Recently, a major transcription system that controls abscisic-acid-independent gene expression in response to dehydration and low temperature has been identified. The system includes the DRE/CRT (dehydration-responsive element/C-repeat) cis-acting element and its DNA-binding protein, DREB/CBF (DRE-binding protein/C-repeat binding factor), which has an AP2 domain. DREB/CBF contains two subclasses, DREB1/CBF and DREB2, which are induced by cold and dehydration, respectively, and control the expression of various genes involved in stress tolerance. Recent studies are providing evidence of differences between dehydration-signaling and cold-stress-dehydration-signaling cascades, and of cross-talk between them.
Addresses
*Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; e-mail: [email protected]
†Biological Resources Division, Japan International Research Center
for Agricultural Sciences (JIRCAS), Ministry of Agriculture, Forestry and Fisheries, 2-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan; e-mail: [email protected]
Current Opinion in Plant Biology2000, 3:217–223 1369-5266/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved. Abbreviations
ABA abscisic acid aba ABA-deficient abi ABA-responsive ABRE ABA-responsive element
ATHK1 ARABIDOPSIS TWO-COMPONENT HISTIDINE KINASE bp base pairs
CaMV cauliflower mosaic virus CBF C-repeat-binding factor cor cold-regulated CRT C-repeat
DRE dehydration-responsive element DREB DRE-binding protein
erd early responsive to dehydration
EREBP ethylene-responsive element binding protein
HOS HIGH EXPRESSION OF OSMOTICALLY SENSITIVE kin cold-inducible
lti low-temperature induced rd responsive to dehydration sfr sensitivity to freezing
Introduction
Among abiotic environmental stresses, drought and low temperature affect plant growth most seriously. Plants respond to dehydration and low temperature with a num-ber of physiological and developmental changes. Molecular and cellular responses to these stresses have been analyzed extensively at the biochemical level: vari-ous kinds of proteins and smaller molecules, including sugars, proline, and glycine betaine, accumulate; in addi-tion, many genes are induced by both dehydration and
cold, but some respond either only to drought or only to cold. These observations suggest the existence of several cellular signal transduction pathways between the percep-tion of stress signals and gene expression.
Drought and high salinity cause plants to produce high lev-els of ABA; exogenous application of ABA also induces a number of genes that respond to dehydration and cold stress [1]. Nevertheless, the role of ABA in low-tempera-ture-responsive gene expression is not clear. Several reports have described genes that are induced by dehydra-tion and low temperature but that do not respond to exogenous abscisic acid (ABA) treatment [1–4]. It is likely, therefore, that both ABA-independent and ABA-depen-dent signal transduction cascades exist [1,4]. One of the transcription systems that function independently of ABA in both dehydration- and low-temperature-responsive gene expression has recently been analyzed extensively (for reviews see [1,5,6]).
In 1994, we indentified a cis-acting dehydration-responsive element (DRE) [7]. A similar cis-acting element has also been reported and named C-repeat (CRT) [8] or ‘low-tem-perature-responsive element’ [9]. The DRE/CRT element is involved in both dehydration- and low-temperature-responsive gene expression. Our short review focuses on roles of the DRE/CRT cis-element and its DNA-binding protein, DREB/CBF (DRF-binding protein/C-repeat-binding factor), in the separation of and cross-talk between two stress signals that are involved in stress-induced gene expression in Arabidopsis. We also discuss the role of ABA in dehydration- and cold-induced gene expression. ABA plays important roles in slow and adaptive responses involving dehydration-induced gene expression. However, ABA seems not to be important in cold-induced gene expression and does not accumulate in response to low temperature. ABA does, however, have important roles in slow adaptive processes during dehydration stress.
Similar genes are induced by dehydration and
cold stress
A variety of genes are induced by both dehydration and low temperature, and their mRNA levels are subsequently reduced by release from stress conditions. This suggests that similar biochemical processes function in dehydra-tion- and cold-stress responses. Genes induced in plants that are subjected to these stresses are thought to function not only in protecting cells by producing important meta-bolic proteins and cellular protectants, but also in regulating genes that are involved in transducing the stress response signal [1,2,10,11]. In Arabidopsis, these genes includerd(responsive to dehydration), erd(early responsive to dehydration), cor(cold-regulated), lti(low-temperature induced),
differences and cross-talk between two stress signaling pathways
and kin (cold-inducible). This variety of stress-inducible genes suggests that the responses of plants to dehydration and cold are complex. Some of the stress-inducible genes are overexpressed in transgenic plants that have enhanced stress tolerance, suggesting that their gene products func-tion in stress tolerance [10–13].
Regulation of gene expression by dehydration
and cold stress
Most dehydration-inducible genes also respond to cold stress, and, conversely, most cold-inducible genes respond to dehydration. Analyses of the expression patterns of genes induced by both dehydration and cold have revealed broad variation in the timing of their induction and differ-ences in their responsiveness to ABA [4]. Many of the genes that are induced by exogenous ABA treatment are also induced by cold or dehydration in ABA-deficient (aba) or ABA-insensitive (abi) Arabidopsis mutants [2]. These observations indicate that these genes are not induced by the accumulation of endogenous ABA, but respond to ABA [1,4]. Several ABA-inducible genes require protein biosyn-thesis for their induction by ABA [4], which suggests that at least two independent pathways signal the expression of stress-induced genes in response to endogenous ABA pro-duction. As shown in Figure 1, at least four independent signal pathways function under drought conditions [4]: two are ABA-independent and two are ABA-dependent. In addition, two ABA-independent pathways are also involved in low-temperature-responsive gene expression [1]. There is a common signal transduction pathway between dehydration and cold stress involving the DRE/CRT cis-acting element, and two additional signal transduction pathways may function only in dehydration or in cold response.
The role of the DRE/CRT
cis
-acting element in
ABA-independent gene expression
In aba or abi mutants, many genes are induced by both dehydration and low temperature; this suggests that these genes do not require ABA for their expression under cold or drought conditions but that they do respond to ABA. Among these genes, the expression of two dehydration-and cold-inducible Arabidopsisgenes, rd29A/lti78/cor78and cor15a, has been analyzed in detail (for reviews see [1,5]). The transcription of rd29Ain abi1and aba1mutants sug-gests that cold- and drought-regulated expression does not require ABA. DRE, a 9-base pair (bp) conserved sequence (i.e. TACCGACAT), is an essential cis-acting element for the regulation of rd29Ainduction in the ABA-independent response to dehydration and cold [7]. Similar motifs, called CRT and low-temperature-responsive element, which include the CCGAC motif that forms the core of the DRE sequence, have been found in the promoter region of cold-inducible genes [8,9].
DREB/CBF transcription factors distinguish
between the dehydration and low temperature
stress-signaling pathways
Protein factors that specifically interact with the 9-bp DRE sequence have been detected in nuclear extracts prepared from either dehydrated or adequately watered Arabidopsis plants [7]. Stockinger et al.[14] first isolated a cDNA clone for a DRE/CRT-binding protein using yeast one-hybrid screening; they named this clone CBF1 (CRT-binding fac-tor 1). In yeast, CBF1 functions as a transcription facfac-tor that upregulates DRE/CRT-dependent transcription. It contains a conserved DNA-binding motif (AP2 domain) that is also found in the EREBP (ethylene-responsive element binding protein) family and AP2 protein, which is involved in floral Figure 1
Cellular signal transduction pathways between the initial drought-stress or cold-stress signal and gene expression in Arabidopsis. There are at least six signal transduction pathways: two (b,c) are ABA-dependent and four
(a,d,e,f) are ABA-independent. Stress-inducible genes rd29A/cor78/lti78, rd29B/lti65, rd22, and erd1have been used to analyse the regulation of gene expression and the signalling process [5,40]. abi1, abi2, and era1are involved in ABA signaling [41–44]. hos5functions in DREB2-related dehydration signaling, and sfr6, hos1, and hos2function in DREB1/CBF-related cold signaling [28••,32–34]. esk1is involved in responses to cold via a DRE-independent process [28••]. Thin and thick arrows
represent the minor and major signalling pathways that are involved in dehydration-responsive gene expression, respectively. Broken arrows represent the signalling pathways that are involved in low temperature stress responses.
ABA
Stress response and stress tolerance sfr1
morphogenesis [15,16]. Independently, Liu et al.[17••] iso-lated five independent cDNAs for DRE/CRT-binding proteins using yeast one-hybrid screening, which they named DREBs (DRE-binding proteins). All of the DREBs also contain a conserved AP2 domain. The five cDNA clones that encode DRE/CRT-binding proteins are classi-fied into two groups, DREB1 and DREB2. The groups contain similar AP2 domains but have low sequence similar-ity outside that domain. There are three DREB1 proteins that are encoded by genes that lie in tandem on chromo-some 4 in the order DREB1B, DREB1A, and DREB1C[18]. DREB1B is identical to CBF1. Gilmour et al.[19] also iso-lated two CBF1 homologues named CBF2 and CBF3. There are two DREB2 proteins, DREB2A and DREB2B [17••]. Both DREB1A and DREB2A bind specifically to DRE/CRT and function as transcriptional activators in plant protoplasts, as well as in yeasts. Expression of the DREB1A/CBF3 gene and its two homologues (i.e. DREB1B/CBF1 and DREB1C/CBF2) is induced by low-temperature stress, whereas expression of the two DREB2 genes is induced by dehydration. These results suggest that the DREB1 proteins are involved in cold-specific gene expression, whereas the DREB2 proteins function in dehy-dration-specific gene expression (Figure 2).
The AP2 domain is found in many plant genes, such as EREBP, APETALA2, AINTEGUMENTA and TINY [16]. EREBPs bind to the ethylene-responsive element (i.e. the GCC box, GCCGCC), whereas DREB/CBFs bind to the DRE/CRT core sequence, PuCCGAC. DRE/CRT and the G box contain PuCCGNC as a common sequence [15,17••]. Liu et al. [17••] showed that DREB/CBF and EREBPs have two different amino acids in the AP2 domain, which may confer different specificity for the DNA-binding of cis-acting elements [17••].
Engineering stress tolerance of transgenics by
overexpressing DREB/CBF
Jaglo-Ottosen et al.[20••] found that overexpressing CBF1, under the control of the CaMV 35S promoter, in transgenic Arabidopsisnot only induced strong expression of corgenes, but also improved freezing tolerance. The growth of these transgenics was similar to that of wild-type plants under normal growth conditions. Liu et al.[17••] and Kasuga et al. [21••] also observed that enhanced expression of the target
cor, rd and erdgenes in transgenic Arabidopsis plants that overexpress DREB1A/CBF3(also under the control of the CaMV 35S promoter) produced dwarfed or growth-retarded phenotypes in unstressed conditions. The DREB1A trans-genic plants also had enhanced freezing and dehydration tolerance. The difference in growth retardation caused by overexpression of DREB1A/CBF3and DREB1B/CBF1may be explained by different levels of expression of the two transgenes or the difference in the genes used. In contrast, overexpression of DREB2A cDNA induced weak expres-sion of the target genes under unstressed conditions and caused slight growth retardation of the transgenic plants [17••]. DREB2 proteins are probably post-transcriptionally activated in dry conditions (Figure 2). These results indi-cate that two independent families of DREBs, DREB1/CBF and DREB2, function as trans-acting factors in two separate signal-transduction pathways under cold and dry conditions, respectively (Figure 2).
As discussed above, overproduction of DREB1A/CBF3 cDNAs driven by the 35S CaMV promoter in transgenic plants causes severe growth retardation under normal growth conditions [17••,21••]. Recently Kasuga et al. [21••] found that the DREB1AcDNA driven by the stress-inducible rd29A pro-moter was expressed at a low level in unstressed control conditions and at a high level in plants exposed to
dehydra-A model of the induction of the
rd29A/cor78/lti78gene and cis- and trans -acting elements involved in stress-responsive gene expression. Two cis-acting elements, DRE/CRT and ABRE, are involved in the ABA-independent and ABA-responsive induction of rd29A, respectively. Two different DRE/CRT-binding proteins, DREB1/CBF1 and DREB2, distinguish two different signal transduction pathways in response to cold and drought stresses, respectively [17••]. DRE/CRT-binding proteins contain an AP2 DNA-binding domain, whereas ABRE-binding proteins encode bZIP transcription factors. Thick broken arrows represent a cold signalling pathway. Solid thick arrows and thin broken arrows represent an ABA-independent and an ABA-dependent signalling pathway, respectively, that are involved in the dehydration response.
Modification?
rd29A promoter Cis elements Transcription
Trans elements Signal perception Transduction
DREB1/CBF genes
DREB2 genes ABA signaling
bZIP ABA biosynthesis ABA
independent
CBF/DREB1 DREB2
ABA independent Temperature change
Low temperature
Osmotic change Dehydration
TATA
DRE/CRT ABRE
tion, salt, and cold stresses. The rd29Apromoter minimized the negative effects on the growth of the transgenic plants. Moreover, this stress-inducible promoter enhanced tolerance of drought, salt, and freezing to a greater extent than did the CaMV 35S promoter. The rd29Apromoter::DREB1Asystem is a self-amplifying system that overexpresses DREB1A pro-tein throughout exposure to stress. Greater expression of the DREB1A protein in the rd29A::DREB1Atransgenics results in greater expression of the target genes involved in stress tol-erance [21••]. This system provides some promise for engineering multi-stress tolerance of transgenic crops because plants such as tobacco, Brassica, and rice, have simi-lar transcription systems to that of Arabidopsis.
ABA in dehydration and low-temperature
stress response
In many plants, endogenous ABA levels increase significant-ly in conditions of drought and high-salinity [2–4]. In Arabidopsis, however, ABA levels increase only transiently in response to low-temperature stress before returning to their basal level [1,22]. Many drought- and cold-stress-inducible genes are induced by exogenous ABA treatment. These genes contain potential ABA-responsive elements (ABREs; PyACGTGGC) in their promoter regions [1,2]. In Arabidopsis, the rd29B(or lti65) gene is induced by dehydra-tion and high salinity, but not by cold stress [23]. rd29Bdoes not contain a DRE/CRT but contains two ABREs in its pro-moter [7]. It is controlled downstream of the abi1and aba1 mutations and so endogenous ABA that accumulates in response to dehydration induces its expression. During cold stress, endogenous ABA is not sufficient to induce rd29B. We therefore believe that the ABA-signaling pathway is not important in cold-stress responses. The endogenous ABA accumulation that has been observed during winter may be attributable to the dehydration of plants, which induces
DRE/CRT-dependent gene expression of most cor, lti, and rdgenes to confer stress tolerance (Figure 3).
The rd29A promoter contains not only DRE but also an ABRE (Figure 2). An ABRE cis-acting element and bZIP transcription factors function in ABA-responsive gene expression [7]. The rd29Agene is therefore controlled by three independent regulatory systems [7,17••]. These results indicate that complex molecular responses to vari-ous environmental stresses may be mediated by both complex regulatory systems of gene expression and signal transduction, and by cross-talk between these systems. The bZIP/ABRE system seems to function after the accumula-tion of endogenous ABA in drought condiaccumula-tions (Figure 3).
The biosynthesis of novel protein factors is necessary for the expression of inducible genes in one of the two ABA-dependent pathways (Figure 1). The induction of the Arabidopsis drought-inducible gene rd22 is mediated by ABA and requires protein biosynthesis for its ABA-depen-dent expression [24]. MYC and MYB recognition sequences are essential for the ABA- and drought-responsive expres-sion of rd22, and ABA-inducible MYC and MYB proteins may function cooperatively in the ABA-dependent expres-sion of rd22 [25,26]. This MYC/MYB system may also function in a slow and adaptive stress response process. The different timing of the induction of stress-inducible genes may be explained by the different regulatory systems that function in their promoters, such as DRE/CRT, ABRE, and MYB/MYC (Figure 3).
Genetic analysis of signal transduction in
response to dehydration and cold stress
Many Arabidopsismutants that are either sensitive to or tol-erant of freezing have been isolated, and their phenotypes Figure 3
ABA biosynthesis
Gene expression
Signal perception Rapid and emergency response Slow and adaptive response Protein synthesis
MYC/MYB system Gene expression bZIP–ABRE system Gene expression
Dehydration
Time course Low temperature
DREB2–DRE system CBF–CRT
(DREB1–DRE) system
Current Opinion in Plant Biology
Molecular responses to dehydration and low temperature based on stepwise gene expression. The regulation of DREB/CBF genes in response to dehydration and low temperature occurs early in the stress response. DRE/CRT-dependent transcription follows the response.
freezing-sensitive (sensitivity to freezing [sfr]) mutants and mapped their positions on Arabidopsis chromosomes. Knight et al.[28••] analyzed the expression of kin1, cor15a, and cor78/rd29A, which contain DRE/CRT in their pro-moters. In the sfr6 mutant, unlike wild-type Arabidopsis plants, these genes were not strongly induced in response to osmotic stress and low temperature. In contrast, ATP5CS, CBF1, CBF2, and CBF3, which do not contain DRE/CRT in their promoters, were not affected in the sfr6 mutant. The SFR6 product may be involved upstream of CBF/DREB1 and function as a positive regulator of this element (Figure 1). A transient increase in cellular calcium ion concentration in responses to dehydration and low temperature seems to stimulate cellular signalling process-es. Nevertheless, evidence from calcium measurement in cold conditions suggests that calcium signalling may not be involved in sfr6 signalling.
Xin and Browse [29•] isolated a constitutively freezing-tol-erant mutant named eskimo1 that, without cold acclimatization, has greater freezing tolerance than wild-type plants. The molecular mechanism of the stress tolerance of eskimo1is not known, but proline accumulates in the eskimo1mutant [29•]. This proline may be involved in stress tolerance as proline functions in osmoprotection, in detoxication of active oxygen, and in protection of pro-teins and nucleic acids. Recently, Nanjo et al.[30] showed that the accumulation of proline in transgenics with anti-sense cDNA for proline dehydrogenase provides strong freezing tolerance as well as salt tolerance. In eskimo1, the expression of cor15a, cor47/rd17, and cor78/rd29A remain low under normal growth conditions. This suggests that ESKIMO1 functions in a different signal transduction pathway from the DRE/CRT system (Figure 1).
Genetic analysis of Arabidopsismutants with the rd29A pro-moter::luciferase transgene suggests complex signaling pathways in drought-, salt-, and cold-stress responses. Ishitani et al.[31] isolated mutants that overexpressed rd29A or repressed it in response to dehydration, high salinity, cold and ABA. They propose that the ABA- and stress-signaling pathways are not independent and that the various stress-sig-naling pathways, including ABA-independent and ABA-dependent pathways, are not completely independent. Xiong et al.[32] isolated a hos5mutant that has increased expression of rd29Awhen under osmotic stress but not when experiencing cold stress. Genetic analysis showed that HOS5 is a negative regulator of osmotic-stress-responsive gene expression. Ishitani et al.[33] and Lee et al.[34] isolated hos1 and hos2mutants, respectively, that enhanced the expression of rd29A, cor47, cor15a, and kin1. Non-acclimatized hos1and hos2 mutants were less cold-hardy than wild-type plants. HOS1and HOS2are therefore thought to function as nega-tive regulators of a cold-specific signal transduction pathway (Figure 1). Genetic analysis of these mutants and of stress-resistant or stress-sensitive mutants is likely to provide more information on stress-induced signal transduction [35].
Signal transduction pathways involved in the drought-stress response have been studied in yeast and animal systems [5]. Two-component systems seem to function in sensing osmotic stress in plants as well as in bacteria and yeast [36]. Recently, Urao et al.[37•] isolated an ArabidopsiscDNA that encodes a two-component histidine kinase (ATHK1), which functions as an osmosensor in yeast. ATHK1 has a typical histidine kinase domain, a receiver domain, and two trans-membrane domains in the amino-terminal domain. ATHK1 might function in signal perception during dehydration stress in Arabidopsis, but sensors for cold stress have not yet been identified.
Many genes encoding factors that are involved in signal-transduction cascades are upregulated by dehydration and cold: mitogen-activated protein (MAP) kinases, cal-cium-dependent protein kinases, and enzymes involved in phospholipid metabolism, such as phospholipase C and phosphatidyl-4, 5-phosphate 5-kinase (PIP5) kinase [5,38,39]. These signaling factors might be involved in the amplification of stress signals and in the adaptation of plant cells to drought-stress conditions. No direct evi-dence has, however, been obtained of the functions of these signaling molecules. Transgenic plants that modify the expression of these genes and mutants with disrupt-ed genes will provide more information on the function of their gene products.
Conclusions and perspectives
A major transcription system regulating ABA-indepen-dent gene expression in response to dehydration and cold stress includes a DRE/CRT cis-acting element and its DNA-binding protein, DREB/CBF. The DREB/CBF family of proteins contain two subclasses, DREB1/CBF and DREB2, which are induced by cold and dehydra-tion, respectively, to express various genes involved in stress tolerance. Cross-talk between dehydration and cold occurs at the transcriptional level. ABA plays impor-tant roles in the dehydration-stress response but not, it seems, in the cold-stress response. Genetic analysis of stress-resistant or stress-sensitive mutants, and mutants with the rd29A promoter::luciferase transgene, should provide more information on signal transduction in response to dehydration and cold stress.
Acknowledgements
Our work is supported in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences and the Special Coordination Fund of Science and Technology Agency of Japan.
References and recommended reading
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