This is to certify that the thesis entitled POSSIBLE ROLE OF ABSCISIC ACID IN THE REGULATION OF COLD-RESPONSIVE TRANSCRIPTION FACTORS IN ARABIDOPSIS THALIANA presented by CARRI S. Duncan has been accepted towards fulfillment of the requirements for Master of Science degree in Genetics W/ffik / Major professor ///(J/ Date / / 0" 0.7639 MSU is an Affirmative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE guards 4(22804 6/01 c/CIRC/DaIeDuopss-pts POSSIBLE ROLE OF ABSCISIC ACID IN THE REGULATION OF COLD- RESPONSIVE TRANSCRIPTION FACTORS IN ARABIDOPSIS THALIANA By Carri 8. Duncan A THESIS Submitted to Michigan State University in partial fulfillment of the requirement for the degree of MASTER OF SCIENCE Genetics Program 2002 ABSTRACT POSSIBLE ROLE OF ABSCISIC ACID IN THE REGULATION OF COLD- RESPONSIVE TRANSCRIPTION FACTORS IN ARABIDOPSIS THALIANA By Carri 8. Duncan The CBF family of transcriptional activators has been shown to have an integral role in the cold acclimation response. In wild-type plants, the CBF genes are rapidly induced in response to low temperature, followed by induction of genes under CBF regulation. Plants overexpressing CBF1, CBF2, or CBF3 do not require a cold acclimation period to survive freezing. Very little is known about the mechanism behind the transcriptional regulation of the CBF genes. In this study, involvement of the hormone ABA on the induction of the CBF genes was investigated. The results indicate that the disruption of ABA signaling or bio- synthesis leads to a decrease in CBF and COR78 gene expression, indicating that ABA may have a role in the induction of the CBF genes and consequently CBF-targeted genes. Proteins regulating the expression of CBF2 were investigated by electromobility shift assays. Significantly, nuclear proteins extract from both cold and non-treated plants, appear to bind to a sequence within a region that resembles previously described ABA-responsive elements. Although both cold and non-treated extracts produced similar DNA-protein complexes, the amount of protein required to produce the patterns differed indicating that temperature may affect the levels or activities of the DNA binding proteins. To those in the next generation of youths who persist through tough times and never stop reaching. ACKNOWLEDGEMENTS Since the start of my program at Michigan State University, I have come to know lots of wonderful people who have made my stay here worthwhile both scientifically and personally. First, I’d like to thank Mike Thomashow for giving me the opportunity to work on an interesting topic and for providing a vibrant work environment. When I decided, for personal reasons, to do a PhD. at the ETH in Zurich, he was understanding and made it possible for me to terminate my program with a Master of Science degree. In fact, all members of the Thomashow lab were helpful, but lab members I’d like to thank specifically are Sarah Fowler for being a mentor and teaching me what it means to do science; Sarah Gilmour, Dan Zarka and Heather van Buskirk for their input while writing my thesis; Dcnatella Canella for being a good friend concerning matters inside and outside the lab. Other friends who made East Lansing more enjoyable than I could have ever imagined are Philip, Verna, Ann-Marie, Veronica, and my best friend Chad. During the last few weeks with job hunting, moving within and outside of the country and preparing my defense, my significant other, Gert was helpful in keeping me sane. Thanks Schatzipoo. Most importantly, I would like to thank family members, specifically my uncle Joe for giving me career advice. Also to thank is my mother, the woman who single-handedly spawned my interest in biological science during my early years which subsequently grew into my quest for understanding how living things work. iv TABLE OF CONTENTS Chapter 1: Early Events Involved in the Regulation of the Cold Acclimation Response .................................................................................................. 1 Chapter 2: Investigation of the Role of ABA in the Cold Regulation of the CBF genes Abstract .................................................................................................... 27 Introduction .............................................................................................. 28 Materials and Methods ............................................................................. 32 Results ..................................................................................................... 36 Discussion ................................................................................................ 49 Literature Cited ......................................................................................... 59 Chapter 3: Preliminary Exploration of Signaling Pathways for Cold-Regulated Transcription Factors Summary .................................................................................................. 63 Introduction .............................................................................................. 64 Materials and Methods ............................................................................. 68 Results ..................................................................................................... 71 Discussion ................................................................................................ 76 Literature Cited ......................................................................................... 78 LIST OF TABLES Table 1.1 COR genes and their alternate designation ......................................... 6 Table 3.1. Transcription Factors Used in This Study .......................................... 65 vi LIST OF FIGURES Figure 1 : Calcuim mediated ABA signaling ................................................. 19 Figure 2.1: Effect of mutations in ABA biosynthesis and signal transduction pathways on the cold induced expression of the CBF genes. ....................... 38 Figure 2.2. Effect of the ABA biosynthetic and signaling mutations on COR78 expression levels .............................................................................. 39 Figure 2.3: EMSA of Arabiopsis nuclear extracts from control and cold- treated plants using the 155 bp region of the CBF2 promoter as a probe ...... 42 Figure 2.4: The addition of Aurintricarboxylic acid leads to loss of DNA- protein interactions .......................................................................................... 43 Figure 2.5: Titration of PolydldC and nuclear extract using 155 bp CBF2 promoter fragment .......................................................................................... 47 Figure 2.6: Gel Shift experiment using wild-type and mutated fragments of the 155 bp promoter regions of CBF2 as competitor ...................................... 48 Figure 3.1: Effect of mutations in ABA biosynthesis and signal transduction pathways on the cold induced expression of ZAT12, RAP2. 7, putZF, and RAV1 ........................................................................................... 73 Figure 3.2: The effect of mutations in the ethylene signaling pathway on cold- induced expression of the CBF target genes COR15a, and RAP2.1 ............. 74 Figure 3.3: The cold regulated expression of $72 in the r3393 mutant .......... 75 vii ABA ABI ABRE AP2 ATA AtERF CaM CAS CBF Col Cor CRT/DRE CTR DesA DREB EIN EMSA ER LIST OF ABBREVIATIONS Abscisic acid ABA insensitive ABA responsive element Apetela 2 Aurintricarboxylic acid Arabidopsis thaliana Ethylene Response Factors Calmodulin Cold Acclimation Specific Genes C-repeat binding factor Columbia ecotype Cold-regulated gene C-repeat/Drought responsive element Constitutive triple response Desaturase A gene Drought responsive element binding factor Ethylene insensitive Electromobility shift assay Endoplasmic reticulum esk fad FP GA his HR ICE IP3 Ler LTI NE putZF RAP rss sfr STZ ZAT viii Eskimo mutant Fatty acid desaturase mutant Free probe Gibberillic acid Hookless mutant Hormone receptor lnducer of CBF expression protein lnositol-3-phosphate Lansberg erecta ecotype Low temperature induction gene Mitogen Activated protein Nuclear extract Putative zinc finger Related to AP2 proteins Reduced sensitivity to salt mutation Sensitive to freezing mutant Salt tolerant zinc finger protein Arabidopsis zinc finger protein Chapter 1 Early Events Involved in the Regulation of the Cold Acclimation Response I. Introduction Plants living in temperate climates are confronted with a number of different environmental temperature stresses throughout their lives. They therefore need mechanisms to respond to various adverse temperature conditions such as cold stress. Cold temperatures effect the distribution as well as the quality of plant growth. When challenged with cold, plants engage . mechanisms to protect themselves from freezing damage. This adaptive process, known as cold acclimation, allows plants to withstand freezing temperatures by prior exposure to low non-freezing temperatures. For instance, wann-grown Arabidopsis thaliana plants treated at 4 °C before exposure to freezing temperatures are able to survive to -10 °C, whereas those without prior exposure to low nonfreezing temperatures only survive to -3 °C (Gilmour et al 1988). ll. Physiological Changes in the Cold The effect of cold on the plasma membrane The Membrane and Freeze Damage The rupture of the plasma membrane is the principal cause of freezing injury (Steponkus, 1984). As the temperature drops to freezing temperatures ice forms. Due to the difference in solute concentration between intracellular and extracellular spaces, ice nucleation first occurs in the extracellular space. Intracellular ice formation is usually considered to be lethal so a higher intracellular solute concentration protects the plant by ice first forming in the extracellular space (Levitt, 1980). Lesions can occur as a result of lamellar—to- hexagonal ll phase transitions in the plasma membrane. The formation of the hexagonal ll phase causes loss of the semipermeable nature of the plasma membrane leading to leaky cells. Other forms of membrane damage can occur such as loss of osmotic responsiveness, or expansion-induced lysis (reviewed in Thomashow 1999). In short, freezing causes cellular dehydration, which creates lesions in the plasma membrane. Cold acclimation decreases freeze-induced damage in a number of ways including preventing lamellar-to-hexagonal ll phase transitions (Uemura and Steponkus, 1997). The Membrane and Signaling Not only is the membrane important to protect the plant from freeze damage, but may also be the site at which temperature sensing first occurs. 2 Recent studies in the cyanobacterium Synechoscystis suggest that membrane fluidity is an early event involved in low temperature sensing (Vigh et al., 1993). A drop in temperature leads to the rigidification of membrane phospholipids. Studies using Synechocystis as a model of monitoring the affect of increase membrane rigidity found that increased hydrogenation of membranes led to the expression of a desaturase gene, DesA (Vigh et al., 1993). Based on these data, DesA may be a part of a feedback loop in maintaining membrane in a fluid state. Another study by lshizaki-Nishizawa et al. (1996) ties in the state of the membrane to the cold response using another desaturase, A9 desaturase, cloned from the cyanobacterium Anacystis nidulans and transformed into tobacco. The transgene leads to reduced levels of saturated fatty acids in the plant, and increases its chilling tolerance at 1°C (lshizaki-Nishizawa et al. 1996). The importance of membrane fluidity was also demonstrated in a study (Miguel et al 1993) using fatty acid desaturase (fad) mutants with reduced polyunsaturated fatty acids. The fad mutants behave like wild-type at 22°C but die at 6°C (Miguel et al 1993). The state of the membrane even affects the transcription of cold regulated genes. Fluidization of the membrane prevents freezing tolerance, cold acclimation specific (CA5) gene expression and calcium ion influx (Orvar et al 2000). These two studies show that a feedback loop using desaturases could be involved in signaling cold perception is perceived in certain plants. Biochemical Changes As mentioned earlier, solute concentrations in the cell protect the plant from lethal ice crystals forming in the intracellular spaces. The solute concentration of the cell is not constant. Changes associated with cold acclimation lead to a number of intracellular biochemical changes in the plant. Changes in proline content and simple sugars in the cell have been associated with cold acclimation (Guy at al., 1992; Wanner and Juntilla, 1999). Studies have shown a positive correlation between the acculation of proline and soluble sugars to the enhancement of freezing tolerance (Nanjo et al., 1999; Wanner and Juntilla, 1999). Furthermore, overexpression of a transcription factor involved in the development of freezing tolerance, CBF3, leads to increase proline and sucrose levels, which are biochemical changes associated with cold acclimation (Gilmour et al., 2000). Accumulation of other compatible solutes also occurs in the cell in response to various abiotic stresses including cold (reviewed in Chen and Gusta, 2002).These metabolic changes help protect the plant from the potentially deleterious effects of freeze-induced injury. III. Transcriptional Regulation of the Cold Acclimation Response A. Cold regulated genes. In 1985, Guy at al. provided the first evidence that changes in gene expression occur during cold acclimation. Since then, many genes have been 4 found to be expressed during cold-treatment. A large number of these genes encode proteins which share certain characteristics such as the ability to remain soluble after boiling and a simple amino acid composition. Included in this group are the COR genes (T able 1.1). The CRT/DRE and Abscisic acid responsive element (ABRE) promoter elements impart the cold and drought responsiveness of these genes (Yamaguchi-Shinozaki and Shinozaki 1994, Baker et al. 1996). The function of proteins in the COR gene family is largely unknown. COR15a, located in the stromal compartment of the chloroplast, was found to enhance the freezing tolerance of the chloroplast by preventing the formation of deleterious hexagonal ll phase lipids in the plasma membrane (Artus et al. 1996, Steponkus et al. 1998). Table 1.1 COR genes and their alternate designation. COFi gene Alternative designation Reference COR78 Lti78, R029a Nordin et al. 1993 Kurkela and Franck, 1990 Kurkela and Borg-Franck, 1 992 COR6.6/COFI6.6a Kin1/Kin2 com 5 None COR47 Rd17 Welin et al., 1994 B.Transcriptional Regulation of the COR genes by CBF The promoters of the COR genes contain CRT/DRE, that is involved in cold regulated gene expression (Yamaguchi-Shinozaki and Shinozaki 1994, Baker et al. 1994). The first transcription factor found to bind this element was identified by a yeast-one-hybrid assay (Stockinger et al. 1997). The 24kDa protein, named CBF1 for C-repeat binding factor, is a part of a small gene family that includes CBF2 and CBF3 (Gilmour et al. 2000 ). All three members are located in tandem on chromosome 4 in Arabidopsis and contain AP2 DNA binding domains (Stockinger et al. 1997, Gilmour et al. 1998). The constitutive overexpression of CBF1 in Arabidopsis was shown to cause the constitutive expression of the COR genes and enhance the freezing tolerance of nonacclimated plants, bypassing the need for cold acclimation to protect the plant against freezing damage (Jaglo-Ottosen et al., 1998). At present, it is unknown how CBF genes are transcriptionally regulated. Gilmour et al. (1998) have proposed that a transcription factor called lnducer of QBF Expression, ICE, is present at warm temperature and its activation leads to the cold-regulated induction of the CBF genes (Fig. 1). C.ABA—dependent vs. ABA-independent transcriptional regulation ABA (Abscisic Acid) has been implicated in the cold acclimation response (discussed in detail below). However, the CBF family of transcription factors defines a pathway in cold acclimation that does not need ABA to function. The CRT/DRE element is not ABA responsive (Yamaguchi-Shinozaki and Shinozaki 1994). Furthermore, in ABA mutants, the transcription of the COR genes is still able to occur with a cold temperature stimulus indicating that ABA is not required for cold-inducible expression of the COR genes (Gilmour and Thomashow, 1991) The role of ABA in the cold is still unclear. Some would argue that the effects of ABA on cold acclimation do not prove ABA is a part of the regulation of the cold acclimation response. Three main points supporting ABA regulation in the cold (in bold), could be counter-argued by those who do not believe ABA is involved in cold signaling (italicized): 1. Endogenous ABA levels increase when plants are exposed to the cold. This response is only transient and is not important in maintaining the freezing tolerance of the plant. 2. Exogenous application of ABA leads to enhanced freezing tolerance. The COR genes have ABREs so are being activated by applied ABA that is at concentration far higher than endogenous ABA levels. This is an artificial response that would not happen in nature. 3. ABA mutants are defective in their freezing tolerance; therefore, ABA normally plays a role in the development of freezing tolerance. 8 Abscisic acid is involved in a number of development and physiological plant processes. It would not, therefore, be surprising when ABA biosynthesis or signaling is defective, a number of pleiotrophic effects can occur that are not part of normal plant physiology. To address the question of whether the ABRE is contributing to the cold- responsive induction of the COR genes, the COR78 promoter which contains the CRT/DRE and ABRE could be used. By mUtating the CRT/DRE and leaving the ABRE intact, one could test the cold responsiveness of a COR78 construct. Studies have shown a link between ABA and cold responsiveness, by using mutants isolated by lshatani and colleagues (1997) which are altered in their ABA and cold responsiveness. They found a single mutation that leads to high expression of RDZQa/COR78 in response to cold and osmotic stress (hOSoold/osm)1 but not ABA. They also found a mutation affecting ABA and cold alone. They conclude that an ABA-dependent and independent pathway does exist, but that they are not completely separate. They believe they converge and crosstalk, although there appears to be no evidence in this study that crosstalk between ABA-independent and ABA-dependent pathways occur. This study does however, show that one mutation can affect the transcriptional responsiveness of a gene for both cold and ABA, which could mean it is a pathway totally ABA- dependent. The studies presented in Chapter 2 and 3 attempt to address the role of ABA in the regulation of genes involved in the cold temperature response. D. Parallel and Convergent Signaling Pathways M The isolation of mutants affected in their freezing tolerance has led to insights in the signaling pathways involved in the cold acclimation response. The constitutive freezing tolerance mutant, esk1, is unaffected in COR gene expression, suggesting ESK1 defines a pathway involved in freezing tolerance distinct from the CRT/DRE activation pathway (Xin and Browse, 1998). The sensitive-to- freezing mutant sfr6 does affect the COR genes, but does not affect CBF transcription (Knight et al., 1999). This suggests that sfr6 is important for the activation of the COR genes, and may act as a coactivator with CBF. The hos1 -1 mutant leads to early flowering and superinduction of CBF, therefore H081 is assumed to be a negative regulator of CBF (Lee et al. 2001 ). Other Cold Regulated Transcription Factors Transcriptome analysis of Arabidopsis plants overexpressing CBF1, CBF2, and CBF3 and wild-type plants has allowed a comparison to be made between what genes are under the regulation of the CBFs and those which are not. In addition, genes found to be cold-regulated, but not under the regulation of the CBF genes have also been compared by genome-wide expression analysis (Fowler and Thomashow, 2002). The Fowler and Thomashow study has given insight on cold signaling pathways independent to that of the CBF genes, and could subsequently lead to finer analysis to determine which cold signaling pathways are likely to play an important role in how plants develop freezing tolerance. 10 IV. Role of ABA in the Cold Acclimation Response A. Enhancement of Freezing Tolerance Complex signaling provides a means to integrate the sensing of external stimuli into a signal transduction pathway ultimately leading to induction of the plant’s cold response. The early events involved in cold sensing are not understood. As mentioned earlier, ABA has been implicated in cold temperature stress. Major contributions implicating ABA in the cold acclimation response was work that focused on the cold tolerance of two cultivars of alfalfa exposed to exogenous ABA- cv. Ranger, which is able to acclimate, and cv. Hairy Peruvian, which is not able to acclimate (Walden, 1975; Riken et al., 1976). When cv. Hairy Peruvian was grown in a nutrient solution containing ABA, it increased in its ability to cold acclimate (Riken, 1976). A study using the same cultivars found that ABA caused a reduction of glberrilic acid activity concluding that ABA may lead to an increase in cold tolerance by decreasing the activity of GA as an inhibitor of cold acclimation (Waldman et al., 1975). More evidence supporting the involvement of ABA in the cold acclimation response are studies showing that endogenous ABA levels increase in response to low temperature (Riken et al., 1976; Dale and Campbell, 1981) and that exogenously applied ABA leads to enhanced freezing tolerance (Riken, 1976; Chen et al., 1983). An early study showing the degree to which ABA increases 11 freezing tolerance without acclimation was done by Chen and Gusta (1983). Suspension cell cultures of winter wheat, winter rye, and bromegrass were tested under four conditions: ABA treatment, cold-acclimated no ABA, cold-acclimated combined with ABA treatment, and a non-treated control. The ABA treated cells were able to survive at a lower temperature than cold-treated alone (-30°C versus -20°C respectively) In all three cell types. Untreated cells survived to -9°C for wheat and rye and -8°C for bromegrass. Another study using potato as a model found that endogenous ABA levels increased during cold acclimation in a cold hardy species. Moreover, endogenous ABA levels in potato, a frost sensitive plant, did not increase in response to cold treatment (Chen et al., 1983). These studies led to the idea that ABA is regulating the cold acclimation response. Subsequent studies support Chen’s hypothesis; specifically those which demonstrate that ABA regulates the expression of low-temperature responsive genes (discussed below). These early studies led ABA to become a focus for studying the development of freezing tolerance in plants. B.Transcriptional Changes Associated with ABA The parallel between cold inducible genes and ABA changes in the cold led to the idea that the cold acclimation response is regulated by ABA through its action on ABA-dependent genes. The promoter element necessary and sufficient for transcription of ABA-dependent gene response is termed ABRE which contains a core sequence of ACGT (Guiltinan et al 1990, Michel et al. 1993). The 12 ACGT core sequence is also found in G-boxes which are responsive to other environmental signals; therefore, it is important to note that the sequences flanking ACGT are probably what lead to specificity of this promoter element. Genes regulated through ABA that are cold responsive have been identified. Namely, the cold-induced expression of RABfB and LTI is mediated through ABA and ABA signaling (Lang and Palva, 1992; Nordin et al. 1991; Welin et al., 1995). The COR genes contain this promoter element and are ABA induced. Also, ABA, through the action of cADPR, leads to vacuolar calcium release (Wu et al. 1997). The ABA mediated activation of COR78 and COR6.6 requires calcium (T ahtiharju et al. 1997, Trewavas and Malho 1998) and cADPR can substitute for ABA and calcium in the induction of these two genes (Wu et al. 1997). ABI1 encodes a phosphatase type 20, and is involved in ABA signaling and contains a calcium binding EF—hand motif. The flux in calcium could lead to calcium binding ABI1 which leads to further ABA signal transduction (Leung et al. 1994). It is, therefore, possible that ABA is activating the COR genes via calcium regulation of cADPR and ABI1. V. A change in calcium is one of the earliest signaling events Although the mechanisms involved in the early events of the plant’s ability to sense cold are poorly understood, calcium is believed to be one of the earliest events in cold signaling. There are studies that have contributed to how cold sensing and the transduction of that signal takes place (Knight et al. 1991, Knight et al., 1996, Monroy and Dhindsa, 1995, Saijo et al., 2000). An increase in 13 cytosolic calcium is associated with the plant sensing a cold temperature stimulus (Knight et al. 1991 ). Calcium plays an important role as a second messenger in various cell types and is the convergence point from which a number of signals disperse. Various approaches have been used to find the role of calcium in the cold acclimation response. Transgenic plants containing the calcium sensitive reporter aequorin exhibit a large cytoplasmic calcium influx when exposed to cold shock (Knight et. al 1991). Moreover, the use of chemicals that negatively and positively affect calcium influx has led to the conclusion that calcium is required for the activation of cold acclimation specific genes in alfalfa (Monroy and Dhindsa 1995), but is required for expression of COR6.6 and KIN 1 , genes containing the CRT/DRE promoter element in Arabidopsis (Knight et al., 1996). It seems that the calcium response has properties specific to the nature of the stimulus, and that distinct calcium pools are used for different signal transduction events (Malho et. al 1998). One possibility of how these fluxes occur could relate to a study in onion where it was found that a mechanosensitive calcium channel is modulated by temperature (Ding and Pickard 1993). In a different study (Plieth et al., 1999), the temperature was gradually dropped from 18°C to 4°C. Although they saw a calcium response immediately after 1°C temperature drop from 18°C to 17°C, the calcium spike was at its highest at around 5°C which is consistent with findings of Ding and Pickard (1993). It was observed, however, that it is the change in temperature that leads to calcium flux rather than the absolute temperature (Plieth et al. 1999). They noted that the 14 calcium channels are unaffected by temperature unless there is a change in membrane tension. Studies by Suzuki and colleagues (2002) demonstrate that the cold sensor is a membrane associated histidine kinase protein which sense changes in the fluidity of membranes in Synechocystis. A more rigid membrane in colder conditions may increase tension on the sensor leading to the activation of calcium channels. Once calcium enters the cell, it is unclear how the cold signal is transduced. A similar system involving a membrane associated protein in Arabidopsis could exist. The direct relationship between cold and the cytoskeleton has been shown to exist in studies where cold leads to actin filament reorganization, and the addition of CD, a chemical permeating the plant’s membrane leading to_the destabilization of actin microfilaments, results in reduced freezing tolerance in alfalfa (Orvar et al., 2000). Moreover, cold temperatures have been shown to activate Mitogen-activated protein kinases (MAPKs) In alfalfa. The cold-regulated MAPK in alfalfa called SAMK is inhibited cytoskeleton destabilizers or inhibitors of calcium influx. Arabidopsis ton mutants, which have constitutively disorganized microtubules, showed higher calcium channel activities in root and mesophyll cells (T hion et al. 1998). All these data taken together could mean that a mechanosensitive membrane embedded calcium channel is responsive to cytoskeletal changes and changes in the fluidity of the plasma membrane. These cold induced changes may regulate the signal transduction pathway which ultimately leads to the 15 activity of calcium flow into the cell and may play an important role in the development of freezing tolerance. Signaling events after calcgrm fig lnositol 1,4,5-trisphosphate (IP3) is widely known to mobilize calcium release from internal stores such as the endoplasmic reticulum (ER) and the vacuole as shown in Fig. 1. IP3 and diacylglyceride are produced through the enzymatic action of phospholipase C. lnositol phosphatases are needed to dephosphorylate catabolites of IP3. A mutant defective in an lnositol phoshphatase, fiery1, leads to high levels of ABA/stress induced gene activation (Xiong L et al 2001). This suggests that attenuation of this gene is necessary for normal ABA induced stress response. Calmodulin (CaM) is a protein which is known to regulate downstream protein targets in plants in a calcium dependent manner (Snedden and Fromm 1998). When CaM is increased above endogenous levels, an interaction with the actin cytoskeleton is observed. (T rewavas and Malho 1998). This could mean that there is a feedback signal modulating the influx of calcium because the calcium channel activity, as previously mentioned, is affected by cytoskeletal organization. CaM kinases could regulate the activity of proteins such as MAP kinases or transcription factors, through phosphorylation leading to the transduction of the calcium signal. A low temperature stimulus results in a decrease in phosphatase activity (Monroy et al., 1998), so phosphatases may act to negatively regulate low temperature signal transduction. 16 All these pieces of data taken together suggest that there may be a cold regulated calcium channel embedded or associated with the membrane which is stimulated as the temperature drops because it senses a change in fluidity of the membrane. The channel opens allowing a flux of calcium to enter the cell. Calcium then acts as a second messenger stimulating the activity of phospholipase C (PLC). PLC leads to the production of IP3 which releases internal stores of calcium. This calcium could then bind calmodulin and ABI1 which would in turn regulate the activity of downstream protein targets. These in turn could lead to the calcium dependent activation of cold responsive genes such as LTI78/COR78, RAB18, RDZQa and KIN2 in tomato cells, and KlN1 and KIN2 in Arabidopsis. (T rewavas and Malho 1998, Thuleau et al 1998, Knight et al. 1996, Tahtiharju et al. 1997). Importance of ALBA in the calcium signal transduction An ABA responsive, signaling pathway was shown to increase cytosolic calcium via production of cyclic ADP ribose (cADPR) independent of IP3 (T huleau et al 1998). cADPR activates calcium release channels from the vacuole membrane. If IP3 Ca2+ release is inhibited, the COR genes can still be activated in a calcium dependent manner in tomato cells (Wu et al. 1997). These results would suggest that the ABA pathway activates the COR genes independent of an IP3-regulated pathway. Figure 1 is a proposed schematic of how calcium mediates ABA-dependent and independent cold signaling, and also includes arrows which represents each paper supporting the events discussed 17 above. Dashed arrows represent proposed events, whereas solid arrows correspond to events that have been published. 18 \ [FIERY1 w ................ In ..... I / cADPR: If Ca2 Flux fr ‘8 I. o c e s e COR78, COR6.6, RAB18 and other genes “—2 Fig. 1. Model of low temperature and ABA responsive signaling. A drop in temperature leads to increases in endogenous ABA levels, also exogenously applied ABA increases freezing tolerance. Both link ABA to cold signaling. Additionaly, cold temperatures increase cytosolic calcium concentrations. ABA is also connected with cytosolic calcium Increase. Calcium is required for the cold acclimation response as well as the activation of COR genes. How calcium controls proteins regulating the COR genes, such as CBF and the hypothetical ”ICE” protein is not yet known. The HOS gene negatively regulates CBF, and sfr6 mutants show decrease COR gene transcript levels are known regulatory components of cold signaling. HOS protein may prevent the induction of CBF by inhibiting “ICE”. The SFR6 gene may act to coactivate the COR genes with CBF, or regulate the COR genes post-transcriptionally. 19 Literature suggortiglsolicLarrows In Figgre 1. 1. Thuleau et al 1998: ABA uses cADPR as a 2nd messenger to activate vacuolar Ca2+ release in beets 2. Franklin-Tong et al. 1996: calcium released from the vacuole in response to IP3 plants 3. Knight et al 1991: Cold leads to a sharp increase in cytosolic calcium from external stores 4. Tahtiharju at 1997: Kin1/2 needs calcium in cold 5. Trewavas and Malho 1998: lti78/COR78 and rab18 accumulate in a Ca2+ mediated ABA-dependent manner 6. Wu et al 1997: IP3 inhibited from activating Ca2+ release does not affect ABA induced gene expression 7. Lee et al. 2001: HOSI negatively regulates CBF expression. 8. Knight et al. 1999: sfr6 mutation suppresses Cor gene expression, but not CBF. 9. Stockinger et al. 1997: CBF activates Cor genes via the c-repeat promoter element. 20 LITERATURE CITED Artus NN, Uemura M, Steponkus PL, Gilmour SJ, Lin CT, Thomashow MF. 1996. Constitutive expression of the cold-regulated Arabidopsis thaliana COR 153 gene affects both chloroplast and protoplast freezing tolerance. Proc. Natl. Acad. Sci. USA. 93: 13404-9. Baker SS, Wilhelm KS, Thomashow MF. 1994. The 5’-region of Arabidopsis thaliana COR15a has cis-acting elements that confer cold-, drought-, and ABA- regulated gene expression. Plant Mol. Biol. 24:701-713 Chen THH, Murata N. 2002. Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 2002 5:250-257. Chen H-H, Li PH, Brenner ML 1983. Involvement of abscisic acid in potato cold acclimation. Plant Physiol 71 :362-365. Chen THH, Gusta LV 1983. Abscisic acid - induced freezing resistance in cultured plant cells. Plant Physiol 73:71-75. Dean J, Pickard B. 1993. Modulation of mechanosensitive calcium-selective cation channels by temperature. The Plant Journal 3:713-720. Fowler DB, Breton G, Limin A, Mahfoozi S, Sarhan F. 2001. Photoperiod and temperature interactions regulate low-temperature-induced gene expression in barley. Plant Physiol. 127: 1676-1681. Franklin-Tong V, Drobak B, Allen A, Watkins P, Trewavas A. 1996. Growth of pollen tubes of Papaver Rhoeas is regulated by a slow-moving calcium wave propagated by lnositol 1,4,5-triphosphate. Plant Cell 8:1305-1321. Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF. 2000. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant physiol. 124: 1854- 65. 21 Gilmour SJ, Thomashow MF. 1991. Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol. Biol. 17: 1233-40. Guiltinan M, Marcotte W Jr, Quatrano R. 1990. A plant leucine zipper protein that recognizes an abscisic acid response element. Science 122267-71. Guy CL, Huber JLA, Huber SC. 1992. Sucrose phosphate synthase and sucrose accumulation at low temperature. Plant Physiol 100: 502-508 Guy CL, Niemi KJ, Brambl R. 1985. Altered gene expression during cold acclimation of spinach. Proc. Natl. Acad. Sci. USA. 82: 3673-7. Hunt L, Grey J. 2001. ABA signaling: A messengers Fiery fate. Curr. Biol. 1 1 :R968-970. lshitani M, Xiong L, Stevenson B, Zhu J-K. 1997. Genetic Analysis of Osmotic and Cold Stess Signal Transduction in Arabidopsis: Interactions and Convergence of Abscisic Acid-Dependent and Abscisic Acid-Independent ' Pathways. Plant Cell 9: 1935-1949. lshizaki-Nishizawa O, Fujii T, Azuma M, Sekiguchi K, Murata N, Ohtani T, Toguri T. 1996. Low-temperature resistance of higher plants is significantly enhanced by a nonspecific cyanobacterial desaturase. Nature Biotechnology 14:1003-1006. Knight MR, Campbell AK, Smith SM, Trewavas AJ. 1991. Transgenic plant aequorin reports the effects of touch and cold shock and elicitors on cytoplasmic calcium. Nature. 352: 524-526. Knight H, Trewavas AJ, Knight MR. (1996). Cold calcium signaling in Arabidopisis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell. 8, 489-503 Knight H, Veale EL, Warren GJ, Knight MR. 1999. The sfr 6 Mutation in Arabidopsis Suppresses Low-Temperature Induction of Genes Dependent on the CRT/DRE Sequence Motif. Plant Cell. 11: 875-886 Kurkela S, Franck M1990. Cloning and characterization of a cold- and ABA- Inducible Arabidopsis gene. Plant Mol. Biol. 15:137-44 22 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. Lang V, Palva ET. 1‘ 993. The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh. Plant Mol. Biol. 21 :581 Lee H, Xiong L, Gong Z, lshitani M, Stevenson B, Zhu JK. 2001. The Arabidopsis H081 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning. Genes & Development. 15: 912-924 Leung J, Bouvier-Durand M, Morris PC, Guerrier D, Chefdor F, Giraudat J. 1994. Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase. Science. 264:1448-52. Levitt J. 1980. Responses of plants to environmental stresses. Chilling, freezing, and high temperature stresses. New York. Academic Press, 2"“ ed. McKown R, Kuroki G, Warren G. 1996. Cold responses of Arabidopsis mutants impaired in freezing tolerance. J. Exp. Bot. 47: 1919-1925. Miguel M, James D, Dooner H, Browse J. 1993. Arabidopsis requires polyunsaturated lipids for low temperature survival. Proc Natl Acad Sci USA 90: 6208-6212. Michel D, Salamini F, Bartels D, Dale P, Baga M, Szalay A. 1993. Analysis of a desiccation and ABA-responsive promoter isolated from the resurrection plant Craterostigma plantagineum. Plant J 1993 4:29-40. Monroy AF, Sangwan V, Dhindsa RS. 1998. Low temperature signal transduction during cold acclimation: protein phosphatase 2A as an early target for cold- inactivation. Plant J. 13, 653-660. Monroy AF, Dhindsa RS. 1995. Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25°C. Plant Cell. 7:321 - 331. 23 Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K. 1999. Antisense suppression of proline degradation improves tolerance to freezing and salinity In Arabidopsis thaliana. FEBS Lett 461: 205- 21 0. Orvar BL, Sangwan V, Omar F, Dhinsa R. 2000. Early steps in cold sensing by plant cells: The role of actin cytoskeleton and membrane fluidity. Plant J 23: 785- 794. Plieth C, Hansen UP, Knight H, Knight MR. 1999. Temperature sensing by plants: the primary characteristics of signal perception and calcium response. Plant J 52491-497. Rikin A, Blumenfeld A, Richmond, AE. 1976. Chilling resistance as affected by stressing environments and abscisic acid. Bot Gaz 137: 307-312. Saijo Y, Hata S, Kyozuka J, Shimamoto K, lzui K. (2000). Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J. 23, 319-327 Shen Q, Zhang P, Ho T. 1996. Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in barley. Plant Cell 8:1107-1119 Snedden W, Fromm H, 1998. Calmodulin, calmodulin-related proteins and plant responses to the environment. Trends in Plant Sci 3:299-304. Steponkus PL. 1984. Role of the plasmamembrane in freezing injury and cold acclimation. Annu. Rev. Plant Physiol. 35: 543-584. Steponkus PL, Uemura M, Joseph RA, Gilmour SJ, Thomashow MF. 1998. Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA. 95: 14570-5. Stockinger, EJ, Gilmour SJ, Thomashow MF. 1997. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C- repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA. 94:1035-1040. Suzuki l, Los DA, Murata N. 2000. Perception and transduction of low- temperature signals to induce desaturation of fatty acids. Biochem Soc Trans 24 Dec 6:628-30. Tahtiharju S, Sangwan V, Monroy A, Dhindsa R, Borg M1997. The induction of kin genes in cold-acclimating Arabidopsis thaliana. Evidence of a role for calcium. Planta. 203:442-447 Thion L, Mazars C, Nacry P, Bouchez D, Moreau M, Ranjeva R, Thuleau P. 1998. Plasma membrane depolarization-activated calcium channels, stimulated by microtubuIe-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large and stable activities in ton 2 mutant cells affected in the organization of CORtical microtubules. Plant Journal 16:603-610. Thuleau P, Schoeder, Ranjeva R. 1998. Recent advances in the regulation of plant calcium channels: evidence for regulation by G-proteins, the cytoskeleton and second messengers. Curr Opin Plant Biol 1:424-427 Trewavas A, Malho R. 1998. Ca2+ signaling in plant cells: the big network! Curr Opin Plant Biol 12428-433. Uemura M, Steponkus PL. 1997. Effect of cold acclimation on membrane lipid composition and freeze-induced membrane destabilization. Plant Cold Hardinesss. Molecular Biology, Biochemistry and Physiology. New York: Plenum. pp. 171 -1 79. Vigh L, Los D, Horvath I, Murata N. 1993. The primary signal in the biological of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proc Natl Acad Sci USA 90: 9090-9094. Wanner LA, Junttila O. 1999. Cold-induced freezing tolerance in Arabidopsis. Plant Physiol 120: 391-400. Welin BJ, Olson A, Nylander M, Palva ET. 1994. Characterization and differential expression of dhn/lea/rab-Iike genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Mol. Biol. 26:1 31 -144 Wu Y, Kuzma J, Marechal E, Graeff R, Lee HC, Foster R, Chua NH. 1997. Abscisic acid signaling through cyclic ADP-ribose in plants. Science. 278:2126- 2130. 25 Xin Z, Browse J. 1998. Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc. Natl. Acad. Sci. USA. 95: 7799-7804. Xiong L, Lee Bh, lshitani M, Lee H, Zhang C, Zhu JK. 2001. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev. 15(15):1971-1984. Yamaguchi-Shinozaki K, Shinozaki K. 1993. Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter In transgenic plants. Mol. Gen. Genet. 236, 331 -40. Yamaguchi-Shinozaki K, Shinozaki K. 1994. A novel cis-acting element in an Arabidopsis gene is involved In responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 6(2):251-264. 26 Chapter 2 Investigation of the Role of ABA in the Cold Regulation of the CBF genes Abstract ABA has been shown to be involved In the cold acclimation response. To evaluate the possibility that ABA plays a role in the cold-regulated induction of the CBF family of transcription factors, known mutants with lesions in the abscisic acid (ABA) biosynthetic and signal transduction pathway were evaluated In their ability to Induce the cold-regulated expression of the CBF genes. In the aba1, abi1, and abi2 mutants, the CBF genes were expressed at lower levels In response to low temperature; whereas transcript levels remained sustained In wild-type. Transcript levels of the CBF target gene COR78, were decreased in response to cold in the ABA and ABI mutants as compared to wild-type, but most greatly affected by the abi1 mutation. To further understand the role of ABA regulation of the CBF genes, I examined binding events occurring at the CBF2 promoter during cold treatment. Electrophoretic mobility shift assay (EMSA) experiments were performed using Arabidopsis thaliana nuclear extracts from control and cold-treated plants. A 155bp region of the CBF2 promoter shown to impart cold-regulation was used for the EMSA studies. The results indicate that the 155 bp CBF2 promoter fragment binds to proteinaceous factors In the nuclear extract and that this binding can be competed off by adding an excess of unlabeled 155 bp fragment. An unlabeled 27 fragment in which a putative ABA responsive element (ABRE) was mutated was unable to compete for binding of these proteins. Additionally, no difference was detected in the DNA-binding proteins present In extracts from both cold-treated and control plants. Taken together, these results suggest that the CBF2 gene promoter is bound between -189 and — 35 relative to the start of transcription by protein factors acting at the putative ABRE sequence In Box V, and that this binding occurs in both control and cold-treated plants. Introduction Plants are confronted with a number of environmental stresses In nature including cold stress. Freezing temperatures affect the ability of plants to grow and proliferate; therefore, how plants living In cold regions respond to and subsequently protect themselves against freezing temperatures Is critical for their survival. Many plants develop freezing tolerance upon exposure to low- nonfreezing temperatures. This process, known as cold acclimation, Involves the expression of the cold-regulated (COR) genes whose products accumulate after a cold temperature stimulus. Many of these genes share a common promoter regulatory sequence known as the C-repeat (CRT)/ dehydration responsive element (DRE) (Baker et al., 1994). The CBF (C-repeat binding factor)/DREB1 (DRE-binding protein) protein family are transcriptional activators which bind the CRT/DRE and play a key role In the process of cold acclimation (T homashow, 2001). The CBF transcription factors are themselves cold-regulated, but do not appear to be autoregulated (Gilmour et al., 1998). The CBF genes comprise a small gene family which Includes CBF 1, CBF2, and CBF3 (Gilmour et al., 1998). 28 The constitutive overexpression of CBF 1, CBF2, and CBF3 in Arabidopsis leads to constitutive expression of the COR genes and results in enhance freezing tolerance of nonacclimated plants, bypassing the requirement for a cold treatment to protect the plant against freezing damage (JagIo-Ottosen et al. 1998, Liu et al, 1998, Kasuga et~al., 1999, Gilmour et al., 2000). At present, the temperature sensing mechanism and the signal transduction pathway which regulate CBF expression are unknown. ABA, however, has been implicated in the cold acclimation response and therefore Is an interesting candidate as an upstream regulator of the CBF pathway (Chen et al., 1983). Application of exogenous ABA enhances freezing tolerance, ABA mutants are less freezing tolerant than wild-type, and endogenous ABA increases transiently in response to low temperature (Chen et al., 1983; Nordin et al., 1991; Gilmour and Thomashow, 1991). Studies examining the responsiveness of the CBF genes to exogenously applied ABA have demonstrated CBF 1, CBF2, and CBF3 are unresponsive to this application (Medina et al., 1999). Moreover, experiments conducted by Gilmour and Thomashow (1991) implicate an ABA independent pathway through which the cold-regulated expression of the COR genes are Induced. Interestingly, however, Zarka (2001) found that the promoter of CBF2 Is responsive to ABA when fused to the GUS reporter gene, although endogenous levels of CBF2 transcript only subtly increase as compare to a high increase of GUS transcripts. To clarify whether the cold responsiveness of CBF2 is mediated through ABA signaling, the expression of CBF2 was monitored using ABA biosynthetic and 29 signaling mutants. Mutations affecting ABA1, ABI1 and ABI2 were selected to determine whether mutations in ABA biosynthesis or signaling could alter expression of CBF genes. ABA1 encodes a zeaxanthin epoxidase involved In ABA biosynthesis (Tan et al., 1997). ABI1 and ABI2 encode homologous phosphatases type 20 which are involved In ABA signal transduction (Leung et al., 1997). The abi1 and abi2 mutants are insensitive to abscisic acid (Merlot et al., 2001). The transcript levels of cold-regulated transcription factors was analyzed by hybridizing probes derived from the transcription factors of Interest with RNA blots of cold-treated wild-type and mutant plants. To gain Insight on which sequences in the CBF2 promoter regulate its cold responsiveness, Zarka (2001) has performed experiments to identify the cold responsive element present In its promoter. The smallest region of the CBF2 promoter that has currently been identified as sufficient to confer cold temperature responsiveness is a 155 bp region from -189 through -35 relative to the start of transcription (Zarka, 2001). A dimer of this promoter fragment fused to a minimal S35 Califlower mosaic virus promoter fused to the beta- glucuronidase (GUS) reporter gene is sufficient to drive cold-responsive GUS expression. When the 155 bp promoter fragment was further deleted, the reporter gene was no longer cold responsive (Zarka, 2001). This indicates that a promoter element(s) responsible for the cold-regulated expression of CBF2 lies within this 155 bp region. The 155 bp region overlaps with regions of homology Identified between the promoter of CBF2 and the promoters of two other genes in its family - CBF1 and CBF3 (Shinwari et al., 1998). These conserved promoter 30 sequences, referred to as Box I - Box Vl, may play an Important role in the regulation of the CBF genes. To complement these promoter deletion experiments, electrophoretic mobility shift assay (EMSA) using the 155 bp region of the CBF2 promoter as a probe were performed to detect whether proteins in nuclear extracts can bind to the 155 bp region. In addition, EMSA experiments using subfragments of the 155bp element harboring a mutation in the putative ABRE sequence were carried out to determine the Importance of the ABRE In the binding of nuclear proteins. Additional competition experiments using fragments mutated in other consensus sequences were also performed. The cold-induced expression of the CBF genes occurs in the presence of cyclohexamide, a chemical which inhibits protein translation (Qin, 2002). Therefore, proteins that are already present at warm temperatures lead to events which regulate CBF transcription. These protein(s) may regulate CBF expression by an activator binding or a repressor leaving the CBF promoters at low temperatures. The EMSA experiments were designed to determine whether there are differences In binding between cold-treated and control plants, and to discover which nucleotides of the CBF2 promoter are bound in either treatment. 31 Materials and Methods Plant Material and Growth Conditions Arabidopsis thaliana ecotype Landsberg erecta (Ler) and Columbia (Col) were used in this study. Seeds of aba1-1, abi1-1, abi2-1, (Ler background) ctr1- 2, ein2 and hIs1-1 (Col background) were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Seeds were surface sterilized and aseptically grown on Petri plates containing Gamborg’s BS medium (lnvitrogen, USA) solidified with agar. To break dormancy and synchronize germination, seeds were Incubated at 4°C for 3 days. The seedlings were grown in controlled environmental conditions under constant fluorescent light (100-150 uEm'zs") at 22-24°C. Ten to twelve day old seedlings were harvested from plates and immediately placed in liquid nitrogen to be used as material for RNA analysis. Cold treated plants were placed at 4°C under ~25 pEm'zs'1 continuous fluorescent light for various amounts of time as indicated in the figures. RNA Extraction and Northern Blot Analysis Frozen tissue samples were ground in liquid nitrogen, and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). To examine the low-temperature induction of the CBF genes and other transcription factors In wild type and mutant plants, RNA analysis of seedlings that had been treated at 4°C for 0, 2, 4 and 24 hours or 0, 1, 2, and 4 hours were used. Total RNA 32 was fractionated by electrophoresis through a 1.5% denaturing agarose gel containing 2.2 M formaldehyde (Sambrook et al., 2001). Transfer of denatured RNA to a positively charged nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) was performed as described by Sambrook et al., (2001). Prehybridization was carried out for 3 hours at 42°C In buffer containing 50% (v/v) formamide, 5X SSPE, 5X Denhart’s solution, 1% (w/v) sodium dodecyl sulfate, 10% (w/v) dextran sulfate, and 20(1ng of sheared salmon sperm DNA (Sambrook, 2001). Full-length DANA inserts for use as probes for detecting the expression of CBF2, were excised from plasmids provided by Sarah Gilmour. The CBF2 probe used in this study cross-hybridizes with CBF1 and CBF3. These fragments were extracted from 1% (w/v) agarose gels (QlAquick Gel Extraction Kit, Qiagen, Valencia CA) and labeled by random priming using 32P-dCTP (Random Priming DNA Labeling Kit, GIbco BRL, Grand Island, NY). Ovemight hybridization of these probes was performed under the same conditions as prehybridization after the addition of the labeled probe. Membranes were washed three times with 2X SSPE, 0.5% (w/v) SDS at 42°C and three times with 0.1 X SSPE/0.5% (w/v) SDS at 55°C, then exposed either to Kodak XAR-S X-ray film at -80°C a phosphor Imaging screen (Kodak, Rochester, NY) for 3 hours to overnight. Screens were scanned on a phosphorimager using Quantity One imaging software (Bio-Rad Laboratories, Hercules, CA). 33 EMSA Analysis EMSAs were carried out as previously described (Stockinger et al., 1997). Nuclear extracts (1 ug/ul) that were present in the lab from control or cold-treated (4°C for 4 hours) Arabidopsis plants were Incubated with a radio-labeled 155 bp DNA fragment of CBF2 promoter (4 ng). To show the binding specificity of the probe, wild-type 155 bp promoter fragments or 50 bp mutated subfragments of the 155 bp region were included as unlabeled competitors in these reactions. The sequences of the wild-type and mutated oligonucleotides are indicated below. The 50 bp subfragment of the 155 bp CBF2 promoter region is highlighted in bold. Subfragment F is a dimer of the Box Vl sequence conserved among CBF1, CBF2, and CBF3. 155 hp fragment CAAGATGGGTCAAAGGACACATGTCAGATTCTCAGTGATTGACAGCCTT GATAATI'ACAAAACCGTGGGATCGCTI'AGCTG'ITTCTTATCCACGTGGCA TTCACAGAGACAGAAACTCCGGC Subfragment A GATAA‘I'I'ACAAAACCGTGGGATCGCTI'AGCTG'I'ITCTTATCCACGTGGCA Subfragment B GATAATTACAAAACCGTGGGATCGCTTAGCTGTI'I’C'I'I'ATCCACGTGGCA Subfragment C GATAATI'ACAAAACCGTGGGATCGCTI'AGCTGTTTCTI'ATCCACGTGGCA Subfragment D GATAATTACAAAACCGTGGGATCGC‘I‘I'AGCTGTITC'I‘I’ATCCACGTGGCA 34 Subfragment E GATAATTACAAAACCGTGGGATCGCTTAGCTGTTI'CTTATCCACGTGGCA Subfragment F CAGAGACAGAAACTCCGCAGAGACAGAAACTCCG Samples were electrophoresed through a 12% (w/v) polyacrylamide (30:1 acrylamide:bisacrylamide) nondenaturing gel in 0.5x TBE at 4°C. Gels were dried on filter paper (Whatman) and exposed to film (Kodak, USA). G box motif CAAG ATGGGTCAAA GGA CA CA TGTCAGATTCTCAGTG ATTGACAGCCTI' put ABRE Box V GATAATTACAAAACCGTGGGATCGC'I'TAGCTGTTTC‘ITA TCCdACg rgGCAl Box v1 ECAICAGAGACAGAAACTCCQCG'ITCGACCCCACAAATATCCAAATATC TTCCGGCCAAMCAGCAAGCTCTCACTCCACCA'I'I'TCTATAAEFTCAAA CACTI'ACCTGAATI'AGAAAAGAAAGATAGATAGAGAAATAAATATI'TATCATA CCATACAAAAAAAAGACAGAGATCTI'CTACTI'ACTCTACTCTCATAAACC'ITA TCCAG'ITTCTTGAAACAGAGTACTCTCATAAACCTI'ATCCAGTTI'CTTGAAAC AGAGTACTCTTCTGATCAATG The 155 bp sequence of the CBF2 promoter region from -189 through the start of translation contains a number of sequence motifs. The sequence in bold repre- sents the 155 bp region used in this study. The arrow represents the start of transcription. Box V and VI are conserved sequences shared in CBF 1, CBF2, and CBF3 (Shinwari et al., 1998).The putative light responsive G-box is underlined and in italics. 35 Results Expression Studies of CBF Genes To determine whether the CBF genes are cold-regulated in an ABA- dependent manner, accumulation of mRNAs of these transcription factors was examined in the aba1, abi1 and abi2 mutants. CBF expression was examined In ABA biosynthetic and signaling mutants as well as in wild-type Arabidopsis plants treated for 0, 1.5 or 2, 4 and 24 hours at 4°C as Indicated (Fig. 2.1A). In the wild- type plants, CBF mRNA levels reach maximum expression at 1 and 2 hours, then drop by 4 hours of cold-treatment. In contrast to wild-type expression, CBF expression levels are decreased over the course of the cold-treatment in aba 1, abi1, or abi2 mutant plants (Fig. 2.1A). In an independent experiment shown in Fig. 2.1 B, CBF expression again appears to be reduced in aba1, abi1 and abi2 mutant plants that were cold-treated. CBF mRNA levels are more highly expressed in wild-type plants that were cold-treated for 0, 2, 4 and 24 hours at 4°C in comparison to aba 1, abi1, and abi2 mutants subjected to the same treatment. Particularly, expression of the CBF genes was most drastically reduced at 4 and 24 hours In aba 1, abi1 and abi2, as compared to wild-type. Transcript levels of CBF 1, CBF2, or CBF3 (unable to determine which CBF since the CBF2 probe cross-hybridizes with CBF1 and CBF3) were as high in aba1, abi2 and abi2 mutants suggesting that ABA biosynthesis and signaling Is required for the full induction of cold-regulated CBF mRNA levels of at least one of the CBF genes In the In wild-type plant (Fig. 2.1). The effect of lesions in the 36 ABA biosynthetic and signal transduction pathways on the cold-regulated Induction of the CBF target gene COR78 was also examined. Wild-type COR78 levels responding to cold, are highly abundant at 4 hours and remained sustained over the course of the cold-treatment (Fig 2.2). Although a similar expression pattern was observed for COR78 in the aba 1, abi 1, and abi2 mutants, mRNA levels were reduced as compared to wild-type. The abi1 mutation leads to the most severe reduction of COR78 mRNA levels. 37 aba1-1 abi1-1 abi2-1 Wt Ler 01.5424 01.54 24 01.54 24 01.5 424 A L 11111 ~13 * 111» ~11- ]~»w-1J CBF aba1-1 abi1-1 abi2-1 Wt Ler 02 424 02424 02424 02424 .1 ii“ . . g - ‘. . _- . ‘1. gt: :5,» A' .1 .3’ 1 . . - 1 , . H . - . n 1 . . 'gg ~ 1. "\‘ ~‘n CBF . r 1. .. I14. _ \ u.“ ”1 ‘ , . . .- \.‘ 1 f“' ’x . ' -. - \k \:1'1‘- 1’ I 1 ' 1 V ;. . ., 1 I ,- 1! _1 53.1»: ._ * 31 '3'. --: 19 . «:7 1.1:? _ , , 9.1-,- . :5 .« ¢.r .3. ... x. . ' .1~‘\'( i. , ' , ,. "‘ ‘ 3. ‘- ‘ 1’» ' ‘3‘1 ‘ - ‘ j. ’ A .‘ ‘ ‘ K I . {I '..‘A ‘ 1,, ‘\‘. ‘ I ‘ . ‘ ‘ ‘ ' ‘ ' , -» 1 ., ~.~ , 1:. 3* ,w; . , .93 was 1. 1, .. - 4 .‘ ..- 1. * ‘ . v. ,11 ‘ ._ ‘ \ 1 . y . I \ . ~ - I 1 . 1- .. .‘ , ‘5 .1. ~ + .‘ynv .7 ‘ ‘ 1 3'. 7» 1 . 1 -.,. ‘ A“; 7‘1 ‘ ‘ 1. - Fig. 2.1 Effect of mutations In ABA biosynthesis and signal transduction pathways on the cold induced expression of the CBF genes. Plants grown at 22-24°C were transferred to 4°C for cold treatment for the times indicated. RNA was extracted and transferred to a nylon membrane. Northern blots were prepared and hybridized with CBF2 probe. A: Northern hybridization analysis of RNA isolated from wild-type, aba 1, abi 1, and abi2 Arabidopsis plants that were cold treated for 0, 1, 2, or 4 hours at 4°C as indicated and hybridized with CBF2 probe. B: Northern blot of RNA Isolated from wild-type, aba1, abi1, and abi2 Arabidopsis plants that were cold treated for 0, 2, 4 or 24 hours at 4°C as indicated probed with CBF2. The ethidium bromide rRNA serves as a loading control. 38 aba1-1 abi1-1 abi2-1 Wt Ler Time (h) 02424 02424 02424 024 24 at4oc .52..» 1‘. .; 1. .11." ‘ . ‘ . I 34511.} , . . Ir“ ‘2 ' 1 00378 Fig. 2.2. Effect of the ABA biosynthetic and signaling mutations on COR78 expression levels. Plants were grown at 22-24°C were transferred to 4°C for cold treatment for the times indicated (h). RNA was extracted from wild-type, aba1, abi 1, and abi2 mutant plants then transferred onto a nylon membrane. Northern hybridization analysis of cold-induced COR78 transcript levels was then examined. Ethidium bromide stained rRNA serves as loading control. 39 Detection of Proteins Binding to the CBF2 Promoter Preliminary EMSA Experiments EMSA experiments were performed to detect whether proteins present In Arabidopsis nuclear extracts bind the 155 bp element of the CBF2 promoter, and specifically to determine whether the putative ABRE sequence in the 155bp element Is bound by these proteins. In a preliminary experiment, nuclear extracts from control and cold treated plants were tested to find whether factors would bind the fragment and lead to a complex of slower mobility than the probe alone. A smear of shifted complexes with the labeled probe was observed (Fig. 2.3). The most likely explanation Is that the shift observed in Fig. 2.3 is due to one or more nuclear proteins binding the probe. However, It was a possibility that the binding which led to the shift was due to a nonproteinaceous factor. To rule out this possibility, aurintricarboxylic acid (ATA), a chemical that disrupts protein-DNA interaction (Blumenthal and Landers, 1973), was added to the extracts to find whether a shift would occur In its presence. The extracts with and without ATA added were then analyzed by gel-shift assays (Fig. 2.4). When ATA was added to the sample containing the CBF2 probe mixed with nuclear extract, no retarded fragments were observed, but retardation was still evident In samples without ATA (Fig. 2.4). This suggests that the shifts observed when nuclear extracts are mixed with the 155 bp fragment are caused by protein(s) binding the CBF2 promoter fragment. Titgation of Nonspecific Competitor 4O To inhibit nonspecific binding of proteins in crude nuclear extracts to the labeled probe, polydldC was used as nonspecific competitor DNA. Due to Its composition of Inosine and cytosine residues, It does not bind tightly to sequence-specific DNA binding proteins such as transcription factors. To find the optimal concentration of polydldC needed for subsequent experiments, titration experiments were carried out (Fig. 2.5a). The amount of polydldC which competed all but three complexes was 1pg and was determined to be the optimal amount of polydldC required to compete off nonspecific proteins binding to the 155 bp fragment. When less than 1ug of polydldC is used in the reaction, the promoter fragment can be occupied by nonspecific proteins in the nuclear extract resulting in a large smear of shifted probe. At 11.19, polydldC prevents nonspecific proteins from binding, so no smear is observed (Fig. 2.5a). Therefore, 1ug of polydldC was used for subsequent experiments. 41 Lane Fig. 2.3 EMSA of Arabiopsis nuclear extracts from control and cold-treated plants using the 155 bp region of the CBF2 promoter as a probe. Nuclear extracts from plants grown at 22°C (N) and nuclear extracts from plants grown at 22°C then cold- treated at 4°C for 4hrs (C) were mixed with CBF2155bp probe. Lane 1 contains CBF2 probe alone. Lane 2 contains probe incubated with nuclear extract from non- treated plants. Lane 3 contains CBF2 probe mixed with nuclear extract from plants cold-treated at 49 for 4hours. 42 ATA- -+-+ - NNCC Fig. 2.4. The addition of Aurintricarboxylic acid leads to loss of DNA-protein interactions EMSA of Arabidopsis nuclear extract from control (N) and nuclear extract from plants cold treated at 4°C for 4 hours (C) with and without Aurinatricarboxylic acid (ATA). Lane1 is CBF2 155bp alone. Lane 2 contains the probe mixed with nuclear extract from control plants. Lane 3 contains the probe mixed with nuclear extract from control plants and ATA. Lane 4 contains the probe mixed with nuclear extract from plants cold treated at 4°C for 4 hours. Lane 5 contains the probe mixed with nuclear extract from cold- treated plants and ATA. 43 EMSA analysis of control vs. coIg-treated_plants Proteins present at warm temperatures regulate the expression of the CBF genes. Differences in CBF gene expression before and after cold-induction could be due to a reorganization of regulatory proteins at the CBF promoters. To test the hypothesis that proteins bind differentially to the CBF promoters In plants that are exposed to low temperature and those that are not, nuclear extracts from control versus cold-treated plants were analyzed using EMSA. When 2119 of nuclear extract per binding reaction was used, nuclear extract from control plants generated a different binding pattern than the nuclear extract from cold-treated plants (Fig. 2.5b). In these lanes, a smear of very low mobility was observed in extracts from control plants, but the more distinct shifted complexes remained In the cold-treated extracts (Fig. 2.5b); however, in extracts from both control and cold treated plants, the shifts produced from increased nuclear extracts led to an increased proportion of low mobility protein-DNA complexes. When 1pg of nuclear was used In the binding reaction, the low mobility smear is not observed and distinct bands appear In lanes corresponding to both control and cold-treated extracts. The presence of higher mobility complexes at lower protein concentrations may Indicate that there are multiple binding sites which are not fully occupied when 1pg of protein is used. The mobility of the protein-DNA complexes may depend on how many and which sites of the DNA probe are bound. Localization of Binding in the 155 bp Fragment Further EMSA experiments were performed to determine whether the shifts seen in Figures 2.3, 2.4 and 2.5 were specific to the 155 bp fragment, and If so, to locate the protein binding site within the fragment using wild-type and mutated subfragments of the 155 bp region as competitors. To answer both questions, EMSA analysis was performed using similar conditions to previous experiments with the addition of unlabeled 155 bp promoter fragments and subfragments of the 155 bp promoter as competitors. The concentration the 155 bp competitor DNA used was far less than the concentration of needed to compete off the binding with polydldC (1 OOng). The unlabeled 155 bp probe completely competed off the binding (Fig. 3.6). Therefore, the shifts observed In previous experiments are concluded to be due to specific binding of proteins to the 155 bp fragment. The promoter fragment containing the mutated ABRE-like sequence within Box VI (fragment D) was unable to compete off any of the three shifts, suggesting that the ABRE and/or Box VI is important for these DNA-protein complexes to occur. If the ABRE is responsible, it would then be expected that fragments containing the wild-type ABRE sequence would be able to compete off these shifts. All other fragments (A, B, C, and E) contain the wild-type ABRE were able to compete off the three shifts to varying degrees. Surprisingly, the promoter fragment containing only a dimer of Box VI was able to compete off shifts one and two and to some degree three. This was unexpected because it does not contain the ABRE. The promoter fragment which contains a Box VI that 45 is completely mutated could compete off the shifts as well. Unless there are sequences similar to Box VI found elsewhere on fragment C, this result is inconsistent with the competition caused by fragment F. That fragment C was able to compete off the shift suggests that Box Vl Is unimportant for binding, yet the competition observed for fragment F suggests Box VI is Important for binding. 46 Control Cold NE Control _& 1 2 1 2 NE (HQ/Ill) PolydldC . .1. ~ (.1234 56 7 8911011 1111 (a) (b) Fig. 2.5. Titration of PolydldC and nuclear extract using 155 bp CBF2 promoter fragment. (a) Lane 1 is the CBF2 probe without nuclear extract (FP). Lanes 2-6 contain 155 bp probe, Arabidopsis nuclear extracts, and decreasing amounts of polydldC: 10ug/ul, 1ug/ul, 0.1ug/ul, 0.01 ug/ul, and 0.001 ug/ul. Lanes 7-11 also contains the same concentration of nuclear extracts, but contain nuclear extracts from plants treated at 4°C for 4 hours.Arrows indicate the three shift complexes formed due to protein- DNA probe Interaction. (b) EMSA performed using 1ug/ul and 2pg/ul of nuclear extracts (NE) from control plants and cold-treated plants at 4°C. Arrows indicate the three shifted complexes formed due to protein-DNA probe interaction. FP Indicates the unbound 155 bp probe. 47 FP ABCDEF ‘1 Box v [E] Box VI Fig. 2.6. Gel Shift experiment using wild-type and mutated fragments of the 155 bp promoter regions of CBF2 as competitor. A: fragment A is the 155 bp sequence; fragment B is -120 through -70 of the promoter containing box V and VI wild-type, fragment C is Box VI mutated, fragment D is the -120 throught -70 with the ABRE sequence located near Box V mutated; fragment E is -189 through -176 of the promoter fragment mutated; fragment F is a dimer of the Box VI sequence. B: EMSA assays were performed using nuclear extract from control Arabidopsis plants mixed with competitor fragments (40ng). The probe was the 155 bp CBF2 promoter fragment labeled with 32P. Lanes are labeled according to the fragment used as competitor. The FP lane indicates unbound probe without nuclear extract. 48 Discussion Effect of ABA Mgtations on CBfiF and COR Gene Expression The Induction of genes by low temperature can be either dependent or independent of ABA (T homashow 1999). The CBF genes have been found to be unresponsive to exogenous ABA (Medina et al., 1999, Gao et al., 2002). Interestingly, the CBF2 promoter when fused to the GUS reporter gene has been found to be transcriptionally responsive to exogenous ABA (Daniel Cook and Daniel Zarka, unpublished data). These results could indicate that ABA-induced CBF2 transcripts are not stable as compared to CBF2 induction due to low temperatures. The change In the stability of CBF2 mRNA may mark a divergence In the control mechanisms Involved in regulating CBF expression. Moreover, the results from the competition assays in Fig. 3.5 implicate the putative ABRE as Important for the binding of Arabidopsis nuclear proteins to the CBF2 promoter (discussed in detail below). Taken together, ABA is likely Involved in the regulation of the CBF genes. The results in Fig. 2.1 indicate that the CBF genes can be cold-Induced In mutants defective In ABA biosynthesis and signaling. However, these results also showed that lesions in ABA 1, ABI1, and ABI2 lead to decreased cold-induced expression of the CBF genes. These results suggest that CBF regulation Is responsive to cold through both ABA-independent and ABA-dependent pathways. It Is not clear from this study which of the CBF genes is affected due to the fact that CBF2 probe used In this study to cross-hybridizes with CBF1 and 49 CBF3. Repeating these experiments using gene specific probes is necessary to determine how mutations In the ABA signaling pathway affect the cold induction of the individual CBF genes. An alternative interpretation of these results is that CBF expression Is completely dependent on ABA but the genes were induced in the aba 1, abi 1, and abi2 mutants because the mutations are leaky- that is, these genes are not completely knocked out in their expression. Mutations In aba1, abi1, and abi2 also Inhibit the cold-regulated induction of a downstream target of the CBF genes, COR78. The decreased transcript levels of COR78 could be due to the ABA mutations Inhibiting the expression of the CBF genes which in turn are not able to activate the COR78. The decreased transcription could also be due in part to the inability of ABA responsive transcription factors to activate COR78 via its ABRE (Fig. 2.2). The drastic affect seen in the abi1 mutant, but not seen In abi2 at first glance was unexpected. The wild-type genes for ABI1 and ABI2 both encode phosphatases type 20, but the mutant alleles chosen In this study are quite different. The abi1 mutant is a dominant negative mutant and abi2 Is a recessive mutant. The abi1 mutant could cause a gain of function in the mutated gene product, resulting in decreased COR78 expression levels, which is not observed in the abi2 mutant. Related Studies In contrast to the study presented here, Gilmour and Thomashow (1991) found that the cold responsiveness of COR47, COR78, and COR6.6 was unaffected In the abi1 and abi2 mutants, and slightly decreased in the abaf 50 mutant. However, ABA induced expression of these genes was found to be deficient in the abi1 mutant (Gilmour and Thomashow, 1991). This shows that ABA responsiveness of the COR genes is due to a separate pathway from the responsiveness of these genes to cold. These data indicate that there is a divergence in the ABA and cold signaling pathways. However, in the Gilmour study, only one time point at twenty-four hours was examined, whereas In this study, a number of time points ranging from zero to twenty-four hours cold treatment were examined. The twenty four hour time point showed different results between both studies. In this study, COR78 expression was most diminished in the abi1 mutant, but also decreased in aba1 and abi2 mutants. The Gilmour and Thomashow (1991) results indicated that the aba1 mutation lead to a two-fold decrease In the cold induction of COR78, whereas abi1 and abi2 and no apparent affect on COR78 expression. A possible explanation for differences seen between these two studies is that growth conditions used In both studies were not the same. The Gilmour and Thomashow study used potted plants grown in soil as opposed to plants grown on Gambourg’s medium on plates. ABA and Cold-regulated Pathways lshitani and colleagues (1997) Identified mutants that are affected in their response to both cold and ABA indicating that there is a point at which these two signaling pathways converge In controlling the response of the RDZQA/COR78 promoter (lshitani et al., 1997). Their results suggest that ABA has a role in cold 51 sensing, and that two separate signaling pathways, ABA-independent and ABA- dependent, crosstalk. The exact mechanism of how a single mutation affects both cold and ABA-Induced RDZQA expression Is not clear. In addition, other studies support the involvement of ABA-dependent Induction of the cold-regulated RAB18. The experiments in this study examining the expression of CBF in the aba1, abi1 and abi2 mutants show that the expression of the CBF genes Is affected, but not obliterated in the mutants. COR genes also appear to be affected In the ABA biosynthetic and signaling mutants. This study demonstrates a convergence between the ABA-Independent and ABA-dependent pathways at the CBF genes. EMSA Experiments Sgpport Expression Studies Using EMSA analysis, a cold responsive region of the CBF2 promoter was examined. The 155 bp region found to be sufficient for cold regulation (Zarka 2002) was shown to be specifically bound by nuclear proteins (Fig. 3.5) possibly at multiple binding sites on the promoter (Fig. 3.4b). Extracts from both control and cold treated plants gave a similar pattern of shifted complexes, which could reflect proteins that are present at the promoter before and after the exposure of the plant to cold (Fig. 3.4a and Fig. 3.4b). In Fig. 3.4b, the pattern of shifted bands was dependent upon the concentration of nuclear extract used in the binding reaction. This may be due to the presence of multiple binding sites on the 155 bp fragment, such that at lower protein concentrations, there are not enough proteins to bind all sites on all DNA fragments. It could also represent multiple 52 proteins at a given site. In both cases, the addition of more protein leads to various complexes of different mobilities. In the cold, less protein appears to be bound after the cold treatment. This could indicate the loss of a repressor at the CBF2 promoter upon the shift from warm to cold temperatures. Altogether, the experiments highlighted in Figures 3.43 and 3.4b show that there are proteins present in the nuclear extracts of both control and cold- treated Arabidopsis plants specifically binding to the 155 bp region (Fig. 3.4). A change in the phosphorylation state or some other protein modification could lead to a change in protein binding affinity to the promoter, or could affect the regulation of CBF2 by a cold-Induced conformational change of proteins bound to Its promoter. Alternatively, differences between the presence of proteins binding the promoter In control and cold-treated nuclear extracts may not be detected If the interaction of proteins are affected by the reaction conditions in these EMSA experiments. Changes at the promoter, therefore, may not be accurately reflected under the conditions set in this EMSA analysis. The results from the competition assays in Fig. 3.5 suggest that Box V containing the putative ABRE is Important for the binding of Arabidopsis nuclear proteins binding to the CBF2 promoter. The conclusion from EMSA using dimers of Box VI (fragment F) as a competitor is unclear, because it was able to compete off bands 1 and 2, suggesting that Box VI in the CBF2 promoter is being bound by proteins leading to the shifts observed as bands 1 and 2 (Fig 3.5). However, when Box VI is mutated, as in fragment C, there is no change in the band shift compared to that produced for fragment F. Unless there are 53 sequences similar to Box VI found elsewhere on fragment C, this result Is Inconsistent with the data suggesting that Box Vl is unimportant for binding. The EMSA data implicate the ABRE as a binding site for proteins at the CBF2 promoter which is consistent with the expression studies in Chapter 2, which indicate that mutations in ABA1, ABI1, and ABI2 genes inhibit the full expression of CBF2, that is, ABA may activate CBF2 through the ABRE-like sequence found in its promoter. When the ABA biosynthetic or signaling pathway Is mutated, or the ABA responsive element in the CBF2 promoter is mutated, it prevents regulatory proteins from activating CBF2 expression in an ABA dependent manner. In this case, ABA-Independent pathways are acting to activate CBF2 expression. However, other data show that the ABRE sequence Is not required for the cold responsiveness of the CBF2 promoter when fused to the GUS reporter gene (D. Zarka 2001). Previous reports have shown the CBF genes to be unresponsive to exogenous ABA (Medina et al, 1999). However, a recent experiment has shown that CBF is In fact responsive to application of exogenous ABA (Daniel Cook and Daniel Zarka, unpublished results), but expression levels are low compared to other ABA responsive genes. Taken together, these data suggest that there are ABA responsive proteins that bind the ABRE in the CBF2 promoter. Presumably, other factors also bind the CBF2 promoter at other regulatory sites which is why partial expression is able to occur In the ABA mutants.The region of the CBF2 promoter responsive to cold has proved difficult to define, possibly because there are multiple binding sites for transcription factors which could act as activators or repressors. 54 An alternative hypothesis could also be put forward based on the EMSA data presented here. There are studies suggesting that the CBF genes could be light regulated. Teppennan et al. (2001) demonstrated that CBF1 and CBF2 are transcriptionally regulated by phytochrome A by comparing the expression of these genes in wild-type and phyA Arabidopsis mutant plants. In addition, another study shows that light affects the induction kinetics of CBF 1, CBF2, and CBF3 (Kim et al., 2002). The putative ABRE and part of Box V in the CBF2 promoter maps exactly to an 11bp sequence motif Identical to the light responsive G-box found In Arabidopsis (Kim et al., 2002). Taken together, this information could mean that proteins binding the CBF2 promoter depend on elements In the promoter that is light responsive. Whether CBF2 is transcriptionally regulated through the putative G-box or the putative ABRE could be tested by mutating part of the G-box sequence, while leaving the ABRE intact. Zarka (2002) was unable to delete the promoter of CBF2 to less than 155 bp and keep the regions necessary that are transcriptionally responsive to cold. It is possible that there are a number of elements In the CBF2 promoter that work together which enable a complex of proteins to bind to different sites. Once a smaller cold responsive region of the promoter is found, DNA footprinting experiments could help resolve the question of how many binding sites exist and where these sites are in the CBF2 promoter. Other EMSA experiments done Independently of those presented in this work support the competition results. Namely, Lahong Sheng (unpublished data) has confirmed binding of bands 1 and 2 to the ABRE sequence. Both studies 55 also found no substantial differences In DNA-protein complexes formed in control and cold-treated plants. This may be an indication that proteins present before the plant is exposed to low temperatures are activated to stimulate transcription by a cold temperature stimulus. An additional protein(s) may also bind to the CBF2 promoter but Is undetectable under the conditions of these experiments. However, with increasing protein concentrations, a different binding pattern emerges between proteins from cold and nontreated plants. This could indicate that the same sites are bound under cold and nontreated plants, but different proteins bind with different affinity at these site between cold and nontreated conditions. Narrowing the cold-responsive element to fewer base pairs may be necessary, because multiple proteins may be binding the 155bp region. Once a smaller cold-responsive region of the promoter is found, other approaches such as yeast-one-hybrid or southwestern analysis, could prove useful in finding not only which specific region binding occurs In the CBF2155 bp promoter fragment, but also what protein Is involved. Future Directions Based on the results of these studies, I conclude that ABA is likely to play a role in the regulation of the CBF genes. To further understand the mechanisms behind this regulation, the proteins regulating the CBF genes In an ABA- dependent manner should be characterized. It has been proposed by Gilmour et al. (1998) that a protein named ICE (lnducer of CBF expression), present at temperatures higher than acclimating temperatures, becomes activated which 56 leads to it regulating the expression of the CBF genes. There may be ABA- Independent (ICE) and dependent (ICE-A1) proteins regulating the CBF genes. Alternatively, ABA specific coactivators binding ICE could determine which pathway ICE is regulated. To find ICE or ICE-A1, yeast-one-hybrid assays could be performed to determine what protein binds the putative ABRE of CBF2 promoter, and once found, one could then ask what role this protein plays in the ABA pathway. Another approach to understanding the role of ICE would be to find where in the plants It localizes. To determine the trafficking of the ABA- dependent protein, lCE-GFP fusion proteins could be used to find where ICE localizes under various conditions. Also, what other proteins (coactivators) are Involved in aiding ICE to activate the CBF genes? Assays to detect protein- protein Interactions such as the yeast-2-hybrid assay could be performed to answer this question. Also, finding where CBF expression is most affected in ABA mutants could give insight on new processes that ABA regulates. Such mRNA localization studies could be achieved by performing in situ hybridization experiments. The results of this study do not Indicate whether CBF genes are affected transcriptionally or post-transciptionally. To determine whether the rate of transcription is changed due to the ABA mutations, nuclear run-on assays could be carried out. Importance of C F The Importance of the ABRE in the cold response is unclear in genes that also containing a CRT/DRE which is activated by the CBF genes. Testing the 57 cold response of a promoter containing a mutated CRT/DRE could shed light on the role of the ABRE in the cold responsive of genes containing both stress responsive elements. There may also be compensatory pathways that aid the CBF genes, such as the pathway involved in regulating the gene SFR6 (see Chapter 1). It would, therefore, be interesting to see the effect of a knockout of the CBF genes on the freezing tolerance of the plant. This could be accomplished by deleting the region of chromosome four encompassing CBF 1, CBF2, and CBF3. A functional knock-out of CBF genes might also be accomplished by overexpressing a nonfunctional CBF containing a mutated activation domain, which could outcompete the native CBF protein. Experiments along these lines could clarify the importance of the CBF pathway In the regulation of cold acclimation and also shed light on other pathways which may converge or act In parallel to contribute to the enhancement of freezing tolerance. 58 Literature Cited Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR. 1999. EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science. 284:2148-52. Artus, N.N., M. Uemura, P.L. Steponkus, S.J.Gilmour, C Lin, and MF. Thomashow. 1996. Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance. Proc Natl Acad Sci USA 93: 13404-13409. Baker SS, Wilhelm KS, Thomashow MF. 1994. The 5’-region of Arabidopsis thaliana COR15a has cIs-acting elements that confer cold-, drought-, and ABA- regulated gene expression. Plant Mol. Biol. 24:701-713 Bleecker A, Kende H. 2000. Ethylene: a gaseous signal molecule in plants. Annu Rev Cell Biol 16:1 -18 Blumenthal T, Landers TA. 1973. The Inhibition of nucleic acid-binding proteins by aurintricarboxylic acid. Clark KL, Larson PB, Wang X, Chang C. 1998. Association of the Arabidopsis CTR1 Raf-like kinase with the ET R1 and ERS1 ethylene receptors. PNAS USA 95: 5401-5406. Foster R, Chua N-H. 1990 An Arabidopsis mutant with deregulated ABA gene expression: implications for negative regulator function. Plant Journal 17:363- 372. Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF. 2000. Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol. 124:1854-1865. Gilmour SJ, Thomashow MF. 1991. Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol. Biol. 17, 1233-40. Gilmour, S.J., D.G. Zarka, E.J. Stockinger, M.P. Salazar, J.M. Houghton and MF. Thomashow. 1998. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step In cold-induced COR gene expression. Plant J. 16: 433-443. Gong Z, Koiwa H, Cushman MA, Ray A, Bufford D, Kore-eda S, Matsumoto TK, Zhu J, Cushman JC, Bressan RA, Hasegawa PM. 2001. Genes that are uniquely 59 stress regulated in salt overly sensitive (sos) mutants. Plant Physiol. 126:363- 375. Guo MJ, Allard G, Byass L, Flanagan A, Singh J. 2002. Regulation and characterization of four CBF transcription factors from Brassica Napus. Plant Molecular Biology. 49: 459-471. lshitani M, Xiong L, Stevenson B, Zhu JK.1997. Genetic analysis of osmotic and cold stress signal transduction In Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways. Plant Cell. 9:1 935-1 949. Kasuga M, Liu 0, Miura S, YamaguchI-Shinozaki K, Shinozaki K. 1999. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-Inducible transcription factor. Nat Biotechnol. 17(3):287-91. Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A., and Ecker, JR. 1993. CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72: 427-441. Kim HJ, Kim YK, Park JY, Kim J. 2002. Light signaling mediated by phytochrome plays an important role in cold-induced gene expression through the C- repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. Plant Journal 29:693-704. Kim JC, Lee SH, Cheong YH, Yoo CM, Lee SI, Chun HJ, Yun DJ, Hong JC, Lee SY, Lim CO, Cho MJ. 2001. A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants. Plant Journal 25:247-259. Knight H, Veale EL, Warren GJ, Knight MR. 1999. The sfr 6 Mutation In Arabidopsis Suppresses Low-Temperature Induction of Genes Dependent on the CRT/DRE Sequence Motif. Plant Cell. 11, 875-886. Lehman A, Black R, Ecker JR. 1996. HOOKLESSI, an ethylene response gene, Is required for differential cell elongation in the Arabidopsis hypocotyl. Cell 85:1 83-94. Leung J, Merlot S, Giraudat J. 1997. The Arabidopsis ABSCISIC ACID- INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 20 involved In abscisic acid signal transduction. Plant Cell 9:759- 71. Ma H, Yanofsky MF, Meyerowitz EM. 1990. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis 60 thaliana. Proc Natl Acad Sci USA 87:3821-3825 Medina J, Bargues M, Terol J, Perez-Alonso M, Salinas J. 1999. The Arabidopsis gene family is composed of three genes encoding AP2 domain proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiol. 119: 463-469. Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J. 2001. The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signaling pathway. Plant Journal 25:295-303. Shinwari Z, Kazuo N, Miura S, Kasuga M, Seki M, Yamaguchi-Shinozaki K, Shinozaki K. 1998. An Arabidopsis Gene Family Encoding DRE/CRT binding proteins In low-temperature-responsive gene expression. BBRC 250: 161 -170. Stockinger, E.J., S.J. Gilmour and MF. Thomashow. 1997. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA 94: 1035-1040. Tan BC, Schwartz SH, Zeevaart JAD, McCarty DR. 1997. Genetic control of abscisic acid biosynthesis In maize. Proc Natl Acad Sci USA 94: 12235-12240. Teppennan JM, Zhu T, Chang HS, Wang X, Quail PH. 2001 Multiple transcription-factor genes are early targets of phytochrome A signaling. Proc Natl Acad Sci USA Jul 31 ;98(16):9437-42. Thomashow MF. 1999. Role of cold-responsive genes in plant freezing tolerance. Plant Physiol 1 18(1):1-8. Thomashow MF. 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant. Physiol. 50, 571 -99. Thomashow MF. 2001. So what's new in the field of plant cold acclimation? lots! . Plant Physiol. 125(1):89-93. Ullah H, Chen JG, Young JC, lm KH, Sussman MR, Jones AM. 2001. Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science. 292:2066- 2069. Wang XQ, Ullah H, Jones AM, Assmann SM. 2001.G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science15z2070- 2072. 61 Yu XM, Griffith M, Wiseman SB. Ethylene induces antifreeze activity in winter rye leaves. 2001. Plant Physiol. 126:1232-1240. Zarka, D. 2001. Studies on low temperature induced gene regulation and freezing stress tolerance in Arabidopsis. Unpublished dissertation, Michigan State University, East Lansing, MI, USA. 62 Chapter 3 Preliminary Exploration of Signaling Pathways for Cold- Regulated Transcription Factors Summary Plants living in changing environmental temperatures represented as seasons must be able to adjust to varying temperatures, including those that are suboptimal. It therefore would not be surprising if plants rallied a number of different signaling factors involved in multiple pathways to protect themselves from the cold. The purpose of this study was to explore the regulation of a number of transcription factors found to be upregulated early and/or have high expression levels for over a course of 7 days by cold temperatures. Those chosen were CBF2, RAV1, RAP2. 7, STZ, ZAT12, and a gene encoding a putative zinc finger (putZF). In addition to their interesting expression patterns in the cold, very little is known about the regulation of the genes chosen. Results from preliminary experiments examining the affect of ABA and ethylene signaling mutants as well as the uncharacterized rssQa mutant were as follows. The cold- Induced expression of RA V1, RAP2. 7, ZAT12, and putZF was examined in ABA biosynthetic and signaling mutants. Expression levels of RAP2.7and putZF were not sustained at wild-type levels In the abi1 mutant; whereas ZAT12 expression appeared unaffected by the ABA biosynthetic and signaling mutations. RA V1 expression was also like wild-type in the abi2 mutant. COR15a and RAP2.7 are 63 both CBF target genes. Their expression was examined In ethylene signaling mutants and was found to be similar in expression to wild-type. Finally, the cold- regulated expression of CBF2, RA V1, STZ, ZAT12 and COR15a was examined in the rssQa mutant which is altered in its response to salt and osmotic stress. The mRNA levels of CBF2, COR15a, ZAT12, STZ and RA V1 were similar to wild-type. Introduction Transciptome profiling of cold-treated Arabidopsis plant led to the identification of a number of transcription factors in Arabidopsis whose transcript levels increased in response to cold (Fowler and Thomashow 2002). Genes of particular interest were transcription factors that were expressed very early and/or remained highly expressed over the seven day cold-treatment, namely, CBF2, RAV1, RAP2.7, STZ, ZAT12, and a gene encoding a putative zinc finger (putZF). The large and sustained upregulation of these transcription factors make these genes interesting candidates for further study since they may play an important role In regulating how the plant responds to the cold. See Table 3.1 for the transcription factors used In this study. Table 3.1. Transcription Factors Used In This Study ABA Drought AGI Reference Gene Name Encodes response Responsive Number AP2-like Not Okamuro RAP2.7 protein studied Yes At2g28550 et al. 1997 Putative zinc Putative zinc finger Not finger protein studied Not studied At4g39860 -- Zinc finger Not Yamada et ZAT12 protein studied Not studied At5959820 al. 2002 Zinc finger Kleinow et STZ/2A T10 protein No Yes ABO3073O al. 2000 AP2-like Not Okamuro RAP2.1 protein studied Not studied At1fi6768 et al. 1997 Hormones and the Cold Stress Response ABA has been implicated in plant stress responses and specifically in the cold acclimation response (Chen et al 1983). Application of exogenous ABA enhances freezing tolerance, endogenous ABA Increases transiently in response to low temperature, and ABA signaling and biosynthetic mutants are less freezing tolerant than wild-type (Chen et al., 1983, Mantyla et al., 1990, Gilmour and Thomashow, 1991). To resolve the question of whether the cold responsiveness of the transcription factors ZAT12, RAW, putZFand RAP2.7 Is mediated through ABA, the expression of these genes in cold-treated Arabidopsis plants was analyzed in mutants defective In ABA biosynthesis and signaling. In addition, the promoter regions of the transcription factors In this study were analyzed using 65 the database on the PIantCARE website to Identify putative ole-acting promoter elements through which ABA might regulate these genes (data not shown). To test the hypothesis that ABA regulates the cold acclimation response by regulating the transcription of cold Induced genes, experiments monitoring the expression of a number of cold-regulated transcription factors were performed using ABA mutants. Mutations affecting ABA 1, ABI1 and ABI2 were selected to determine whether mutations In ABA biosynthesis or signaling could alter expression of cold regulated genes. ABA1 encodes a zeaxanthin epoxidase Involved in ABA biosynthesis (Tan et al., 1997). ABI1 and ABI2 encode homologous phosphatases type 2C which are Involved In ABA signal transduction (Leung et al., 1997). The abi1 and abi2 mutants are insensitive to abscisic acid. The transcript levels of cold regulated transcription factors was analyzed by hybridizing probes derived from the transcription factors of interest with RNA blots of cold-treated wild-type and mutant plants. Ethylene is Involved In a number of plant responses, including cold stress (Bleecker and Kende, 2000). A number of studies on the role of ethylene Involvement in the cold response suggest that endogenous ethylene and ethylene signaling components are Involved in the cold stress response. First, studies In rye have shown ethylene production to Increase when plants were exposed to 5°C. , and when nonacclimated plants were exposed to ethylene, antifreeze protein activity increased in leaves (Yu et al., 2001). Second, in a study using Arabidopsis as a model, transcription factors responsive to ethylene signaling, AtERFs, were also found to be cold-Induced, and cold induction of 66 these transcription factors was lost In an ethylene insensitive mutant (Fujimoto et al., 2000). Finally, ethylene was found to be involved in the chilling tolerance of tomato (Ciardi, et al., 1997). Together, these data implicate the involvement of ethylene synthesis and signaling pathways in regulating the cold-temperature stress response in plants. In order to investigate whether ethylene is involved in the regulation of certain low temperature responsive transcription factors, I examined the effect of lesions in the genes involved in ethylene signaling on the transcription of a select number of cold regulated genes encoding transcription factors. To test the effect of ethylene mutations on the ability of plants to cold acclimate, whole plant freeze tests were performed on mutants in ethylene signaling. The cold Induction of CBF responsive genes, COR15a and RAP2.1, was examined In the ein2-1, ctr1- 1, and hIst-1 mutants. CTR (Constitutive Triple Response) encodes a Raf-like ser/thr kinase which may act as a part of a MAP kinase cascade in ethylene signaling that interacts with ethylene receptors (Bleecker and Kende 2000, Clark et al., 1998). A mutation in the CTR1 gene causes a constitutive ethylene responsive phenotype in Arabidopsis plants. A loss of function mutation in EIN2 results in ethylene insensitivity, although, the function of EIN2 is unknown (Alonso et al., 1999). The HOOKLESS (HLS) gene shows similarity to a diverse group of N-acetyltransferases, is ethylene responsive and has decreased expression in the ein2 mutant (Lehman et al., 1996). 67 In addition to the ABA and ethylene signaling pathway as candidates regulating the expression of genes chosen in this study, the affect of the rssQa mutation on transcription factors chosen for this study was also examined. The rssQa mutant was found to have the ability to germinate under high salt conditions. Also, rssQa plantlets accumulate less proline than wild-type when exposed to high salt concentration or Increased osmotic pressure (Wemer and Finkelstein, 1994). This mutation therefore affects stress responses of the plant at both germination and during vegetative stages. Specifically, the reduced proline levels of the plant make it interesting to test whether this plant is generally altered in its ability to respond to stress. In this study, the cold-responsiveness of RAV1, RAP2.7, STZ, ZAT12, putZF and the CBF genes was examined in the rssQa mutant. The gene defective In rss9a has not yet been characterized. Materials and Methods Plant Material and Growth Conditions Arabidopsis thaliana ecotype Landsberg erecta (Ler) and Columbia (Col) were used in this study. Seeds of aba1-1, abi1-1, abi2-1, (Ler background) ctr1- 2, ein2 and hlsf-1 (Col background) were obtained from the Arabidopsis Biological Resource Center at Ohio State University. Seeds were surface sterilized and aseptically grown on Petri plates containing Gamborg’s BS medium (lnvitrogen, USA) solidified with agar. To break dormancy and synchronize germination, seeds were Incubated at 4°C for 3 days. The seedlings were grown in controlled environmental conditions under constant fluorescent light (100-150 68 uEm'zs") at 22-24°C. Ten to twelve day old seedlings were harvested from plates and immediately placed in liquid nitrogen to be used as material for RNA analysis. Cold-treated plants were placed at 4°C under ~25 pEm'zs'1 continuous fluorescent light for various amounts of time as indicated in the figures. RNA Extraction and Northern Blot Analysis Frozen tissue samples were ground In liquid nitrogen, and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). To examine the low-temperature induction of ZAT12, RAV1, putZFand RAP2.7 In wild type and mutant plants, RNA analysis of seedlings that had been treated at 4°C for 0, 2, 4and 24 hours or 0, 1, 2, and 4 hours were used. Total RNA was fractionated by electrophoresis through a 1.5% denaturing agarose gel containing 2.2 M formaldehyde (Sambrook et al., 2001). Transfer of denatured RNA to a positively charged nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ) was performed as described by Sambrook et al., (2001). Prehybridization was carried out for 3 hours at 42°C in buffer containing 50% (v/v) formamide, 5X SSPE, 5X Denhart’s solution, 1% (w/v) sodium dodecyl sulfate, 10% (w/v) dextran sulfate, and 20pg/ml of sheared salmon sperm DNA (Sambrook, 2001). Full-length cDNA inserts for use as probes for detecting the expression of CBF2, ZAT12, RAV1, STZ, RAP2.7, RAP2.1 and the putative zinc finger protein gene (putZF) were excised from plasmids provided by Sarah Gilmour and Jonathan Vogel. The CBF2 probe used In this study cross-hybridizes with CBF1 69 and CBF3. These fragments were extracted from 1% (w/v) agarose gels (QlAquick Gel Extraction Kit, Qiagen, Valencia CA) and labeled by random priming using 32P-dCTP (Random Priming DNA Labeling Kit, GIbco BRL, Grand Island, NY). Overnight hybridization of these probes was performed under the same conditions as prehybridization after the addition of the labeled probe. Membranes were washed three times with 2X SSPE, 0.5% (w/v) SDS at 42°C and three times with 0.1 X SSPE/0.5% (w/v) SDS at 55°C, then exposed either to Kodak XAR-5 X-ray film at -80°C a phosphor Imaging screen (Kodak, Rochester, NY) for 3 hours to overnight. Screens were scanned on a phosphorimager using Quantity One imaging software (Bio-Rad Laboratories, Hercules, CA). Whole plant freeze tests. Wild-type Arabidopsis ecotype Columbia and Landsberg, and ein2-1, ctr1- 2, hIs1-1 and rssQa mutants were grown in Petri plates for 12 days on Gamborg’s minimal medium without sucrose under constant fluorescent light (100-150 pEm‘ 2s") at 22-24°C. Cold acclimated seedlings were treated at 4°C for 7days under constant fluorescent light (~25 uEm'zs"). Plates containing control and cold-acclimated plants were transferred to a freezer set at -2°C In darkness. After two hours, ice was added to the plate to nucleate freezing. The plates were maintained at -2°C for an additional 14 hours. The temperature of the freezer was then adjusted to -5°C for 24 hours. The temperature of the freezer was then increased to +4°C where it remained a further 24 hours. The plates were transferred to a growth chamber for two days 70 at 22°C under continuous light at 100-150 uEm'zs'1 after which time the plants were scored for survival. Results _A_ffect of A_BA Mptations To determine whether RAP2.7, putZF, ZAT12 and RAV1 are cold-regulated in an ABA-dependent manner, accumulation of mRNAs of these transcription factors was examined in the aba 1, abi1 and abi2 mutants. In the wild-type plants, RAP2.7and putZF mRNA levels reached maximum expression at 2 hours, then remained elevated through 24 hours of cold-treatment. In contrast to wild-type expression, RAP2.7 and putZF do not display sustained expression levels through 24 hours of cold-treatment in abi1 mutant plants (Fig. 3.1A). In both the ABA mutants as well as in wild-type Arabidopsis plants, RNA levels of ZAT12 exhibited the same pattern of expression when RNA loading Is taken into account (Fig. 3.1A). However, the expression pattern of ZAT12 in wild-type at 24 hour cold-treatment differs from that observed in the genome wide analysis and Northern hybridization experiments conducted by Fowler and Thomashow (2002). In their study, ZAT12 expression remained high through the 24 hour cold treatment. The difference could be due to the different ages and ecotypes used between this study and that conducted by Fowler and Thomashow. The cold induced expression levels of RAV1 were not appreciably different between the abi2 mutant and wild-type plants (Fig. 3.1 B). Role of Ethylene in the Plant Stress Response 71 The effect of mutations in the ethylene signal transduction pathway genes- CTR1 and HLS- on the cold induction of the CBF target genes COR 15A and RAP2.1 was tested by northern analysis. In wild-type plants, transcript levels of both genes are abundant at 24 hours cold-treatment as well as in ethylene signaling mutant plants (Fig. 3.2). This suggests that mutations In ethylene signaling genes appeared to have no effect on the transcription of COR15a and FIA P2. 7. Effect of RSSQa mutation To test whether the expression of certain‘cold responsive genes was affected due to the rssQa mutation, CBF, COR15, ZAT12, $72 and RAV1 expression was examined in the rssQa mutant- a mutant which can tolerate higher salt concentrations than wild-type during germination (Wemer and Finkelstein, 1993). (Fig. 3.3). In the wild-type plants, the CBF genes, ZAT12 , 872 and RAV1 mRNA levels reach maximum expression at 1 hour, then remained elevated through 4 hours of cold-treatment. CBF, ZAT12, STZ, and RAV1 expression are similar to wild-type, although appear higher in the rssQa mutant due to uneven loading. COR15a transcript levels increase after wild-type plants are exposed to 4 hours in the cold, and its expression may be more enhanced in the rssQa mutant. The enhanced COR15a mRNA levels may be due to uneven loading (Fig. 3.3). Repeat experiments are needed to determine whether the affects on the transcription of these genes due to the rssQa mutation are reproducible. 72 A aba1-1 _a_bi1-1 abi2-1 Wt Ler . 02424024240242402424138990111t 31911131121!!! n1! ' Q’s ZAT12 mafia-1:91 w... *111 we.” RAP2.7 [1 fififlrwfé ~ ”E11 efifij putZF B abi2-1 _VJIEL. o 1 2 4 2 4 Time(h)at4°C .. , , . RAV1 eIF4A Fig. 3.1 Effect of mutations In ABA biosynthesis and signal transduction pathways on the cold Induced expression of ZAT12, RAP2.7, putZF, and RAV1. Plants grown at 22-24°C were transferred to 4°C for cold treatment for various lengths of time (h). RNA was extracted and transferred onto a nylon membrane. Northern blots were prepared and probed with ZAT12, RAP2. 7, putZF, and RAV1 genes. A: Northern hybridization analysis of RNA isolated from cold-treated wild- type, aba 1, abi1, and abi2 mutant Arabidopsis plants probed with ZAT12, RAP2.7, and putZF. rRNA serves as an Internal loading control. B: Northern hybridization analysis of RNA Isolated from wild-type and abi2-1 mutant plants were cold-treated at the times Indicated. eIF4A serves as an internal loading control. 73 Wt Ler ctr1-1 hls 1-1 . —— —— ——- T h t 02424 0242402424 4129‘” 9 ' COR15a ‘ RAP2.1 r~~-~U~~~ ”WV” ”*7" 7. rRNA rRNA on membrane Fig. 3.2 The effect of mutations in the ethylene signaling pathway on cold- induced expression of the CBF target genes COR15a, and RAP2. 1. Plants were grown at 22-24°C were transferred to 4°C for cold treatment for the times Indicated (h), then RNA was isolated. Northern blots were prepared and probed with COR15a and RAP2. 1. Northern blot of RNA isolated from wild-type, ctr1 and hls1 mutant Arabidopsis plants that were cold-treated for various lengths of times at 4°C as indicated probed with COR15a and RAP2. 1. The ethidium bromide stained rRNA pictured here on an agarose gel and on the membrane serves as a loading control. 74 Wt Ler r3593 0 1 2 4 012 4 Time(h)at4°C 7"”? ”W car-'2 I V s. ' - COR15a 1 “W ZAT12 RAV1 STZ elF4A Fig. 3.3. The cold regulated expression of STZin the rssQa mutant. Plants were grown at 22-24°C and were transferred to 4°C for cold treatment for the times Indicated (h). RNA was extracted from wild-type and rssQa mutant plants, then transferred to a nylon membrane. RNA blots were then probed with CBF2, COR15a STZ, ZAT12 and RAV1. eIF4a served as a loading control. 75 Discussion The results in Fig. 3.1 may suggest that RAP2.7and putZF expression levels are not sustained at wild-type levels in the abi1 mutants. If repeated and the same result is obtained, it could implicate ABI1 in sustaining the response of certain transcription factors. ABI1 encodes a type 20 phosphatase which may act as a positive regulator that prevents the attenuation of the ABA signal perhaps by preventing mRNA degradation or by positively regulating transcription. Alternatively, the dominant mutation in the abi1 mutant could cause processes to occur which do not occur in the aba1 mutant, such that a negative regulator in the abi1 mutant causes the reduced mRNA levels of RAP2.7 and putZF. Although the ABA signaling and biosynthetic mutations appeared to have not affected the cold-regulated expression of ZAT12, two possibilities could cause this to be an incorrect conclusion. First, the ABA mutant contains 30% of the ABA levels as those in wild-type, which may be enough for signaling to occur, making ZAT12 ABA-dependent. Because aba1 is a leaky mutation, a more accurate reflection of the role of ABA1 would be to test these genes in null mutants. Mutations leading to defective ethylene signaling appear to have no effect on the cold-regulated transcription of CBF target genes. COR15a and RAP2.1 both showed high expression at 24 hours cold-treatment in both ethylene signaling mutants and wild-type plants. It is therefore unlikely that their direct transcriptional regulators, the CBF genes, are affected in the ethylene signaling mutants tested. However, examining the expression of the CBF genes directly 76 will determine whether the CBF genes are transcriptionally regulated through ethylene signaling. Although the rssQa mutation alters the Arabidopsis stress response, this mutation appeared to have no affect on the cold-regulated expression levels of CBF2, COR15a, ZAT12, STZ, and RAV1 (Fig. 3.3). Future Directions Aside from the CBF genes, nothing is known about the importance of the transcription factors examined in this study in the development of freezing tolerance. To determine whether HAP2.7, ZAT12, or putZF what role these transcription factors play in cold acclimation, knockouts or transgenic plants overexpressing these genes could be used to determining the importance of these genes in the cold acclimation response, and may add to the understanding of how the cold acclimation network is connected. 77 Literature Cited Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR. 1999. EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science. 284:2148-52 Bleecker A, Kende H. 2000. Ethylene: a gaseous signal molecule in plants. Annu Rev Cell Biol 16:1-18. Chen THH, Gusta LV 1983. Abscisic acid - induced freezing resistance in cultured plant cells. Plant Physiol 73:71 -75. Ciardi, JA, Deikman, J, Orzolek MD. (1997). Increased ethylene synthesis enhances chilling tolerance in tomato. Physiol. Planta. 101 :333-340. Fowler S, Thomashow MF. 2002. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell. 14:1675-1690. Fujimoto SY, Ohta M, Usui A, Shinshi H, Ohme-Takagi M. 2000. Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression. Plant Cell. 12:393-404. Gilmour SJ, Thomashow MF. 1991. Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol. Biol. 17, 1233-40. Gong Z, Koiwa H, Cushman MA, Bay A, Bufford D, Kore-eda S, Matsumoto TK, Zhu J, Cushman JC, Bressan RA, Hasegawa PM. 2001. Genes that are uniquely stress regulated in salt overly sensitive (sos) mutants. Plant Physiol. 126:363- 375. Kleinow T, Bhalerao R, Breuer F, Umeda M, Salchert K, Koncz C. 2000. Functional identification of an Arabidopsis snf4 ortholog by screening for heterologous multicopy suppressors of snf4 deficiency in yeast. Plant J. 23(1):115-22. Lippuner V, Cyert M, Gasser C. 1996. Two classes of plant cDNA clones differential complement yeast calcineurin mutants and increase salt tolerance of wild-type yeast. J Biol. Chem. 271 :12859-12866. Mantyla E, Lang V, Palva ET. 1995. Role of abscisic acid in drought-inducible proteins of alfalfa. J. Biol. Chem. 267:15367-74. 78 Okamuro JK, Caster B, Villarroel R, Van Montagu M, Jofuku KD. 1997. The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci U S A 94(13):7076-81. Tan BC, Schwartz SH, Zeevaart JAD, McCarty DR. 1997. Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA 94: 12235-12240. Werner J and Finkelstein R. (1995). Arabidopsis mutants with reduced response to NaCI and osmotic stress. Physiol. Plant. 93:659-666. Yamada,K., Chan,M.M., Chang,C.H., Dale,J.M., Deng,J.M., Hsuan,V.W., Lee,J.M., Quach,H.L., Tang,C., Toriumi,M., Wallender,E.K., Wong,C., Wu,H.C., Yu,G., Yuan,S., Bowser,L., Carninci,P., Chen,H., Cheuk,R., Hayashizaki,Y., lshida,J., Jones,T., Kamiya,A., Karlin-Neumann,G., Kawai,J., Kim,C., Lam,B., Lin,J., Miranda,M., Narusaka,M., Nguyen,M., Palm,C.J., Sakurai,T., Satou,M., Seki,M., Shinn,P., Southwick,A., Shinozaki,K., Davis,R.W., Ecker,J.R. and Theologis,A. 2002. Arabidopsis Open Reading Frame (ORF) Clones (unpublished). RIKEN Genomic Sciences Center Genbank direct submission. Yu XM, Griffith M, Wiseman SB. 2001. Ethylene induces antifreeze activity in winter rye leaves. Plant Physiol. 126:1232-40. 79 ll 9i