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217
Molecular responses to dehydration and low temperature:
differences and cross-talk between two stress signaling pathways
Kazuo Shinozaki* and Kazuko Yamaguchi-Shinozaki†
Recently, a major transcription system that controls abscisicacid-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 dehydrationsignaling and cold-stress-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 Biology 2000, 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 number of physiological and developmental changes.
Molecular and cellular responses to these stresses have
been analyzed extensively at the biochemical level: various kinds of proteins and smaller molecules, including
sugars, proline, and glycine betaine, accumulate; in addition, 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 perception of stress signals and gene expression.
Drought and high salinity cause plants to produce high levels 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-temperature-responsive gene expression is not clear. Several
reports have described genes that are induced by dehydration 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-dependent 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-temperature-responsive element’ [9]. The DRE/CRT element
is involved in both dehydration- and low-temperatureresponsive 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-repeatbinding 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 dehydration- 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 metabolic 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
include rd (responsive to dehydration), erd (early responsive to
dehydration), cor (cold-regulated), lti (low-temperature induced),
218
Physiology and metabolism
Figure 1
Dehydration
Signal
perception
Low temperature
Temperature
change
Osmotic
change
?
?
aba
Signal
transduction
Transcription
factor
ABA
?
(a)
MYC/MYB
(b)
abi1, abi2
era1
bZip
(c)
?
hos5
? sfr1
hos1
hos2
DREB2 CBF/DREB1
(d)
esk1
?
(e)
Cis element
?
MYCR/MYBR
ABRE
DRE/CRT
Gene
expression
erd1
rd22
rd29B
rd29A/lti78, cor15a
(f)
?
Stress response and stress tolerance
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. Stressinducible genes rd29A/cor78/lti78,
rd29B/lti65, rd22, and erd1 have been used
to analyse the regulation of gene expression
and the signalling process [5,40]. abi1, abi2,
and era1 are involved in ABA signaling
[41–44]. hos5 functions in DREB2-related
dehydration signaling, and sfr6, hos1, and
hos2 function in DREB1/CBF-related cold
signaling [28••,32–34]. esk1 is 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 dehydrationresponsive gene expression, respectively.
Broken arrows represent the signalling
pathways that are involved in low temperature
stress responses.
Current Opinion in Plant Biology
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 function 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 differences 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 biosynthesis 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 production. 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 dehydrationand cold-inducible Arabidopsis genes, rd29A/lti78/cor78 and
cor15a, has been analyzed in detail (for reviews see [1,5]).
The transcription of rd29A in abi1 and aba1 mutants suggests 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 rd29A induction 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 coldinducible 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 factor 1). In yeast, CBF1 functions as a transcription factor 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
Molecular responses to dehydration and low temperature Shinozaki and Yamaguchi-Shinozaki
219
Figure 2
A model of the induction of the
rd29A/cor78/lti78 gene and cis- and transacting 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.
Low temperature
Dehydration
Temperature change
Osmotic change
Signal perception
Transduction
ABA
independent
ABA
independent
ABA biosynthesis
Transcription
DREB1/CBF DREB2 genes
Modification?
genes
Trans elements
rd29A promoter
Cis elements
CBF/DREB1
DREB2
DRE/CRT
ABA signaling
bZIP
ABRE
TATA
Current Opinion in Plant Biology
morphogenesis [15,16]. Independently, Liu et al. [17••] isolated 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 classified into two groups, DREB1 and DREB2. The groups
contain similar AP2 domains but have low sequence similarity outside that domain. There are three DREB1 proteins
that are encoded by genes that lie in tandem on chromosome 4 in the order DREB1B, DREB1A, and DREB1C [18].
DREB1B is identical to CBF1. Gilmour et al. [19] also isolated 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 lowtemperature 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 dehydration-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
Arabidopsis not only induced strong expression of cor genes,
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 erd genes 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 transgenic plants also had enhanced freezing and dehydration
tolerance. The difference in growth retardation caused by
overexpression of DREB1A/CBF3 and DREB1B/CBF1 may
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 expression 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 indicate 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 DREB1A cDNA driven by the stress-inducible rd29A promoter was expressed at a low level in unstressed control
conditions and at a high level in plants exposed to dehydra-
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Physiology and metabolism
Figure 3
Low temperature
CBF–CRT
(DREB1–DRE) system
Gene expression
DREB2–DRE system
Dehydration
ABA biosynthesis
bZIP–ABRE system
Gene expression
Protein synthesis
MYC/MYB system
Gene expression
Time course
Signal perception
Rapid and emergency response
Slow and adaptive response
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.
ABA biosynthesis is induced by dehydration and activates two
regulatory ABA-dependent gene expression systems. One is the
bZIP/ABRE system; the other is the MYC/MYB system, which requires
do novo production of the MYB and MYC proteins in response to ABA.
tion, salt, and cold stresses. The rd29A promoter 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 rd29A promoter::DREB1A system
is a self-amplifying system that overexpresses DREB1A protein throughout exposure to stress. Greater expression of the
DREB1A protein in the rd29A::DREB1A transgenics results
in greater expression of the target genes involved in stress tolerance [21••]. This system provides some promise for
engineering multi-stress tolerance of transgenic crops
because plants such as tobacco, Brassica, and rice, have similar transcription systems to that of Arabidopsis.
DRE/CRT-dependent gene expression of most cor, lti, and
rd genes to confer stress tolerance (Figure 3).
ABA in dehydration and low-temperature
stress response
In many plants, endogenous ABA levels increase significantly 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 dehydration and high salinity, but not by cold stress [23]. rd29B does
not contain a DRE/CRT but contains two ABREs in its promoter [7]. It is controlled downstream of the abi1 and 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
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 rd29A gene is therefore controlled by
three independent regulatory systems [7,17••]. These
results indicate that complex molecular responses to various 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 accumulation of endogenous ABA in drought conditions (Figure 3).
The biosynthesis of novel protein factors is necessary for the
expression of ABA-inducible genes in one of the two ABAdependent pathways (Figure 1). The induction of the
Arabidopsis drought-inducible gene rd22 is mediated by
ABA and requires protein biosynthesis for its ABA-dependent expression [24]. MYC and MYB recognition sequences
are essential for the ABA- and drought-responsive expression of rd22, and ABA-inducible MYC and MYB proteins
may function cooperatively in the ABA-dependent expression 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 Arabidopsis mutants that are either sensitive to or tolerant of freezing have been isolated, and their phenotypes
Molecular responses to dehydration and low temperature Shinozaki and Yamaguchi-Shinozaki
have been analyzed in detail. Warren et al. [27] isolated five
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 promoters. 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 processes. 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-tolerant mutant named eskimo1 that, without cold
acclimatization, has greater freezing tolerance than wildtype plants. The molecular mechanism of the stress
tolerance of eskimo1 is not known, but proline accumulates
in the eskimo1 mutant [29•]. This proline may be involved
in stress tolerance as proline functions in osmoprotection,
in detoxication of active oxygen, and in protection of proteins and nucleic acids. Recently, Nanjo et al. [30] showed
that the accumulation of proline in transgenics with antisense 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 Arabidopsis mutants with the rd29A promoter::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-signaling pathways, including ABA-independent and
ABA-dependent pathways, are not completely independent.
Xiong et al. [32] isolated a hos5 mutant that has increased
expression of rd29A when 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 hos2 mutants, respectively, that enhanced the expression
of rd29A, cor47, cor15a, and kin1. Non-acclimatized hos1 and
hos2 mutants were less cold-hardy than wild-type plants.
HOS1 and HOS2 are therefore thought to function as negative regulators of a cold-specific signal transduction pathway
(Figure 1). Genetic analysis of these mutants and of stressresistant or stress-sensitive mutants is likely to provide more
information on stress-induced signal transduction [35].
221
Signal perception and signal transduction
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 Arabidopsis cDNA 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 transmembrane 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 signaltransduction cascades are upregulated by dehydration
and cold: mitogen-activated protein (MAP) kinases, calcium-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 evidence has, however, been obtained of the functions of
these signaling molecules. Transgenic plants that modify
the expression of these genes and mutants with disrupted genes will provide more information on the function
of their gene products.
Conclusions and perspectives
A major transcription system regulating ABA-independent 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 dehydration, respectively, to express various genes involved in
stress tolerance. Cross-talk between dehydration and
cold occurs at the transcriptional level. ABA plays important 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.
Sequencing of the Arabidopsis genome will be completed
by the end of 2000, which means that the structure of all
25,000 Arabidopsis genes will soon be determined. All
stress-inducible genes can then be identified by the systematic analysis of gene expression in microarrays. In the
next decade, we think it important to develop novel methods to analyze the complex networks that control the stress
responses of higher plants. A reverse genetic approach, as
well as classical forward genetics, will become more important for understanding not only the functions of
stress-inducible genes but also the complex signaling
processes of the dehydration- and cold-stress responses.
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Physiology and metabolism
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
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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EREBP/AP2 DNA binding domain, separate two cellular signal
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Warren G, McKown R, Martin AL, Teutonico R: Isolation of mutations
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•• Arabidopsis suppresses low-temperature induction of gene
dependent on the CRT/DRE sequence motif. Plant Cell 1999,
11:875-886.
Genetic and molecular analysis of freezing-sensitive mutants indicated that
the SFR6 product may be involved upstream of the CBF/DREB1 system
and function as a positive regulator. This mutant is useful in analyzing the signaling cascade that controls the CBF/DREB1 system.
29. Xin Z, Browse J: eskimo1 mutants of Arabidopsis are constitutively
•
freezing-tolerant. Proc Natl Acad Sci USA 1998, 95:7799-7804.
The authors report the freezing-tolerance (without cold acclimatization) of
the eskimo1-IMO-mutant. ESK1 functions in a different signaling process
from that of the DRE/CRT system. The accumulation of proline in eskimo1
suggests an important role for proline in freezing tolerance.
30. Nanjo T, Kobayashi M, Yoshiba Y, Kakubari Y, Yamaguchi-Shinozaki K,
Shinozaki K: Antisense suppression of proline degradation
improves tolerance to freezing and salinity in Arabidopsis
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31. Ishitani M, Xiong L, Stevenson B, Zhu JK: Genetic analysis of
osmotic and cold stress signal transduction in Arabidopsis:
interactions and convergence of abscisic acid-dependent and
abscisic acid-independent pathways. Plant Cell 1997, 9:1935-1949.
32. Xiong L, Ishitani M, Lee H, Zhu JK: HOS5—a negative regulator of
osmotic stress-induced gene expression in Arabidopsis thaliana.
Plant J 1999, 19:569-578.
33. Ishitani M, Xiong L, Lee H, Stevenson B, Zhu JK: HOS1, a genetic
locus involved in cold-responsive gene expression in Arabidopsis.
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gene expression and freezing tolerance in an Arabidopsis
thaliana mutant. Plant J 1999, 17:301-308.
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Urao T, Yakubov B, Satoh R, Yamaguchi-Shinozaki K, Seki M,
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1999, 11:1743-1754.
The first report of the osmosensor histidine kinase ATHK1 in Arabidopsis.
ATHK1 has similar structure and functions to those of yeast osmosensor
223
Sln1. This evidence suggests an important role for the two-component system in osmotic stress perception.
38. Mizoguchi T, Ichimura K, Shinozaki K: Environmental stress
response in plants: the role of mitogen-activated protein kinases
(MAPKs). Trends Biotechnol 1997, 15:15-19.
39. Mikami K, Katagiri T, Iuchi S, Yamaguchi-Shinozaki K, Shinozaki K:
A gene encoding phosphatidylinositol-4-phosphate 5-kinase is
induced by water stress and abscisic acid in Arabidopsis thaliana.
Plant J 1998, 15:563-568.
40. Nakashima K, Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K:
A nuclear gene, erd1, encoding a chloroplast-targeted Clp
protease regulatory subunit homolog is not only induced by water
stress but also developmentally up-regulated during senescence
in Arabidopsis thaliana. Plant J 1997, 12:851-861.
41. Gosti F, Beaudoin N, Serizet C, Webb AA, Vartanian N, Giraudat J:
ABI1 protein phosphatase 2C is a negative regulator of abscisic
acid signaling. Plant Cell 1999, 11:1897-1910.
42. Pei ZM, Ghassemian M, Kwak CM, McCourt P, Schroeder JI: Role of
farnesyltransferase in ABA regulation of guard cell anion
channels and plant water loss. Science 1999, 282:287-290.
43. Grill E, Himmelbach A: ABA signal transduction. Curr Opin Plant
Biol 1998, 1:412-418.
44. Bonetta D, McCourt P: Genetic analysis of ABA signal transduction
pathways. Trends Plant Sci 1998, 6:231-235.
Molecular responses to dehydration and low temperature:
differences and cross-talk between two stress signaling pathways
Kazuo Shinozaki* and Kazuko Yamaguchi-Shinozaki†
Recently, a major transcription system that controls abscisicacid-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 dehydrationsignaling and cold-stress-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 Biology 2000, 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 number of physiological and developmental changes.
Molecular and cellular responses to these stresses have
been analyzed extensively at the biochemical level: various kinds of proteins and smaller molecules, including
sugars, proline, and glycine betaine, accumulate; in addition, 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 perception of stress signals and gene expression.
Drought and high salinity cause plants to produce high levels 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-temperature-responsive gene expression is not clear. Several
reports have described genes that are induced by dehydration 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-dependent 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-temperature-responsive element’ [9]. The DRE/CRT element
is involved in both dehydration- and low-temperatureresponsive 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-repeatbinding 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 dehydration- 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 metabolic 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
include rd (responsive to dehydration), erd (early responsive to
dehydration), cor (cold-regulated), lti (low-temperature induced),
218
Physiology and metabolism
Figure 1
Dehydration
Signal
perception
Low temperature
Temperature
change
Osmotic
change
?
?
aba
Signal
transduction
Transcription
factor
ABA
?
(a)
MYC/MYB
(b)
abi1, abi2
era1
bZip
(c)
?
hos5
? sfr1
hos1
hos2
DREB2 CBF/DREB1
(d)
esk1
?
(e)
Cis element
?
MYCR/MYBR
ABRE
DRE/CRT
Gene
expression
erd1
rd22
rd29B
rd29A/lti78, cor15a
(f)
?
Stress response and stress tolerance
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. Stressinducible genes rd29A/cor78/lti78,
rd29B/lti65, rd22, and erd1 have been used
to analyse the regulation of gene expression
and the signalling process [5,40]. abi1, abi2,
and era1 are involved in ABA signaling
[41–44]. hos5 functions in DREB2-related
dehydration signaling, and sfr6, hos1, and
hos2 function in DREB1/CBF-related cold
signaling [28••,32–34]. esk1 is 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 dehydrationresponsive gene expression, respectively.
Broken arrows represent the signalling
pathways that are involved in low temperature
stress responses.
Current Opinion in Plant Biology
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 function 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 differences 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 biosynthesis 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 production. 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 dehydrationand cold-inducible Arabidopsis genes, rd29A/lti78/cor78 and
cor15a, has been analyzed in detail (for reviews see [1,5]).
The transcription of rd29A in abi1 and aba1 mutants suggests 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 rd29A induction 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 coldinducible 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 factor 1). In yeast, CBF1 functions as a transcription factor 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
Molecular responses to dehydration and low temperature Shinozaki and Yamaguchi-Shinozaki
219
Figure 2
A model of the induction of the
rd29A/cor78/lti78 gene and cis- and transacting 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.
Low temperature
Dehydration
Temperature change
Osmotic change
Signal perception
Transduction
ABA
independent
ABA
independent
ABA biosynthesis
Transcription
DREB1/CBF DREB2 genes
Modification?
genes
Trans elements
rd29A promoter
Cis elements
CBF/DREB1
DREB2
DRE/CRT
ABA signaling
bZIP
ABRE
TATA
Current Opinion in Plant Biology
morphogenesis [15,16]. Independently, Liu et al. [17••] isolated 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 classified into two groups, DREB1 and DREB2. The groups
contain similar AP2 domains but have low sequence similarity outside that domain. There are three DREB1 proteins
that are encoded by genes that lie in tandem on chromosome 4 in the order DREB1B, DREB1A, and DREB1C [18].
DREB1B is identical to CBF1. Gilmour et al. [19] also isolated 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 lowtemperature 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 dehydration-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
Arabidopsis not only induced strong expression of cor genes,
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 erd genes 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 transgenic plants also had enhanced freezing and dehydration
tolerance. The difference in growth retardation caused by
overexpression of DREB1A/CBF3 and DREB1B/CBF1 may
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 expression 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 indicate 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 DREB1A cDNA driven by the stress-inducible rd29A promoter was expressed at a low level in unstressed control
conditions and at a high level in plants exposed to dehydra-
220
Physiology and metabolism
Figure 3
Low temperature
CBF–CRT
(DREB1–DRE) system
Gene expression
DREB2–DRE system
Dehydration
ABA biosynthesis
bZIP–ABRE system
Gene expression
Protein synthesis
MYC/MYB system
Gene expression
Time course
Signal perception
Rapid and emergency response
Slow and adaptive response
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.
ABA biosynthesis is induced by dehydration and activates two
regulatory ABA-dependent gene expression systems. One is the
bZIP/ABRE system; the other is the MYC/MYB system, which requires
do novo production of the MYB and MYC proteins in response to ABA.
tion, salt, and cold stresses. The rd29A promoter 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 rd29A promoter::DREB1A system
is a self-amplifying system that overexpresses DREB1A protein throughout exposure to stress. Greater expression of the
DREB1A protein in the rd29A::DREB1A transgenics results
in greater expression of the target genes involved in stress tolerance [21••]. This system provides some promise for
engineering multi-stress tolerance of transgenic crops
because plants such as tobacco, Brassica, and rice, have similar transcription systems to that of Arabidopsis.
DRE/CRT-dependent gene expression of most cor, lti, and
rd genes to confer stress tolerance (Figure 3).
ABA in dehydration and low-temperature
stress response
In many plants, endogenous ABA levels increase significantly 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 dehydration and high salinity, but not by cold stress [23]. rd29B does
not contain a DRE/CRT but contains two ABREs in its promoter [7]. It is controlled downstream of the abi1 and 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
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 rd29A gene is therefore controlled by
three independent regulatory systems [7,17••]. These
results indicate that complex molecular responses to various 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 accumulation of endogenous ABA in drought conditions (Figure 3).
The biosynthesis of novel protein factors is necessary for the
expression of ABA-inducible genes in one of the two ABAdependent pathways (Figure 1). The induction of the
Arabidopsis drought-inducible gene rd22 is mediated by
ABA and requires protein biosynthesis for its ABA-dependent expression [24]. MYC and MYB recognition sequences
are essential for the ABA- and drought-responsive expression of rd22, and ABA-inducible MYC and MYB proteins
may function cooperatively in the ABA-dependent expression 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 Arabidopsis mutants that are either sensitive to or tolerant of freezing have been isolated, and their phenotypes
Molecular responses to dehydration and low temperature Shinozaki and Yamaguchi-Shinozaki
have been analyzed in detail. Warren et al. [27] isolated five
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 promoters. 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 processes. 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-tolerant mutant named eskimo1 that, without cold
acclimatization, has greater freezing tolerance than wildtype plants. The molecular mechanism of the stress
tolerance of eskimo1 is not known, but proline accumulates
in the eskimo1 mutant [29•]. This proline may be involved
in stress tolerance as proline functions in osmoprotection,
in detoxication of active oxygen, and in protection of proteins and nucleic acids. Recently, Nanjo et al. [30] showed
that the accumulation of proline in transgenics with antisense 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 Arabidopsis mutants with the rd29A promoter::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-signaling pathways, including ABA-independent and
ABA-dependent pathways, are not completely independent.
Xiong et al. [32] isolated a hos5 mutant that has increased
expression of rd29A when 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 hos2 mutants, respectively, that enhanced the expression
of rd29A, cor47, cor15a, and kin1. Non-acclimatized hos1 and
hos2 mutants were less cold-hardy than wild-type plants.
HOS1 and HOS2 are therefore thought to function as negative regulators of a cold-specific signal transduction pathway
(Figure 1). Genetic analysis of these mutants and of stressresistant or stress-sensitive mutants is likely to provide more
information on stress-induced signal transduction [35].
221
Signal perception and signal transduction
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 Arabidopsis cDNA 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 transmembrane 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 signaltransduction cascades are upregulated by dehydration
and cold: mitogen-activated protein (MAP) kinases, calcium-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 evidence has, however, been obtained of the functions of
these signaling molecules. Transgenic plants that modify
the expression of these genes and mutants with disrupted genes will provide more information on the function
of their gene products.
Conclusions and perspectives
A major transcription system regulating ABA-independent 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 dehydration, respectively, to express various genes involved in
stress tolerance. Cross-talk between dehydration and
cold occurs at the transcriptional level. ABA plays important 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.
Sequencing of the Arabidopsis genome will be completed
by the end of 2000, which means that the structure of all
25,000 Arabidopsis genes will soon be determined. All
stress-inducible genes can then be identified by the systematic analysis of gene expression in microarrays. In the
next decade, we think it important to develop novel methods to analyze the complex networks that control the stress
responses of higher plants. A reverse genetic approach, as
well as classical forward genetics, will become more important for understanding not only the functions of
stress-inducible genes but also the complex signaling
processes of the dehydration- and cold-stress responses.
222
Physiology and metabolism
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.
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