Published online 19 June 2002 Molecular genetic analysis of cold-regulated gene transcription C. Viswanathan and Jian-Kang Zhu* Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA Chilling and freezing temperatures adversely affect the productivity and quality of crops. Hence improving the cold hardiness of crop plants is an important goal in agriculture, which demands a clear understanding of cold stress signal perception and transduction. Pharmacological and biochemical evidence shows that membrane rigidiŽcation followed by cytoskeleton rearrangement, Ca21 inux and Ca21-dependent phosphorylation are involved in cold stress signal transduction. Cold-responsive genes are regulated through C-repeat/dehydration-responsive elements (CRT/DRE) and abscisic acid (ABA)-responsive element ciselements by transacting factors C-repeat binding factors/dehydration-responsive element binding proteins (CBFs/DREBs) and basic leucine zippers (bZIPs) (SGBF1), respectively. We have carried out a forward genetic analysis using chemically mutagenized Arabidopsis plants expressing cold-responsive RD29A promoter-driven luciferase to dissect cold signal transduction. We have isolated the Žery1 ( fry1) mutant and cloned the FRY1 gene, which encodes an inositol polyphosphate 1-phosphatase. The fry1 plants showed enhanced induction of stress genes in response to cold, ABA, salt and dehydration due to higher accumulation of the second messenger, inositol (1,4,5)- triphosphate (IP3). Thus our study provides genetic evidence suggesting that cold signal is transduced through changes in IP3 levels. We have also identiŽed the hos1 mutation, which showed super induction of cold-responsive genes and their transcriptional activators. Molecular cloning and characterization revealed that HOS1 encodes a ring Žnger protein, which has been implicated as an E3 ubiquitin conjugating enzyme. HOS1 is present in the cytoplasm at normal growth temperatures but accumulates in the nucleus upon cold stress. HOS1 appears to regulate temperature sensing by the cell as cold-responsive gene expression occurs in the hos1 mutant at relatively warm temperatures. Thus HOS1 is a negative regulator, which may be functionally linked to cellular thermosensors to modulate cold-responsive gene transcription. Keywords: low temperature; signalling; CRT/DRE; abscisic acid-responsive element; FRY1; HOS1 1. INTRODUCTION Frost tolerance is essential for temperate crops like winter wheat, while in tropical crops like rice, maize, soybean, cotton and tomato, productivity and quality are affected by even non-freezing low temperatures. Hence engineering cold-tolerant crop plants is one of the cherished goals in agriculture. To achieve this, a thorough understanding of cold stress signal perception and transduction in plant cells, which lead to cold acclimatization, is required. Thanks to the advent of molecular biology, which has propelled the research in this area over the past two decades, today some of the events of cold signal perception, transduction and cold acclimation are deŽned at the molecular level (for recent reviews, see Shinozaki & YamaguchiShinozaki 2000; Browse & Xin 2001; Thomashow 2001; Zhu 2001). In nature, low temperature stress is often accompanied by dehydration (or osmotic stress), as low temperature may limit water uptake by the roots, while freezinginduced ice formation in the apoplast (due to low solute concentration) causes reduction in water potential, which leads to movement of water into the apoplast from the symplast. This process causes severe dehydration. In addition, a minor change in osmotic potential occurs due to its temperature dependency (osmotic potential is CiRT, where C is the concentration of solutes, i is the ionization constant, R is the gas constant and T is the absolute Adjustment is a way of life for sessile and poikilothermic land plants that endure environmental stresses such as low or high temperatures, water deŽcit and salinity. These abiotic stresses not only limit the temporal and spatial distribution of plants but also adversely affect the productivity and quality of agriculturally important crops. Plant body temperature changes with ambient temperature, although a few plants can control their temperature by several degrees above (through alternate oxidase respiration) or below (through transpirational cooling) the ambient temperature. Most temperate plants can acquire tolerance to freezing temperatures by prior exposure to low nonfreezing temperatures, a process called cold acclimatization. This is achieved by the expression of many genes, change in the membrane lipid composition, accumulation of compatible osmolytes (proline, betaine, polyols and soluble sugars), transient rise in ABA and reduction or cessation of plant growth (Levitt 1980). Tropical and sub-tropical plants are incapable of cold acclimatization. * Author for correspondence (jkzhu@ag.arizona.edu) . One contribution of 15 to a Discussion Meeting Issue ‘Coping with the cold: the molecular and structural biology of cold stress survivors’. Phil. Trans. R. Soc. Lond. B (2002) 357, 877–886 DOI 10.1098/rstb.2002.1076 877 Ó 2002 The Royal Society 878 C. Viswanathan and J.-K. Zhu Cold signalling temperature). Decrease in turgor pressure is known to induce biosynthesis of the plant stress hormone ABA. Hence, depending upon the level of cold stress, in addition to cold stress, dehydration and ABA-mediated signalling operate to regulate freezing tolerance. In plants, gene expression is regulated by both developmental and environmental cues. For example, some of the dehydration-responsive genes are also expressed developmentally during late embryogenesis. Cold stress can vary in severity (how low the temperature is), rate of stress development (change in temperature per unit time), duration and uctuations (diurnal and seasonal). So it is logical to expect plants to have various mechanisms for sensing and transduction, which means that there are multiple cold stress perception and transduction modules and interactions at different nodes in these signal transduction modules. This review focuses on recent developments in cold stress signalling and gene regulation in higher plants. 2. MEMBRANE RIGIDIFICATION: A MECHANISM OF COLD SENSING Membrane uidity and protein structural stability and exibility are determined by the composition of building block molecules and their interacting environment. Temperature is one of the important environmental cues that inuence the membrane uidity and protein structural stability and exibility. Hence these can be the primary candidates as biological thermometers. It is logical to expect that plants have thermosensors in the expressed state, irrespective of developmental and environmental cues, so that stress can be sensed at once. The folding kinetics of Escherichia coli cold shock protein A is temperature dependent (Leeson et al. 2000). The CBF1, a transcriptional activator involved in cold-regulated gene expression in Arabidopsis thaliana, undergoes cold-induced denaturation (when the temperature changes from 25 to 4 °C) in both N-terminal and acidic regions in vitro (Kanaya et al. 1999). Changes in the structure of protein can alter its ability for protein–protein interaction, i.e., its ability to form a multimer of its own or with other proteins, which plays an important role in gene regulation. Although so far no such low temperature sensor has been identiŽed, these studies form a prima facie case for considering the possibility of protein denaturation-dependent low temperature sensors in plants. Change in membrane uidity is one of the immediate effects of cold stress and hence the plasma membrane is proposed as a primary sensor of low temperature (Levitt 1980). The Žrst evidence for this hypothesis is in cyanobacterium Synechocystis PCC6803, where Palladium (Pd)catalyzed plasma membrane rigidiŽcation activated the expression of the cold-inducible fatty acid desaturase A (desA) gene (Vigh et al. 1993). Ca21 inux into the cytosol is an early event in cold acclimatization (Knight et al. 1991; Monroy et al. 1993; Plieth et al. 1999); blocking the Ca21 inux by Ca21 channel blockers inhibited cold acclimatization at 4 °C, and Ca21 channel agonist (Bay K8644) or Ca21 ionophore could induce cold acclimatization even at 25 °C in Alfalfa (Medicago sativa) (Monroy et al. 1993; Monroy & Dhindsa 1995) and Arabidopsis (Tähtiharju et al. 1997). Actin microŽlament re-organization has been implicated in Ca21 inux in hepatocytes Phil. Trans. R. Soc. Lond. B (2002) (Yamamoto 1989) and tobacco protoplasts (Mazars et al. 1997). However, the temporal relationship between membrane rigidiŽcation, actin microŽlament reorganization and Ca21 inux was not known. Örvar et al. (2000) have demonstrated that membrane uidity may indeed act as a thermosensor in Alfalfa cell suspension cultures using pharmacological agents and CAS30 gene expression and cold acclimatization as end markers. At 25 °C, Ca21 inux, CAS30 expression and cold acclimatization could be achieved in alfalfa cells by treatment with membrane rigidiŽer (DMSO) and actin microŽlament destabilizer (cytochalasin D). Conversely, treatment with membrane uidizer (Benzyl alcohol) and actin microŽlament stabilizer ( Jasplakinolide) inhibited Ca21 inux, CAS30 expression and cold acclimatization even at 4 °C. Reorganization of actin microŽlaments in cold signalling is downstream of membrane rigidiŽcation and above Ca21 inux, as cold or DMSO induced CAS30 expression and cold acclimatization is inhibited by the treatment of cells with Jasplakinolide (Örvar et al. 2000). Further strength to this proposal was provided by the study of Sangwan et al. (2001) in intact seedlings of Brassica napus transgenic plants carrying a BN115 promoter-driven GUS reporter gene. The transgene was induced at 25 °C by treatment of the leaves with membrane rigidiŽer (DMSO), microŽlament destabilizer (Latrunculin B) and microtubule destabilizer (oryzalin/colchicine), while the transgene was not expressed even at 0 °C in plants treated with membrane uidizer (Benzyl alcohol), microŽlament stabilizer ( Jasplakinolide) and microtubule stabilizer (taxol). Gadolinium (Gd31), a mechanosensitive calcium channel blocker, could inhibit cold-, DMSO-, Latrunculin B-, oryzalin- and colchicines-induced reporter gene expression (Sangwan et al. 2001). Cold-induced membrane rigidiŽcation is thought to occur in distinct microdomains of the plasma membrane (Murata & Los 1997). Hence in higher plants, cold-induced rigidiŽcation at microdomains on the plasma membrane may lead to cytoskeleton rearrangement, induction of stretch-sensitive Ca21 channels and increase in cytosolic Ca21 that triggers cold-induced gene expression and cold acclimatization (Örvar et al. 2000; Sangwan et al. 2001). Isolation and characterization of a cold-inducible TaADF further supports the involvement of cytoskeleton rearrangement during cold signalling. TaADF expression is strongly induced by cold but not by ABA, dehydration, heat or NaCl and the level of expression correlates with increase in cold acclimatization and the genotypic differences in cold acclimatization. TaADF is phosphorylated by a 52 kDa protein kinase in a temperature-dependent manner. Because this TaADF is not detected at the normal growth temperature (24 °C) of wheat and is expressed at a signiŽcant level only after 2 days of cold stress, this speciŽc TaADF may not be actively involved in the initial process of cold perception but may be involved in cold acclimatization (Ouellet et al. 2001). 3. REGULATION OF CA21 INFLUX The involvement of Ca21 in cold signal transduction is demonstrated in many studies (Knight et al. 1991; Monroy et al. 1993; Monroy & Dhindsa 1995; Tähtiharju et al. 1997; Plieth et al. 1999; Örvar et al. 2000; Sangwan et Cold signalling al. 2001). Hence it is essential to analyse how this Ca21 inux occurs and what are the downstream components that translate speciŽc Ca21 signatures during cold stress. The regulators of Ca21 channels in cells are IP3, cADPR and nicotinic acid adenine dinucleatide ribose. Stretch/mechanosensitive Ca21 channels are also involved in Ca21 inux. Mechanosensitive Ca21 channels are activated by cytoskeleton rearrangement during cold stress to induce Ca21 inux in alfalfa cell suspension culture (Örvar et al. 2000) and in seedlings of B. napus (Sangwan et al. 2001). Single-cell microinjection in tomatoes showed that cADPR could activate cold-regulated genes RD29A (responsive to dehydration) and KIN2 (cold inducible). Microinjection of ABA (50 m M) or cADPR (1 m M) could induce the RD29A/KIN1 promoter-driven GUS gene, while ADP-ribosyl cyclase (an enzyme that synthesizes cADPR) or Ca21 can replace the ABA and cADPR in reporter gene induction. Conversely, induction of ABAresponsive reporter genes was inhibited by 8-aminocADPR (a structural analogue of cADPR) or EGTA (a Ca21 chelator) (Wu et al. 1997). In B. napus seedlings, cADPR treatment at 25 °C could induce the BN115 promoter-driven GUS reporter gene, BN115 transcripts and increase freezing tolerance (Sangwan et al. 2001). Thus pharmacological and biochemical evidence indicates that cADPR acts as a signal molecule in activating calcium channels to cause Ca21 inux during cold stress. Microinjection of heparin, a speciŽc inhibitor of IP3 receptors, could block the IP3-induced RD29A/KIN1 promoter-driven GUS reporter gene in tomatoes but not ABA-mediated expression of the reporter gene. Hence the effect of IP3 was thought to be a secondary response in the regulation of RD29A and KIN1 (Wu et al. 1997). However, the involvement of IP3 in cold, ABA, salt and dehydration signal transduction was demonstrated by Zhu and his colleagues by using a molecular genetic approach. Xiong et al. (2001) used chemically mutagenized Arabidopsis transgenic plants with the RD29A promoter-driven luciferase (Luc) to dissect cold signal transduction and isolated a mutant fry1 that showed enhanced induction of cold-regulated genes (RD29A, KIN1, COR15A, COR47A and ADH ) in response to cold, ABA, salt and dehydration stresses. The fry1 mutant constitutively expressed the cold-responsive genes at low abundance and the coldresponsive genes were super-induced under abiotic stresses. The fry1 mutant is hypersensitive to these abiotic stresses, in other words, the level of stress required to induce cold-responsive genes is lower than that required for WT plants. RD29A promoter-driven Luc reporter gene expression at 15 °C was similar to that of the WT at 0 °C. Similarly, the ABA and NaCl requirements were 0.1 m M and 10 mM, respectively, in fry1 as compared with 100 m M ABA and 250 mM NaCl required by the WT for the same level of RD29A-Luc expression. The fry1 mutation was mapped to chromosome Žve and isolated by map-based cloning. Sequence comparison of FRY1 revealed that it encodes a protein homologous to a bifunctional enzyme with 39(29), 59-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. The FRY1 protein was found to be mainly acting as an inositol polyphosphate 1-phosphatase, which functions in the catabolism of IP3. Northern analysis revealed that FRY1 is expressed in every tissue constitutively, but the Phil. Trans. R. Soc. Lond. B (2002) C. Viswanathan and J.-K. Zhu 879 expression is signiŽcantly higher in leaves. Analysis of the IP3 content revealed that the fry1 mutant accumulated signiŽcantly higher levels of IP3 even in unstressed conditions when compared with WT plants. ABA induced a signiŽcant increase in IP3 within 1 min in WT, which returned to the basal level within 10 min. However, the fry1 mutant maintained its basal level of IP3 at 1 min after ABA treatment, but accumulated a signiŽcantly higher level over 30 min. This shows that ABA induces a transient increase in cellular IP3 in intact seedlings of Arabidopsis and that FRY1 is involved in the regulation of IP3 levels during signal transduction. Although cold-responsive genes are super-induced in the fry1 mutant, the fry1 plant is defective in cold acclimatization and germination is highly sensitive to ABA and NaCl. This shows that the coldresponsive gene regulation and cold acclimatization processes can be unlinked. Thus the fry1 study provides, to our knowledge, the Žrst genetic evidence of the involvement of IP3 in ABA and abiotic signal transduction in plants. Cold-responsive genes are regulated through CBFs and bZIP transacting factors. The expression of CBF2 (which is a transcriptional activator of cold-responsive genes) is similar in the WT and the fry1 mutant at 1.5 and 3 h of cold stress. In the WT, CBF2 expression decreased drastically after 3 h to a minimum level. However, in the fry1 mutant the CBF2 transcript was 1.8 times higher than the WT level after 6 h of cold treatment. Hence FRY1 is a negative regulator of cold-responsive gene expression through modulating IP3 levels, which may also regulate the cold-induced transient changes in the transcript level of CBF2 (Xiong et al. 2001). 4. SENSORS OF CA21 SIGNATURES Intracellular Ca21 signatures are sensed by the calcium sensor family of proteins like calmodulin and CDPKs (Zielinski 1998). An antagonist (W7) of the CDPKs could inhibit cold-responsive gene expression and cold acclimatization in alfalfa (Monroy et al. 1993) and Arabidopsis (Tähtiharju et al. 1997). In rice (Oryza sativa L. cv. Don Juan), a constitutively expressed membrane-bound CDPK has been characterized. Cold stress (12–18 h) signiŽcantly increases the auto-phosphorylation and kinase activity of this rice CDPK, thus showing a post-translational regulation of CDPK by cold stress (Martṍn & Busconi 2001). Recently, a new family of calcium sensors called CBL proteins was identiŽed in Arabidopsis, which are similar to the regulatory B subunit of calcineurin and the neuronal calcium sensors in animals (Liu & Zhu 1998; Kudla et al. 1999). AtCBLs are small Ca21 binding proteins that themselves do not have any enzyme activity but act through protein kinases. Involvement of CBL proteins in salt stress signal transduction has been demonstrated by genetic analysis and molecular cloning of SOS3, which activates the protein kinase SOS2. SOS2 in turn activates SOS1, a plasma membrane Na1/H1 antiporter (Halfter et al. 2000; Ishitani et al. 2000; Liu et al. 2000; Shi et al. 2000; Guo et al. 2001). Another member of the Arabidopsis CBL family, AtCBL1, is highly inducible by cold, drought and wounding (Kudla et al. 1999). Using yeast two-hybrid screening, a target protein for AtCBL1 was identiŽed from Arabidopsis, named as CIPK1. CIPK1 encodes a 49 kDa protein and is expressed constitutively 880 C. Viswanathan and J.-K. Zhu Cold signalling in all tissues. CIPK1 interacts with AtCBL1 in a Ca21dependent manner and EGTA (a Ca21 chelator) could inhibit this interaction (Shi et al. 1999). The requirement of reversible phosphorylation of preexisting proteins for cold acclimatization has been demonstrated in alfalfa and Arabidopsis (Monroy et al. 1993, 1997, 1998; Tähtiharju et al. 1997). Hence the role of protein kinases and phosphatases has been explored in cold signal transduction. Is there a speciŽc set of protein kinases and phosphatases, which perceive cold-speciŽc Ca21 signatures? If these protein kinases and phosphatases are involved in cold signal transduction, they should show a cold-regulated activation/inhibition. Tomato seedlings microinjected with protein kinase inhibitor (K252a) could inhibit ABA/cADPR/Ca21-induced RD29A and KIN2 expression, while microinjection with protein phosphatase inhibitor (okadaic acid) stimulated RD29A and KIN2 expression even in the absence of ABA treatment (Wu et al. 1997). Similar responses to the inhibitors of protein kinases and phosphatases were observed in alfalfa CAS15 expression (Monroy et al. 1998). Treatment with inhibitors of tyrosine kinases (genistein), protein kinase C (H7) and phosphoinositide kinases (wortmannin) on B. napus seedlings carrying the BN115 promoter-driven GUS gene prevented reporter gene expression and freezing tolerance even after cold treatment, while treatment with inhibitors of protein phosphatases 1 (okadaic acid) and 2A (calyculin A) could induce the reporter gene at 25 °C and conferred freezing tolerance (Sangwan et al. 2001). Low temperature causes Ca21-dependent, rapid and dramatic decrease in protein phosphatase 2A in alfalfa (Monroy et al. 1998). Thus it appears that the Ca21 signal is transduced by protein kinases/phosphatases to regulate cold-responsive genes during cold acclimatization. Cold stress (4 °C)induced increase in the expression of AtPP2CA reached a maximum by 12 h and remained high afterwards. Arabidopsis transgenic plants expressing AtPP2CA in antisense showed that regulation of cold-responsive genes (RAB18, RCI2A/LTI6, RD29A/LTI78) was cold stress-dependent similar to the WT, but they are super-induced during cold stress in AtPP2CA antisense plants and conferred better freezing tolerance. Also the cold-responsive gene expression and cold acclimatization were accelerated in AtPP2CA antisense plants, i.e. less time of cold stress was required when compared with that of the WT. As the expression pattern of CBF1, CBF2, CBF3 and DREB2 was unaltered in AtPP2CA antisense plants, the enhanced expression of cold-responsive genes is not mediated through the CRT/DRE element (Tähtiharju & Palva 2001). The expression of RAB18 and RCI2CA (rarely cold inducible) is regulated by an ABA-dependent pathway through ABREs (LaÊ ng & Palva 1992). Hence AtPP2CA is a negative regulator of cold stress through ABA-dependent pathways (Tähtiharju & Palva 2001). MAPKs are serine/threonine protein kinases that play key roles in integrating multiple intracellular signals transmitted by various second messengers. A MAPK cascade consists of three protein kinases. Inactive MAPKKKs are activated by a stress signal messenger; upon activation, they activate MAPKKs by phosphorylation at conserved serine and serine/threonine (SXXXS/T motif). Activated MAPKKs activate MAPKs by phosphorylating MAPK at both tyrosine and threonine residues in the TXY motif. Phil. Trans. R. Soc. Lond. B (2002) MAPKs enter the nucleus to regulate appropriate transacting factors. Thus activated, MAPKs can regulate speciŽc gene expression. In plants, many MAPK family members have been cloned and proposed to be involved in environmental stress responses (Mizoguchi et al. 1997). A MAPK cascade regulated by cold and dehydration independently of ABA has been characterized in alfalfa. Expression of the alfalfa MAPK gene, MMK4, was strongly induced by cold and drought stress within 45 min, while salt, heat and ABA did not alter the transcript’s level. Although the steady state level of protein was unaltered, kinase activity of MMK4 was enhanced by cold and dehydration ( Jonak et al. 1996). Arabidopsis ATMPK3 (MAPK) gene expression is highly induced by cold, NaCl and touch. AtMPK3 expression reached a very high level within 10 min of cold stress, while the induction by NaCl and touch was less sensitive (Mizoguchi et al. 1996). In Arabidopsis, H2O2 can activate a speciŽc MAPKKK, ANP1, which initiates the phosphorylation cascade involving cold stress-regulated AtMPK3. Transgenic tobacco constitutively expressing NPK1, an orthologue of ANP1 showed enhanced tolerance to cold, drought and ABA (Kovtun et al. 2000). Hence an ANP1 cascade involving AtMPK3 might be involved in cold signal transduction. In alfalfa, a gene encoding a negative regulator of MAPKKK, a mitogen protein phosphatase type 2C, has been cloned and proposed to act as a negative regulator of cold-, drought-, touch- and wound-induced MAPK cascade (Meskiene et al. 1998). In Arabidopsis, AtMPK4 (MAPK), AtMPK6 (MAPK) and a 44 kDa MAPK are activated by phosphorylation within 5 min by cold, dehydration, wound and touch but not by ABA and heat (Ichimura et al. 2000). Using yeast two-hybrid analysis, a possible MAPK cascade comprising ATMEKK1 (MAPKKK), MEK1 (MAPKK)/ATMKK2 (MAPKK) and ATMPK4 (MAPK) has been proposed (Mizoguchi et al. 1998). How cold-induced calcium signatures activate MAPKKKs and what are the target proteins of cold stressactivated MAPK await further studies. 5. RECEPTOR PROTEIN KINASES Receptor protein kinases (which include two-component histidine kinases, receptor-like protein kinases and G-protein associated kinases) play active roles in environmental stress signal transduction. In Synechocystis PCC6803, a two-component histidine kinase HIK33 has been identiŽed. Autophosphorylation of HIK33 occurs upon sensing the cold-induced membrane rigidity and subsequently transfers a phosphate group to HIK19, then to RER1. RER1 induces the expression of fatty acid desaturase gene (desB) (Suzuki et al. 2000, 2001). Although AtHK1, a two-component regulator, has been proposed to function as osmosensor in Arabidopsis (Urao et al. 2000), so far no two-component system involved in cold stress signalling has been identiŽed in higher plants. Involvement of a G-protein was demonstrated in ABA signal transduction in stomata (Wang et al. 2001), but involvement of G-proteins and associated receptors in cold stress signal transduction are not known. In Arabidopsis, the receptor-like protein kinase, RPK1, contains a putative amino terminal signal sequence domain, an extracellular domain with leucine-rich repeat sequences, a Cold signalling membrane-spanning domain and a cytoplasmic protein kinase domain. RPK1 gene expression is rapidly induced by cold, salt and dehydration stress and the expression is ABA-independent as the dehydration-induced expression is not impaired in the ABA biosynthesis mutant (aba1) and ABA-insensitive mutants (abi1-1, abi2-1 and abi3-1) (Hong et al. 1997). Whether this RPK1 participates in cold signal perception or transduction is not known. 6. REGULATION OF COLD-RESPONSIVE GENES Cold acclimatization is accomplished by the expression of many cold-regulated genes (reviewed by Thomashow 1999; Shinozaki & Yamaguchi-Shinozaki 2000; Browse & Xin 2001; Zhu 2001). In Arabidopsis, these genes are called rd (responsive to dehydration), erd (early responsive to dehydration), lti (low-temperature induced), kin (coldinduced) and cor (cold-regulated). These genes are also induced by dehydration (due to water deŽcit or high salt) and ABA, and can be collectively called cold-responsive genes. Cold-responsive gene expression studies in ABA deŽcient (aba) and ABA-insensitive (abi) mutants of Arabidopsis demonstrated that expression of some coldresponsive genes is mediated by both ABA-independent and ABA-dependent pathways (Kurkela & Franck 1990; Nordin et al. 1991; Horvath et al. 1993; YamaguchiShinozaki & Shinozaki 1993; Ingram & Bartels 1996). To understand the mechanism of regulation, the promoter region of RD29A (= COR78/LTI78) gene of Arabidopsis was analysed by Yamaguchi-Shinozaki & Shinozaki (1994) and they identiŽed DRE or CRT, a cis-element with CCGAC as its core sequence. CRT/DRE-related motifs have also been identiŽed in the promoters of genes regulated by osmotic, low temperature and salt stress, including COR15a, KIN1, COR6.6/KIN2, RAB18 and RD17/COR47 in Arabidopsis (Kurkela & Franck 1990, 1992; LaÊ ng & Palva 1992; Baker et al. 1994). These DRE elements are not involved in ABA-dependent gene expression (Wang et al. 1995; Shinwari et al. 1998) because these genes are expressed in aba and abi mutants of Arabidopsis. Hence it is thought that cold-regulated gene expression occurs through ABA-independent pathways. However, a cold-induced transient increase in intracellular ABA was observed in many plant species. Analysis of promoter regions of RAB18, LTI65, RD29A and RD29B revealed the presence of ABREs, PyACGTGGC (Nordin et al. 1993; Welin et al. 1994; YamaguchiShinozaki & Shinozaki 1994). Cold-responsive accumulation of RAB18 and LTI65 transcripts is severely impaired in aba1 or abi1 mutants. Hence cold-responsive regulation of these genes may occur through an ABAdependent pathway (LaÊ ng & Palva 1992; Nordin et al. 1993). By chemical mutagenesis of transgenic Arabidopsis plants carrying the RD29A promoter-driven luciferase reporter gene, Ishitani et al. (1997) have isolated mutants with hyper-expression or diminished expression of RD29A in response to both cold and ABA, and thus demonstrated that cold- and ABA-dependent regulatory pathways crosstalk at some nodes of signal transduction. Phil. Trans. R. Soc. Lond. B (2002) C. Viswanathan and J.-K. Zhu 881 7. CRT/DRE-DEPENDENT REGULATION OF COLDRESPONSIVE GENES An important step towards understanding of the coldresponsive gene regulation was isolation of a gene encoding a CRT/DRE-binding protein, called CBF1, from Arabidopsis by Stockinger et al. (1997). Later, Žve independent genes encoding DREBs were isolated from Arabidopsis using a yeast one-hybrid screening. Similar to CBF1, these DREBs also contain an APETELA2/ ethylene-responsive element binding protein DNA binding domain. These DREBs are classiŽed into two classes: DREB1 (DREB1A, DREB1B & DREB1C) and DREB2 (DREB2A & DREB2B) (Liu et al. 1998). CBF1 homologues, namely CBF2 and CBF3, have also been cloned from Arabidopsis (Gilmour et al. 1998). Both DREB1 and DREB2 can speciŽcally bind to the CRT/DRE elements and transactivate cold-responsive genes in yeast and Arabidopsis protoplasts. Expression of DREB1A (= CBF3) and its homologues, DREB1B (= CBF1) and DREB1C (= CBF2), is induced by low temperature stress, while expression of DREB2A and DREB2B is induced by dehydration and salt stresses (Liu et al. 1998). Thus two independent families of DREB proteins, DREB1 and DREB2, function as transcriptional factors in low temperature and dehydration signal transduction pathways, respectively, to activate CRT/DRE cis-elements. Constitutive overexpression of CBFs under the control of the CaMV35S promoter induced cold-responsive gene expression strongly and also imparted acquired freezing tolerance to the transgenic Arabidopsis without prior cold treatment ( Jaglo-Ottosen et al. 1998; Liu et al. 1998; Kasuga et al. 1999). Over-expression of CBF3 driven by the RD29A promoter resulted in a constitutive low-level expression of cold-regulated genes and enhanced expression under cold, dehydration and salt stresses in transgenic Arabidopsis (Kasuga et al. 1999). These studies provided functional evidence for the involvement of CBFs in cold signal transduction; they act as nodes of cross-talk between cold, dehydration and salt stress signalling pathways and also offer a promising approach to engineer multi-stresstolerant transgenic plants of agronomic value. Towards this step, Thomashow and his colleagues have overexpressed the Arabidopsis CBF genes in canola (B. napus) and found that the expression of CRT/DRE-regulated genes increased freezing tolerance in both acclimatized and non-acclimatized canola plants ( Jaglo et al. 2001). Recently, Stockinger et al. (2001) have shown that transcriptional activation of CRT/DRE cis-elements by CBFs involves a chromatin structure modifying transcriptional adaptor complex consisting of Ada2, Ada3 and GCN5 (histone acetyltransferase). These proteins are constitutively expressed in Arabidopsis in all tissues with the highest expression in leaves, and cold stress did not alter the expression of the genes. 8. ABRE-MEDIATED REGULATION OF COLDRESPONSIVE GENES The transient increase in ABA during cold stress and enhancement of freezing tolerance by exogenous application of ABA indicate that ABA must be playing a critical role in cold acclimatization. Cold- and cold stress- 882 C. Viswanathan and J.-K. Zhu Cold signalling mediated dehydration lead to an increase in endogenous ABA, which might regulate cold-responsive genes through the ABRE cis-elements (PyACGTGGC), as these elements have been identiŽed in the promoters of COR15a (Baker et al. 1994), RD29A (Yamaguchi-Shinozaki & Shinozaki 1994) and COR6.6 (Wang et al. 1995). Gene expression through ABREs is regulated by bZIPtransacting proteins in plants. Cold-regulated bZIP proteins have been identiŽed in Arabidopsis (Lu et al. 1996; Choi et al. 2000), rice (Aguan et al. 1993) and maize (Kusano et al. 1995). In Arabidopsis, three cold-induced C2H2 zinc Žnger proteins, AZF1, AZF3 and STZ, have been cloned (Sakamoto et al. 2000). Four ABRE binding factors (ABF1, 2, 3 & 4) have been cloned from Arabidopsis using a yeast one-hybrid system. All four ABFs are induced by ABA, while the induction of ABF1 is speciŽc to cold and ABA. ABFs could transactivate ABRE-driven reporter gene expression in yeast (Choi et al. 2000). However, no transgenic plants have been developed to show the role of these bZIP and C2H2 zinc Žngers in coldresponsive gene regulation and cold acclimatization. Recently, Kim et al. (2001) have cloned a novel coldinducible zinc Žnger protein from soybean, SCOF1, using an mRNA differential display technique. The SCOF1 contains two C2H2-type zinc Žngers and a putative nuclear localization signal, KRKRSKR. The SCOF1 expression pattern differs from DREB1 in the following ways: (i) SCOF1 is weakly constitutive but DREB1 is cold-inducible; (ii) DREB1 is induced by cold (4 °C) within 40 min, reaches maximum expression by 2 h and then slowly decreases to a minimum level by 24 h at 4 °C (Liu et al. 1998; Shinwari et al. 1998), while SCOF1 induction occurs at 3 h at 4 °C and then the level of transcript tends to increase even up to 72 h (Kim et al. 2001); (iii) SCOF1 could be weakly induced by exogenous application of ABA, while DREB1 expression is not induced by ABA. These temporal sequences in the expression pattern of DREB1 and SCOF1 indicate that the initial induction of cold-responsive gene expression by DREB1 is synergistically increased by SCOF1 during cold stress (after 3 h when DREB1 decreases). The SCOF1:GUS fusion protein revealed that SCOF1 is a nuclear protein. Over-expression of SCOF1 under the control of the constitutive CaMV35S promoter in Arabidopsis resulted in constitutive expression of cold-responsive genes (COR15a, COR47 and RD29B) and constitutive freezing tolerance. However, SCOF1 did not directly bind to ABRE or DRE/CRT motifs. Transactivation experiments in Arabidopsis protoplasts revealed that SCOF1 enhanced the DNA binding activity of SGBF1, a bZIP transcription factor (Kim et al. 2001). SGBF1 is cold- and ABA-inducible (Hong et al. 1995). Thus SCOF1 interacts with SGBF1 to regulate the cold-responsive gene expression through activation of ABRE in ABA-dependent pathways of cold stress signal transduction (Kim et al. 2001). 9. REGULATION OF CBFs/DREBs AND bZIP TRANSACTING FACTORS The DREB1 and DREB2 genes are expressed only under stress. DREB1A was induced with in 1 h at 4 °C and the expression peaked at 2 h at 4 °C. DREB2 was induced by 250 mM NaCl within 10 min, but reached its Phil. Trans. R. Soc. Lond. B (2002) maximum at 5 h of stress in Arabidopsis (Liu et al. 1998). Hence the question arises, how are these CBF genes regulated by cold and dehydration stresses/ABA? Analysis of promoter regions of DREB1-family genes of Arabidopsis revealed that the 59 upstream regions contain motifs similar to G-box and ABRE sequences (T/CACGTGG/TC), and to MYB (C/TAACNA/G) and MYC (CANNTG) recognition motifs. Because DREB1 genes were not induced by ABA, the G-box motifs do not function as ABREs (Shinwari et al. 1998). Arabidopsis cold-induced C2H2 zinc Žnger proteins, AZF1, AZF3 and STZ, also have MYB and MYC cis-acting motifs. The transcript level of these proteins reached a maximum within 30 min of cold stress (Sakamoto et al. 2000). One of the largest families of transcription factors in Arabidopsis is the MYBR2R3 family, which contain two imperfect repeats of the MYB motif (Riechmann et al. 2000). The MYB motif consists of a helix-turn-helix structure with three regularly spaced tryptophan residues. An Arabidopsis cDNA encoding a MYB homologue, AtMYB2, was cloned from a cDNA library of dehydrated rosette plants. AtMYB2 was induced by ABA, salt and dehydration stresses, and disappeared upon rehydration. An AtMYB2 promoter-driven GUS reporter could be activated by dehydration and salt stresses in transgenic Arabidopsis (Urao et al. 1993, 1996). AtMYB2 proteins have been shown to transactivate the RD22B promoter-driven GUS reporter in Arabidopsis leaf protoplast (Abe et al. 1997). However, to our knowledge, there is no evidence so far that MYB transacting factors are involved in the regulation of CBFs/bZIPs expression through MYB-related cis-elements present in their 59 upstream regions. 10. GENETIC DISSECTION OF COLD SIGNAL TRANSDUCTION A classical genetic approach on freezing tolerance led to the identiŽcation of sfr (sensitive to freezing) mutants in Arabidopsis (Warren et al. 1996). In the sfr6 mutant, the cold-induced expression of KIN1, COR15a and RD29A was abolished, and also osmotic stress and ABA-induced expression of KIN1 was inhibited. However, the expression of CBF1, CBF2, CBF3 and ATP5CS1 was not inuenced by the sfr6 mutation. Hence SFR6 speciŽcally affects the transactivation of DRE/CRT by CBFs (Knight et al. 1999). It appears that sfr6 is also involved in ABREregulated gene expression, as ABA and salt stress could not induce KIN1. Cloning and characterization of sfr6 may shed further light on the regulation of cold-responsive genes. IdentiŽcation of the esk1 (constitutively freezingtolerant) mutant of Arabidopsis revealed proline accumulation as a possible mechanism of acquired freezing tolerance (Xin & Browse 1998). The esk1 mutant did not differ in its cold-responsive gene expression from the WT, but maintained a 30-fold higher proline level due to higher expression of the P5CS gene when compared with the WT plants in normal growing conditions. In esk1, cold acclimatization leads to differential expression of cold-responsive genes, i.e. RD29A, COR47 and COR15a expression were similar to that of the WT and RAB18 expression was enhanced by three to fourfold, while COR6.6 expression was signiŽcantly reduced. Further understanding of how Cold signalling these cold-responsive genes are differentially regulated needs the molecular cloning of ESK1. Genetic analysis of chemically mutagenized Arabidopsis transgenic with RD29A promoter (which contains both CRT/DRE and ABRE)-driven luciferase revealed that cold, drought, salt and ABA stress signalling pathways interact at different nodes of signal transduction (Ishitani et al. 1997). The hos1 (high expression of osmotically responsive genes) mutation resulted in super-induction of RD29A, COR47, COR15a, KIN1 and their transacting factors (CBF2 and CBF3) at 4 °C. In WT plants, these genes are also induced by ABA, high salt, or polyethylene glycol in addition to cold, but the hos1 mutation only enhances their expression under cold stress (Ishitani et al. 1998). The expression of CBFs is transient in the WT, while in the hos1 mutant CBFs mRNA abundance was maintained at a much higher level even up to 24 h during cold stress. Hence HOS1 negatively regulates the coldresponsive genes by modulating the expression level of the CRT/DRE binding factors. Molecular cloning and characterization revealed that HOS1 encodes a ring Žnger protein, which has been implicated as an E3 ubiquitin conjugating enzyme. HOS1 is constitutively expressed, shows a drastic decrease within 10 min of cold stress and recovers back to the basal level after 1 h of cold stress. HOS1 protein is present in the cytoplasm at normal growth temperatures and accumulates in the nucleus upon cold stress. The hos1 mutation also affected the thermosensing mechanism, as is evident from the fact that RD29A expression occurs at relatively warmer temperatures (Lee et al. 2001). The cold-induced Ca21 signature outputs depend on the rate of stress development (change in temperature per unit time). The hos1 mutant reached a maximum level of RD29A expression within 10 h at 0 °C while WT plants reached maximal level of expression only at 24 h at 0 °C, which indicates that the rate of output signal from the cellular thermosensor is much higher in the hos1 mutant. These results show that, being a constitutively expressed protein, HOS1 may be closely interacting with cellular thermosensors to modulate thermosensing and the rate of signal output from the cellular thermosensor (Lee et al. 2001). Cold-responsive genes are regulated by both ABA-independent and ABAdependent pathways during cold stress. The expression of RD29A:luc showed a threefold increase if the Arabidopsis plants were treated with ABA after 44 h at 0 °C. Hence, at low temperatures, ABA acts synergistically with the cold signal (Xiong et al. 1999). However, in hos1 and hos2 mutants of Arabidopsis, cold-responsive genes are superinduced by cold stress but their expression pattern is unaltered by ABA or salt stress, indicating that colddependent signal transduction is speciŽcally altered by these mutations (Ishitani et al. 1998; Lee et al. 1999). Although the signal ow through cold signal transduction modules has increased, it did not inuence the signal through the ABA-dependent transduction module. Overexpression of cold-responsive genes in transgenic plants achieved through over-expression of CBFs or the antisense AtPP2CA gene conferred better freezing tolerance. By contrast, the super-induction of cold-responsive genes was not sufŽcient to provide cold acclimatization in hos2 mutants. Because the expression kinetics of the P5CS gene in hos2 mutants was similar to the WT, proline concenPhil. Trans. R. Soc. Lond. B (2002) C. Viswanathan and J.-K. Zhu 883 tration in the cell is not responsible for decreased capacity of the hos2 mutant to cold acclimatization. Hence it appears that HOS2 is a negative regulator of cold signal transduction required for developing cold acclimatization. 11. CONCLUSIONS AND FUTURE PERSPECTIVES Combined use of genetics and molecular approaches has begun to shed light on cold signal transduction modules and their components. Pharmacological and biochemical evidence shows that membrane rigidiŽcation followed by cytoskeleton rearrangement, Ca21 inux and Ca21-dependent phosphorylation are involved in cold stress signal transduction. Genetic evidence provided by Arabidopsis fry1 mutants indicates that change in IP3 level is an important component of cold signalling. Protein dephosphorylation negatively regulates the coldresponsive genes, as evident from AtPP2CA antisense transgenic plants. Cold-responsive genes are regulated through CRT/DRE and ABRE cis-elements by transacting factors CBFs/BREBs and bZIPs (SGBF1), respectively. Constitutive over-expression or stress promoter-driven expression of these transacting factors induced coldresponsive genes and freezing tolerance in transgenic plants. Genetic evidence showed that HOS1 is a negative regulator of CBFs/DREB1-dependent cold-regulated genes and is a modulator of the cellular thermosensor’s sensitivity to temperature. Still, the components of Ca21mediated signal transduction into the nucleus and their spatial and temporal positions in cold signalling need to be deŽned genetically. 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GLOSSARY ABA: abscisic acid ABRE: ABA-responsive element AtCBL: A. thaliana calcineurin B-like bZIP: basic leucine zipper cADPR: cyclic adenosine 59diphosphate ribose CAS30: cold acclimation-speciŽc protein CBF: C-repeat binding factor CBL: calcineurin B-like CDPK: calcium-dependent protein kinase CIPK1: CBL-interacting protein kinase 1 CRT: C-repeat DMSO: dimethyl sulphoxide DRE: dehydration-responsive element DREB: dehydration-responsive element binding protein esk1: eskimo1 fry1: Žery1 IP3: inositol (1,4,5)-triphosphate MAPK: mitogen activated protein kinase MAPKK: MAPK kinase MAPKKK: MAPK kinase kinase RER1: response regulator 1 SCOF1: soyabean cold-inducible factor 1 SGBF1: soyabean G-box binding factor 1 TaADF: Triticum aestivum actin depolymerizing factor WT: wild-type