Bushman E, landy A (1988) Autonomous DNA ginding domains of integlase recognize two different sequence families. Cell 54, 923- 29 Dynan W, Tjian R (1985) Control of eukaryotic messenger RNA synthesis by sequence specific DNA binding proteins. Nature 316, 774-777 Eissenberg JC, Elgin SCR (1991) Boundary functions in the control of gene expression. Trends in Genetics 7, 335-340 Elliott R, Dickey L, White M, Thompson W (1989) Cir-acting elements for light regulation of pea ferredoxin I gene expression are located within the transcribed sequences. The Plant Cell 1, 691-698 Finkelstein RR, Sommerville CR (1990) Three classes of abscisic acid (ABA)- insensitive mutations of Arabidopsis define genes that control overlapping subsets of ABA responses. Plant Physiol. 94, 1172-1179 Gatenby AA (1992) Protein folding and chaperonins. Plant Mol. Biol. 19, 677-687 Gilmour SJ, Hejela RK, Thomashow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol. 87, 745-750 Gilmour SJ, Thomashow MF (1991) Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol. Biol. 17, 1233-1240 Gordon-Kamm WJ, Steponkus P (1984) The influence of cold acclimation on the behavior of the plasma membrane following osmotic contraction of isolated protoplasts. Protoplasms 123, 161-173 Gordan-Kamm WJ, Steponkus P ( 1984) The behavior of the plasma membrane following osmotic contraction of isolated protoplasts: Implications in freezing injury. Protoplasma 123, 83-94 Greenblatt J (1991) Roles of TFIID in transcription initiation by polymerase 11. Cell 66, 1067-1070 Gronemyer H (1992) Control of transcriptional activation by steroid hormone receptors. The FASEB Journal 6, 2524-2529 Guiltinan MJ, Marcotte WR, Quatrano RS (1990) A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250, 267-270 Guy C (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann. Rev. of Plant Physiol. 41, 187-223 19 Hajela R, Horvath DP, Gilmour SJ, Thomashow MF (1990) Molecular cloning and expression of cor (cold-regulated) genes in Arabidopsis thaliana. Plant Physiol. 93, 1246-1252 Horikoshi M, Hai T, Lin Y, Green M, Roeder R (1988) Transcription factor ATF interacts with TATA factor to facilitate establishment of a preinitiation complex. Cell 54, 1033-1042 Hunter T, Karin M (1992) The regulation of transcription by phoshorylation. Cell 70, 375-387 Inges C, Shales W, Cress S, Treizenberg S, Greenblatt J (1991) Reduced binding of TFIID to transcriptionally compromised mutants of VP16. Nature 351, 588-590 Johnson P, McKnight S (1989) Eucaryotic transcriptional regulatory proteins. Ann. Rev. Biochem. 58, 799-839 Karin M, Smeal T (1992) Control of transcription factors by signal transduction pathways: the beginning of the end. TIBS 17, 418-422 Yamaguchi-Shinozaki K. and Shinozaki K. (1992) Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet. in press. Klein-Hitpass L, Tsai S, Weigel N, Allan G, Riley D, Rodriguez R, Schrader W, Tsai M, O'Malley B (1990) The progesterone receptor stimulates cell-free transcription by enhancing the formation of a stable preinitiation complex. Cell 60, 247-257 Klimczak LI, Schindler U, Cashmore T (1992) DNA binding activity of the Arabidopsis G-box binding factor GBFl is stimulated by phosphorylation by casein kinase II from broccoli. The Plant Cell 4, 87-98 Koeller D, Casey J, Hentze M, Gerhardt E, Lee-Nien C, Klausner R, Harford J (1989) A cytosolic protein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA. PNAS 86, 3574-3578 Kurkela S, Borg-Franck M (1992) Structure and expression of kin2, one of two cold- and ABA-induced genes of Arabidopsis thaliana. Plant Mol. Biol. 19, 689-692 Lam E, Chua N—H (1991) Tetramer of a 21-base pair synthetic element confers seed expression and transcriptional enhancement in response to water stress and abscisic acid. J. Biol. Chem. 266, 17131-17135 Lang V, Heino P, Palva ET (1989) Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh. Theorétical and Applied Genetics 77, 729-734 Layboum JP, Kadonaga JT ( 1991) Role of Nucleosomal cores and histone H1 in regulation of transcription by RNA polymerase II. Science 254 238,244 Levitt J (1980) Responses of plants to environmental stress. Chilling, freezing, and high temperature stresses. Ed 2, Academic Press, New York 20 Liebold EA, Munro HN (1988) Cytoplasmic protein binds to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs. PNAS 85, 2171-2175 Luehrsen K, Walbot V (1991) Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol. Gen. Genet. 255, 81-93 Malter J (1989) Identification of an AUUUA-specific messenger RNA binding protein. Science 246, 664-666 Meyerowitz EM (1989) Arabidopsis, a useful weed. Cell 56, 263-269 Mohapatra SS, Poole RJ, Dhindsa RS (1988) Abscisic acid regulated gene expression in relation to freezing tolerance in alfalfa. Plant Physiol. 87, 468-473 Muller H, Sogo J, Schaffner W (1989) An enhancer stimulates transcription in trans when attached to the promoter via a protein bridge. Cell 58, 767-777 Mullner E, Kuhn L (1988) A stem loop in the 3‘ untranslated region mediates iron dependent regulation of the transferrin receptor mRNA stability in the cytoplasm. Cell 53, 815-825 . Mullner E, Neupert B, Kuhn L (1989) A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin mRNA. Cell 58, 373-382 Mundy J, Yamaguchi-Shinozaki K, Chua N-H (1990) Nuclear proteins bind conserved elements in abscisic acid-responsive promoter of a rice rab gene. PNAS 87, 1406- 1410 Myer V, Lee S, Steitz J (1992) Viral small nuclear ribonuclearproteins bind a protein implicated in messenger RNA destabilization. PNAS 89, 1296-1300 Nordin K, Pekka H, Palva E (1991) Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh. Plant Mol. Biol. 16, 1061-1071 Odea K, Salinas J, Chua N-H (1991) A tobacco bZip transcription activator (TAP-l) binds to a G-box-like motif conserved in plant genes. The EMBO Journal 10, 1793- 1802 Payvar F, DeFranco D, Firestone G, Edgar B, Wrang O (1983) Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell 35, 381-392 Pugh BF, Tjian R (1991) Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes and Dev. 5, l935=~1945 Ross J (1989) The turnover of messenger RNA. Scientific American Apr. pp48-55 21 Roy A, Meisteremst M, Pogmonec P, Roeder R (1991) Cooperative interaction of an initiator-binding transcription initiation factor and the helix-loop-helix activator USF. Nature 354, 245-248 Sakai A, Larcher W. (1987) Frost survival of plants; response and adaptation to freezing stress. Spring-Verlag, New York Sawadogo M, Setenac A (1990) RNA polymerase B(II) and general transcription factors. Ann. Rev. Biochem. 59, 711-754 Schindler U, Terzaghi W, Beckmann H, Kadesch T, Cashmore T (1992) DNA binding site preferences and transcriptional activation properties of the Arabidopsis transcription factor GBFl. The EMBO Journal 11, 1275-1289 Schindler U, Menkens AE, Beckmann H, Ecker JR, Cashmore T (1992) Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZip proteins. The EMBO Journal 11, 1261-1273 Shapiro D, Blume J, Nielson D (1987) Regulation of messenger RNA stability in eucaryotic cells. Bio. Essays 6, 221-226 Sharp P (1992) TATA-binding protein is a classless factor. Cell 68, 819-821 Shaw G, Kamen R (1986) A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659-667 Shyru A-B, Greenberg M, Belasco G (1989) The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Genes and Dev. 3, 60-72 Steponkus PL (1984) Role of the plasma membrane in freezing injury and cold acclimation. Annu. Rev. Plant Physiol. 35, 543-585 Steponkus P, Lynch D (1989) Freeze/thaw induced destabilization of the plasma membrane and the effects of cold acclimation. I. Bioenerg. Biomembr. 21, 21-41 Struhl K, (1997) Promoters, activators proteins, and the mechanisms of transcriptional initiation in yeast. Cell 49, 295-297 Takahashi K, Vigneron M, Matthes H, Wildman A, Zenke M, Chambon P (1989) Requirement of stereospecific alignments for initiation from simian virus 40 early promoter. Nature 319, 121-126 Taylor I, Workman J, Schuetz T, Kingston R (1991) Facilitated binding of GAL4 and heat shock factor to nucleosomal templates: Differential function of DNA-binding- domains. Genes and Dev. 5, 1285-1298 Thomashow M. (1990) Molecular genetics of cold acclimation in higher plants. In Scandalios JG, and Wright TRF, eds. Advances in genetics, Vol. 28, Genetic Responses to Environmental Stress. Academic Press, New York 22 Thomashow M, Gilmour S, Hajela R, Horvath D, Lin C, Guo W (1990) Studies on cold acclimation in Arabidopsis thaliana. IN, AB Bennett, SD O'Neill, eds, Horticultural Biotechnology. Wiley-Liss, New York, pp305-314 Treisman R, Maniatis T (1985) Simian virus40 enhancer increases the number of RNA polymerase II molecules on linked DNA. Nature 315, 72-75 Tseng M-J, Li PH (1990) Alterations of gene expression in potato (Solanum commersoniz) during cold acclimation. Plant Physiol. 78, 538-547 Wang W, Gralla J, Carey M (1992) The acidic activator GAL4-AH can stimulate polymerase II transcription by promoting assembly of a closed complex requiring TFIID and TFIIA. Genes and Dev. 6, 1716-1727 Weintraub H, Davis R, Tapscott S, Thaymer M, Kraus M, Benezera R, Blackman T, Turner D, Rupp R, Hollenberg S, Zhuang Y, Lassar A (1991) The myoD gene family: Nodal point during specification of the muscle cell lineage. Science 251, 761-766 Weiser CJ (1970) Cold resistance and injury in woody plants. Science 169, 1269- 1278 Williams ME, Foster R, Chua N-H (1992) Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. The Plant Cell 4, 485-496 23 Chapter 2 CLONING AND EXPRESSION OF COLD- REGULATED GENES FROM ARABIDOPSIS T HALIANA SUMMARY Previous work has demonstrated specific changes in the population of mRNAs in Arabidopsis thaliana during incubation at 5°C (a treatment known to acclimate plants to low temperatures and increase freezing resistance). Consequently, four clones (pHH28, pHH29, pHH67, and pHH7.2) were isolated from a cDNA library made from mRNA of cold-treated Arabidopsis. These clones represented distinct mRNAs (of sizes 2.5, 0.55, 0.65, and 1.1 kb) which specifically accumulated 10-30 fold during a three day cold treatment. These cgld-regulated (cor) mRNAs also accumulate in response to drought stress. They do not however, accumulate in response to heat shock. The car messages accumulate in plants placed at temperatures of about 10°-12°C for three days. Nuclear runon experiments suggests that three of these mRNAs (represented by clones pHH28, pHH29, and pHH7.2) are primarily post-transcriptionally regulated. The mRNA represented by clone pHH67 appears to be primarily transcriptionally regulated. The implications for signal transduction concerning these results are discussed. 24 INTRODUCTION Many plant species have the ability to "cold harden" or acclimate to freezing temperatures when first exposed to low, non-freezing temperatures. Such cold acclimated plants become increasingly freezing tolerant and are better able to survive frost (Levitt, 1980; Sakai and Larcher, 1987). In addition to this cold acclimation process (Graham and Patterson, 1982), ABA application at ambient temperatures has been shown to bring about increased freezing tolerance in intact plants as well as in cell. and tissue cultures (Rikin er al., 1975; Chen et al., 1979; Chen and Gusta, 1983; Keith and McKersie, 1986; Orr et al., 1986; Reaney and Gusta, 1987). ' Several physiological and biochemical changes are associated with cold acclimation. Changes in membrane composition and organization, such as increased membrane fluidity and desaturation of membrane fatty acids are believed to play important roles in maintaining cellular integrity (Sakai and Larcher, 1987). Other cellular changes like alterations in levels of free sugars, amino acids and soluble proteins have been reported to be associated with and to have important roles in cold acclimation in general and freezing tolerance in particular (Levitt, 1980). However, the exact roles of these changes during cold acclimation are not yet fully understood. Some of the sugars, amino acids and proteins that accumulate during cold acclimation may physically help prevent freezing, but no direct cause and effect relationship has been clearly established. Endogenous levels of ABA have been shown to increase during cold acclimation and prior to development of freezing tolerance (Kasperska-Palacz et al., 1977; Daie and Campbell, 1981; Chen et al, 1983). It has even suggested that cold induces an 25 increase in endogenous ABA levels which then acts as the stimulus for development of cold hardiness, a hypothesis which remains to be tested directly (Chen et al., 1983). Cold hardiness is a heritable trait (see Thomashow, 1989 for a review). Many investigators have observed accumulation of new mRNAs and novel polypeptides during cold acclimation and prior to development of freezing tolerance (Graham and Patterson, 1982; Cloutier, 1983; Guy and Haskell 1989; Kurkela er al., 1988; Gilmour er al., 1988). These studies have relied both on in vitro translation of isolated messages as well as in viva labelling of polypeptides. Also, cytoplasmic protein synthesis inhibitors like cycloheximide have been observed to block cold acclimation (Chen et al, 1983; Trunova, 1982). However, chloramphenicol,a chloroplast protein synthesis inhibitor has no effect on cold acclimation. All of these observations support the earlier suggestions that regulation of nuclear gene activity may be involved in cold acclimation (Weiser, 1970). However, the specific genes, and the mechanisms and levels of regulation of action remain unknown. To establish clearly a relationship between gene expression and the increase in freezing tolerance, the genes in question must be isolated, identified, and characterized. It has previously been established (Kurkela er al., 1988; Gilmour er al., 1988) that Arabidapsis thaliana, like many other herbaceous plants, can cold acclimate. I decided to use Arabidopsis for further investigations on the molecular biology of cold acclimation in plants. Arabidapsis is a small crucifer with a short life cycle (about six weeks), has a small genome of about 70 million hp (with very little repetitive DNA), and is amenable to gene transfer and regeneration of transgenic plants (Meyerowitz, 1989). The availability of many well characterized mutants further adds to its utility in molecular genetic studies. Finally, since Arabidopsis is a crucifer, what is learned from studies on it may be generally applicable to other, economically important 26 crucifers such as rapeseed mustards, and Brassica aleracea (broccoli, cauliflower, cabbage and a host of others). In this chapter, the isolation and molecular cloning of several cold-regulated (car) genes from a cDN A library made from cold acclimated Arabidapsis is reported. Initial characterization of some of these cDNA clones is presented, and the environmental factors that may play a role in their regulation are explored. Finally, the level (transcriptional or post—transcriptional) of regulation of these car genes is examined. MATERIALS AND METHODS Growth and treatment of plants Plants and calli were grown as previously described (Gilmour er al., 1988). Plant material was flash frozen in liquid nitrogen, pulverized to a fine powder and stored at -80°C until used. To study the effects of various temperatures or other stresses, plants grown under constant illumination at 23°C for 18 - 20 days were used. To determine the minimum temperature at which the car genes were induced, plants were moved to growth chambers preset to 5, 7, 10, 12, 15 or 17°C. For all other experiments plants were moved to growth chambers set at 5°C and incubated for the desired times. For time course of cold-induction studies, samples were withdrawn after 1 and 4 hours and at subsequent 4 hour intervals until 24 hours, then at 3, 7 and 14 days, frozen in liquid nitrogen and stored at -80°C. Similarly, for the time course of deacclimation, plants that had remained in cold for 3, 7, or 14 days were returned to growth chambers set at 23°C and samples removed after 1 and 4 hours and at subsequent 4 hourly intervals to 24 hours and then at 3, 7, and 14 days, frozen in liquid nitrogen and stored at -80°C. ABA treated plants were sprayed to runoff with a 100 27 uM solution of (+/-) ABA (mixed isomers obtained from Sigma Chem. Corp.). Pots were covered with plastic wrap and placed under continuous fluorescent light. Samples were taken at various times up to 24 hours. Heat stressed plants were moved to a chamber set at 37°C and incubated for 3 hours before harvesting and storage at -80°C. Drought stress was induced by withholding water for up to 7 days. RNA extraction and northern analysis Poly A mRNA from Arabidopsis was extracted as previously described (Gilmour et al. , 1988). Initial northern analyses were done on poly A+ mRNA from ecotypes Columbia and Landsberg erecta to ascertain the universality of the induction of the selected messages. All later northern analyses used total RNA from Landsberg plants. Preparation and screening of cDNA library Double stranded cDNA used for making a library in EcoRI-cut lambda ZAP was synthesized essentially by the method of Gubler and Hoffman (1983) using AMV reverse transcriptase (Promega). Double stranded (DS) cDNAs were methylated with EcoRI methylase and ligated to EcoRI linkers (NEB). These were then cut with EcoRI, and fractionated on a Sepharosc CL4-B column using established, published methods (Maniatis et al. , 1982). DS cDNA fractions 0.5 kb and larger were pooled, ligated to EcoRI cut, phosphotased lambda ZAP, and the recombinant phage packaged in vitro with Packagene (Promega) extract. The resulting phage particles were plated on E. cali BB4 cells supplied with lambda ZAP (Stratagene). At least 5 x 105 primary recombinant plaques were obtained, amplified one cycle and frozen for storage at - 80°C in 7% DMSO (Maniatis et al., 1982). All subsequent plating was done by 28 scraping a small amount of phage from the frozen stock without any secondary amplification to prevent any possible loss of recombinants. Differential screening was done on plaque lifts on Nytran (Schleicher & Schull) and at least half the library equivalent was screened in one experiment, using 32P labeled cDNA probes made from control or cold acclimated plant or callus mRNA. Probed filters were sandwiched between Cronex lighting plus (DuPont) intensifying screens and Kodak AR5 X-ray film at -80°C. Selected plaques were purified, rescreened as mentioned in 'results' and recombinant cDNA 'excised' out of the phage in plasmid bluescript SK' using the biological rescue recommended by Stratagene. Genomic blots and Southern analysis of cDN As Southern analysis was performed on EcoRI cut recombinant plasmids to establish the relationships of the isolated clones. Southern analysis was also done on restricted genomic DNA from Arabidapsis plants to identify genomic segments and to determine the copy number of these genes. Northern analysis was performed after formaldehyde gel electrophoresis (Maniatis et al., 1982) using poly A mRNA or total RNA isolated as above. Nytran (Schleicher & Schull) was used for Southern and northern blotting. Nuclear isolation and runon transcription . Isolation of nuclei for runon transcription employed the method of Feinbaum (1987). Runon transcription using 32P labelled UTP was done as described by Ausubel er al., (1987), RNA was purified and used to probe slot blots of gel purified isolated inserts of DNA from selected recombinant plasmids. 29 RESULTS Cloning of cold-regulated genes Cold acclimation in Arabidapsis is accompanied by stable changes in the mRNA population as demonstrated by one or two dimensional gels of in vitra translation products (Gilmour et al. , 1988; Kurkela er al. , 1988). Differential screening of a lambda Zap/cDNA library made from cold acclimated Arabidapsis mRNA yielded about twenty cold-regulated cDNA clones. Southern hybridization indicated that four different genes (or gene families) were represented by these 20 clones. A representative clone for each gene (or gene family) was selected and used to probe northern blots of poly A+ or total mRNA from control, cold acclimated and deacclimated plants of A. thaliana ecotype Landsberg (see Table 2.1). Four discrete mRNA species ranging in size from 0.5 to 2.4 kb were found to be cold-regulated (Figure 2.1). A non-cold-regulated gene represented by pHH25 was isolated to use as a control. It is evident from these experiments that the recombinants selected represent messages which are either absent or present at very low basal levels in both control and deacclimated plants but accumulate significantly during cold adaptation. Cold-regulated genes are induced at temperatures between 10°C and 15°C In order to determine the maximum temperature at which these four cold- regulated RNAs start to accumulate, plants were incubated for three days in growth chambers set at temperatures ranging from 17°C to 5°C. Northern blots of RNA isolated from these plants were probed using the inserts from selected cold-regulated cDNA clones. The results demonstrate that the threshold temperature for inducing the cor genes is reached at between 10 and 15°C (Figure 2.2). 30 Table 2.1 General Characteristics of cor cDNA Clones Clone Insert Transcript Representation Designation Size Size in Library‘ kbp k0 °/e pHH7.2 ‘l .1 1.4 0.02 pHH28 1.3 2.5 0.03 pHH29 0.6 0.6 0.1 pHH67 0.7 0.7 0.1 ' Inserts from the various cor cDNA clones were ”P-labeled (10). ' hybridized with plaque lifts of the cDNA library, and the percentage of the plaques displaying hybridization estimated. 31 Figure 2.1 Northern analysis of car gene transcripts. Total RNA (5 ug) isolated from Arabidapsis leaves and stems was fraqg'onated on formaldehyde agarose gels, transferred to nytran, and hybridized with P-labled cDNA inserts as indicated. N, Nonacclimated; A, acclimated at 5°C for 3 days; D, deacclimated (3 days at 5°C then 1 day at 22°C). Transcript sizes are given in kb. 32 BNIIa amxxo < Z I. m~zxa azrn mazze Figure 2.1 2 33 Figure 2.2 Northern analysis of car gene transcripts after plants were plants were placed at various temperatures (as indicated) for three days. 10 ug of total RNA was fractionated on formaldehyde agarose gels, blotted to Nytran, and hybridized to nick translated plasmids containing the indicated car cDNAs. 34 pHH67 I... Figure 2.2 35 Cold-regulated genes are controlled both transcriptionally and post- transcriptionally The level at which the selected cold-regulated (car) genes are regulated was investigated using nuclear runon transcription assays. This study demonstrates that the gene represented by the cDNA clone pHH67 is transcribed at a very low level in control plants. Its transcription increases dramatically when the plant is placed at 5°C for three days (Table 2.2). This result suggests a strong transcriptional component in its regulation. However, the increase in the rate of transcription is insufficient to explain the levels to which the RNAs corresponding to pHH28, pHH29, and pHH7 .2 accumulate (Table 2.2). Also, it is interesting to note that even under control conditions, where the level of accumulation of these cold-regulated messages is nearly zero, the genes corresponding to pHH28, and pHH7.2 are transcribed at a rate at least as high as any. of the other control genes in these experiments (Figure 2.3, and other data not shown). These data suggest that the genes represented by pHH28, pHH7 .2, and pHH29 are regulated at a post-transcriptional level. Cold-regulated genes are induced by drought stress, but not by heat shock When plants are subjected to freezing temperatures, one major form of injury is directly related to cellular dehydration caused by extra-cellular ice formation (Steponkus, 1984), it was of interest to determine if drought stress would also induce any of these cold-regulated genes. To do this, water was withheld from two week old plants, their relative water content was determined, and their RNA was extracted. Northern analysis demonstrated that drought stress is also capable of inducing all four cor genes (Figure 2.4). 36 Table 2.2 Relative Transcription Rates and Transcript Levels a! cor Genes in Cold Acclimared Versus Nonacclimated Plants cDNA Clone Parameter pHH7.2 pill-£28 pHH29 pHH67 pill-£25 401d W. h cold-acclimated” Transcription rate' 1 .2 2 0.2 1.8 at 0.3 2.4 t 1.1 9.6 t 2.3 1.7 t 0.3 Transcript level' 8.6 2 1.9 22 2: 5.0 11 t 4.3 . 25 t 3.4 0.8 t 0.03 ' Three independent sets ol nuclei were isolated from control and 3 d cold-acclimated plants and the relative transcription rates were detennined by nuclear run-on transcription assays as described it 'Materials and Methods.’ Values are the mean (2 55) fold increase in transcription rate it cold-acclimted plants. 'Total RNA was isolated from control and cold-ecclinulted plants (either leaves and stems or whole plants) and the steady state transcript levels of the indlcated genes were detennined by Northern analysis as described in “Materials and Methods.’ Values are the mean (3 se) fold increase i1 transcript level in cold-acefimated plants. n - 4 for the cor genes: n - 2 tor pHH25. Two of the determinations for the cor genes and one lor pHH25 were from the plants used in the transcription rate assays.Thevalueslorthecorgenesareminimunestirnatessincemeydonotindudedetenninations where transcripts were not detected in control plants (Le. cases in much the herease was 'infinite'). 37 Figure 2.3 A representative example of data from the nuclear runon assays. Run- ons: Nuclei were isolated from cold treated angl control plants, and transcription was allowed to continue in vitro in the presence of 2P-labled UTP. Equivalent number of counts from both acclimated (A), and nonacclimated (N), were used to probe slot blots of isolated insert of the various cor genes and the gene for the small subunit of Rubisco (rch) as a control. Northerns: Total RNA was isolated from the same batch of plants from which the given nuclei preparations were made. RNA was n out on 3 formaldehyde agarose gel, blotted to Nytran, and hybridized to P-labled insert DNA from the various cor genes and the rch gene. 38 aceuztoz ”Evan—ac <fi z _ < _ z 2253.... on:1n_ «NIT-n ocean. Figure 2.3 39 Figure 2.4 Effect of drought treatment on accumulation of cor gene mRNA. Drought treatment was accomplished by withholding water from the plants. The relative water contents (RWC) of the plants were measured. Total RNA was extracted from the plants and “big from each sample was fractionated on a formaldehyde gel and hybridized to the P-labled isolated inserts from the indicated plasmids. 4O RWC (96) 93 so 70 43 pHHl-z - -- " pHH28 a” pHH29 C ” pHH67 pHH25.. Figure 2.4 41 Because both drought stress and cold stress cause osmotic perturbations, the question arose as to if any osmotic stress could cause an increase in accumulation of cor gene message. To study this possibility, plants were watered with a 400 mM NaCl solution. Plants were visibly wilted after 24 hours. These plants were then harvested and their RNA extracted. Northern analysis was done to determine the amount of cor gene mRNA accumulated in response to the salt stress (Figure 2.5). The results demonstrated that the mRNA from the genes represented by pHH28, pHH7.2, and pHH29 all accumulate in response to salt stress. pHH67 mRNA however, accumulates very little in response to the salt treatment. Because the cor genes are induced by drought as well as by cold, it was of interest to determine if the accumulation of the cold-regulated RNAs was simply the result of a general stress phenomenon. To check this possibility, plants were placed at 37°C (a temperature shown to be capable of inducing a heat shock response in Arabidopsis, (Wu et al. , 1987) for three to four hours, RNA was extracted, and accumulation of the four cor mRNAs was determined by northern analysis. The results from this experiment show that none of these genes are induced by heat shock (Figure 2.6); however, the conditions used for these heat shock treatments were sufficient to induce the gene for hsp20. In addition to heat shock, plants were wounded by dicing with a razor, and floated on water for seven hours. Again, plants were harvested and the level of accumulation of cor gene mRNA was determined by northern analysis (Figure 2.7, lane 1). It was determined that the wounding treatment did not result in accumulation of cor gene mRNAs to levels higher than that of the unwounded, warm controls. Also, plants were wounded mg floated on water at 3°C for seven 42 Figure 2.5 Effect of salt stress on car gene mRNA accumulation. Plants were grown in 1 Liter clay pots in Bacto Potting Mix for 18 days. Plants were then watered with 200 mls of a 0.4 M NaCl solution (salt), or were placed at 5° C overnight (cold), or were left untreated (warm). After 24 hours, plants were harvested and total RNA was extracted for northern analysis. RNA was fractionated on formaldehyde agarose gels, hybridized to polyester backed nitrocellulose, and hybridized to the various cor genes. 43 Warm 0‘ ' 0 Cold *0 m m N 00 Salt . ”.pHH7.2 . pHH67 O . pHH29 ..pHHZS Figure 2.5 44 Figure 2.6 Effect of heat shock on car gene message accumulation. Plants were placed at 37°C for 3 hours, and then harvested for total RNA extractions. RNA was fractionated on formaldehyde agarose gels, hybridized to polyester backed nitrocellulose, and hybridized to the various cor genes. 45 CHS pHH7Q . pHHZB pHH29._ pHH57 pH H25 " hspZU Figure 2.6 46 Figure 2. 7 The effect of wounding on accumulation of cor gene mRNA. Plants were diced with a razor blade and floated on water for seven hours at 22° C (warm/wounded) or at 3° C (cold/wounded) or were left untreated (warm), or were placed at 3° C overnight (cold). Plants were harvested and total RNA was extracted for northern analysis. RNA was fractionated on formaldehyde agarose gels, hybridized to polyester backed nitrocellulose, and hybridized to the various cor genes. 47 W/ wounded C/ wounded 30 ' . pHH28 . pHH7.2 . pHH67 a: ' . pHH29 .... pHH25 Figure 2.7 48 hours in order to determine that the wounding treatment did not prevent accumulation of cor gene message. The results from this treatment demonstrated that the wounded plants were still capable of inducing all of the cor genes except the one represented by pHH67 (Figure. 2.7, lane 2). The wounding and heat shock experiments support the idea that the cor genes are not induced by general plant stress, but are specifically induced by stresses related to cold. Induction of the cor genes in roots, stems, and leaves and at various stages in the life cycle of AmbidOpsis thaliana The tissue specificity of cor gene mRNA accumulation was looked at by northern analysis. Four week old flowering plants were cold treated or left as untreated controls. Plants were harvested and plant parts were separated into roots, stems and flowers, and leaf rosettes, and RNA was extracted. The results of these northerns (Figure 2.8) indicate that the gene represented by pHH28 and pHH29 accumulate in all of the plant parts tested. The gene represented by pHH67 however, was only expressed in green tissues. Recent results have indicated that the gene represented by pHH67 (cor-15) is a chloroplast targeted protein (Lin and Thomashow, 1992). Thus, it is not surprising that it would only be expressed in green tissue. In addition to looking at tissue specificity, the ability of Arabidopsis to express these cor genes at various stages of their life cycle was examined. To do this, plants were cold treated at two weeks of age (which correlated to the 6 leaf stage), three weeks of age (when plants were just beginning to bolt), and at four weeks of age (when the plants were flowering and setting seed). The rosette portion of the plants were harvested, and RNA was extracted for northern analysis. The results of these experiments indicated that all of the cor genes tested accumulated in response to cold at all of the ages tested (Figure 2.9). 49 Figure 2. 8 The tissue specificity of the accumulation of cor gene mORNA was examined. Flowering, pot grown Arabidopsis thaliana were left at 22° C (W for warm control) or were placed at were placed at 5° C for three days (C for cold treated). After treatment, Whole plants were harvested (total) or were separated into roots (R), stems (S), and leaves (L). RNA was extracted, and 10 ug of total RNA was subjected to northern analysis. Blots were probed with nick translated Blue Script plasmid containing cDNA clones of the cor genes tested. _ I _ C C p o - pHH28 I 0 Q pHH67 51 Figure 2.9 The ability of Arabidopsis to express the cor genes in response to cold throughout its life cycle was examined. Plants were harvested at 2 weeks, 3weeks, and 4weeks of age, before (warm), or after (cold) a cold treatment. RNA was extracted, and 10 ug of total RNA was subjected to northern analysis. Blots were probed with nick translated blue script plasmid containing cDNA clones of the cor genes tested. 52 E E P. O O O a 29 £9 E ii 29‘ a 3 3 N m :1“ lWCIWClWCl .- . . pHH28 ‘.' . '.pHH67 ’ I r O . pHH29 ' pHH25 Figure 2.9 53 DISCUSSION Cold acclimation in higher plants is a many-faceted phenomenon. The most distinct manifestation of cold acclimation is increased freezing tolerance. To achieve this important low temperature survival ability, the plants must make numerous physiological changes that are likely to be mediated by the coordinated expression of several genes. It has been found that the rapid cold acclimation in Arabidopsis thaliana is concurrently accompanied by changes in steady state levels of a subset of translatable mRNAs (Gilmour et al., 1988; Kurkela et al., 1988). In vivo polypeptide labeling experiments have demonstrated that the protein products of many of these mRNAs also accumulate at the whole plant level. These observations, together with those in other plants, (Graham and Patterson, 1982; Cloutier, 1983, 84; Guy and Haskell 1989; Kurkela et al., 1988; Gilmour er al. , 1988) clearly demonstrate selectively regulated gene expression during cold acclimation, and suggest a possible role for these genes in the process of cold acclimation. With the goal being to understand the molecular genetic basis of this process, isolation and molecular cloning of cold-regulated genes was a very important first step. Currently, it has not been demonstrated that the cor genes play any role in the cold acclimation process. However, their cloning has provided additional evidence that the proteins they encode may be important in protecting the plant from freezing damage. Perhaps the strongest evidence for this comes from the fact that the cor genes have been evolutionarily conserved and their respective homologues all appear to be induced upon cold-stress (Guo et al., 1992; Singh et al. , unpublished data). It has been shown that several of the cor genes have homologues in Brassica napus (Singh et al. , unpublished data), and one car gene (cor4 7) has significant homology to a 54 cold-regulated gene from wheat (Guo et al., 1992). Also, all of the proteins encoded by the cor genes share two rather peculiar physical properties. They all remain soluble after boiling (Lin et al., 1991), and all are extremely hydrophilic (Gilmour et al., 1990; Horvath, see Chapter 3). Finally, another set of hydrophilic boiling soluble proteins from spinach were shown to protect isolated thylakoid membranes from freezing damage in in vitro assays (Volger and Heber, 1975). Thus, it seems unlikely that the cor genes would be conserved over great physiological distances (i.e. monocots to dicots) or that they would share similar structural motifs if they were not important. Also, since the cor genes and their homologues are cold-regulated, it implies that it is advantageous for plants to express proteins with similar physical characteristics during cold stress. Along with expression during cold stress, the cor genes are also induced by osmotic stresses (such as drought and salt stress), and by exogenous application of abscisic acid (ABA) (Figures 2.4 and 2.5; Hajela er al., 1990; Kurkula et al., 1989). These data, though interesting, are not surprising given earlier findings that both drought stress and exogenous application of ABA have been shown to cause the accumulation of a similar subset of novel proteins (Reviewed by Guy, 1990). ABA and drought stress have been shown to increase the freezing tolerance of otherwise unacclimated plants (Chen et al., 1983;Lang et al., 1989). Finally, severe dehydration is one of the major causes of cellular damage during a freezing stress (Steponkus, 1984). Thus, there appears to be a common link between cold stress and drought stress. ABA has been shown to increase in several plants (including Arabidopsis) in response to both drought and cold stress (Chen et al., 1989; Palva et al., personal communication). Also, ABA has been shown to increase freezing tolerance when exogenously applied to some plants (Chen et al., 1989; Lang et al., 1989). Therefore, it is tempting to speculate that ABA is the primary signal involved in regulating cor 55 gene expression. There are several lines of evidence which suggest that drought, cold and ABA may all regulate cor gene expression via independent signal transduction pathways. It is known that Arabidopsis plants with a mutation that renders them insensitive to exogenous applications of ABA (designated abiI for ABA insensitive 1), were either cold treated, drought stressed, or sprayed with ABA, and the induction of the cor genes was monitored. The results showed that the cor genes were still capable of responding to drought and cold stress, but were not induced by exogenous application of ABA (Nordin et al., 1991; Gilmour and Thomashow, 1991). Since initial analysis of the chi] mutation has shown that the exogenously applied ABA does accumulate in such plants (suggesting that these plants are not deficient in ABA uptake), these studies appear to indicate that the induction of the cor genes in response to both cold and drought is not dependent on the plant's ability to perceive increases in internal ABA levels (Finkelstein and Sommerville, 1990). Such results point to the possibility that the ABA response operates via a different (or partially different) signal transduction pathway than' does the drought or cold responses. It is noted however, that since the nature of the abiI mutation is not known, it is possible that endogenous ABA may still be functioning as an intermediate, and that some part of the pathway involved with the transmission of signal from exogenously applied ABA may be interrupted in the abiI mutants. Since the exact nature of the signal transduction pathway(s) is not yet known, it would be interesting to study the cis acting regulatory sequences of these (and other) car genes in order to isolate sequences that can impart specifically cold, ABA, or drought regulation to a reporter gene. One of the observation that was made during the general characterization of the environmental parameters of the cold-induced accumulation of several of these cor genes was that they appeared to be induced at temperatures between 15°C and 10°C. Interestingly, there are a number of other physiological processes that also demonstrate 56 a temperature threshold within this range, including vemalization (Lang and Melchers, 1947) and stratification (Berrie, 1985). The membranes of several chilling sensitive plants have also been observed to undergo a phase shift from a flexible liquid crystalline state to that of a solid-gel structure at temperatures ranging from 12°C to 12°C (Lyons, 1973). This last observation is particularly interesting since it means that the plasma membrane may provide a physical mechanism for initiating a signal transduction pathway that could result in changes in gene expression. Thus, it is possible that changes in the state of the plasma membrane could be the primary sensor for low temperature sensing in plants. The results of the nuclear runon experiments have suggested that the cor gene represented by pHH67 was primarily regulated at the transcriptional level (Figure 2.3). These data then suggest that the cold regulatory cis acting sequences in this gene should be found within its 5' nontranscribed sequences. In fact, there are recent data that support this conclusion. Constructs have been made that place the promoter for the gene represented by pI-II-I67 in front of a GUS reporter gene. These constructs demonstrate the induction of the GUS reporter gene in response to cold, drought, and ABA thus confirming the implications of the nuclear runon assays (Baker, manuscript in preparation). Nuclear runon results from the other three cor genes (represented by clones pHH28, pHH7.2, and pHH29) suggest that these genes are post-transcriptionally regulated (Figure 2.3). This would suggest that the cis-acting sequences responsible for the cold-regulation of these genes should be found within their transcribed regions. Sequence analysis of the cDNAs from these three cor genes suggests a number of interesting sequence motifs that may play a role in their regulation. The 3' untranslated regions (UTR) of all three of these presumably post-transcriptionally regulated cor genes are very ”U" rich. Stretches of poly "U"s in the 3' UTR of several genes have 57 been shown to be sites of nucleolytic cleavage in both in vitro and in viva systems (Albrecht et al., 1984; Ross, 1989). Also, the sequence AUUUA (of which there are several within the 3'UTR of the genes represented by pHH7.2 and pHI-128) in the 3' UTR of several post-transcriptionally regulated genes from animal systems (c-myc and GM-CFS) has been shown to act as a binding site for proteins implicated in altering the stability of these (and other) mRNAs (Shaw and Kamen, 1986; Myer et al., 1992). However, it should be noted that nuclear runon assays have been known to give results that are difficult to interpret (Thompson and White, 1991). Consequently, more work will need to be done in order to be certain that the cor genes represented by pHH28, pHH29, and pHH7.2 are truly post-transcriptionally regulated. If these genes are truly post-transcriptionally regulated, then the sequences causing these genes to be cold-regulated should be in their transcribed region. Therefore, fusion constructs that place the transcribed portion of these genes under the control of a constitutive promoter, should be expressed in response to cold. Likewise, if these genes are really transcriptionally regulated, then the promoters of these genes should contain the necessary cis acting elements for cold-regulation. Thus, as was the case for the gene represented by pHH67, the promoters of these genes should be able to drive cold- regulated expression of a reporter gene. Acknowledgments I would like to credit and thank Dr. Ravindra Hajela for preparing and helping screen the cDNA library from which the cor genes were cloned. I would also like to thank him for preparation of figure 2.1. 58 Literature Cited " Albrecht G, Krowczynska A, Brawerman G ( 1984) Configuration of B-globin messenger RNA in rabbit reticulocytes. J. Mol. Biol. 178, 881—896 Ausubel FM, Brendt R, Kingston RE, Moore DD, Seidmon JG, Smith JA, Struhl K (1987) Current protocols in molecular biology. Greene Publishing Associates, Wiley Interscience, New York Berrie, (1985) Germination and dormancy. In Advanced Plant Physiology M.B. Wilkins ed. Pitman Publishing Inc. Massechusetts Chen HH, Gavinlertvatana P, Li PH (1979) Cold acclimation of stem cultured plants and leaf callus of solanum species. Bot. Gaz. 140, 142-147 Chen THH, Gusta LV (1983) Abscisic acid-induced freezing resistance in cultured plant cells. Plant Physiol. 73, 71-75 Chen H, Li PH, Brenner ML (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71, 362-365 Cloutier Y (1983) Changes in the electrophoretic patterns of the soluble proteins of winter wheat and rye following cod acclimation and desiccation stress. Plant Physiol. 71, 400403 Daie J, Campbell WF (1981) Response of tomato plants to stressful temperatures. Plant Physiol. 67, 26-69 Feinbaum RL, Ausubel FM (1988) Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol. Cell. 810.. 8, 1985-1992 Freier S, Kierzek R, Jeager J, Sugimoto N, Caruthers M, Neilsin T, Turner D (1986) Improved free energy parameters for predictions of RNA duplex stability. PNAS 83, 9373-9377 Gilmour SJ, Hejela RK, Thomashow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol. 87 , 745-750 Graham D, Patterson BD (1982) Responses of plants to low, non-freezing temperatures: proteins, metabolisms, and acclimation. Annu. Rev. Plant Physiol. 33, 347-372 Gulber U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269 Guo W, Ward R, Thomashow M (1992) Characterization of a cold-regulated gene from wheat related to Arabidopsis COM 7. Plant Physiol. 100, 915-922 59 Guy CL, Haskell D (1989) preliminary characterization of high molecular mass proteins associated with cold acclimation in spinach. Plant Physiol. Biochem. 27, 389-394 Guy CL, Haskell D, Yelenosky G (1988) Changes in freezing tolerance and peptide content of spinach and citrus at 5°C. Cryobiology 25, 264-271 Guy C (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Ann. Rev. of Plant Physiol. 41, 187-223 Kasperska-Palacz A , Dlugokecka E, Breitenwald J, Wcislinska B (1977) Physiological mechanisms of frost tolerance: possible role of protein in plant adaptation to cold. Biol. Plant. 19, 10-17 Keith CN, McKensie BD (1986) The effect of abscisic acid on the freezing tolerance of callus cultures of Lotus comicalatus L. Plant Physiol. 80, 766-770 Koeller D, Casey J, Hentze M, Gerhardt E, Lee-Nien C, Klausner R, Harford J (1989) A cytosolic protein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA. PNAS 86, 3574-3578 Kurkela S, Franck M, Heino P, Lang V, Palva T (1988) Cold-induced gene expression in Arabidopsis thaliana L. Plant Cell Rep. 7, 495-498 Lang A. and Melchers G. (1947) Vernalization und devemalization bei einer zweijahrigen pflanze. Z. Naturf. 2, 444-449 Lang V, Heino P, Palva ET (1989) Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 77, 729-734 Levitt J (1980) Responses of plants to environmental stress. Chilling, freezing, and high temperature stresses. Ed 2, Academic Press, New York Lyons J .M. (1973) Chilling injury in plants. Ann. Rev. Plant Physiol. 24, 445-466 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Meyerowitz EM (1989) Arabidopsis, a useful wwd. Cell 56, 263-269 Mullner E, Kuhn L (1988) A stem loop in the 3' untranslated region mediates iron dependent regulation of the transferrin receptor mRNA stability in the cytoplasm. Cell 53, 815-825 Olien CR (1977) Ice adhesions in relation to freezing stress. Plant Physiol. 60, 499- 503 Orr W, Keller WA, Singh J ( 1986) Induction of freezing tolerance in an embryonic cell suspension culture of Brassica napus by abscisic acid at room temperature. J. Plant Physiol. 126, 23—32 60 Reaney MJT, Gusta LV (1987) Factors influencing the induction of freezing tolerance by abscisic acid in cell suspension cultures of Bromus inermis Leyss and Medicago sativa L. Plant Physiol. 83, 423-427 Rikin A, Waldman M, Richmond AE, Dovrat A (1975) Hormonal regulation of morphogenisis and cold-resistance. J. Exper. Bot. 26, 175-183 Ross J (1989) The turnover of messenger RNA. Scientific American Apr. pp48-55 Sakai A, Larcher W (1987) Frost survival of plants: response and adaptation to freezing stress. Spring-Verlag, New York A Shaw G, Kamen R (1986) A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659-667 Thomashow M (1990) Molecular genetics of cold acclimation in higher plants. In Scandalios JG, and Wright TRF, eds. Advances in genetics, Vol. 28, Genetic Responses to Environmental Stress. Academic Press, New York Thomashow M, Gilmour S, Hajela R, Horvath D, Lin C, Guo W (1990) Studies on cold acclimation in Arabidopsis thaliana. IN, AB Bennett, SD O'Neill, eds, Horticultural Biotechnology. Wiley-Liss, New York, pp305-314 Thompson EF, White M] (1991) Physiological and molecular studies of light-regulated nuclear genes in higher plants. Ann. Rev. Plant Physiol. 42, 423-466 Trunova TI (1982) Mechanisms of winter wheat hardening at low temperature . In PH Li, A Sakai, eds, Plant cold hardiness and freezing stress, Vol 2. Academic Press, New York, pp 41-54 Volger HG, Heber U (1975) Cryoprotective leaf proteins. Biochem. Biophys. Acta. 412, 335-349 Wilson CC (1948) The effect of some environmental factors on the movements of guard cells. Plant Physiol. 23, 5-18 Wright STC, Hiron RWP (1972) The accumulation of abscisic acid in plants during wilting and under other stress conditions. In DJ Can, ed., Plant Growth Substances. New York Springer-Verlag. 61 Chapter 3 Regulation of the Arabidopsis cor78 gene SUMMARY In order to gain a better understanding of the regulation of the apparently post- transcriptionally regulated cor genes from Arabidopsis, the genomic clone of the cor78 gene was obtained and a portion of it sequenced. The 5' portion of this clone was used as a probe to isolate a near full length cDNA clone corresponding to this gene. Sequence analysis of both clones indicated that this gene has three introns and should encode a highly hydrophilic, 79 kD polypeptide. In vitro transcription and translation of the near full length cDNA, however, demonstrated that the protein encoded by cor78 runs as 160 kD on an SDS-PAGE gel. A series of constructs containing portions of the genomic clone were prepared to determine which regions of the cor78 gene were responsible for its cold-regulated expression. Analysis of the expression of these constructs in transgenic Arabidopsis indicated that sequences responsible for the cold-regulation of the cor78 gene are contained within its promoter. Implications of this result with regards to previous nuclear runon data are discussed. Histological staining for cor78 promoter driven GUS expression demonstrated that the cor78 promoter is active throughout most of the plant during cold treatment. Experiments that followed the kinetics of cor78 induction and repression during and after a three hour cold treatment demonstrated a lag period of approximately two hours prior to the 62 induction and 45 min prior to the inactivation of the cor78 gene. The implications for the mechanisms involved in signal transduction are discussed. INTRODUCTION Many plants indigenous to temperate regions of the earth are exposed to extremes in low temperature. Thus, it is not surprising that some plants have evolved means by which they can sense temperature drops and alter their biochemistry in order to prevent or minimize damage caused by freezing. In fact it is known that many plants, if first subjected to low but non-freezing temperatures, are able to become more tolerant to freezing stress (Levitt, 1980), a process called cold acclimation. Much is known about the changes in plant biochemistry that occur during the cold acclimation process. Many plants show a buildup of small soluble molecules such as proline and sugars (Sakai and Larcher, 1987). Often changes in the composition of membrane lipids also accompany the plant's response to the cold (Steponkus et al. , 1988). Finally, there are data to suggest that protein synthesis is necessary for the cold acclimation process to occur (Kasperkas-Palacz et al. , 1977). In fact, there are specific changes in the protein profile of plants that have undergone cold acclimation (reviewed in Thomashow, 1990). To gain a better understanding of the molecular basis of cold acclimation, four genes from Arabidopsis thaliana that encode proteins which accumulate specifically in plants undergoing cold acclimation have been recently cloned. These four gold- ;egulated (cor) genes, designated cor6. 6, cor15, cor47, and cor78 (previously referred to as genes represented by clones pHH29, pHH67, pHH7 .2, and pHH28 respectively (Hajela et al. , 1990), all encode boiling soluble proteins that were observed to accumulate during conditions that induce cold acclimation (Lin et al. , 1990). Other 63 environmental stimuli that affect their regulation have been studied, and it has been learned that many of these cor genes can be induced by ABA and by osmotic stress (Hajela er al., 1990). Also, work using ABA deficient and insensitive mutants has demonstrated the possibility for separate but converging signal transduction pathways in the regulation of cor genes by these environmental stimuli (Gilmour et al., 1991; Nordin et al., 1991). However, although there is growing evidence on the signal transduction pathways that operate to alter the expression of these genes, little is known about the location of sequences within these genes that are necessary for its regulation. Nuclear runon assays have indicated that at least one cor gene, cor15, is primarily transcriptionally regulated, suggesting that the sequence(s) important for its regulation is located within this gene's promoter (Hajela et al., 1990). This hypothesis was recently confirmed by promoter fusion studies (Baker et al., manuscript in preparation). Nuclear runon assays also indicated that cor6. 6, cor47, and cor78 are primarily post-transcriptionally regulated. As such, it would be predicted that the important cis-acting elements will be found in the transcribed portion of these genes (Hajela et al. , 1990). In order to learn more about the nature of the cis-acting sequences within cor genes and to gain insight into the signal transduction pathways that may function in their regulation, a genomic and a near full length cDNA clone of the Arabidopsis cor78 gene have been cloned and characterized. Further, the kinetics of cor78 transcript accumulation under inducing and repressing conditions were 3 studied. In order to localize the important cis-acting elements, constructs using these clones and the GUS reporter gene plasmids developed by Jefferson et al. (1987) have been prepared and their expression followed in transgenic Arabidopsis. The GUS reporter gene was used to determine tissue and cell type specificity for one of the cold-regulated constructs in the transgenic plants. 64 MATERIALS AND METHODS Plant Material and Treatments Arabidopsis thaliana L. (Heyn) ecotypes RLD and Landsberg erecta were generally grown in pots for 2 to 3 weeks in controlled environment chambers at 21°C 2 5'1). For most cold-regulation under constant light (approximately 100 umole m’ experiments, plants were transferred to a controlled environment chamber at 5°C (constant light) for various lengths of time. For ABA, drought and some cold treatments, plants were grown in petri plates on germination medium (GM) supplemented with B-5 vitamins (Valvekes et al., 1988) at room temperature (about 25°C) on a 16 hour day 8 hour night schedule until they reached the 4-6 leaf stage (about two weeks). ABA treatment consisted of plants being sprayed to runoff with 100 uM ABA (mixed isomers, Sigma) in 0.02% (v/v) Tween-20; control plants were sprayed with a solution of 0.02% (v/v) Tween-20. Treated plants were covered to slow evaporation and placed on the lab bench for 4 hours. Drought stress treatments consisted of removing the lid of the petri dish and allowing the plants to dry overnight in the growth chamber, at which point they had become visibly wilted. In the time course cold-regulation studies, A. thaliana ecotypes Landsberg and RLD were grown on petri plates as described above. Plates were then transferred to a cold room at 2°C 2 5'1) for various lengths of time. with constant light (approximately 50 umole m' Isolation and Analysis of Nucleic Acids Plant material was frozen in liquid nitrogen, pulverized using a mortar and pestle, and stored at -80°C prior to extraction of nucleic acids. Total and poly(A+) RNA and genomic DNA was isolated as previously described (Hajela er al., 1990). Plasmid 65 DNA was prepared from E. coli using standard protocols (Maniatis et al., 1982). Northern transfers were prepared and hybridized with [32P]-labeled probes as described previously (Maniatis et al. , 1982). Isolation and DNA Sequencing of Genomic and cDNA Clones for cor78 The insert from cDNA clone pHH28 (Hajela et al., 1990) was gel purified (Sambrook et al., 1989), radiolabeled with 32P and used to screen plaque lifts (Sambrook et al., 1989) of a lambda EMBL3 library of A. thaliana genomic DNA (a SauIIIa partial digest of ecotype Columbia; the library was kindly provided by Dr. Harry Klee, Monsanto) for genomic clones carrying cor78. One such clone, lambda 284, was isolated and an 8.4 kb HindIII fragment (containing 4.4 kb of lambda sequences and 4 kb of Arabidopsis sequences including the entire transcribed portion of cor78 as well as 808 bps of 5'upstream sequences) was subcloned into bluescript. This clone was then cut with SalI which liberated two 2kb fragments corresponding to the 5' and 3' halves of the cor78 gene. Both fragments were subsequently cloned separately into bluescript and designated pDH28-5' and pDH28-3' respectively. A gel isolated insert from pDH28-5' was then used to generate a probe for screening a cDNA library (in lambda ZAP) of A. thaliana (Lin and Thomashow, 1992) for clones that contained near full-length inserts. One chosen for further study contained 2588 bps from +19 to +2607. This recombinant cDNA was 'excised' out of the phage in plasmid bluescript SK' using the biological rescue recommended by Stratagene, thus creating plasmid pBMl. DNA sequencing was done by the dideoxy method of Sanger et al., (1977) using double-stranded DNA templates as described previously (Lin and Thomashow 1992). Hydropathy plots were conducted using a window of nine amino acid residues according to Kyte and Doolittle (1982). TFASTA and WORDSEARCH programs 66 were used to search protein and nucleic acid data bases (EMBL, GenBank, PIR, and NBRF) for previously sequenced proteins and genes. In Vitm Transcription/Translation Reactions pBMl was linearized by digestion with BamHI and the insert was transcribed in vitro with T7 RNA polymerase (Promega) using the T7 promoter carried on the bluescript vector. The resulting transcripts were extracted with PCI (phenol:chloroform:isoamyl alcohol (25:24: 1)) and precipitated with ethanol. Transcription products from these reactions and total RNA isolated from cold-treated and control Arabidopsis thaliana ecotype RLD were translated in vitro using the rabbit reticulocyte lysate system (Promega) containing [358]methionine. Boiling-soluble polypeptides were prepared as described (Lin er al., 1990), and were fractionated by SDS-PAGE (Laemmli, 1970) on 15% (w/v) polyacrylamide gels and visualized by autoradiography. Primer Extension Analysis Primer extension experiments were done according to Sambrook et al. , (1989) with modifications. In a total reaction volume of 30 ul, 100 ug of total RNA from cold-treated (15 hour) A. thaliana ecotype RLD was mixed with 8 ul of 5X M-MLV reverse transcriptase buffer (BRL), and 1 ul of a 0.02 nM stock of a 21 bp primer corresponding to bp +39 to +59 that had been end-labelled with 32P (1-2 x 105 cpm) (Sambrook et al., 1989). The mixture was heated to 70°C for 10 min and placed at room temperature for 2 h. 10 ul of a 4X extension cocktail, consisting of 4 mM DTT, 2.4 mM each dNTP, and 0.2 units InhibitAce (5 Prime -> 3 Prime, Inc.), was added along with 30 units of M-MLV reverse transcriptase and the mixture was incubated for 10-20 min at 42°C. The reaction was stopped by heating to 70°C for 5 min followed 67 by incubation with 10 ul RNase A at 42°C for 30 min. The reaction was extracted twice with PCI and once with CHCl3 and then precipitated with EtOH. The pellet was resuspended in 10 ul of loading buffer (50% formamide, 0.01% bromophenol blue, 0.01% xylene cyanol) and run on an 8% DNA sequencing gel. The gel was dried and autoradiographed for 24-48 hours with an intensifying screen at -80°C. cor78 Constructs and Plant Transformation The construct 78Pgus (pDH78P) contains the promoter region of cor78 from bp -808 to bp +5 inserted into the polylinker of pBIlOl.l (Jefferson et al. , 1987) in such a way as to drive the expression of the GUS reporter gene (see Figure 3.5A). It was made by PCR of the plasmid pDH28-5' using the T7 bluescript sequencing primer as the 5' primer. The synthesized 3' primer was designed to hybridize to bps +5 to -l2, (a BamI-II site was included at the 3' end of this primer in order to facilitate cloning into pB1101.1). This fragment was blunt ended by a Klenow reaction (Maniatis et al., 1982) and sub-cloned into the Smal site of bluescript. Finally, this fragment was cloned into pBIlOl.1 as a BamHI-HindIII fragment, and the junction was confirmed by sequencing using the GUS primer (Clontech Laboratories Inc.). Construct 78Plgus (pDH78Pl) contains a portion of the cor78 genomic clone from bp -808 to bp +250 ligated 5' to the GUS gene from pBIlOl.2 (Jefferson et al., 1987) (see Figure 3.5A). This construct was designed to place the 5'portion of the cor78 gene (as expressed from the cor78 promoter) in the same reading frame as the GUS gene from pBIlOl.2. The 1058bp fragment of DNA from cor78 (-808 to +250) was obtained by an exo-mung bean deletion of the 3' portion of the genomic clone from pDH28-5'. The deleted pDH28-5' was cut with Sall and the resulting fragment of cor78 was ligated into SalI/SmaI cut bluescript (creating plasmid pDH 15K). pDH 68 15K was cut with BamHI and SalI and the gel purified insert was ligated into the SalI/BamHI cut polylinker of pBIlOl .2 to create pDH78PI. Constructs were also made that contained portions of the cor78-3 ' transcribed region (oriented 5' to 3') ligated between the GUS gene from pBIl3l and a nopaline synthase terminator sequence that contained all of the necessary signals for poly A tail addition (see Figure 3.6A). The plasmid pHH28 was subjected to exo-mung bean deletion at its 3' end, and the resulting fragments were religated in the presence of SacI linkers. This resulted in a pool of bluescript-derived plasmids that contained various deletions of the cor78 gene with an added SacI site at their 3' end. The extent of the deletions from several of these plasmids was determined by sequencing. Two such plasmids had deletions ending at bp +2981 and +2983, thus removing the predicted poly A addition signals. One of these clones was cut with SacI thus releasing a 1000 bp fragment which was ligated into SacI cut pBIl3l to create plasmid pDH410C. The orientation of the resulting clones was determined by restriction mapping. The other deleted plasmid was cut with AluI which cut the cor78 sequence at bp +2877 leaving a blunt ended fragment containing the 3'untranslated region (UTR) of cor78 and a portion of the bluescript plasmid. The fragment containing the 3'UTR of cor78 was gel purified and subcloned into another bluescript plasmid that had been cut with Smal creating plasmid pDH 3'. pDH 3' was then cut with SacI and the resulting 250bp fragment was also cloned into SacI-cut pBIl3l creating the plasmid pDH81C. Again, the orientation of the resulting clones was confirmed by restriction mapping. The construct containing the entire transcribed region of the cor78 gene driven by the 358 CaMV promoter 35Scor78 (pDH78T) (see Figure 3.68) was prepared in two steps. First, PCR was used to prepare a fragment containing all of the transcribed sequences of cor78 from pDH28-5'. The 5' primer for the PCR reaction was designed to hybridize to bps -11 to +6 (and included an XbaI site at the 5' end to facilitate 69 cloning into pBIl21 (Jefferson er al. , 1987). The 3' primer was the T3 primer for bluescript. This PCR fragment was blunt ended by treatment with the Klenow fragment of E. coli DNA polymerase, and sub-cloned into Smal cut bluescript. The PCR fragment was subsequently cloned into pBIl2l as an Xbal/EcoRI fragment in such a way as to replace the GUS gene and the nopaline synthase terminator sequences. This construct was confirmed by sequencing across the junction between the CaMV promoter and the cor78 transcriptional start site with the primer used in the primer extension assays described above. The 2 kb Sall fragment that contained the rest of the 3' portion of the gene along with approximately 400 bp of 3' untranslated sequences was cloned into the Sall site of the modified pBIl21 plasmid. The orientation of the cloning was confirmed by restriction mapping of the new plasmid. All constructs were transformed into A. thaliana RLD using the Agrobacterium mediated root transformation protocol (Valvekes et al., 1988), with the exception that kinetin was omitted from the callus inducing medium (CIM) and -napthaleneacetic acid (NAA) was omitted from the shoot inducing medium (SIM). RNase Protection Assays The RNA probe (for the RNase protection assays) was prepared from hand isolated pDH 15K which had been cut with MluI and Sall to linearize the plasmid and remove sequences 5' to bp -113. In vitro transcription of the probe was accomplished using the Maxi-script kit (Ambion Inc.) following the manufacturer's protocols. RNase protection assays were done with the Rapa II kit (Ambion Inc.) following the manufacturer's protocols. 70 Histochemical Staining for GUS Activity. Plants transformed with the plasmids pDH78PI and pBIl21 were grown in pots and cold-treated as described above. Whole plants or plant parts were then placed in GUS staining solution (lOOmM NaPO4, 3mM K3[Fe(CN)6], 10mM EDTA, 0.1% Triton X100, and 2mM 5-bromo-4-chloro-3-indoyl-B-D-glucopyranoside (X-Gluc)). Samples were vacuum infiltrated in the GUS staining solution for at least 20 minutes and then incubated at 37°C overnight. For thin sections, plants were embedded in plastic (De Block and Debrouwer, 1992), and cut into 10 or 20 micron sections using a glass knife. GUS staining was done either before embedding (as described above), or after sectioning by placing the section between a glass slide and a cover slip and allowing a drop of GUS staining solution to seep between them and hydrate the section. The slides were then placed in a humidity chamber and incubated at 37°C overnight. RESULTS Cloning and characterization of cor78 Previous work resulted in the cloning and characterization of pHH28 a partial cDNA for the cor78 (previously c0r160) gene (Hajela er al. , 1990). In order to learn more about the structure and regulation of the cor78 gene, a cDNA clone (isolated by Brett McLamey, which appeared to contain all of the translated sequences), and a genomic clone of the cor78 gene were isolated. Both clones were sequenced (Again with the help of Brett McLamey) (Figure 3.1). Analysis of the data indicated that this gene contained three Short introns, (all less than 100 bps each) and a 68 bp 5' 71 Figure 3.1 Nucleotide sequence and predicted protein sequence of the genomic clone for cor78 . Promoter sequences with probable functions are labeled and underlined. Transcribed but untranslated sequences are denoted in lower case letters. Repeated amino acid sequences within the predicted protein are underlined for repeat 1, double underlined for repeat 2, and bold typed and underlined for repeat 3. The probable poly A tail addition signal is underlined, and the beginning of the poly A tail as determined by sequences from two separate cDN A clones is labeled. 72 GAICTCAAAGTTTGAAAGAAAATTTAITTCTTCGACT CAAAACAAACTTACGAAATTTAGGTAGAACTTATATACATTATATTGTAATTTTTTGTAACAAAATG TTTTTATTAITAITATAGAATTTTACTGGTTAAATTAAAAATGAATAGAAAAGGTGAATTAAGAGGA GAGAGGAGGTAAACATTTTCTTCTATTTTTTCATATTTTCAGGATAAATTATTGTAAAAGTTTACAA GATTTCCAITTGACTAGTGTAAATGAGGAATATTCTCTAGTAAGATCATTATTTCAICTACTTCTTT TATCTTCTACCAGTAGAGGAATAAACAATATTTAGCTCCTTTGTAAATACAAATTAATTTTCGTTCT TGACATCAITCAATTTTAATTTTCGTAIAAAATAAAAGATCAIACCTAITAGAACGATTAAGGAGAA ATACAKTTCGAATGAGAAGGATGTGCCGTTTGTTATAATAAACAGCCACACGACGTAAACGTAAAAI GACCACATGAIGGGCCAATAGACATGGACCGACTACTAATAATAGTAAGTTACAITTTAGGATGGAA Repeat TAAA:AIgAxAQQQAQATQAQIIIQAAAAQAAAAAGGGAAAAAAAGAAAAAATAAATAAAAGATATA Repeat QIASQGASAIQAQIIQSAAAAAGCAAAAAAAAAGATCAAGCCGACACAGACACGCGTAGAGAGCAAA ABR£(?) ATGACTTTGACGTCACACCACGAAAACAGACGCTTTAIAQQTQTCCCTTTATCTCTCTCAGTCTCTC TATA +1 -> TATAAAQTTAGTGAGACCCTCCTCTGTTTTACTC ecaeetetgcaaactegeeeecaetcetcaqga etaeegggtttgattecttctettggatttggaee 5:5 GAT CAA ACA GAG GAA CCA CCA H D Q T E E P P CTC AAC ACA CAC CAG CAG CAC CCA G gtagettcteatttcegeaacttetattttttt L N T R Q O H P teegtgacaatcctctgaatttacttaaacttattgtgetttatggetacag AA GAA GTT GAA E E V 2 CAT CAT GAG AAT.GGT GCG ACT AAG ATG TTT AGG AAA GTA AAG GCT AGA GCT R H E N G A T X H F R K V K A R A AAG AAG TTC AAG AAC AGT CTG ACT AAA CAT GGA CAA AGC AAT GAG CAT GAG x R P R N S L T X a G 0 s N E E E CAA GAT CAT GAT TTG GTT GAA GAA GAT GAT GAT GAT GAC GAG CTA GAA CCT D D H D L V E I D D D D D E L B P GAA GTG ATC GAT GCA CCA G qttectttcttttgtagtttcattceecttatgetcteee E V I D A P eactattggttettceattttgcgtgecattaacggtttggtatctgtatatgceg GC GTA ACA G V T GGT AAA CCT AGA GAA ACT AAT GTT CCA GCA TCG GAG GAA ATT ATT CCA CCA G x P R E T N V P A s E E I I P P GGG ACA AAG GTG TTT CCT GTC GTG TCT TCC GAT TAC ACC AAA CCC ACT GAA G T ' R V P P V V s S D Y T R P T E TCT GTA CCA GTA CAA GAG GCC TCT TAC GGA CAC GAT GCA CCG GCT CAT TCT s V P V D E A S I G H D A P A a 5 GTA AGG ACG ACG TTT ACA TCG GAC AAG GAA GAG AAA AGA GAT GTA CCG ATT V R T T P T S D K 5 E x R D V P I CAT CAT CCT CTG TCC GAA TTG TCA GAC AGA GAA GAG AGT AGA GAG ACT CAT H H P L s E L s D R E E s R E T 8 Figure 3.1 -772 -705 -636 -571 -504 -437 -370 -303 -236 -169 -102 -35 +33 +92 +150 +213 +264 +315 +366 +425 +499 +540 +591 +642 +693 +764 73 CAT GAG TCA TTG AAC ACT CCG GTC TCT CTG CTT TCT GGA ACA GAG GAT GTA H E S L N T P V S L L S G T E D V ACG AGT ACG TTT GCT CCA AGT GGT GAT GAT GAA TAT CTT GAT GGT CAA CGG T S T F A P S G D D E Y L D G 0 R AAG GTC AAC GTC GAG ACC CCG ATA ACG TTG GAG GAA GAG TCG GCT GTT TCA K V N V E T P I T L E E E S A V S GAC TAT CTT AGT GGT GTA TCT AAT TAT CAG TCC AAA GTT ACT GAT CCC ACC D Y L S G V S N Y Q S K V T D P T AAA GAA G gtaagaactttgaccttttaagattgtgtttttctttagtgattatgaatatgtaa: K‘ E eactctgttacqttgtgtttggtttag AA ACT GGA GGA GTA CCG GAG ATT GCT GAG E T G G V P E I A I TCT TTT GGT AAT ATG GAA GTG ACT GAT GAG TCT CCT GAT CAG AAG CCA GGA s P G N H E V T D 2 s P D Q R P G CAA TTT GAA AGA GAC TTG TCG ACG AGA AGC AAA GAA TTC AAA GAG TTT GAT Q P E R D L s T R s K E P K E P D CAG GAC TTT GAC TCT GTT CTC GGT AAG GAT TCG CCG GCT GAA ATT TCC AGG 0 D P D _ s V L G X D S P A X P P G GAA TCA GGA GTT GTT TTC CCG GTG GGC TTT GGT GAC GAG TCA GGA GCT GAG E S G V V P P V G P G D E S G A ; CTG GAA AAA GAT TTT CCG ACG AGA AGT CAT GAT TTT GAT ATG AAG ACT GAA _IL__JL__JL___Q___£ P T R S H D F D M N T 2 ACT GGA ATG GAC ACG AAT TCT CCA TCA AGA AGC CAT GAA TTT GAT CTG AAG T G H D T N S P S R S H P D ACT GAA TCT GGA AAC GAC AAG AAT TCT CCG ATG GGC TTT GGT AGT GAA TCA S G, I! D X N S P H G E' (3 §__“E___§ GGA GCT GAG CTG GAA AAA GAA TTT GAT CAG AAG AAC GAT TCT GGA AGA AAC Q A E L E K I E D Q R N D S G R N GAG TAT TCG CCG GAA TCT GAC GGC GGT TTA GGA GCT CCG TTG GGA GGA ART 3 Y S P E S D G G L G A P L G G N TTT CCG GTG AGA AGT CAT GAG TTG GAT CTG AAG AAC GAA TCT GAT ATC GAC F P V R S a E L Q L E E 5 § D I D AAG GAT GTG CCG ACG GGA TTT GAC GGA GAA CCA GAT TTT CTG GCG AAG GGA K D V P T G P D G E P D F L A K G .AGA CCT GGA TAC GGT GAG GCA TCA GAA GAG GAT AAA TTT CCG GCG AGA AGT R P G Y G E A S E E D X F P A R S GAT GAT GTG GAA GTA GAG ACT GAG CTG GGA AGA GAC CCA AAG ACG GAG ACT D D V E V E T E L G R D P K T E T (TTT GAT CAA TTC TCA CCG GAA CTT TCT CAT CCT AAA GAA AGA GAT GAG TTT L D O F S P E L S H P K E R D E P .AAG GAG TCC AGA GAT GAT TTT GAG GAG ACG AGA GAT GAG AAA ACA GAG GAG K E S R. D D P E E T R D E K T E 2 CCA AAA CAG AGC ACT TAC ACA GAG AAG TTT GCT TCA ATG CTA GGT TAC TCC P R Q S T Y T E K P A S H L G Y S GGA GAA ATT CCG GTG GGA GAT CAA ACT CAA GTG GCG GGA ACT GTT GAT GAG (3 E I P V G D Q T Q V A G T V D E .AGG TTG ACT CCG GTC AAT GAG AAG GAT CAA GAA ACA GAG TCT GCC GTG ACG 1‘ L T P V N E K D Q E T E S A V T ACG AAG TTA CCT ATC TCC GGA GGT GGA AGT GGA GTA GAG GAG CAA CGA GGG 'T K L P I S G G G S G V E E Q R G GAA GAT AAA AGT GTG TCG GGT AGA GAT TAT GTG GCG GAG AAA CTG ACA ACT ‘E I) IN S V S G R D Y V A E K L T T GAA GAA GAA GAC AAA GCC TTT TCT GAT ATG GTT GCC GAG AAA CTT CAG AT? 2: IE 2 D K. A. P’ S D M V A E K L Q I Figure 3.1 cont +795 +846 +897 +948 +1012 +1068 +1119 +1170 +1221 +1272 +1323 +1374 +1425 +1476 +1527 +1578 +1629 +1680 +1731 +1782 +1833 +1884 +1935 +1986 +2037 +2088 +2139 momswxmiutnimtn a W a 74 ccaccamcucacAAGnccunccaccacxnccucrccxcncarc scantttxtrrrxzvzxr 'rc'racccAGAAcccaccarcccmcxcacrcxccccmwmmm srzxaaszzczavnztv mmmmmAammmamanmmmncmmm xecccuvcarxs:_1__¢__¢n ccc ACT GAT car; are AAG ccx GAA me can car 'rcr c'rr GAA GAG ccr CCA Jrnzzxzzgzngzzzap AAA'rCA'rc'rcccrccmccrcc'rcc'rcccxcccxccxccrcucccxm It s s _9___I___I___9__Jl__JL__1L__1L__£___£__;!___A___z___l 'rcccc'rcu'rcccnmcmrcrcumrcamcccrccacrmm _I___2___l___l___!___I___l S P Q S L G S T V V COG GTG CAG AAG GAG CTT taageetatgegeactgaqettttceegtttcactttggngt P ‘V X I L ttatqtqtgttttgtttqecqtctttgatgteftetggtetaettccttgtttgtqtgeaeeaegge -> poly A catttggttggggglttqtthgctttqgettaegeeqttcctcceteccegctactqutctaeeg ngqteeeetcettggetttettcccttceeegttcttegeattettceceggetttecettetqeq ctegteqt Figure 3.1 cont +2190 +2241 +2292 +2343 +2394 +2445 +2506 +2573 +2640 +2707 +2715 75 Figure 3.2 A 32P-end-labeled 20 bp primer that hybridized to the sense strand of the cor78 gene from bp +39 to +59 was mixed with 100 ug of total RNA from cold treated (lane 1) or control plants (lane 2), and extended by MW reverse transcriptase. The product was run out on denaturing poly acrylamide gel. The same primer was used in a sequencing reaction of the cor78 genomic clone for use as a size marker, and the sequence corresponding to the sense strand 1s denoted to the left of the figure. 76 .. . . o A GATClZ .& . TCCCATTC m3 ATAAAAGMJ Figure 3.2 77 nontranslated leader sequence. The start of transcription, as determined by primer extension (Figure 3.2), was located 27 bps 3' to a putative TATA box. The sequences surrounding the start site, CT CACAA, loosely fit the consensus for plant transcription start sites, CTCATCA (Joshi, 1987). Earlier hybrid select and hybrid arrest experiments indicated that the partial cDNA for car78 corresponded to the 160kD boiling soluble cold-regulated protein observed in previous in vitro translations and in viva labeling experiments of mRNA and proteins (respectively) from cold-treated Arabidopsis (Lin et al., 1990; Gilmour et al., 1988). However, sequencing of the near full length car78 cDNA indicated that this gene should encode a protein of 711 amino acids with a molecular weight of 77.9 kD. In order to determine if the cloned gene encoded the observed COR78 protein, RNA from the near full length cDNA was transcribed in vitro, translated, and analyzed by SDS-PAGE (Figure 3.3). The results indicated that this cDNA does indeed encode a boiling soluble protein that runs at l60kD (Lin et al., 1990). Analysis of the predicted structure of the COR78 suggested that the protein it encoded would be very hydrophilic (Figure 3.4) and acidic (pl=4.25). Both of these predictions are in agreement with the observations that this protein is boiling soluble and acidic as determined experimentally by 2-D SDS-PAGE analysis (Gilmour et al. , 1988). A number of repeated amino acid sequences were evident in COR78 (Figure 3.1). A search of the GenBank/EMBL (modified) data base (release70.0/29.0) showed no significant homology with any known proteins. Also, homology plots were performed to compare the amino acid sequence of COR78 with other known COR proteins, and with all of the known molecular chaperones and many plant heat shock genes. Again, no significant homologies were found. Finally, the repeated amino acid sequences within the COR78 protein were checked against the PROSITE data base 78 Figure 3.3 In vitro transcription and translation of pBMl. 10 ul of water as a negative control (designated N), 0.1 ug of RNA from in vitro transcribed pBMl (designated I), 10 ug of tootal RNA from A. thaliana, ecotype Landsberg erecta that had been cold treated at 5° C overnight (designated C), or 10 ug of total RNA from warm controls (designated W) was added to a rabbit reticulocyte lysate 1n the presence of 35S methionine and in vitro translated. Each reaction was boiled, centrifuged, and the supernatant was run out on an SDS-PAGE gel. 79 M01. Wt. (kDa) ES; 300 Ema c835 4160 Figure 3.3 80 Figure 3.4 Hydrophilicity plot of car78. Primary sequence data from the near full length cDNA clone (pBMl) was used to predict the protein structure of COR78. This information was used to generate a hydrophilicity plot of the predicted car78 protein utilizing the MACVECTOR 3.5 program. Data output was used directly for this figure. 81 33:23.3: 700 500 400 300 200 100 Figure 3.4 82 (release 8.0). No similarity to any part of any known protein was found. The promoter of car78 is regulated by cold, drought, and ABA. As an initial step in defining sequences that are important for the regulation of the car78 gene, a construct was prepared such that the promoter region from base pair -808 through +5 (construct 78P) was ligated into pBIlOl. Another construct containing the car78 promoter plus a portion of the car78 coding region from base pair -808 to +250 (construct 78PI, which includes the first intron) was ligated in frame to the GUS gene from pBIlOl.2. These constructs were then used to transform Arabidopsis (Figure 3.5A). Initial characterization by northern analysis indicated that the endogenous car78 gene was induced by cold, ABA, and drought (Hajela et al., 1990). Also, primer extension analysis indicated that when the car78 gene was induced with ABA, the transcription initiation site was the same as that of cold-induced transcription (data not shown). A transgenic line containing the 78Pgus construct was grown on GM plates and either placed at 2°C overnight, sprayed with 100 uM ABA for four hours, drought stressed by leaving the plate lid Open overnight, or left untreated. RNA was extracted, blotted, and probed with a 32P labeled, gel purified fragment comprising the GUS gene (Figure 3.58). The GUS message accumulated 10-30 fold over the controls. In support of these data, the 78P1gus construct also gave rise to a cold-regulated GUS message, but the GUS gene expressed from the CaMV promoter in pB1121 is not cold- regulated (Figure 3.5C). This experiment demonstrates that the car78 promoter appears to be provide sufficient regulation to account for the observed accumulation of the car78 message in cold, drought and ABA treated plants. 83 Figure 3.5A A. thaliana ecotype RLD were transformed with the following plasmids: pB1121, which has the GUS gene under the control of the CaMV 35S promoter (Jefferson er al., 1988), pDH78P (construct 78Pgus), which contains a potion of the car78 gene from bp --808 to bp +5 ligated in front of the GUS gene in the plasmid pBIlOl.l (Jefferson et al., 1988), and pDH78PI (construct 78Plgus), which contains a portion of the car78 genomic clone from bp -808 to bp +250 (containing the first intron of the car78 gene) ligated in frame with the GUS gene from the plasmid pBIlOl.2 (Jefferson et al., 1988). 84 r GUS . /\ pBIlZl [ W I pDH78P I 90178 1 -801 +5 -801 +250 Figure 3.5A 1ij 85 Figure 3.5B Northern analysis of plants carrying the 78Pgus construct after drought, cold, and ABA treatment. A representative transgenic line of Arabidopsis plants that had been transformed with pDH78P were grown in petri dishes and drought stressed (D), left as warm controls (W), placed at 5 C overnight (C), or sprayed with 100 uM ABA (A) prior to harvesting. RNA was extracted, and 10 ug of total RNA was run on Silenaturing formaldehyde agarose gel forflirthem analysis. The probes were either P labeled GUS gene from pB1121, or a P labeled portion of the car78 gene from bp -808 to bp (+250. mucou mDU < U.>>Q 87 Figure 3.5C Northern analysis of transgenic plants carrying promoter sequences of 60178 driving GUS gene expression in the warm and cold. Representative transgenic lines of three week old Arabidopsis, transformed with either pBIlZl (designated 358), pDH78P (designated 78P2), or pDH78P] (designated 78PI3), were placed at 5 °C (C) or left at 22° C (W). RNA was extracted, and lOug of total RNA was run on 32 denaturing formaldehyde agarose gel ffi‘ northern analysis. The probes were labeled GUS gene from pB1121, or a Plabeled portion of the car78 gene from bp -808 to bp +250. 88 w mace mDU u 3 03 u >> 3%: Emu mmm . Figure 3.5C 89 Transcribed sequences do not impart cold-regulated expression to the car78 gene. In order to determin whether that the transcribed region of the car78 gene contained sequences important for cold regulation, constructs containing portions of pHH28 were placed between the GUS gene from plasmid pBIl3l (Jefferson et al., 1987) and the nopaline synthase terminator (constructs gusSIC and gus410C) (Figure 3.6A). Also, the entire transcribed region of the car78 gene, driven by the CaMV 35S promoter from pBIl2l was prepared (construct 355car78). These constructs were transformed into Arabidopsis (Figure 3.63). Transformed lines were isolated and subjected to an overnight cold treatment along with a non-transformed control, and RNA was then isolated from these plants. Northern analysis on plants containing portions of the cDNA on the 3' end of GUS indicated that those constructs were incapable of imparting cold regulation to the reporter gene (Figure 3.6C). RNase protection assays were used to determine if message from construct containing the entire transcribed portion of car78 accumulated preferentially in the cold. The probe used for this experiment was a 32P labeled, in vitro transcribed fragment from the genomic clone (homologous to the sense strand from bp -113 to +250). 117 bps of this probe is protected by the endogenous car78 transcript from the start of transcription to the 5' end of the first intron, and to the corresponding region of the 35Scar78 construct transcript plus the additional 11 bp of 5' sequences contained in this construct. The results of the RNase protection assays (Figure 3.6D) show that in the cold- trested plants carrying the 3SScar78 construct, the message from both the endogenous car78 message and the 35Scar78 construct protect specific fragments. In the warm grown controls however, only the 35Scar78 transcript hybridizes to the probe. Unlike 90 Figure 3.6A Arabidopsis plants were transformed with the following plasmids: pB1131 (Jefferson et al., 1989.) which has a GUS gene driven by the Small subunit of Rubisco followed by the nopaline synthase (Nos) terminator. pB1131 in which a portion of the 3' untranslated region of the car78 gene from the stop codon at bp +2463 up to, but not including, the putativepoly A signal at bp +2582 ligated between the GUS gene and the Nos terminator (designated 81C). pB1131 in which a portion of the transcribed region of the car78 gene from bp +1510 to bp +2580 (again close to, but not including the putative poly A signal) ligated between the GUS gene and the Nos terminator (designated 410C). The poly A signal was left off in these constructs in order to prevent any interference with the poly A signal from the nopaline synthase terminator. Construct from pBIl 3 1 Construct 81C Construct 410C 91 SSuR GUS NosT [Cor78 +2464 +25 82 Cor78 +1511 +2580 Figure 3.6A 92 Figure 3.6B The construct 355cor78 in the plasmid pDH78T contains a portion of the cor78 genomic clone from bp -11 to bp +3000 placed under the control of the CaMV 358 promoter from plasmid pB1121. The insert shows the additional sequences (9 bps from the CaMV 358 gene (italicized), 11 bps from the pBIlOl polylinker that was left after the construction of pBIlZl including the Xbal site, and the 11 bps of 00178 promoter sequences (underlined)) that would be transcribed as part of the 5'U'I'R from the 35Scar78 construct. The transcription of the addition of the llbps from the car78 promoter makes it possible to distinguish between the endogenous car78 transcript and the transcript of 35$car78 construct by using an RNase protection assay with the probe described below. 93 pDH7 8T CaMV Promoter Cor78 Transcribed Region ACACGCGGC GGCACT CT AGA CT G'IT'ITACT C - 1 1 - 1 Figure 3.68 94 Figure 3.6C Arabidopsis thaliana ecotype RLD were transformed with pBIl31 (lanes 1 and 2), pDH81C (lanes 3 and 4), and pDH410C (lanes 5 and 6) and either cold treated (lanes 1, 3, and 5) or left as untreated controls (lanes 2, 4, and 6). 10 ug of total RNA from each was run out on a denaturing ormaldehyde agarose gel and subjected to northern analysis. The probes were P-labeled gel isolated insert of either the GUS gene from pB1121, or a fragment of the car78 gene from -809 to +250. 95 wnpouv mDUV' ......" E >>_ 0 >30 2: HmH U024. UHw Figure 3.6C 96 Figure 3.6D RNase protection assay for detecting 3SScar78 transcripts from warm 01' cold treated transgenic and non-transgenic plants. A. thaliana, ecotype RLD, left as a non-transformed control (RLD), or a representative line which had been transformed with the plasmid pDH78T (7812) were placed at 5°C overnight (C) or left at 22°C (W). Plants wegi harvested and RNA was extracted. 10 ug of total RNA was hybridized to a P-labeled, in vitro transcribed RNA complimentary to the sense strand of the genomic clone from bp -113 to bp +250. The hybridization reaction was then treated with RNases A and T1 and run out on a 8% polyacrylamide sequencing gel. 97 I. l as; - . wNHOUIWmm V I is..- WU >3 U . 31. Nan Dig Figure 3.7D 98 the previous nuclear runon results, the data from these experiments suggest that transcribed sequences are not sufficient for imparting cold regulation to the car78 gene. It is possible however, that the additional 3lbps (9bps from the CaMV promoter, llbps from the polylinker of pBIlOl, and llbps from the 5' nontranscribed region of car78) interfered with the post-transcriptional control sequence(s). Tissue specificity of the car78 promoter Northern blot analysis indicated that car78 should be expressed in roots, stems, and leaves (see Figure A.9). To further define where in the plant the car78 promoter functions, plate grown (Figures 3.8A-C) or pot grown (Figures 3.8D-X) plants containing the 78P1gus construct (which produced greater GUS activity than any of the 77Pgus containing lines) were stained for GUS activity after a cold treatment (Figures 3.7A,D,G,J,M,N,O and W). Plants containing the 3SS/gus construct were used as a positive control (Figures 3.7C,F,I,L,T and U). Staining for GUS in control plants indicated that GUS was not present or was present at very low levels in most of the tissues examined (Figures 3.8, E,H,K,P,Q,R,S,V and X). One exception to this was the trichomes in which there appeared to be significant levels of GUS activity (Figures 3.78, and X). The GUS activity in the trichomes is most probably the result of a localized drought response since the same plant, when sprayed with water and covered in plastic wrap, lost GUS activity in the trichomes within 24 hours (Figure 3.7V). The same plant did demonstrate significant GUS activity in both the trichomes and other leaf cells after drought stress (Figure 3.7W) In cold-treated plants, the stems, leaves, SBpals, and the filament portion of the stamen had high levels of GUS activity (Figure 3.7A,G,J and M). Low levels of activity were seen in petals, and the base and tips of 99 Figure 3.7A The tissue specificity of GUS gene expression from the car78 promoter was examined. Plants containing the car78/gus construct (Columns 1 and 2) or plants containing the 3SS/gus construct (Column 3) were cold treated (Column 1) or left untreated (Columns 2 and 3). Plants were then stained for GUS activity (see Materials and Methods), and photographed under dark field illumination. Row 1 shows two week old plate grown plants. Row 2-4 show selected parts of four week old pot grown plants. Stems Roots Young Plants Flowers 100 pDH78PI pDH78PI pB1121 Warm Cold Figure 3.7 101 Figure 3.7 The tissue specificity of GUS gene expression from the car78 promoter was examined. Plants containing the car78/gus construct or plants containing the 35S/gus construct were cold stressed (cold), drought stressed (drought), sprayed with water and covered with plastic wrap for 24 hours (humid), or left untreated (warm). Plants were then stained for GUS activity (see Materials and Methods), and photographed under dark field illumination. 102 r .10. .. .. ......t”. O ... . a . .3 ,. , 1 a. . u a". . r ..4. . .. . . ,. ,3 . f... . . I ....vnwwIJJV Eco u§w\m§8 EBB .§m\wm,we.o Eco "aamkmceo 8.83 “aawénceo namhfim Lawson—o “aamkngeo Eco “hawkteo ES? “wanking . 833 ”38an8 2:5: ”hawkeuxeo Figure 3.7 cont 103 the siliques. No GUS activity was observed in the anthers, pistils, or seeds. The lack of staining in the body of the siliques appears to be the result of poor infiltration of the GUS staining solution since a similar staining pattern was observed for plants transformed with pB1121. Also, siliques that were sliced with a razor blade turned blue upon staining (data not shown). The roots showed staining in the vascular tissue and pith, but little activity in the epidermis. Again, this same staining pattern was observed with plants containing the 358/ gus construct, and therefore the lack of staining probably does not reflect tissue specificity of GUS expression in response to cold. Temporal regulation of the car78 promoter In order to gain insight into the signal transduction pathway(s) involved in the induction of the car78 gene, it was of interest to study the time course of accumulation and loss of the endogenous car78 transcript as the plants were moved from room temperature to 2°C and then back to room temperature. Therefore, plants were grown on petri plates, each plate representing a sample time point, and all plates were placed in the cold with samples being harvested for up to three hours. The remaining plates were then placed back at room temperature and samples taken for an additional three hours. From earlier results (Hajela er al., 1990), it was observed that car78 mRNA accumulated to approximately one half its maximal levels within 4 hours. Thus it was assumed that the car78 gene would be activated and mRNA would begin to accumulate rather quickly upon a cold treatment. To follow the induction of this gene in the cold over times less than 4 hours, plate grown plants were placed in the cold and plants from a single plate were harvested every hour. RNA from was extracted and subjected to northern analysis. Surprisingly, it was found (Figure 3.8A) that car78 mRNA did not 104 Figure 3.8A A representative northern blot analysis of a time course of the induction and loss of the car78 message during and after a three hour cold treatment. Plants were grown on petri dishes and then placed at 50C for three hours. Plants were harvested at 60, 120, 150, and 180 minutes after the begining of the cold treatment. Plants were then removed from the cold and samples were harvested every 15 minutes for the first hour and then at 90 and 120 minutes. The temperature of the agar surface on the plates were measured with a therrno-couple and are noted under each band. 105 Time in Minutes 60 120 150 ———180/0 cold o<3 o o o o r) o (5 o 104 4 3 152124252525 Figure 3.8A 106 Figure 3. 8B Northern analysis of the kinetics of car78 expression. Plate grown Arabidopsis plants were placed at 3° C for 3 hours and then moved back to room temperature. Plants were harvested every fifteen minutes from 0 minutes to 60 minutes and then every half hour for the last two time points. The surface temperature (in degrees C) of the agar from each experiment was measured with a thermocouple and is denoted above each data point across the upper X axis. RNA was then harvested from each time poi apt! and run out on a gel for northern analysis. Gels were blotted and probed with -Plabeled car78 clone. Hybridization was quantified on a B-scope 3000. The counts were normalized as a percentage of the maximum counts in the hottest lane. The log of the average percent accumulation from two to three separate experiments was then plotted over time. 107 omw .. mop _ our _ .3525. c. 68:. mow om ms cm me. on 9. o b — _ _ _ b _ F.F 1N; com _ comm _ oomN oomN uo_.m~ uo_- 8.: com BEER... Ema some Lo 233.888. momtam mmmcm>< uogremwnoov annalaa 10 501 Figure 3.8B 108 Figure 3. 8C Northern analysis of the kinetics of car78 expression. Plate grown Arabidopsis plants were placed at 3° C overnight and then moved back to room temperature. Plants were harvested every fifteen minutes from 0 minutes to 60 minutes and then every half hour for the last two time points. The surface temperature (in degrees C) of the agar from each experiment was measured with a thermocouple and is denoted above each data point across the upper X axis. RNA was then harvested from each time point, and run out on a gel for northern analysis. Gels were blotted and probed with the car78 clone. Hybridization was quantified on a B-scope 3000. The counts were normalized as a percentage of the maximum counts in the hottest lane. The average of the log of the percent accumulation from two to three separate experiments was then plotted over time. 109 Average Surface Temperature of each plate sampled 22° 23° 24° 25° 25° 25° Log of Relative Accumulation 1.0 - o 20 4o 60 80 100 120 140 Time in Minutes Figure 3.8C 110 accumulate to detectable levels for nearly two hours after the plants had reached 10°C (a temperature previously determined to be capable of inducing car gene accumulation (Thomashow ét al. , 1990). What was perhaps more surprising were the findings that the level of car78 mRNA continued to rise for 45 to 60 minutes after the plants had been returned to normal growing temperatures (Figures 3.8A and B). Interestingly, the lag time observed for the loss of message upon the return of the plants to normal growing temperatures after three hours in the cold was not observed when the plants were left in the cold overnight (Figure 3.8C). Finally, once the loss of the car78 message had begun to occur, it consistently demonstrated a half-life of about 35 minutes in nearly all of the samples tested. DISCUSSION Analysis of the transcribed region of the car78 gene In the 5' untranslated leader of car78, there are two possible ATG codons that may initiate translation (see Figure 1). The first ATG following the start of transcription lacks most of the consensus sequences (AACArflQGC) normally found around active translational initiation sites in plants (Joshi, 1987). Also, translation initiation from the first ATG would not lead to a long open reading frame. The second ATG however provides a much better match to the consensus sequence. Therefore, we believe that it is this second ATG that actually is used to initiate translation. Assuming that the second ATG is the true initiation codon, the protein encoded by car78 is predicted to be 77.9 kD in size. However, when RNA from the full length cDNA was in vitro transcribed and translated, SDS-PAGE analysis indicated that it ran as a 160 kD polypeptide (as does the endogenous car78 gene product). Detergents, high temperature, guanidium sulfate, urea, and formamide, were used in attempts to 111 break apart any possible dimer formed by this protein, but in all cases, the in vitro translation product ran as a. l60kD protein (Gilmour, unpublished results). One possible explanation for why COR78 runs anomalously through an SDS-PAGE gel is that the hydrophilic nature of this protein reduces its affinity for SDS, and thereby resulting in a protein that migrates more slowly through the electrophoretic field (Nelson, 1971; Panyim and Chalkley, 1971). The hydrophilic or acidic nature of COR78 has led to speculation concerning its function. It is possible that the highly charged nature of COR78 may allow it to function as a compatible solute (Carpenter and Crowe, 1988), and thus possibly protect other cellular components during freezing and dehydration stress. Sequences within the car78 promoter may play a role in ABA and drought regulation of this gene Initial characterization of the endogenous car78 gene indicated that, in addition to cold, it was induced by both ABA and drought (Hajela et al., 1990). Northern analysis indicated that the car78 promoter, by itself, is capable of conferring ABA and drought regulation to the GUS reporter gene. Interestingly, sequence data indicated the presence of a G box like element (TACGTG) at position -66 (26 bps 5' to the presumed TATA box). This is of great interest, since G box like elements have been implicated in ABA responsiveness in a number of plant promoters (Lam and Chua, 1991; Mundy et al., 1990; Guiltinan et al., 1990). G-box like elements have been found in the same proximity (25-30bp 5') to the TATA box in the promoters of two other car genes from Arabidopsis (Horvath, unpublished results) and in a car gene from Brassica napus (Singh et al. , personal communication). Interestingly, the G-box from the car78 promoter has been shown to 112 act as a binding site for the DNA binding factor GBFl (Schindler et al., 1992). The location of the G boxes in these car genes may be of significance, since they are much closer to the TATA box than has been observed with the G boxes implicated in ABA responsiveness from other plants (normally found between 189-270 in other species) (Mundy er al., 1990). It is not yet known if the presence and/or position of the G box in the car78 gene is of significance for cold regulation. However, preliminary data has indicated that the region from -240 through the TATA box (which includes both the G Box like element and the direct repeat (described below) is essential for draught activated regulation from this promoter in transgenic tobacco plants (Kazuko et al., unpublished results). Another interesting sequence that was found in the car78 promoter was a 21 bp direct repeat at bp 120 and bp 210. Within this repeat is a 5 bp sequence (CCGAC) that has been found in all of the cold-regulated promoters from both Arabidopsis and Brassica napus found to date (Table 3.1). Interestingly, a short (180 bp) fragment from the promoter of car15 was shown to confer cold-regulation to the CaMV 355-90 minimal promoter in preliminary transient expression studies (McLamey, unpublished results). The only conserved sequence within the 180 bp fragment of carI5 was the 5 bp sequence observed in the repeat of car78. Expression of the car78 constructs and implications of the nuclear runon transcription assays The results of previous nuclear runon experiments (Hajela er al. , 1990) suggested that the car78, car4 7 and car6.6 were post-transcriptionally regulated. Thus one could expect that sequences important for their cold regulation would be located within the transcribed regions of these genes, and the promoter from cor78 would 113 Table 3.1 Location and Sequence of ICE-Box Gene Sequence Position Arabidopsis car78 R1 ACCGACATCAGTT - 1 66 Arabidopsis car78 R2 ACCGACATGAGTT -225 Arabidopsis carl 5a R1 GCCGACATACATT - 1 8 3 Arabidopsis carl 53 R2 GCCGACCIGC'ITI -3 60 Arabidopsis car15b GCCGACCTCTITI‘ - 196 [Consensus RCCGACMI'NNTT Conserved sequence found in the promoter of several car genes. Two copies of this element are found in car78 (R1 and R2 respectively). Two copies are found in carISa (Rl and R2) and one copy in car15b. The position of each element is listed relative to the known (car78 and carISa) or putative (carI5b) transcriptional start sites of each gene. The consensus sequence for this ”Induced by Cold Element” (ICE-Box), is shown. 114 be active constitutively. However, the results of the car78/GUS fusion constructs appear to contradict this. For these two results to be compatible, it would have to be concluded that the sequences between +1 and +5 contain all the necessary information for cold regulation. In light of the data which demonstrated that the car78 transcribed region (containing the sequences from +1 to +5) was not cold-regulated when placed under the control of the constitutive CaMV 35$ promoter (see Figure 3.7D), this possibility seems unlikely. Although it is possible that the 31 bps of additional sequences 5' to the normal transcription start site interfered with the regulation of this construct, the most likely interpretation of these data is that the sequences controlling cold regulation are not contained in the transcribed region of car78. It was also possible (although unlikely) that there were sequences that caused the car78 gene to be constitutively expressed, but that these sequences were not included in the car78 promoter/GUS constructs. In order to test this possibility, nuclear runon assays were done on transgenic plants containing the 78P/gus constructs (Data not shown). Preliminary results from these nuclear runon experiments.show that the car78/gus constructs are regulated in the same manner as the endogenous car78 gene. This then suggests that all of the information necessary for controlling the GUS reporter gene in the same manner as the endogenous car78 (i.e., making it also appear to be constitutively expressed in nuclear runon assays), are contained within the region between -808 to +5 of the car78 gene. Since it appears that the promoter region of the car78 gene is responsible for its cold regulation, the nuclear runon results indicating that this gene is expressed constitutively may provide some interesting insight into how this gene is regulated. One possible explanation for the observed accumulation of car78 message in nuclei 115 from control Arabidopsis, is that there is a poised polymerase present on the car78 promoter which is released during the isolation procedure. Such poised polymerases have been observed on the Hsp 70 promoter from Drasaphila (Rougvie and Lis, 1988), and it has been demonstrated that certain treatments (such as high potassium concentrations or detergents), could release the polymerase and allow transcription in vitro. Although the nuclear isolation procedure for Arabidopsis would not expose the nuclear to either of these conditions, the possibility for the release of a polymerase still exists since the nuclear isolation procedures for Arabidopsis (which uses ether to dissolve the nuclear membrane) and Drasaphila (which uses nonidet P40) are quite different. Recent work on the car78 gene indicates that it may be part of a multi-gene family (Yamaguchi-Shinozaki and Shinozaki, 1992). Thus it is possible that one or more of the members of this family are constitutively transcribed, but others are inducible. Consequently, if I happened to clone one of the transcriptionally regulated capies of car78, then the discrepancy between the nuclear runon results and the results from the construct data need not be mutually exclusive. Another explanation for the observed nuclear runon results may be that some treatment during the nuclear isolation procedure activates the car78 promoter. Since it is known that drought stress can induce the car78 gene, it is possible that during the time when the Arabidopsis plants are being diced, the loss of turgor pressure caused by the physical disruption of the cell membranes was enough of a stimulus to cause the car78 promoter to be activated. In light of recent studies that have shown that the car78 gene is expressed at high enough levels to be detected by northern analysis in less than 20 minutes upon dehydration (Kazuko et al., 1992), this appears to be the most likely explanation. 116 Implications of the data from the temporal expression of car78. The results from the time course studies on the accumulation and loss of the car78 mRNA demonstrated that there was a lag period of about 2 hours between the time the plants experienced inducing conditions (10°C) and the time it took before car78 mRNA accumulated to detectable levels. Assuming that there is no significant effect on the general rate of transcription due to the cold per se, these data suggest that there is some complex or slow metabolic pathway involved in the induction process of car78 (thus negating the possibility of having a trans-acting factor that directly senses cold). If however, the cold does have a general effect on the rate of transcription, then it is possible that there is some trans-acting factor that directly senses the cold, and binds to the promoter once the inducing temperature is reached (thus activating the gene). However, the RNA polymerase may have to undergo some modification in order to adjust to transcription in the cold before reaching full activity. Therefore, the lag period prior to induction of car78 could be the result of the time it takes to modify the RNA polymerase rather than the time it takes to activate the car78 promoter. Although no short term measurements of transcription rates have been reported for plants undergoing a cold stress, such studies have been done in E. cali. In these experiments, it was determined that the rate of transcription did fall in bacteria undergoing a cold shock, but that the rates recovered to near normal levels within 15 minutes (Mackow and Chang, 1989). It should be however, relatively easy to determine if transcription from the car78 promoter is inhibited for any significant length of time by the cold per se. Work by Yamaguchi-Shinozaki and Shinozaki., (1992) has shown that drought stress is capable of inducing car78 in as little as 20 minutes. Therefore, one should be able to determine if the cold has a direct inhibitory effect on the accumulation of the car78 transcript by simultaneously drought 117 stressing and cold stressing plants. It is noted here however, that such an experiment would be conclusive only if it gave positive results. If it turned out that the drought treatment was not capable of inducing car78 in the cold, no conclusion could be immediately drawn since the cold treatment could directly alter the effectiveness of the drought treatment (i.e. by slowing evaporation). Another interesting finding from the time course experiments was the observed difference in the loss of car78 mRNA between plants that were left in the cold for ! three hours, and those that were left in the cold overnight. Plants left in the cold for three hours increased or at least maintained significant levels of car78 mRNA for up to 60 minutes after the plants were returned to room temperature. Since the plants had I reached non-inducing temperatures within the first 15 minutes after were removed from the cold, the car78 promoter was apparently active for at least 45 minutes under non- inducing conditions. In contrast, the car78 message started to disappear after only 15 minutes when plants were left in the cold overnight prior to returning them to room temperature. These results suggest that there is some difference in the way the car78 gene is regulated after experiencing three hours of cold as opposed to 12 hours of cold. There are two possible models that may explain these differences. One implies that the mechanisms involved in the initial induction of the gene are different than those that are involved in maintaining expression over longer periods of time. The other implies that there is more than one mechanism that is affecting car78 accumulation shortly after induction. It is possible that the processes involved in the induction of this gene (perhaps the buildup of a particular metabolite as the result of a cold sensitive enzyme) activate a signal transduction pathway. This could lead to a number of changes including the induction of a trans-acting factor that is critical for its own transcription and that of the 00178 gene. In this scenario the lag in the loss of the 118 car78 RNA in 3 hour cold-treated plants would be the result of the time it would take to reduce the amount of the critical metabolite back to non-inducing levels. The second scenario would be that there is some trans-acting factor that is activated by cold temperatures and inactivated at warmer temperatures. However, during the initial stages of the cold stress, there are several other physiological events occurring, such as an increase in ABA levels, that can affect the induction of the car78 gene. Increases in ABA levels during the initial stages of cold acclimation have been ! documented in several systems (Daie and Campbell, 1981; Chen et al, 1983). Therefore, the lag in the loss of the car78 mRNA in 3 hour cold-treated plants could be the result of continued induction by ABA, rather than a carry-over of some mechanism i that was specifically responsive to temperature per se. Finally, the results from these time course studies demonstrate that the car78 message has an unusually short half-life (on the order of 35 minutes), and that the car78 promoter is essentially inactivated in less than 15 minutes after reaching noninducing conditions (at least in plants that were cold-treated overnight). These two observations together suggest that the expression of this gene very tightly controlled, and thus it may be deleterious for the plant to express it under optimal growing conditions. If this protein affects the way water interacts with important structures within the cell, such as membranes or critical enzymes, (as discussed in Chapter 1) then the COR78 protein may cause significant problems in unstressed plant cells. The rapidity of the loss of the car78 message brings with it a number of other implications and potentially interesting side studies. If the car78 promoter is indeed turned off as rapidly and completely as it seems to be, it may make for a very useful expression system for studying the half lives of other RN As. In the same vein, it should be of general interest to determine what sequences or structures, contained within the car78 message, are responsible for its apparent instability. In fact, the 119 constructs DH81C, and DH410C may be useful in looking for such instability sequences. One of these constructs (pDH8lC) carries just the 3'UTR of the car78 mRNA added to the 3'UTR of GUS. Given the relative AU richness of this sequence, (AU richness being generally associated with mRNA stability, Ross, 1989), it will be of interest to see if these sequences (in either orientation) alter the stability of the GUS message. 120 Literature Cited Carpenter JF, Crowe JH (1988) The mechanism of cryoprotection of proteins by solutes. Cryobiology 25 , 244-255 Chen HH, Li PH, Brenner ML (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71 ,362-365 Daie J, Campbell WF ( 1981) Response of tomato plants to stressful temperatures. Plant Physiol. 67, 26-69 De Block M, Debrouwer D (1992) In-situ enzyme histochemistry on plastic-embedded plant material. The development of an artifact-free -glucuronidase assay. The Plant Journal 2(2), 261-266 Gilmour SJ, Hajela RK, Thomashow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol. 87, 745-7750 Gilmour SJ, Thomashow MF (1991) Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol. Biol. 17, 1233-1240 Guiltinan MJ, Marcotte WR, Quatrano RS (1990) A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250, 267-270 Hajela R, Horvath DP, Gilmour SJ, Thomashow MF (1990) Molecular cloning and expression of car (cold-regulated) genes in Arabidopsis thaliana. Plant Physiol. 93, 1246-1252 Jefferson RA, Kavanaugh TA, Bevan MW (1987) GUS fusions: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal 6, 3901-3907 Joshi CP (1987) An inspection of the domain between putative TATA box and translation start sites in 79 plant genes. Nucl. Acids Research 15, 6643-6653 Kasperska-Palacz A , Dlugokecka E, Breitenwald J, Wcislinska B (1977) Physiological mechanisms of frost tolerance: possible role of protein in plant adaptation tocold. Biol. Plant. 19, 10-17 Yamaguchi-Shinozaki K, Shinozaki K (1992) Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol. Gen. Genet. in press. Kyte, Doolittle (1982) A simple method for displaying the hydropathic nature of a protein. J. Mol. Biol. 15, 105-132 Laemmli UK (1977) Cleavage of structural proteins during the assembly of the head of bacteriOphage T4. Nature 227, 680-685 121 Lam E, Chua N-H (1991) Tetramer of a 21-base pair synthetic element confers seed expression and transcriptional enhancement in response to water stress and abscisic acid. J. Biol. Chem. 266, 17131-17135 Levitt J (1980) Responses of plants to environmental stress. Chilling, freezing, and high temperature stresses. Ed 2, Academic Press, New York Lin CT, Guo W, Everson E, Thomashow M (1990) Cold acclimation in Arabidopsis and wheat. Plant Physiol. 94, 1087-1083 Lin CT, Thomashow (1992) DNA sequence analysis of a complimentary DNA for cold-regulated Arabidopsis gene carI5 and characterization of the CORI5 polypeptide. Plant Physiol. 99, Mackow ER, Chang FN (1989) Correlation between RNA synthesis and ppGpp content in Escherichia cali during temperature shifts. Mol. Gen. Genet. 192, 5-9 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Mundy J, Yamaguchi-Shinozaki K, Chua N-H (1990) Nuclear proteins bind conserved elements in abscisic acid-responsive promoter of a rice rab gene. PNAS 87, 1406- 1410 \ Nelson CA (1971) The binding of detergents to proteins. J. Biol. Chem. 246, 3895- ' 3901 Nordin K, Pekka H, Palva E (1991) Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh. Plant Mol. Biol. 16 1061-1071 1K Panyim S, Chalkley R ( 1971) The molecular weights of vertebrate histones exploiting a modified sodium Dodecyl sulfate electrophoretic method. J. Biol. Chem. 246, 7557- 7560 Rougvie AE, Lis JT (1988) The RNA polymerase II molecule at the 5' end of the uninduced hsp70 genes of D. melanogaster is transcriptionally engaged. Cell 54, 795-804 Ross J (1989) The turnover of messenger RNA. Scientific American Apr. pp 48-55 Sakai A, Larcher W. (1987) Frost survival of plants; response and adaptation to freezing stress. Spring-Verlag, New York Sambrook J, Fritsh EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Ed2. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Schindler U, Terzaghi W, Beckmann H, Kadesch T, Cashmore T (1992) DNA binding site preferences and transcriptional activation pr0perties of the Arabidopsis transcription factor GBFl. The EMBO Journal 11, 1275-1289 122 Steponkus P, Uemura M, Balsmo R, Arvinte T, Lynch D (1988) Transformation of the cryobehavior of rye protOplasts by modification of the plasma membrane lipid composition. PNAS 85 , 9026-9030 Thomashow M, (1990) Molecular genetics of cold acclimation in higher plants. In Scandalios JG, and Wright TRF, eds. Advances in genetics, Vol. 28, Genetic Responses to Environmental Stress. Academic Press, New York Thomashow M, Gilmour S, Hajela R, Horvath D, Lin C, Guo W (1990) Studies on cold acclimation in Arabidasis thaliana. In AB Bennett, SD O'Niell, eds, Horticultural Biotechnology. Wiley-Liss, New York, pp305-314 Valvekes D, Lijsebettens MV, Van Montagu M (1988) Arabidasis thaliana root ' explant transformation and regeneration. PNAS 85, 5536-5572 ! 123 Summary and Conclusions The results from this body Of work have helped establish the basic groundwork and provided the tools (namely the car genes themselves) for future studies into how plants sense the cold, and the biochemical events that occur in order to bring about altered gene expression in response to low temperature. The fact that cold (or some physiological manifestation thereof) can have an effect on gene expression has been directly demonstrated by the cloning and study of the four cold-regulated genes. Further, data have been presented which provides strong evidence that there exist cis-acting promoter elements that can impart cold regulation to normally non—cold- regulated genes (such as GUS). Finally, the results from the studies on the kinetics of car78 induction and loss provide some evidence to suggest models concerning aspects of the signal transduction process(es) that may be Operating in the induction Of car78. Briefly, it is possible (and perhaps most likely) that cold temperatures operate via a lengthy or slow signal transduction cascade. Alternatively, there could be a trans-acting factor that changes to an active conformation in a direct response to cold per se. However, for the data to be consistent with this last hypothesis, RNA polymerase 11 must be relatively inactive for nearly two hours after the plants reach inducing temperatures. Much work however, is left to be done to further define the cold sensing mechanism(s) and the involvement Of other genes and their proteins in the signal transduction pathway(s). There is now a some evidence that suggests that ABA levels increase during cold stress in Arabidopsis (Palva; Borg-Franck, personal communication). However, there is also some evidence to suggest that ABA may be Operating independently of cold (and of drought) in the activation the car genes (Nordin 124 et al. , 1991; Gilmour and Thomashow 1991). Since G-box or G-box like sequences have been strongly correlated with ABA regulation, and all of the known car genes from rice, rape and Arabidopsis contain G-box like sequences in their promoters (Mundy et al., 1990; Singh et al, personal communication; Horvath et al., unpublished results), it is likely that these sequences are having some effect on the ability of the car genes to be expressed in response to ABA. In support of this, it has been shown that multimers of a particular G-box like sequence (hex3) has been shown to impart ABA and drought regulation to the CaMV 358 minimal promoter in tobacco (Lam and Chua, 1991). This demonstrates, at least in this case, that the G-box may be sufficient for ABA and drought regulation. However, in other deletion studies on light regulated genes, the G-box was shown to be necessary, but was not sufficient for appropriate light regulation of a reporter gene (Donald and Cashmore, 1990). Also, there are two known Arabidopsis car genes (carISB and kinI) that appear to have G-box like elements in their promoters, but which do not appear to be responsive to drought stress (Kurkela and Franck, 1991). Consequently, it is Of considerable interest to determine if these G-box like sequences in the car gene promoters are necessary and/or sufficient for cold regulation. Given these results, and the results concerning the expression of the car genes in response to cold and drought in abiI mutant lines (Nordin et al., 1991; Gilmour and Thomashow, 1991) it seems likely that the G-box like sequences will at least be necessary (if not sufficient) for the ABA regulation Of the car genes, but that they will not be sufficient (although they may prove necessary) for the regulation of these genes by cold per se. Along with the G-box like sequences in the car gene promoters, there is also another short GC rich sequence (CCGAC) that appears to be conserved. Not only is this sequence conserved among the car genes, it is the only conserved sequence that is present in a 180bp fragment of the car15 promoter that appears to confer cold 125 regulation to the CaMV 358 -90 promoter as determined by preliminary in vitro assays (McLamey, unpublished results). Therefore, it will be interesting to determine if multimers of this sequence can confer cold regulation to a minimal promoter, or if mutations or deletion of this sequence can prevent cold-regulated expression in the context of any of the known car promoters. In addition to looking at specific sequences and their presumed trans-acting factors, the constructs that have car promoters driving GUS gene expression should provide excellent tools for studying other components of the signal transduction pathway that affect the car genes. Plants carrying these constructs can be (and have been) mutagenized and screened for altered GUS gene expression, (either constitutive expression under control temperatures, or non-inducible expression in the cold) (Wilhelm, unpublished results). Such experiments should lead to the characterization and eventual isolation of the genes involved in the signal transduction pathway. Also, any presumed epistatic interactions Of the resulting mutants should allow the elucidation of the steps and branch points in the signal transduction pathway(s). From a more physiological aspect, the cloning of the car genes make it possible to directly test the role (if any) that these genes have in protecting the plant from freezing damage. By freezing (without prior acclimation) plants that constitutively express these genes at warm temperatures, it should be possible to determine if any particular car gene can, by itself, provide any protection against freezing stress. Also, utilizing antisense technology to reduce the function of any particular car gene, or, if one were to Obtain a mutant (see above) that was incapable of expressing any of the car genes, it should be possible to determine if one or more of these genes are necessary for cold acclimation. 126 Finally, sequence data has shown that all of the Arabidopsis COR proteins are very hydrophilic in nature (Thomashow et al., 1990). This finding is supported by the fact that they all remain soluble upon boiling (Lin et al., 1990). This similarity suggests some possible commonality in the function of the COR proteins. However, much needs to be done in order to gain a better understanding of what the COR proteins are doing within the cells. Additional information gained concerning the intracellular location of these proteins, and the other types of molecules they associate % or interact with (i.e. membranes, specific enzymes, etc.) could add immensely to the ‘ understanding of what these proteins do, and to their role (if any) in protecting the plants from freezing stress. i In the long term, the isolation of the car genes and promoters may well prove quite valuable in the field of genetically engineered crop improvement. There are a number of agronomically important crop species (particularly in the family Solanacea) that are chilling tolerant but freezing sensitive and which have close relatives that are freezing tolerant (Chen et al., 1983). Thus it is possible that these species have lost one or more of their own car genes, or the ability to express them. Therefore, it may be possible to add one or more of the Arabidopsis car genes (or genes necessary for their expression) to one of these crops and Obtain individuals that are better able to handle freezing temperatures. In plants in which the car promoters are functional, (such as they are likely to be in Brassica napus particularly given the sequence similarities within the car gene promoters of both Brassica napus and Arabidopsis), it should be possible to obtain lines that express any given gene in response to cold temperatures. For instance, it should be possible to place any gene, (such as genes that may be desirable to express during cold storage of produce), under the control of a car promoter. Therefore, the car promoters may prove to be useful tools for crop improvement. 127 At least one of the car promoters has several advantages that make it amenable to various studies in basic plant biology. It has been established that the car78 promoter appears to rapidly shift from an induced state to a non-induced state when the plant is returned to warm temperatures (see Figure 3.108). Also, it has been established that there are few changes in mRNA populations that occur in response to cold (Gilmour et al., 1988). Therefore, the car promoters should be useful in expression systems where expression under reasonably non-disruptive conditions, and/or rapid repression is desirable. Finally, there is recent evidence for cross-talk between separate signal transduction pathways (as reviewed by Nishizuka, 1992), also, there may be several signal transduction pathways that play a role in the regulation of the car genes (as discussed above). Consequently, these initial studies in the molecular biology of cold acclimation and cold-regulated gene expression, may provide a starting point for studies on the interaction and communication between the various signal transduction pathways involved in environmental sensing. The experiments and fields of study discussed above should add greatly to our understanding of plant gene regulation, biochemistry, and physiology. Literature Cited Brown, Ryan (1984) Isolation and characterization of a wound-induced trypsin inhibitor from alfalfa leaves. Biochemistry 23, 3418-3422 Chen HH, Li PH, Brenner ML (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71,362-365 Donald RGK, Cashmore (1990) Mutation of either G box or I box sequences profoundly affects expression from the Arabidasis rch-IA promoter. EMBO Journal 9, 1717-1726 Gilmour SJ, Hajela RK, Thomashow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol. 87, 745-7750 .‘Nishizuka Y, (1992) Signal transduction: crosstalk. TIBS 17, 367 128 Gilmour SJ, Thomashow MF (1991) Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol. Biol. 17, 1233-1240 Kurkela S, Borg-Franck M (1992) Structure and expression of kin2, one of two cold- and ABA-induced genes of Arabidopsis thaliana. Plant Mol. Biol. 19, 689-692 Lam E, Chua N-H (1991) Tetramer of a 21-base pair synthetic element confers seed expression and transcriptional enhancement in response to water stress and abscisic acid. J. Biol. Chem. 266, 17131-17135 Lang V, Heino P, Palva ET (1989) Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 77, 729-734 Lin CT, Guo W, Everson E, Thomashow M (1990) Cold acclimation in Arabidopsis and wheat. Plant Physiol. 94, 1087-1083 Mundy J, Yamaguchi-Shinozaki K, Chua N-H (1990) Nuclear proteins bind conserved elements in abscisic acid-responsive promoter of a rice rab gene. PNAS 87, 1406- 1410 Nordin K, Pekka H, Palva E (1991) Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh. Plant MOl. Biol. 16 1061-1071 Thomashow M, Gilmour S, Hajela R, Horvath D, Lin C, Guo W (1990) Studies on cold acclimation in Arabidopsis thaliana. IN, AB Bennett, SD O'Neill, eds, Horticultural Biotechnology. Wiley-Liss, New York, pp305-314 129 APPENDIX Correlation of freezing tolerance to accumulation of car gene transcripts For some time, it has been known that when the plants undergo cold acclimation, car genes expression is induced. Also, other work had demonstrated that environmental conditions other than cold could bring about the induction of the car genes (Chen et al., 1983). For instance, it was shown that exogenous application of ABA to plate grown Arabidopsis plants could both induce the car genes and increase the cold hardiness of the treated plants (Lang et al. , (1989). It was of interest then to determine if any other environmental conditions that bring about the induction of the car genes would also be able to increase the freezing tolerance in plants. A series of experiments were undertaken to correlate the levels of freezing tolerance to the expression of the car genes. To do this, the level of freezing tolerance in plants was determined after they were subjected to treatments known to induce the car genes (5°C, 10°C, drought stress, and salt stress), or to decrease the expression of already induced car genes (deacclimation). In initial studies on the freezing tolerance of Arabidopsis using electrolyte leakage as an indicator of tissue damage caused by freezing, it was determined that unacclimated Arabidopsis exhibited a loss of 50% (Lt50 for lethal temperature 50) of their electrolytes at around -3°C, but that Arabidopsis which had been acclimated at 5°C for three days exhibited an Lt50 at about -5°C to -7°C (Gilmour et al., 1989). Similar results were obtained in these experiments (Figures A. 1-4). The data Obtained from plants that were exposed to 10°C, or drought stress or salt stress, did not appear 130 to significantly change the USO from that Of the controls (Figures A. 1-3). This data however, does not suggest that these plants are better able to survive a freezing stress. It should be noted, that in both the drought stressed and the salt stressed plants, the initial electrolyte leakage at -l°C was abnormally high (around 40%) as compared to the cold-treated and control plants (which averaged around 20%), suggesting that these plants were significantly damaged by the stresses (Figures A.2 and A.3). It should also be noted that both drought and salt stressed plants exhibited 100% electrolyte leakage at temperatures significantly lower than the controls (-8°C as compared to -6°C) and only one degree lower than that of plants which were fully acclimated (3 days at 5°C) (figures A.2 and A.3). This last observation may indicate that drought and salt stress may indeed increase the freezing tolerance of Arabidopsis. Therefore, taken together with the results of the ABA treatment from Palva (1989), these results are consistent with the idea that the expression of the car genes correlates with an increase in the freezing tolerance. In contrast to these results, treatment of the plants at 10°C for two days did not appear to have a significant effect on the freezing tolerance of the plants (Figure A. 1) Since it is likely that these plants were expressing the car genes, it appears that simple accumulation of the cor mRNA does not always correlate with increased freezing tolerance. In fact, it is common practice to acclimate plants for greater than three days in order to get very reproducible differences in electrolyte leakage between acclimated and nonacclimated plants (Artus and Gilmour, unpublished observations). It should also be noted, however, that although the car genes were likely to be induced, the actual accumulation of the COR proteins during any of these experiments was not determined. Results from earlier experiments indicated that the level of car gene message in deacclimating plants begins to drop within 1 hours and return to control levels within 131 F Cgure A. l Freezing tolerance of salt stressed plants. Plants were cold treated at 5C for 3 days (0), left untreated (I), or watered with 200 mls of a 0. 4 M NaCl solution and allowed to sit overnight (A). Leaves were then removed at random, and the opercent Of electrolyte leakage (relative to the same leaf samples after freezing to - 80° C) was measured after freezing to various temperatures according to the methods of Gilmour et al.,1988). 120 100 on O b O ‘70 Electrolyte Leakage O 132 1' f l I I -2 -4 —6 -8 -10 Temperature + Nonacclimated + Acclimated + Salt Stressed Figure A.l 133 Figure A.2 Freezing tolerance of drought stressed plants. Plants were cold treated at 5 C for 3 days (0), left untreated (I), or were left unwatered until they reached a relative water content below 68% (4). Leaves were then removed at random, and the percent of electrolyte leakage (relative to the same leaf samples after freezing to -80°C) was measured after freezing to various temperatures according to the methods of Gilmour et al., 1988). 134 120 <1) 8 x 100- CD 3 so- 0) 3., so- 9 +5 40- .9 Lu ao- \° ° 0 -2 -4 -6 -8 -10 -12 Temperature + Nonacclimated + Acclimated + Drought Stressed Figure A.2 135 Figure A. 30 Freezing tolerance of plants cold acclimated at 10°C. Plants were cold treated at 5° C for 3 days (4), left untreated (I), or were placed at 10° C for 2 days (A). Leaves were then removed at random, and() the percent of electrolyte leakage (relative to the same leaf samples after freezing to -800 C) was measured after freezing to various temperatures according to the methods of Gilmour et al.,1988). 136 ..A N o O O on O A: ‘ '1.3. 13.1 1.. .. “.013 A O % Electrolyte Leakage O . I I 'T I -4 -6 -8 -1o -12 Temperature + Nonacclimated + Acclimated + 2 Days at 10 C O 1 N Figure A.3 137 Figure A. 4 Freezing tolerance of plants after cold acclimation for various lengths of time, and for various lengths of time after deacclimation. Plants were cold treated at 5 0C for from 0 to 20 days, (solid line), or moved from the cold after 14 days and left to deacclimate (dotted line). Leaves were then removed at random, and the percent of electrolyte leakage (relative to the same leaf samples after freezing to -80° C) was measured after freezing to various temperatures according to the methods of Gilmour er al., 1988). The point at which plants from each treatment lost 50% of their electrolytes (LT50) was then plotted over time. n 'ILQ .71: a P" e5" 11. 7:31: of W 5 LT50 1°C! 138 ACCLIHATION 2 4 6 8101214161820 ‘ DAYS Figure A.4 ~—l ;~-¢ 139 8 hours after plants are moved out of the cold (Hajela et al. , 1990). Therefore, it was of interest to determine if the loss of car gene activity correlated with the loss of freezing tolerance. The results in this experiment demonstrated that deacclimating plants still maintain a significant level of freezing tolerance up to two days after they were moved back normal growing temperatures (Figure A.4). This may suggest that the expression of the car genes is not absolutely necessary for the maintenance of freezing tolerance. However, the stability Of the car gene products is not known. Therefore, it is possible that the COR proteins remain at relatively high levels for some time after the plants are returned to normal growing temperatures. Further studies should be done to determine the half-life of the COR proteins, in order to clarify this conflict. Kinetics of expression of the car78/ GUS gene construct Once it was determined that the region of car78 between -808 and +5 could impart cold regulation to a GUS reporter gene (see Figure 3.5B and 3.5C), it was of interest to determine if these sequences could also impart the same kinetics of induction and loss of the GUS reporter gene as was seen with the endogenous car78. Consequently, an Arabidopsis line carrying the 78Pgus construct was grown on plates for 18 days and then placed at 5°C for either 3 hours or overnight. Plates were placed back at room temperature and samples were taken at various times for RNA extractions and northern analysis (Figure A.5-8). Preliminary results indicated that indeed, similar kinetics of induction and loss of the GUS message could be obtained by placing the GUS gene under the control of the car78 promoter (Figure A.5-8. However, a few minor differences in the 140 Figure A. 5 Northern analysis of the kinetics of car78 expression. Plate grown Arabidopsis plants containing the car78/gus construct were placed at 3° C for 3 hours and then moved back to room temperature. Plants were harvested every fifteen minutes from 0 minutes to 60 minutes and then every half hour for the last two time points. RNA was then harvested from each time pointsfnd run out on a gel for northern analysis. Gels were blotted and probed with P- labeled car78 clone. Hybridization was quantified on a B-scope 3000. The counts were normalized as a percentage of the maximum counts in the hottest lane. The log of the percent accumulation was then plotted over time. mm“ ijm MM, new“ gdW clone. neat rcent Log of Relative Accumulation 1.8 1.6 1.4 1.2 141 20 r T I I 40' 60 80 100 Time in Minutes Figure A5 120 140 142 Figure A. 6 Northern analysis of the kinetics of GUS expression. Plate grown Arabidopsis plants containing the car78/gus construct were placed at 3° C for 3 hours and then moved back to room temperature. Plants were harvested every fifteen minutes from 0 minutes to 60 minutes and then every half hour for the last two time points. RNA was then harvested from each time pointsfn d run out on a gel for northern analysis. Gels were blotted and probed with P- labeled GUS gene isolated from pBIlOl. Hybridization was quantified on a B-scope 3000. The counts were normalized as a percentage of the maximum counts in the hottest lane. The log of the percent accumulation was then plotted over time. 143 C.’ .Q q—o £9. E 3 ll 3 hours < .“ll 0 :10 tilt g liar. . £9 16150.3it (D 51313“ D: slog 0:11: _ O U) 3 o e 9 1 j T M I T T O 20 40 60 80 100 120 140 Time in Minutes Figures A.6 144 Figure A. 7 Northern analysis of the kinetics of car78 expression. Plate grown Arabidopsis plants were placed at 3° C overnight and then moved back to room temperature. Plants were harvested every at various times. RNA was then extracted from each time point, and run out on a gel for northern analysis. Gels were blotted and probed with the car78 clone. Hybridization was quantified on a B-scope 3000. The counts were normalized as a percentage of the maximum counts in the hottest lane. The log of the percent accumulation was then plotted over time. late grow to room . then 6111515 were 110m" 3° )6 30(1) The hottest 1106 Log of Relative Accumulation 145 100 200 Time in Minutes Figures A.7 300 146 Figure A. 8 Northern analysis of the kinetics of GUS expression. Plate grown Arabidopsis plants were placed at 3° C overnight and then moved back to room temperature. Plants were harvested every at various times. RNA was then extracted from each time point, and run out on a gel for northern analysis. Gels were blotted and probed with the GUS gene isolated from pBIlOl. Hybridization was quantified on a B- scope 3000. The counts were normalized as a percentage Of the maximum counts in the hottest lane. The log of the percent accumulation was then plotted over time. 147 116 gm“ ‘.0 1001n :hen timid ‘, “are 31013: ’3‘; land-fled 0“,; um COWS 1; )\'Cr lime. Log of Relative Accumulation 1.0v-11-1rvrvv1-r1.r,.. O 20 4O 60 80 100120140 Time in Minutes Figures A.8 148 accumulation and loss curves were detected. In this one experiment, it appeared that the GUS message only continued to accumulate for 30 minutes after the plants were moved back to normal growing temperatures following a three hour cold treatment (Figure A.6). The endogenous car78 gene, however, continued to accumulate for 45 to 60 minutes in the same plant samples (Figure A.5). Further experiments would need to be done in order to determine if this slight difference in induction was repeatable. Another difference that was observed was that the loss of the GUS message appeared to be significantly slower (Figure A.8) than that from the endogenous car78 (Figure A.7). Given that the half-life Of the car78 message is unusually short, it is not surprising that the GUS gene may be more stable and therefore have somewhat different kinetics of degradation. Literature Cited Chen H, Li PH, Brenner ML (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71,362-365 Gilmour SJ, Hajela RK, Thomashow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol. 87, 745-7750 Hajela R, Horvath DP, Gilmour SJ, Thomashow MF (1990) Molecular cloning and expression of car (cold-regulated) genes in Arabidopsis thaliana. Plant Physiol. 93, 1246-1252 Lang V, Heino P, Palva ET (1989) Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 77, 729-734 Lin CT, Thomashow (1992) DNA sequence analysis of a complimentary DNA for cold-regulated Arabidopsis gene car15 and characterization of the COR15 polypeptide. Plant Physiol. 99, "Illillllllllllllllllllli