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161 Molecular responses to drought and cold stress Kazuo Shinozaki* and Kazuko Yamaguchi-Shinozakif A variety of plant genes are induced by drought and cold stress, and they are thought to be involved in the stress tolerance of the plant. At least five signal transduction pathways control these genes: two are dependent on abscisic acid (ABA), and the others are ABA-independent. A novel cis-acting element involved in one of the ABA-independent signal transduction pathways has been identified. In addition, a number of genes for protein kinases and transcription factors thought to be involved in the stress signal transduction cascades have been shown to be induced by environmental stresses. Addresses *Laboratory of Plant Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan; e-maih [email protected] tBiological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Ministry of Agriculture, Forestry and Fisheries, 2-10hwashi, Tsukuba, Ibaraki 305, Japan Current Opinion in Biotechnology 1996, 7:161-167 © Current Biology Ltd ISSN 0958-1669 Abbreviations ABA abscisic acid ABRE ABA-responsive element bZlP basic region leucine zipper CDPK Ca2+-dependent protein kinase DRE dehydration-responsive element LTRE low temperature responsive element MAPK mitogen-activated protein kinase MAPKK MAPK kinase MAPKKK MAPKK kinase P5C A 1 -pyrroline-5-carboxylate SH Src-homology Introduction Plants respond to conditions of drought or cold stress through a number of physiological and developmental changes. Many genes that respond to drought or cold stress at the transcriptional level have been described recently [1-3]. The majority have been shown to be induced by both drought and cold stress, but some are responsive only to drought and others only to cold stress [2,3]. The molecular responses of plant genes to drought or cold stress raise several interesting questions. How do plant cells sense the loss of water or the decrease of temperature? How are the stress signals transduced into cellular signals and transmitted to the nucleus? How is gene transcription affected by these stress signals? And, finally, how do the gene products function in stress tole rance ? Abscisic acid (ABA) is produced under such environmental stresses, and it is important in the tolerance of plants to drought, high salinity, and cold [4]. Many genes that respond to drought and/or cold stress are also induced by the exogenous application of ABA [1,2,4,5]. It appears that dehydration triggers the production of ABA, which, in turn, induces various genes. Cis-acting and trans-acting factors involved in ABA-induced gene expression have been extensively analyzed [2,5,6]. Several reports have described genes that are induced by drought and cold stress, but are not responsive to exoge- nous ABA treatments [2,3,5]. These findings suggest the existence of both ABA-independent and ABA-dependent signal-transduction cascades between the initial signal of drought or cold stress and the expression of specific genes. Analyses of the promoters of drought-inducible and cold-inducible genes have revealed a novel cis-acting element that is involved in the ABA-independent response to conditions of dehydration, low temperature, or high salt [7°°]. Several genes encoding factors involved in signal transduc- tion cascades, such as protein kinases, transcription factors, and phospholipase C, are induced by drought and cold stress; this suggests that these factors may function in the signal transduction pathways between initial water stress signals and gene expression. The present review focuses both on recent progress in the study of the expression of drought-inducible and cold-inducible genes and on the signal transduction cascades under such environmental stresses. Function of drought/cold stress inducible genes Many genes have been demonstrated to respond to drought and/or cold stress in various plant species. Functions for a lot of the encoded products have been predicted from sequence homology with known functional proteins. Genes induced under stress conditions are thought to function in protecting cells from water deficit or temperature change by the production of several different gene products: water channel proteins involved in altering cellular water potential [2,8-12]; the enzymes required for the biosynthesis of various osmoprotectants such as sugars, proline and betaine [2,11-13,14",15°,16]; lipid desaturases for membrane modification [2,3,11,12]; protective proteins such as late embryogenesis abundant (LEA) proteins, osmotin, antifreeze proteins, chaperones, and mRNA-binding proteins [2,3,11,12,17,18°]; thiol pro- teases, Clp protease, and ubiquitin, which are required for protein turnover [2,11,12,18°,19,20]; the detoxifica- tion proteins, such as glutathione S-transferase, soluble epoxide hydrolase, catalase, and ascorbate peroxidase [2,11,12,21,22"°,23"]; and protein kinases, phospholipase C, and transcription factors, which are involved in further

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Page 1: Molecular responses to drought and cold stress

161

Molecular responses to drought and cold stress Kazuo Shinozaki* and Kazuko Yamaguchi-Shinozakif

A variety of plant genes are induced by drought and cold stress, and they are thought to be involved in the stress tolerance of the plant. At least five signal transduction pathways control these genes: two are dependent on abscisic acid (ABA), and the others are ABA-independent. A novel cis-acting element involved in one of the ABA-independent signal transduction pathways has been identified. In addition, a number of genes for protein kinases and transcription factors thought to be involved in the stress signal transduction cascades have been shown to be induced by environmental stresses.

Addresses *Laboratory of Plant Molecular Biology, The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan; e-maih [email protected] tBiological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Ministry of Agriculture, Forestry and Fisheries, 2-10hwashi, Tsukuba, Ibaraki 305, Japan

Current Opinion in Biotechnology 1996, 7:161-167

© Current Biology Ltd ISSN 0958-1669

Abbreviations ABA abscisic acid ABRE ABA-responsive element bZlP basic region leucine zipper CDPK Ca2+-dependent protein kinase DRE dehydration-responsive element LTRE low temperature responsive element MAPK mitogen-activated protein kinase MAPKK MAPK kinase MAPKKK MAPKK kinase P5C A 1 -pyrrolin e-5-carboxylate SH Src-homology

I n t r o d u c t i o n Plants respond to conditions of drought or cold stress through a number of physiological and developmental changes. Many genes that respond to drought or cold stress at the transcriptional level have been described recently [1-3]. The majority have been shown to be induced by both drought and cold stress, but some are responsive only to drought and others only to cold stress [2,3]. The molecular responses of plant genes to drought or cold stress raise several interesting questions. How do plant cells sense the loss of water or the decrease of temperature? How are the stress signals transduced into cellular signals and transmitted to the nucleus? How is gene transcription affected by these stress signals? And, finally, how do the gene products function in stress tole rance ?

Abscisic acid (ABA) is produced under such environmental stresses, and it is important in the tolerance of plants

to drought, high salinity, and cold [4]. Many genes that respond to drought and/or cold stress are also induced by the exogenous application of ABA [1,2,4,5]. It appears that dehydration triggers the production of ABA, which, in turn, induces various genes. Cis-acting and trans-acting factors involved in ABA-induced gene expression have been extensively analyzed [2,5,6].

Several reports have described genes that are induced by drought and cold stress, but are not responsive to exoge- nous ABA treatments [2,3,5]. These findings suggest the existence of both ABA-independent and ABA-dependent signal-transduction cascades between the initial signal of drought or cold stress and the expression of specific genes. Analyses of the promoters of drought-inducible and cold-inducible genes have revealed a novel cis-acting element that is involved in the ABA-independent response to conditions of dehydration, low temperature, or high salt [7°°].

Several genes encoding factors involved in signal transduc- tion cascades, such as protein kinases, transcription factors, and phospholipase C, are induced by drought and cold stress; this suggests that these factors may function in the signal transduction pathways between initial water stress signals and gene expression. The present review focuses both on recent progress in the study of the expression of drought-inducible and cold-inducible genes and on the signal transduction cascades under such environmental stresses.

F u n c t i o n o f d r o u g h t / c o l d s t r e s s i n d u c i b l e g e n e s Many genes have been demonstrated to respond to drought and/or cold stress in various plant species. Functions for a lot of the encoded products have been predicted from sequence homology with known functional proteins. Genes induced under stress conditions are thought to function in protecting cells from water deficit or temperature change by the production of several different gene products: water channel proteins involved in altering cellular water potential [2,8-12]; the enzymes required for the biosynthesis of various osmoprotectants such as sugars, proline and betaine [2,11-13,14",15°,16]; lipid desaturases for membrane modification [2,3,11,12]; protective proteins such as late embryogenesis abundant (LEA) proteins, osmotin, antifreeze proteins, chaperones, and mRNA-binding proteins [2,3,11,12,17,18°]; thiol pro- teases, Clp protease, and ubiquitin, which are required for protein turnover [2,11,12,18°,19,20]; the detoxifica- tion proteins, such as glutathione S-transferase, soluble epoxide hydrolase, catalase, and ascorbate peroxidase [2,11,12,21,22"°,23"]; and protein kinases, phospholipase C, and transcription factors, which are involved in further

Page 2: Molecular responses to drought and cold stress

162 Plant biotechnology

regulation of signal transduction and gene expression [2,11,24"°,25°,26°°,27°,28-30,31"]. Some stress-inducible genes have been over-expressed in transgenic plants, pro- ducing a stress-tolerant phenotype of the plant, indicating that the gene products function in stress tolerance. Some stress-inducible genes do not always function in stress tolerance, however. The functions of the genes induced by drought, high salinity and cold stress have been reviewed [2,3,11-13]. Original reports that have appeared in 1994 and 1995 are cited in this review.

Regulation of gene expression by drought and cold stress Most of the drought-inducible genes also respond to high-salinity stress, but some of them do not respond to cold stress, and vice versa [2,3]. The expression patterns of genes induced by drought and cold stress were analyzed by northern blot analysis. Results indicate broad variations in the timing of induction of these genes and that some genes respond to ABA, whereas others do not [32-34]. ABA-deficient mutants were used to analyze cold-inducible and drought-inducible genes that respond to ABA [2,3]. Several genes were induced by exogenous ABA treatment, but they were also induced by cold or drought in ABA-deficient (aba) or ABA-insensitive (abt) Arabidopsis mutants. These observations indicate that these genes do not require an accumulation of endogenous ABA under cold or drought conditions, but do respond to ABA [2,3,19,34-37]. Analysis of the expression of ABA-inducible genes showed that several

Figure 1

genes require protein biosynthesis for their induction by ABA, suggesting that at least two independent pathways exist between the production of endogenous ABA and gene expression under stress conditions [38].

As shown in Figure 1, at least three independent signal pathways function under drought conditions [39°]: two are ABA-dependent (pathways II and III) and one is ABA-independent (pathway IV). There are also at least two independent cold stress signal transduction pathways [19,34-36]: one is ABA-dependent (pathway III) and the other is ABA-independent (pathway IV). These pathways overlap with those of the drought response [5,19,34-36]. In addition, two other signal transduction pathways may function only in drought response (pathway I) or in cold response (pathway V) [40,41°°]. Therefore, at least five independent signal transduction pathways mediate drought or cold responses (Fig. 1). The existence of complex signal transduction pathways in the responses of plants to drought and cold stress is strong evidence of a molecular basis for these complex physiological responses.

Absc is ic acid respons ive g e n e express ion u n d e r d r o u g h t and cold s t ress The levels of endogenous ABA increase significantly in many plants under drought and high-salinity conditions [4]. ABA levels also increase, at least transiently, in response to low-temperature stress [3,42]. Many drought- inducible and cold-inducible genes are induced by ex- ogenous ABA treatment. These genes contain potential

Signal transduction pathways between initial drought-stress and/or cold-stress signals and gene expression. At least five signal transduction pathways exist (I-V): two are ABA-dependent (11 and III), and three are ABA-independent (I, IV and V). Protein synthesis is required in one of the ABA-dependent pathways (11). There are at least four signaling pathways (I, II, III and IV) that function under drought conditions (indicated by solid arrows), and three pathways (111, IV and V) that function under cold stress (indicated by dashed arrows). ABRE is involved in one of the ABA-dependent pathways (111), and DRE is involved in one of the ABA-independent pathways (IV). (See text for further details.)

ii i"

~iiiii ~ iii~ III

?

0 [l

ABRE DRE Gene expression

Temperature change

D D D D B

?

D

© t 996 Current Opinion in Biotechnology

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Molecular responses to drought and cold stress $hinozaki and Yamaguchi-Shinozaki 163

ABA-responsive elements (ABREs) in their promoter regions [2,3]. The ABRE (PyACGTGGC, where Py is a pyrimidine) functions as a c/s-acting DNA element involved in ABA-regulated gene expression [5,6]. The G box resembles the ABRE motif and functions in the regulation of plant genes in a variety of environmental conditions, such as red light, UV light, anaerobiosis, and wounding. Recently, nucleotides around the ACGT core motif have been shown to be involved in determining the binding specificity of basic region leucine zipper (bZIP) protein [5,6]. Furthermore, a coupling element is required to specify the function of the ABRE, constituting an ABA-responsive complex [43**].

The induction of an Arabidopsis drought-inducible gene, rd22, is mediated by ABA, but requires protein biosynthe- sis for its ABA-dependent expression [38]. A 67 bp region of the rd22 promoter is essential for its ABA-responsive expression and contains several conserved motifs of DNA-binding proteins, such as MYC and MYB, but no ABREs [39*]. Recently, a cDNA for a transcription factor MYC homolog has been cloned by the south-western method, using the 67 bp DNA as a probe. This suggests that a drought-inducible MYC homolog may function in the ABA-inducible expression of rd22 (H Abe, K Yamaguchi-Shinozaki, K Shinozaki, unpublished data). A promoter region has been identified that functions in ABA-inducible expression of the CDeT27-45 gene in Craterostigma plantagineum [44*]. In addition, an ABA- inducible DNA-binding activity to this region was de- tected in nuclear extracts. These results indicate that drought-inducible or ABA-inducible transcription factors may function in the ABA-responsive gene expression un- der drought stress and that two independent transcription systems mediate ABA-responsive gene expression: one is controlled by ABA directly through ABRE, and the other may require de novo synthesis of ABA-inducible transcription factors.

Abscisic acid independent gene expression under drought and cold stress Several genes are induced by both cold and drought in aba or abi Arabidopsis mutants. This suggests that these genes do not require ABA for their expression under cold or drought conditions, but do respond to ABA [19,34-37]. These genes include rd29A (also termed lti78 and cor78), kin1, cor6.6 (kin2), and cor47 (rd17) [3]. Among these genes, the expression of a drought-inducible gene for rd29A/lti78/cor78 was analyzed [36,45,46]. Two genes, rd29A and rd29B/lti65, were found to be located in tandem in an 8 kbp region of the Arabidopsis genome and encode hydrophilic proteins [36,45]. The transcription of rd29A in Arabidopsis abil and abal mutants suggests that cold-regulated and drought-regulated expression does not require ABA. These observations indicate that at least two separate regulatory systems function in gene expression during drought and cold stress. One of the cis-acting elements responsible for the dehydration-induced and

cold-induced expression of rd29A was identified at the nucleotide sequence level in transgenic plants [7**].

A 9bp conserved sequence, TACCGACAT, termed the dehydration-responsive element (DRE), is essential for the regulation of the induction of rd29A under drought, low-temperature, and high-salt stress conditions, but does not function as an ABRE. The DRE sequence is not found in the rd29B promoter, which responds to ABA treatment, but not to cold stress [7°*]. Nordin et al. [451 reported conserved motifs (ACCGACA), including the DRE, as putative low-temperature responsive elements. DRE-related motifs have been reported in the promoter regions of cold-inducible and drought-inducible genes such as kinl, cor6.6, and cor47/rdl 7 [7°°,47]. Baker et al. [48] also reported a similar motif (C-repeat; TGGCCGAC) in the promoter region of cold-inducible cor15a [48]. The 5bp CCGAC core sequence was found in the promoter regions of the cold-inducible Brassica gene, BNllS, and designated the low temperature responsive element (LTRE) [49]. These results suggest that DRE- related motifs are involved in drought-responsive and cold-responsive, but ABA-independent, gene expression. A protein factor that specifically interacts with the 9bp DRE sequence was detected in nuclear extract prepared from either dehydrated or untreated Arabidopsis plants [7°°]. Cloning of the cDNA for a DRE-binding protein is in progress; this should provide a better understanding of the regulation of drought-inducible and cold-inducible genes.

Genes for transcription factors that are induced by drought or cold stress In higher plants, genes for several transcription factors have been shown to be induced by environmental stresses or signals. These include genes encoding MYB-related and bZIP-related transcription factors. A cDNA, designated Atmyb2, that encodes an MYB-related protein was cloned by screening a cDNA library prepared from dehydrated Arabidopsis plants [29]. The Atmyb2 gene is rapidly induced by dehydration stress. High-salt conditions and the application of exogenous ABA also result in the induction of Atmyb2, although Atmyb2 does not respond to cold or heat stress. Bacterially expressed ATMYB2 protein binds to the MYB recognition sequence, which suggests that the drought-inducible MYB homolog can also control drought-inducible and ABA-inducible gene expression.

Kusano and colleagues [30,31 °] have isolated a cold- inducible gene encoding a bZIP transcription factor from rice (lipl9) and maize (mliplS) plants. These genes also respond to high salt and to exogenous ABA treatment. The mLipl5 protein can bind to the hexamer sequence, ACGTCA, and to a G-box-like sequence. The mLipl5 protein binds to the promoter region of the cold-inducible alcohol dehydrogenase gene in maize [31°]. These stress-inducible transcription factors are thought to function in the regulation of stress-inducible genes, which

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164 Plant bioteehnology

respond to drought or cold stress rather slowly after the production of these transcription factors.

Signal transduction under drought and cold stress Signal transduction cascades between initial stress signals and the expression of various genes, as well as the signaling molecules that function in the cascades, have not been extensively studied, and these are attractive research subjects for the next decade. Conversely, the ABA-regulated closure of stomata and the dehydration-de- pendent, cold-dependent or ABA-dependent responses of guard cells have been extensively analyzed at physiological levels [5,6,50]. Transient elevations of cytoplasmic Ca z+ have been observed in response to cold and to ABA treatment [3,6]. An influx of CaZ+ ions into the cytoplasm occurs under cold conditions and is thought to function in the induction of cold-inducible genes [41°°]. The addition of a Ca 2+ ionophore or a Ca 2÷ agonist to non-acclimated cells has been shown to cause an influx of extracellular Ca2+ ions and induce the expression of cold-inducible cas genes in alfalfa cells grown in culture.

ABA is important in plant responses to drought and cold stress. The role of ABA in stress-related signal transduction has been analyzed with ABA-insensitive mutants in various species. Among them, maize vpl and Arabidopsis abil and abi3 are extensively characterized and their genes have been cloned. The vpl and abi3 genes are expressed in seeds and encode homologous transcriptional activators [6,50,51]. VP1 protein requires an ABRE for ABA-responsive expression of the EM gene [52,53], but requires a Sph motif for the induction of the C1 gene during seed development [6]. The ABI1 and ABI2 gene products appear to function mainly in vegetative tissues and also participate, to some extent, in seed development [4-6]. Thus, ABI1 and ABI2 are important in the signal transduction pathways involved in drought and cold stress. The abil gene has been cloned by chromosome walking and shown to encode a protein that is related to type 2C protein serine/threonine phosphatases; it also includes a putative CaZ+-binding domain (EF hand) [54"°,55"]. The ABI1 gene product functions in stomata closure, and study of the abil mutants reveals a wilty phenotype. The sensitivity of guard cell K ÷ channels to ABA is suppressed by abil [56°]. These results suggest that protein phosphorylation has a role in ABA-responsive signaling during dehydration. ABA signaling has recently been reviewed [6,50].

Stress-inducible genes encoding factors involved in signal transduction pathways In higher plants, many genes involved in signal trans- duction pathways, such as those encoding calmodulins, G proteins, protein kinases, and transcription factors, are induced by environmental signals or stresses. The genes for several protein kinases and for phospholipase C are

also induced by drought and cold stress. PCR fragments prepared from the conserved regions, were used as probes for cloning these cDNAs by screening cDNA libraries prepared from dehydrated Arabidopsis plants. A cDNA for phospholipase C, AtPLC1, has been isolated from dehydrated Arabidopsis [24"']. AtPLC1 contains the X and Y domains of phospholipase C and an EF hand structure, which resembles the 8 type of phospholipase C. Two cDNAs for the Ca2+-dependent protein kinases (CDPKs), AtCDPK1 and AtCDPK2, have also been isolated from Arabidopsis [25"]. Northern blot analyses have shown that these genes are rapidly induced by drought and salt stresses. Likewise, the gene for CDPK from alfalfa is also induced by cold stress [41"°]. In animals, phospholipase C digests phosphatidylinositol 4,5-bisphosphate to generate two secondary messengers, inositol 3-phosphate and diacylglycerol. Inositol 3-phosphate induces the release of Ca2+ into the cytoplasm, which, in turn, causes various responses in the cytoplasm. These observations suggest that inositol 3-phosphate and CaZ+ may function as secondary messengers in the signal transduction pathways under drought and cold conditions.

A wheat ABA-inducible gene for protein kinase (PKABA1) is induced by dehydration, cold, and osmotic stresses [27°]. An Arabidopsis root specific gene encoding a protein kinase (ARSK1) is also induced by dehydration, ABA, and high salt [28]. In addition, a salt-regulated tobacco gene and two cold-inducible Arabidopsis genes encode proteins homologous to the 14-3-3 proteins, a family of protein kinase inhibitors; furthermore, these proteins activate ADP ribosyltransferase and inhibit protein kinase C [57,58]. These results further suggest that phosphorylation has a role in the stress response, that the level of factors involved in signal transduction pathways arc controlled by environmental signals or stresses, and that these factors probably regulate the efficiency of signal transduction in higher plants.

Mitogen-activated protein kinase (MAPK) is involved in the signal transduction pathways associated with growth factor dependent cell proliferation and with stress responses in yeast and animals. In plants, many genes for protein kinases involved in MAPK cascades have been identified. In Arabidopsis, more than nine genes encode MAPKs, and there are at least four subfamilies of MAPK based on phylogenetic analysis [26°°,59]. One of the MAPK genes, ATMPK3, is induced at the mRNA level by drought, cold, and high salinity [26°°]. Moreover, two genes for protein kinases involved in the MAPK cascade, ATIIIEKK1 (which encodes MAPK kinase kinase [MAPKKK]) and ATPK19 (which encodes ribosomal $6 kinase), are also induced by low temperature, high salt, and dehydration [26°°,60]. MAPKKK and ribosomal $6 kinase are upstream and downstream factors of MAPK, respectively. These observations suggest that one of the MAPK cascades may function in the signal transduction pathways under cold, high salt, and dehydration conditions

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Molecular responses to drought and cold stress Shinozaki and Yamaguchi-Shinozaki 165

[26"°]. In yeast, one of the MAPK cascades (i.e. the HOG1 cascade) functions in the high-osmolarity response.

Signal perception On the basis of the variety of regulatory systems involved in the expression of drought/cold-inducible genes, there appear to be several signal transduction pathways. Water deficit occurs not only during drought, but also during cold conditions, and probably causes turgor stress at the cellular level [1-3]. A change in the osmotic potential across a plasma membrane, caused by turgor stress, may be one of the triggers of the stress response at the molecular level. Changes in membrane fluidity caused by low temperature may also be triggering the stress response [61]. Catalytic hydrogenation of membrane lipids, which may decrease membrane fluidity, induces the cold-inducible desaturase A gene. An oxidative burst may occur under drought/cold-stress conditions, which may trigger stress signaling [22°',23"]. Cold stress induces the genes for mitochondrial catalasc-3 isozyme [22"'], ascorbate peroxidase, and superoxide dismutase [23°]; it also elevates the enzyme activities of catalase-3 and guaiacol peroxide dismutase [22"]. Drought stress induces genes for detoxification enzymes such as glutathione S-transferases [18 °] and soluble epoxicide hydrolase [21].

In yeast, exposure to high osmolarity activates a MAPK cascade that includes PBS2 MAPK kinase (MAPKK) and HOG1 MAPK. The SLN1 gene acts early in the hyperosmolarity stress response and encodes a prokaryote- type 'two-component' histidine kinase, Slnlp [62"',63"]. Slnlp functions as a 'sensor' protein, phosphorylating and inhibiting Ssklp, a 'response regulator' protein. At high osmolarity, unphosphorylated Ssklp activates SskZp or Ssk22p (which are MAPKKKs) [63"']. Activated Ssk2p/Ssk22p activates Pbs2p (a MAPKK) by Scr-Thr phosphorylation, which then activates Hoglp (a MAPK) by Thr-Tyr phosphorylation. Recently, another transmem- brane 'osmosensor', Sholp, has been reported [63**]. Sholp activates Pbs2p through an interaction between the Sholp Src-homology (SH)3 domain and the Pbs2p SH3-binding site, and then activates the PBS2-HOG1 MAPK cascade. A similar osmosensing mechanism may function in higher plants in response to a water deficit. In higher plants, another 'two-component' histidine kinase, ETR1, is a receptor in ethylene signal transduction [64]. CTR1, which encodes a RAF-related protein kinase and a component of the MAPK cascade, functions downstream from ETR1. Several ETR1 h,m.logs are found in Arabidopsis expressed sequence tag cDNAs, one of which may function as an osmosensor-like Slnlp.

Conclusions Many drought-induced and cold-induced genes have been cloned in a variety of plants. Structural analyses of their gene products suggest that they function cooperatively in drought and cold tolerance. The transcriptional regulatory

regions of the drought-induced and cold-induced genes have been analyzed to identify several cis-acting and trans-acting elements that are involved in drought-induced and cold-induced gene expression. Analyses of the ex- pression of stress-inducible genes indicate the presence of multiple signal transduction pathways between the initial stress signals and gene expression; this explains the complex stress response observed after exposure of plants to drought and cold stress. Many genes for factors involved in the signal transduction cascades are regulated by these stress signals. Molecular analyses of these factors should provide a better understanding of the signal transduction cascades under drought and cold stress.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

= of special interest 0o of outstanding interest

1. Skriver K, Mundy J: Gene expression in response to abscisic acid and osmotic stress. Plant Cell 1990, 2:503-512.

2. Bray EA: Molecular responses to water deficit. Plant Physiol 1993, 103:1035-1040.

3. Thomashow MF: Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. In Arabidopsis. Edited by Meyrowitz E, Sommerville C. Cold Spring Harbor: Cold Spring Harbor Press; 1994:807-834.

4. Davies WJ, Jones HG: Abscisic Acid: Physiology and Biochemistry. Oxford: BIOS Scientific; 1991.

5. Chandler PM, Robertson M: Gene expression regulated by abscisic acid and its relation to stress tolerance. Annu Rev Plant Physio/ Plant Mo/ Bio/1994, 45:113-141.

6. Giraudat J, Parcy F, Bertauche N, Gosti F, Leung J, Morris P-C, Bouvier-Durand M, Vartanian N: Current advances in abscisic acid action and signaling. Plant Mo/Bio/1994, 26:1557-1577.

7. Yamaguchi-Shinozaki K, Shinozaki K: A novel c/s-acting element * - in an Arabidopsis gene is involved in responsiveness to

drought, I,w-temperature, or high-salt stress. Plant Cell 1994, 6:251-264.

The promoter region of the drought-inducible and cold-inducible Arab/d,p- sis rd2gA/Iti78/cor?8 gene is analyzed using transgenic plants. A novel c/s-acting element, DRE (TACCGACAT), is identified, which functions in drought-response and cold-response, but not in ABA-responsive expression. DRE-related motifs are found in the promoters of many drought-inducible and cold-inducible genes. Nuclear factor, which hinds to DRE, is detected. These results indicate that DRE-related motifs play important roles in gene expression under drought and cold stress.

8. Chrispeels M J, Agre P: Water channel proteins of plants and animal cells. Trends Biochem Sci 1994, 19:421-425.

9. Yamada S, Katsuhara M, Kelly WB, Michalowski CB, Bohnert H J: A family of transcripts encoding water channel proteins: tissue-specific expression in the common ice plant. Plant Cell 1995, 7:1129-1142.

10. Jones JT, Mullet JE: Developmental expression of a turgor- responsive gene that encodes an intrinsic membrane protein. Plant Mol Bio11995, 28:983-996.

11. Bohnert H J, Nelson DE, Jensen RG: Adaptations to environmental stresses. Plant Cell 1995, 7:1099-1111.

t 2. Bartels D, Nelson D: Approaches to improve stress tolerance using molecular genetics. Plant Cell Environ 1994, 17:659-667

13. Delauney A J, Verma DPS: Proline biosynthesis and osmoregulation in plants. Plant J 1993, 4:215-223.

14. K/shot PBK, Hong Z, Miao GH, Hu CAA, Verma DPS: • Overexpression of Al-pyrroline.5-carboxylate synthetase

increases proline production and confers osmotolerance in transgenic plants. Plant Physio/1995, 108:1387-1394.

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1 66 Plant biotechnology

Over-expression of mothbean Z~l-pyrroline-5-carboxylate (P5C) synthetase results in the accumulation of proline in transgenic tobacco, which indicates that P5C synthetase is the rate-limiting factor in proline synthesis. Over-pro- duction of proline confers osmotolerance in transgenic plants, which sug- gests that proline acts as an osmoprotectant.

15. Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T, Yamaguchi- • Shinozaki K, Wada K, Harada Y, Shinozaki K: Correlation

between the induction of a gene for Al-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thai/aria under osmotic stress. Plant J 1995, 7:751-760.

An Arabidopsis gene for P5C synthetase is demonstrated to be induced by dehydration, high salt and ABA treatment, whereas a gene for P5C reductase is not induced. Simultaneous accumulation of proline with P5C synthetase mRNA is observed under stress conditions, which suggests that P5C synthetase plays a principal role in the biosynthesis of proline under osmotic stress.

16. Ishitani M, Nakamura T, Han SY, Takabe T: Expression of the betaine aldehyde dehydrogenase gene in badey in response to osmotic stress and abscisic acid. Plant Mo/Bio/1995, 27:307-315.

17. Goday A, Jensen AB, Culianez-Maci& FA, AIb& MM, Figueras M, Serratosa J, Torrent M, P&ges M: The maize abscisic acid- responsive protein Rab17 is located in nucleus and interacts with nuclear localization signals. Plant Cell 1994, 6:351-360.

18. Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K: Cloning of cDNA • for genes that are early-responsive to dehydration stress

(ERDs) in Arabidopsis thaliana L.: identification of three ERDs as HSP cognate genes. Plant Mol Bio/1994, 25:791-798.

16 cDNAs for genes that are 'early-responsive' to dehydration before the accumulation of ABA are cloned from Arabidopsis. These include heat-shock protein cognates, a ubiquitin, an ATP-dependent protease regulatory subunit (CIp A) homolog, and glutathione S-transferase homologs.

19. Williams J, Bulman M, Hyttly A, Phillips A, Neill S: Characterization of a cDNA from Arabidopsis thai/aria encoding a potential thiol protease whose expression is induced independently by wilting and abscisic acid. Plant Mo/ Bio/1994, 25:259-270.

20. Jones JT, Mullet JE: A salt- and dehydration-inducible pea gene, Cyp15a, encodes a call-wall protein with sequence similarity of cysteine proteases" Plant Mo/Bio/1995, 28:1055-1065.

21. Kiyosue H, Beetham J, P/not, F, Hammock BD, Yamaguchi- Shinozaki K, Shinozaki K: Characterization of an Arabidopsis cDNA for a soluble °pox/de hydrolase gene that is inducible by auxin and water stress. Plant J 1994, 6:259-269.

22. Prased TK, Anderson MD, Martin BA, Stewart CR: Evidence • • for chilling-induced oxidative stress in maize seedlings and

a regulatory role for hydrogen peroxide. Plant Cell 1994, 6:65-74.

One of the chilling acclimation-responsive (car) genes is shown to encode the mitochondrial catalase-3 isozyme. Hydrogen peroxide levels are elevated during both acclimation and chilling of seedlings. During acclimation, per- oxide signals the induction of antioxidant enzymes such as catalase-3 and guaiacol oxidase.

23. Mittler R, Zilinskas BA: Regulation of pea cytosolic ascorbate • peroxidase and other ant/oxidant enzymes during the

progression of drought stress and following recovery from droughL Plant J 1994, 5:397-405.

The mRNAs for ascorbate peroxidase and cytosolic Cu/Zn superoxide dis- mutase are shown to increase during drought stress and are at higher levels following recovery from drought. Their enzyme activity as well as catalase activity also increases under drought. These enzymes may function in scav- enging hydrogen peroxide which is generated during drought stress.

24. Hiyayama T, Ohto C, Mizoguchi T, Shinozaki K: A gene encoding ,,• a phosphatidylinositol-specific phospholipase C is induced by

dehydration and salt stress in Arabidopsis thaliana. Proc Nat/ Acad Sci USA 1995, 92:3903-390?.

A cDNA for phospholipase C (AtPLC1) is cloned from dehydrated Arabidop- sis plants. The recombinant AtPLC1 protein hydrolyzes phosphatidylinositol 4,5-bisphosphate and this activity is dependent on Ca 2+, as is observed for mammalian phospholipase C proteins. The AtPLC1 gene is induced by dehydration, high salinity and low temperature, which suggests that AtPLC1 functions in the signal transduction pathways under these stress conditions.

25. Urao T, Katagiri T, Mizoguchi T, Yamaguchi-Shinozaki K, • Hayashida N, Shinozaki K: Two genes that encode Ca 2+-

dependent protein kinases are induced by drought and high- salt stresses in Arabidopsis thaliana. Mol Gen Genet 1994, 224:331-340.

Two cDNAs for Ca2+-dependent protein kinases (AtCDPK1 and AtCDPK2) are cloned from dehydrated Arabidopsis plants. The recombinant AtCDPK1

protein revealed Ca2+-dependent protein kinase activity. The AtCDPK1 and AtCDPK2 genes are induced by dehydration and high salinity, but not by low temperature, which suggests that AtPLC1 may function in the signal transduction pathways of dehydration.

26. Mizoguchi T, Irie K, Hirayama T, Hayashida N, Yamaguchi- • • Shinozaki K, Matsumoto K, Shinozaki K: A gene encoding a MAP

kinase kinase kinase is induced simultaneously with genes for a MAP kinase and an $6 kinase by touch, cold and water stress in Arabidopsis thaliane. Proc Nat/Acad Sci USA 1996, 93:765-769.

A cDNA for Arabidopsis MAPKKK (AtMEKK1) is cloned, which could com- plement the yeast stel 1 mutant. The ATMEKK1 gene is induced significantly not only by cold, high salinity and dehydration, but also by touch. Genes encoding MAPK and ribosomal $6 kinase are simultaneously induced by the same stimuli, which suggests that MAPK cascade may be involved in signal transduction pathways under these stress conditions.

2?. Holappa LD, Walker-Simmons MK: The wheat abscisic acid- - responsive protein kinase mRNA, PKABA1, is up-regulated

by dehydration, cold tamparature, and osmotic stress. Plant Physiol 1995, 108:1203-1210.

An ABA-inducible gene for a protein kinase (PKABA1) is also induced by dehydration, cold and osmotic stress. High levels of the PKABA 1 mRNA are observed in field-grown plants under cold winter conditions, but not under warm summer conditions, which suggests that PKABA1 functions as part of a general environmental stress response.

28. Hwang I, Goodman HM: An Arabidopsis thaliana root-specific kinase homolog is induced by dehydration, ABA, and NaCl. Plant J 1995, 8:37-43.

29. Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K: An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 1993, 5:1529-1539.

30. Aguan K, Sugawara K, Suzuki N, Kusano T: Low temperature- dependent expression of a rice gene encoding s protein with a leucine-zipper motif. Mol Gen Genet 1993, 240:1-8.

31. Kusano T, Berberich T, Harada M, Suzuki N, Sugawara K: A • maize DNA-blnding factor with a bZlP motif is induced by low

temperature. Mol Gen Genet 1995, 248:507-517. Themlip15 gene encoding a bZIP DNA-binding factor is induced by cold, high salinity and ABA, but not by heat and drought. The tuLIP15 protein can bind to the h/stone hexamer motif, ACGTCA, and the promoter region of the cold-inducible maize adhl gene, which suggests that the mLIP15 protein may control some cold-inducible genes in maize.

32. Guerrero FD, Jones JT, Mullet JE: Turgor-responsive gene trancription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Me/Bio/1990, 14:11-26.

33. Yamaguchi-Shinozaki K, Koizumi K, Urao S, Shinozaki K: Molecular cloning and characterization of 9 cDNAs for genes that are responsive to desiccation in Arabidopsis thaliana: sequence analysis of one cDNA clone encodes a putative transmembrane channel protein. Plant Cell Physio/1992, 33:217-224.

34. Nordin K, Heine P, Palva ET: Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh. Plant Mo/ Bio/1991, 16:1061-1071.

35. Gilmour S J, Thomashow MF: Cold acclimation and cold- regulated gene expression in ABA mutants of Arabidopsis thaliana. Plant Mol Biol 1991, 17:1 233-1240.

36. Yamaguchi-Shinozaki K, Shinozaki K: Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Mol Gen Genet 1993, 236:331-340.

37. Gosti F, Bertauche N, Vartanian N, Giraudat J: Abscisic acid- dependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana. Mo/ Gen Genet 1995, 246:10-18.

38. Yamaguchi-Shinozaki K, Shinozaki K: The plant hormone abscisic acid mediated the drought-induced expression but not the seed-specific expression of rd22, a gene responsive to dehydration stress in Arabidopsis theliana. Mo/ Gen Genet 1993, 238:1 ?-25.

39. Iwasaki 1", Yamaguchi-Shinozaki K, Shinozaki K: identification of a • ds-regulatory region of a gene in Arabidopsis thaliana whose

induction by dehydration is mediated by abscisic acid and requires protein synthesis. Mol Gen Genet 1995, 247:391-398.

Page 7: Molecular responses to drought and cold stress

Molecular responses to drought and cold stress Shinozaki and Yamaguchi-Shinozaki 167

A promoter region of a dehydration-responsive and ABA-responsive rd22 gene is analyzed in transgenic tobacco. A 67 bp fragment contains cis-regu- latory regions that control dehydration-responsive and ABA-responsive gene expression. A novel cis-acting element other than ABRE probably functions in the ABA-responsive expression of rd22.

40. Wilhelm KS, Thomashow MF: Arabidopsis theliana cor15b, a homologue of cor15a, Is strongly responsive to cold and ABA, but not drought. Plant Mol Bio11993, 23:1073-1077.

41. Monroy AF, Dhindsa RS: Low-temperature signal transduction: ** induction of cold acclimation-specific genes of alfalfa by

calcium at 25"C. Plant Cell 1995, 7:321-331. The relationship between Ca 2+ influx and accumulation of cold acclimation- specific (cas) genes of alfalfa is analyzed. Ca 2+ chelators or Ca 2+ channel blockers inhibit the induction of cas genes at 4"C, whereas Ca 2+ ionophore or Ca 2+ channel agonist causes the influx of extracellular Ca 2+ and induces the expression of cas genes at 25"C. These results suggest that cold- induced Ca 2+ influx plays an essential role in cold acclimation. Two se- quences corresponding to Ca2+-dependent protein kinases are isolated, one of which is induced by cold stress.

42. I_~ng V, M~ntyl~ E, Welin B, Sundberg B, Palva ET: Alterations in water status, endogenous abscisic acid content, and expression of rab18 gene during the development of freezing tolerane in Arabidopsis thaliana. Plant Physiol 1994, 104:1341-1349.

43. Shen CI, Ho THD: Functional dissection of an abscisic acid • . (ABA)-inducible gene reveals two independent ABA-responsive

complexes each containing a G-box and a novel c/s-acting element. Plant Cell 1995, 7:295-307.

Promoter analysis of barley ABA-inducible HVA22 gene shows that ABRE3 (TGTACGTGGC) is necessary, but not sufficient, for an ABA response, and a novel coupling element (CE1 ; TGCCACCGG) is required to form an ABA response complex with ABRE3. The interaction between G-box sequences and coupling element type sequences is thought to determine the specificity in ABA-responsive, light-responsive, and coumaric acid responsive expres- sion.

44. Nelson D, Salamini F, Barrels D: Abscisic acid promotes novel • DNA-binding activity to a desiccation-related promoter of

Craterostigma plentagineum. Plant J 1994, 5:451-458. Two types of DNA binding activity, detected in nuclear proteins from a toler- ant callus, are shown to interact with the promoter regions of ABA-inducible and desiccation-inducible CDeT27-45 gene. Formation of two out of the four protein-DNA complexes is increased by ABA treatment and are prevented by protein synthesis inhibitors, whereas the formation of the others remains constant during the treatments. DNA footprint analysis reveals a novel mo- tif, other than ABRE, that is required for ABA-responsive and desiccation- responsive expression.

45. Nordin K, Vahala T, Palva E-T: Differential expression of two related, low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh. Plant Mol Bio/1993, 21:641-653.

46. Horvath DP, McLarney BK. Thomashow MF: Regulation of Arabidopsis theliana L.(Heyn) cor78 in response to low temperature. Plant Physic)/1 g93, 103:1047-1053.

47. Wang H, Datla R, Georges F, Loewen M, Cuter A J: Promoters from kin1 and cor6.6, two homologous Arabidopsis thai/aria genes: transcriptional regulation and gene expression induced by low temperature, ABA osmoticum and dehydration. Plant Mol Biol 1995, 28:605-617.

48. Baker SS, Wilhelm KS, Thomashow MF: The 5'-region of Arabidopsis thaliana cor 15a has c/s-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Mol Bio11994, 24:701-713.

49. White TC, Simmonds D, Donaldson P, Singh J: Regulation of BN115, a low-temperature-responsive gene from winter Brass/ca napus. Plant Physiol 1994, 106:917-928.

50. Giraudat J: Abscisic acid signaling. Curr Opin Cell Bio/1995, 7:232-238.

51. Parcy F, Valon C, Raynal M, Gaubier-Comella P, Delseny M, Giraudat J: Regulation of gene expression programs during Arabidopsis seed development: roles of the AB13 locus and of endogenous abscisic acid. Plant Cell 1 gg4, 6:1567-1582.

52. Hattori 1", Terada 1", Hamasuna S: Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1 : analysis

of c/s-acting promoter elements required for regulation by abscisic acid and VP1. Plant J 1995, 7:913-925.

53. Vasil V, Marcotte WR, Rosenkrans L, Cocciolone SM, Vasil IK, Guatrano RS, McCarty DR: Overlap of viviparous1 (VP1) and abscisic acid response elements in the Em promoter: G-box elements are sufficient but not necessary for VP1 transact/vat/on. Plant Cell 1 g95, 7:1511-1518.

54. Leung J, Bouvier-Durand M, Moris PC, Guerrier D, Chefdor F, e. Giraudat J: Arabidopsis ABA response gene abil: features

of a calcium-modulated protein phosphatase. Science 1994, 264:1448-1452.

Reports the cloning of the Arabidopsis abil gene by chromosome walk- ing and functional analyses in transgenic plants. The abil gene encodes a putative serine/threonine phosphatase 2C that contains a putative EF-hand structure.

55. Meyer K, Leube MP, Grill E: A protein phosphatase 2C involved ** in ABA signal transduction in Arabidopsis thaliana. Science

1 g94, 264:1452-1455 . Reports findings similar to those in [54"'].

56. Armstrong F, Leung J, Grabov A, Brearley J, Giraudat J, Blatt MR: • Sensitivity to abscisic acid of guard-cell K + channels is

suppressed by abil-1, a mutant Arebidopsis gene encoding a putative protein phosphatase. Proc Nat/Acad Sci USA 1995, 92:9520-9524.

ABA modulates the activities of K + and anion channels in guard cells. Trans- genic tobacco that has been transformed with abil-I dominant mutant gene shows wilty mutant phenotypes. Voltage clamp analysis is used to monitor the influence of the abil-1 mutation on K + and anion channels in guard cells of the transgenic tobacco. The results implicate ABI1 as part of a phosphatase/kinase pathway that modulates the sensitivity of K + channels to ABA-evoked signal cascades.

57. JariUo JA, Capel J, Leyva A, Martnez-Zapater JM, Salinas J: Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators. Plant Mol B/el 1994, 25:693-704.

58. Chen Z, Fu H, Liu D, Chang PFL, Narasimhan M, Ferl R, Hasegawa PM, Bressan RA: A NaCI-regulated plant gene encoding a brain protein homolog that activates ADP ribosyltransferase and inhibits protein kinase C. Plant J 1994, 6:729-740.

59. Mizoguchi T, Hayashida N, Yamaguchi-Shinozaki K, Kamada H, Shinozaki K: ATMPKs: a gene family of plant MAP kinases in Arabidopsis thaliena. FEBS Lett 1993, 336:440-444.

60. Mizoguchi T, Hayashida N, Yamaguchi-Shinozaki Y, Kamada H, Shinozaki K: Two genes that encode ribosomal-protein S6 kinase homologs are induced by cold or salinity stress in Arabiddopsis thaliana. FEBS Left 1995, 358:199-204.

61. Vigh L, Los D, Horvath I, Murata N: The primary signal in biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of desA gene in Synechocystis PCC6803. Proc Nat/Acad Sci USA 1993, 90:9090-9094.

52. Maeda T, Wurgler-Murphy SM, Salto H: A two-component *. system that regulates an osmosensing MAP kinase cascade

in yeast. Nature 1994, 396:242-245. This paper and [63 °°] describe osmosensors and signal transduction cas- cades in yeast at high osmolarity. These authors show that a two-component system, composed of the transmembrane sensor histidine kinase Sin 1 p and the cytoplasmic regulator Sskl p, functions as an osmosensor that regulates the PBS2-HOG1 MAPK cascade.

63. Maeda T, Takehara M, Saito H: Activation of yeast PBS2 MAPKK ** by MAPKKKs or by binding of an SH3-containing osmosensor.

Science 1995, 269:554-558. Demonstrates that Pbs2p MAPKK is activated by two independent signals that emanate from distinct osmosensors. Pbs2p is activated by the MAP- KKKs Ssk2p/Ssk22p that are under the control of the SLN1/BSK1 two- component osmosensor. Pbs2p is also activated by putative transmembrane osmosensor Sholp by a mechanism of protein-protein interaction at the SH3 domain.

64. Ecker JR: The ethylene signal transduction pathway in plants. Science 1995, 268:667-675.