Molecular responses to drought, salinity and frost:common and different paths for plant protectionMotoaki Seki�y, Ayako Kameiy, Kazuko Yamaguchi-Shinozakiz andKazuo Shinozaki�y§

Drought, high salinity and low temperature are major

environmental factors that limit plant productivity. Plants

respond and adapt to these stresses in order to survive.

Signaling pathways are induced in response to environmental

stress and recent molecular and genetic studies have revealed

that these pathways involve many components. In this review,

we highlight recent findings on the gene expression associated

with stress responses and the signaling pathways that are either

common or specific to the response.

Addresses�Plant Mutation Exploration Team, Plant Functional Genomics Research

Group, RIKEN Genomic Sciences Center, 3-1-1 Koyadai, Tsukuba

305-0074, JapanyLaboratory of Plant Molecular Biology, RIKEN Tsukuba Institute,

3-1-1 Koyadai, Tsukuba 305-0074, JapanzBiological Resources Division, Japan International Research Center for

Agricultural Sciences, Ministry of Agriculture, Forestry and Fisheries,

2-1 Ohwashi, Tsukuba, Ibaraki 305-0851, Japan§e-mail: sinozaki@rtc.riken.go.jp

Current Opinion in Biotechnology 2003, 14:194–199

This review comes from a themed issue on

Plant biotechnology

Edited by Csaba Koncz

0958-1669/03/$ – see front matter

� 2003 Elsevier Science Ltd. All rights reserved.

DOI 10.1016/S0958-1669(03)00030-2

AbbreviationsABA abscisic acid

ABF ABRE-binding factor

ABRE ABA-responsive element

AREB ABRE-binding protein

AtNCED Arabidopsis 9-cis-epoxycarotenoid dioxygenase

bZIP basic domain leucine zipper

CBF C-repeat-binding factor

CRT C-repeat

DRE dehydration-responsive element

DREB DRE-binding protein

ERF ethylene-responsive element binding factor

IntroductionPlant growth is greatly affected by environmental abiotic

stresses, such as drought, high salinity and low tempera-

ture. These stresses induce various biochemical and phy-

siological responses in plants, which respond and adapt in

order to survive. Several genes have been studied that

respond to drought, salt or cold stress at the transcriptional

level [1–3,4��]. The products of these stress-inducible

genes have been classified into two groups: those that

directly protect against environmental stresses and those

that regulate gene expression and signal transduction in

the stress response. The first group includes proteins that

probably function by protecting cells from dehydration,

such as the enzymes required for the biosynthesis of

various osmoprotectants, late embryogenesis abundant

proteins, antifreeze proteins, chaperones, and detoxifica-

tion enzymes. The second group of gene products includes

transcription factors, protein kinases, and enzymes

involved in phosphoinositide metabolism. Stress-induci-

ble genes have been used to improve the stress tolerance of

plants by gene transfer [1–3]. It is important to analyze the

functions of stress-inducible genes not only to understand

the molecular mechanisms of stress tolerance and the

responses of higher plants, but also to improve the stress

tolerance of crops by gene manipulation. Hundreds of

genes are thought to be involved in abiotic stress responses

[2,4��,5–7,8��]. In this review, we highlight recent studies

on gene expression in response to environmental stress

and on the signaling pathways that are either common or

specific to the stress response.

Regulation of gene expression by drought,high-salinity and cold stressMany genes that are induced by osmotic stress have been

identified [2,3,4��,5–7,8��]. Although the signaling path-

ways responsible for the activation of these genes are

largely unknown, transcriptional activation of some stress-

responsive genes is well understood, owing to studies on

the RD29A/COR78/LTI78 (responsive to dehydration/

cold-regulated/low-temperature-induced) gene. The pro-

moter of this gene contains both an ABRE (abscisic acid

responsive element) and a DRE/CRT (dehydration-

responsive element/C-repeat) [9]. ABRE and DRE/

CRT are cis-acting elements that function in abscisic acid

(ABA)-dependent and ABA-independent gene expression

in response to stress, respectively. Transcription factors

belonging to the ERF/AP2 (ethylene-responsive element

binding factor/apetala 2) family that bind to DRE/CRT

were isolated and termed DREB1A/CBF3 (DRE-binding

protein/C-repeat-binding factor), DREB1B/CBF1 and

DREB1C/CBF2 [10,11]. The genes encoding these tran-

scription factors are induced early and transiently in

response to cold stress, and these transcription factors, in

turn, activate the expression of target genes. Similar tran-

scription factors (DREB2A and DREB2B) are induced by

dehydration stress and promote the expression of various

genes involved in drought stress tolerance [11]. Sakuma

194

Current Opinion in Biotechnology 2003, 14:194–199 www.current-opinion.com

et al. [12] precisely analyzed the DNA-binding specificity

of DREB1A/CBF3 and DREB2 and demonstrated that the

core sequence of DRE is the 6 base pair A/GCCGAC

sequence. The ability of DREB1/CBF to activate the

DRE/CRT class of stress-responsive genes was further

demonstrated by the observation that overexpression or

enhanced inducible expression of DREB1/CBF could

activate the target genes. Overexpression of DREB1/

CBF also increased the tolerance of transgenic plants to

freezing, drought and salt stresses [2,11,13,14], suggesting

that the system is important for the development of stress

tolerance in plants. The DREB1/CBF pathway has a key

role in regulating ABA-independent gene expression in

response to drought and cold stress [2]. Taji et al. [15�]showed that the galactinol synthase gene (AtGolS) is a

target of DREB1A/CBF3. Transgenic Arabidopsis plants

overexpressing the AtGolS2 gene accumulated galactinol

and raffinose, showed a reduced transpiration rate, and

were more tolerant to drought stress than control plants.

Kim et al. [16�] also reported that cold-induced gene

expression through DRE/CRT is greatly enhanced by a

signal generated by light; the primary photoreceptor

involved in this light signaling was identified as phyto-

chrome B.

Several basic leucine zipper (bZIP) transcription factors

that can bind to ABRE and activate the expression of

ABRE-driven reporter genes have been isolated: AREB1/

ABF2, AREB2/ABF4, AREB3, ABF1 and ABF3 [17�,18].

AREB1/ABF2 and AREB2/ABF4 need ABA for full acti-

vation; the activities of these transcription factors were

reduced in the ABA-deficient mutant aba2 and ABA-

insensitive mutant abi1-1, but were enhanced in the

ABA-hypersensitive era1 (enhanced response to ABA)

mutant. ABA is probably required for the ABA-dependent

phosphorylation of the proteins and their activation in the

pathway [17�]. Recently, Kang et al. [19] reported that

constitutive overexpression of ABF3 or AREB2/ABF4 in

Arabidopsis resulted in ABA hypersensitivity, reduced

transpiration rate and enhanced drought tolerance.

Changes in phenotypes for loss-of-function mutants have

not yet been reported for any DREB/CBF or AREB/ABFgenes. This may be due to functional redundancy

between the family members, and hence it may be

necessary to combine loss-of-function mutants for two

or more members to see the phenotype.

The induction of the drought-inducible gene RD22 is

mediated by ABA and requires protein biosynthesis for its

ABA-dependent expression [2,20]. A MYC transcription

factor, RD22BP1 (also known as AtMYC2), and a MYB

transcription factor, ATMYB2, were shown to bind ciselements in the RD22 promoter and activate RD22 in a

cooperative manner [20].

Many drought- and/or ABA-inducible genes encoding

various transcription factors have been reported. Among

them, the homeodomain-containing transcription factor

ATHB6 functions as a negative regulator downstream of

ABI1 in the ABA signal transduction pathway [21].

Using gene expression profiling to identifystress response genesGene expression profiling using cDNA microarrays or

gene chips is a useful approach for analyzing the expres-

sion patterns of genes under conditions of drought, cold

and high-salinity [22�,23��,24�,25,26��,27]. These meth-

ods can also be used to identify the target genes of stress-

related signaling components. Moreover, by combining

the expression data with the genomic sequence data,

potential cis-acting DNA elements could be analyzed

[22�,23��,24�,26��]. We used a full-length cDNA micro-

array containing �1300 Arabidopsis full-length cDNAs to

identify drought- or cold-inducible genes and to establish

target genes of DREB1A/CBF3 [22�]. From the 1300

genes analysed, we identified 12 DREB1A/CBF3 target

genes, all of which contained DRE or DRE-related

CCGAC core motif sequences in their promoter regions

[22�]. Recently, a new full-length cDNA microarray

containing �7000 independent Arabidopsis full-length

cDNAs was used to identify 299 drought-inducible genes,

54 cold-inducible genes, 213 high salinity inducible genes

and 245 ABA-inducible genes [23��,24�]. Information on

each stress-inducible gene is available at http://www.gsc.

riken.go.jp/Plant/index.html. Venn diagram analysis indi-

cated the existence of greater crosstalk between drought

and high-salinity stress signaling processes than between

cold and high-salinity stress signaling processes [23��].Furthermore, many ABA-inducible genes were shown to

be induced after drought and high-salinity stress treat-

ments and more crosstalk was seen between ABA and

drought responses than between ABA and cold responses

[24�]. These results support our previous model on the

overlap of gene expression in response to drought, high-

salinity, cold and ABA [2]. Among the cold-inducible

genes identified, nine did not contain DRE or DRE-

related CCGAC core motifs in their promoters, suggest-

ing the existence of novel cis-acting elements involved in

cold-inducible gene expression [23��]. Among the genes

induced by drought, cold or high-salinity, we found 40

(corresponding to �11% of all stress-inducible genes

identified) transcription factor genes, suggesting that

various transcriptional regulatory mechanisms function

in the drought, cold or high-salinity stress signal transduc-

tion pathways [23��,24�]. Stress-inducible transcription

factors were identified from a wide range of protein

families: six from the DREB family, two ERF family

members, ten zinc finger proteins, four WRKY family

members, three MYBs, two basic helix-loop-helix pro-

teins, four members of the bZIP family, five NAC family

members, and three homeodomain transcription factors.

These transcription factors probably regulate various

stress-inducible genes either cooperatively or separately.

Functional analysis of these stress-inducible transcription

Plant molecular responses to stress Seki et al. 195

www.current-opinion.com Current Opinion in Biotechnology 2003, 14:194–199

factors should provide more information on signal trans-

duction in response to drought, cold and high-salinity.

Recently, Fowler and Thomashow [27] identified 306

cold-regulated genes and 41 DREB/CBF-regulated

genes using Affymetrix Gene Chips. Several differences

between our results and those of Fowler and Thomashow

exist. These may be due to diferences in expression

profiling methods, the ecotypes used and plant growth

conditions.

Identifying the molecular components ofsignaling pathwaysSeveral signal transduction and stress tolerance mutants

have been identified using genetic approaches and bio-

chemical analyses [4��,8��,28,29,30�]. In a genetic screen

using a firefly luciferase reporter gene (LUC) under the

control of the RD29A promoter, Zhu and colleagues

isolated several Arabidopsis mutants with altered induc-

tion of stress-responsive genes under conditions of

drought, high-salinity, cold and ABA treatments [31].

Compared with wild-type RD29A–LUC plants, mutants

either exhibited a constitutive (cos), high (hos) or low (los)level of RD29A–LUC expression in response to various

stress or ABA treatments [31]. These mutants might be

involved in the activation of the DRE/CRT class of

genes. The occurrence of mutations with differential

responses to stress or ABA or combinations of the stimuli

suggested that there is a close relationship between the

cold, drought, salinity, and ABA signal transduction path-

ways [31]. The characterization and cloning of some of

the mutations have provided new insight into the

mechanisms of stress and ABA signal transduction

[4��,8��]. The loci of the mutations fiery1 (fry1) [32��],hos1 [33�], los1 [34], los2 [35], los5/aba3 [36�], los6/aba1[37] and sad1 (supersensitive to ABA and drought) [38�]have been cloned. Their roles in stress signaling were

discussed in recent reviews [4��,6,7,8��].

The Arabidopsis salt overly sensitive mutants (sos1, sos2,

sos3 and sos4) were identified by genetic screening for

seedlings that were hypersensitive to salt stress [39,40�,41�,42]. The sos1, sos2 and sos3 mutants are hypersensitive

to salt stress, but activation of the DRE/CRT class of

genes seems to be unaffected. SOS signaling appears to

be relatively specific for the ionic aspect of salt stress and

is calcium-dependent. The targets of this type of signal-

ing are ion transporters that control ion homeostasis under

salt stress. Functional studies in yeast and plants have

shown that SOS1 is activated by the SOS3–SOS2 complex

[43��,44]; Zhu has summarized recent advances in the

SOS signaling pathway [4��,45]. Rus et al. [46�] identified

T-DNA insertion mutations in AtHKT1 (Arabidopsis high-

affinity Kþ transporter) which functionally disrupt its

expression and suppress the Naþ hypersensitive pheno-

type of the sos3 mutant. These results indicate that

AtHKT1 is a salt tolerance determinant that controls

Naþ entry and high-affinity Kþ uptake.

Reverse genetic approaches, such as transgenic analyses,

are also useful for studying the function of the signaling

components [1,8��,30�,47]. Improvement of stress toler-

ance has been reported by the overexpression of the ABA

biosynthesis gene, AtNCED3 [48]. Overexpression of

constitutive, active PKS18 (an SOS2-like protein kinase)

in which a threonine residue was substituted with aspar-

tic acid, resulted in hypersensitivity to ABA in seed

germination and seedling growth, whereas silencing

the kinase gene using double-stranded RNA interfer-

ence conferred ABA insensitivity [49]. Silencing an

SOS3-like calcium-binding protein (SCaBP5) and an

SOS2-like protein kinase (PKS3) caused ABA hypersen-

sitivity in seed germination, seedling growth, stomatal

closing and gene expression, suggesting that these pro-

teins act as negative regulators that specifically modulate

ABA signal transduction [50].

Conclusions and perspectivesMolecular and genetic approaches are starting to shed

light on the components of signal transduction pathways

induced in response to drought, cold and high-salinity

stress. A major transcription system regulating ABA-inde-

pendent 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 contains two subclasses, DREB1/CBF

and DREB2, which are induced by cold and drought,

respectively, to express various genes involved in stress

tolerance. AREB/ABF genes that can bind to ABRE and

activate the expression of ABRE-driven reporter genes

have also been isolated. Expression profiling has been

used to identify more than 300 genes induced by drought,

cold or high salinity, and 40 stress inducible transcription

factor genes. Genetic and biochemical approaches have

also proved useful to dissect the signal transduction path-

ways induced in response to stress.

The availability of the Arabidopsis genome sequence will

not only greatly facilitate the isolation of mutations iden-

tified by genetic screening, but will offer many other

useful opportunities to study stress signal transduction.

Genome-wide expression profiling of stress-resistant or

stress-sensitive mutants and plants with mutations in the

stress signal transduction pathways should help to iden-

tify more genes that are regulated at the transcriptional

level by the signaling components. Moreover, full-length

cDNAs [51��] are useful resources for transgenic analyses

(e.g. overexpression, antisense suppression, and double-

stranded RNA interference) and biochemical analyses to

study the function of the encoded proteins. T-DNA- or

transposon-knockout mutants also offer the opportunity

to study gene function. Genome-wide protein interaction

studies will help to identify the interactions between

signaling components and will allow signal networks to

be constructed. The information generated by focused

studies of gene function in Arabidopsis will be the

196 Plant biotechnology

Current Opinion in Biotechnology 2003, 14:194–199 www.current-opinion.com

springboard for a new wave of strategies to improve the

dehydration, salt and cold tolerance of agriculturally

important crops.

AcknowledgementsWe thank Kyonoshin Maruyama for his helpful comments. This work wassupported in part by a grant for Genome Research from RIKEN, the Programfor Promotion of Basic Research Activities for Innovative Biosciences, theSpecial Coordination Fund of the Science and Technology Agency, and aGrant-in-Aid from the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MECSST) to KS. This work was also supported in partby a Grant-in-Aid for Scientific Research on Priority Areas (C) ‘GenomeScience’ from MECSST to MS.

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest��of outstanding interest

1. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ: Plant cellularand molecular responses to high salinity. Annu Rev Plant PhysiolPlant Mol Biol 2000, 51:463-499.

2. Shinozaki K, Yamaguchi-Shinozaki K: Molecular responses todehydration and low temperature: differences and cross-talkbetween two stress signaling pathways. Curr Opin Plant Biol2000, 3:217-223.

3. Thomashow MF: Plant cold acclimation: freezing tolerancegenes and regulatory mechanisms. Annu Rev Plant Physiol PlantMol Biol 1999, 50:571-599.

4.��

Zhu JK: Salt and drought stress signal transduction in plants.Annu Rev Plant Biol 2002, 53:247-273.

This paper provides an excellent summary of studies on salt and droughtstress signal transduction.

5. Shinozaki K, Yamaguchi-Shinozaki K: Molecular responses todrought stress. In Molecular Responses to Cold, Drought, Heatand Salt Stress in Higher Plants. Edited by Shinozaki K, Yamaguchi-Shinozaki K. Austin, TX: RG Landes; 1999:11-28.

6. Xiong L, Zhu JK: Abiotic stress signal transduction in plants:molecular and genetic perspectives. Physiol Plant 2001,112:152-166.

7. Xiong L, Zhu JK: Molecular and genetic aspects of plantresponses to osmotic stress. Plant Cell Environ 2002, 25:131-139.

8.��

Xiong L, Schumaker KS, Zhu JK: Cell signaling during cold,drought, and salt stress. Plant Cell 2002, (Suppl):S165-183.

A recent review of the common characteristics of stress signaling trans-duction in plants and recent studies on the functional analysis of signalingcomponents.

9. Yamaguchi-Shinozaki K, Shinozaki K: A novel cis-acting elementin an Arabidopsis gene is involved in responsiveness todrought, low temperature or high-salt stress. Plant Cell 1994,6:251-264.

10. Stockinger EJ, Gilmour SJ, Thomashow MF: Arabidopsis thalianaCBF1 encodes an AP2 domain-containing transcriptionactivator that binds to the C-repeat/DRE, a cis-acting DNAregulatory element that stimulates transcription in response tolow temperature and water deficit. Proc Natl Acad Sci USA 1997,94:1035-1040.

11. Liu X, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-ShinozakiK, Shinozaki K: Two transcription factors, DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellularsignal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, inArabidopsis. Plant Cell 1998, 10:1391-1406.

12. Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K: DNA-binding specificity of the ERF/AP2 domain ofArabidopsis DREBs, transcription factors involved indehydration- and cold-inducible gene expression. BiochemBiophys Res Commun 2002, 290:998-1009.

13. Jaglo-Ottosen KR, Gilmour SJ, Zarka DG, Schabenberger O,Thomashow MF: Arabidopsis CBF1 overexpression induces corgenes and enhances freezing tolerance. Science 1998,280:104-106.

14. Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K:Improving plant drought, salt, and freezing tolerance by genetransfer of a single stress-inducible transcription factor.Nat Biotechnol 1999, 17:287-291.

15.�

Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M,Yamaguchi-Shinozaki K, Shinozaki K: Important roles of drought-and cold-inducible genes for galactinol synthase in stresstolerance in Arabidopsis thaliana. Plant J 2002, 29:417-426.

This study provided the direct evidence that raffinose family oligosac-charides have an important role in Arabidopsis abiotic stress tolerance.Transgenic Arabidopsis plants overexpressing the AtGolS2 gene accu-mulated galactinol and raffinose, showed a reduced transpiration rate,and were more tolerant to drought-stress than control plants.

16.�

Kim HJ, Kim YK, Park JY, Kim J: Light signaling mediated byphytochrome plays an important role in cold-induced geneexpression through the C-repeat/dehydration responsiveelement (C/DRE) in Arabidopsis thaliana. Plant J 2002,29:693-704.

The authors show that the cold-induced gene expression through DRE/CRT is greatly enhanced by a signal generated by light. The primaryphotoreceptor responsible for the activation of cold-stress signalling inresponse to light was identified as phytochrome B. The study provideddirect evidence that the integration of signals generated by both light andtemperature are involved in the DREB1/CBF cold signaling pathway.

17.�

Uno Y, Furihata T, Abe H, Yoshida R, Shinozaki K, Yamaguchi-Shinozaki K: Arabidopsis basic leucine zipper transcriptionfactors involved in an abscisic acid-dependent signaltransduction pathway under drought and high-salinityconditions. Proc Natl Acad Sci USA 2000, 97:11632-11637.

The authors identified the three cDNAs (AREB1/ABF2, AREB2/ABF4 andAREB3) encoding bZIP-type ABRE-binding proteins using the yeast one-hybrid approach. The results suggested that both AREB1/ABF2 andAREB2/ABF4 function as transcriptional activators in the ABA-induciblegene expression of RD29B, and that ABA-dependent post-transcriptionalactivation of AREB1/ABF2 and AREB2/ABF4, probably by phosphoryla-tion, is necessary for their maximum activation by ABA.

18. Choi H, Hong JH, Ha J, Kang JY, Kim SY: ABFs, a family of ABA-responsive element binding factors. J Biol Chem 2000,275:1723-1730.

19. Kang JY, Choi HI, Im MY, Kim SY: Arabidopsis basic leucinezipper proteins that mediate stress-responsive abscisic acidsignaling. Plant Cell 2002, 14:343-357.

20. Abe H, Yamaguchi-Shinozaki K, Urao T, Iwasaki T, Hosokawa D,Shinozaki K: Role of Arabidopsis MYC and MYB homologs indrought- and abscisic-acid-regulated gene expression.Plant Cell 1997, 9:1859-1868.

21. Himmelbach A, Hoffmann T, Leube M, Hohener B, Grill E:Homeodomain protein ATHB6 is a target of the proteinphosphatase ABI1 and regulates hormone responses inArabidopsis. EMBO J 2002, 21:3029-3038.

22.�

Seki M, Narusaka M, Abe H, Kasuga M, Yamaguchi-Shinozaki K,Carninci P, Hayashizaki Y, Shinozaki K: Monitoring theexpression pattern of 1300 Arabidopsis genes under droughtand cold stresses using a full-length cDNA microarray.Plant Cell 2001, 13:61-72.

This paper reports the gene expression profiling of Arabidopsis plantsectopically expressing DREB1A/CBF3 using cDNA microarrays. Out of1300 genes examined, 12 were identified as targets of DREB1A/CBF3.The authors demonstrate that the full-length cDNA microarray is a usefultool with which to analyze the expression pattern of Arabidopsis genes inresponse to drought and cold stress. The microarray was also used toidentify target genes of stress-related transcription factors and potentialcis-acting DNA elements by combining the expression data with genomicsequence data.

23.��

Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A,Nakajima M, Enju A, Sakurai T et al.: Monitoring the expressionprofiles of 7000 Arabidopsis genes under drought, cold, andhigh-salinity stresses using a full-length cDNA microarray.Plant J 2002, 31:279-292.

Using 7000 Arabidopsis full-length cDNA microarrays, the authors iden-tified 277 drought-inducible, 53 cold-inducible, and 194 high-salinity

Plant molecular responses to stress Seki et al. 197

www.current-opinion.com Current Opinion in Biotechnology 2003, 14:194–199

stress-inducible genes. Among the stress-inducible genes, the authorsfound 40 transcription factor genes, suggesting that various transcriptionalregulatory mechanisms function in the drought, cold or high-salinity stresssignal transduction pathways. Expression profiling indicated greater cross-talk between drought and high-salinity stress signaling processes thanbetween cold and high-salinity stress signaling processes.

24.�

Seki M, Ishida J, Narusaka M, Fujita M, Nanjo T, Umezawa T,Kamiya A, Nakajima M, Enju A, Sakurai T et al.: Monitoring theexpression pattern of ca. 7000 Arabidopsis genes under ABAtreatments using a full-length cDNA microarray.Functional Integrative Genomics 2002, 2:282-291.

Using a microarray containing 7000 Arabidopsis full-length cDNAs, theauthors identified 245 ABA-inducible genes. The results showed thatmany ABA-inducible genes were induced under conditions of droughtand high-salinity, and that there is more crosstalk between drought andABA responses than between ABA and cold responses.

25. Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K,Galbraith D, Bohnert H: Gene expression profiles during theinitial phase of salt stress in rice. Plant Cell 2001, 13:889-905.

26.��

Chen W, Provart NJ, Glazebrook J, Katagiri F, Chang HS, Eulgem T,Mauch F, Luan S, Zou G, Whitham SA et al.: Expression profilematrix of Arabidopsis transcription factor genes suggests theirputative functions in response to environmental stresses.Plant Cell 2002, 14:559-574.

The authors report a microarray study on the expression of 402 Arabi-dopsis transcription factors in plants exposed to a range of stresses. Thestudy demonstrated overlap in the expression of several transcriptionfactors in different stress signaling pathways.

27. Fowler S, Thomashow MF: Arabidopsis transcriptome profilingindicates that multiple regulatory pathways are activatedduring cold acclimation in addition to the CBF cold responsepathway. Plant Cell 2002, 14:1675-1690.

28. Browse J, Xin Z: Temperature sensing and cold acclimation.Curr Opin Plant Biol 2001, 4:241-246.

29. Knight H, Knight MR: Abiotic stress signalling pathways:specificity and cross-talk. Trends Plant Sci 2001, 6:262-267.

30.�

Finkelstein RR, Gampala SSL, Rock CD: Abscisic acid signaling inseeds. Plant Cell 2002, 14:S15-S45.

A recent review of ABA signaling that emphasizes genetic and molecularinteractions among ABA response loci, developmental regulators andother signaling components.

31. Ishitani M, Xiong L, Stevenson B, Zhu JK: Genetic analysis ofosmotic and cold stress signal transduction in Arabidopsis:interactions and convergence of abscisic acid-dependent andabscisic acid-independent pathways. Plant Cell 1997,9:1935-1949.

32.��

Xiong L, Lee BH, Ishitani M, Lee H, Zhang C, Zhu JK: FIERY1encoding an inositol polyphosphate 1-phosphatase is anegative regulator of abscisic acid and stress signaling inArabidopsis. Genes Dev 2001, 15:1971-1984.

This report describes the map-based cloning of FRY1, which encodes aninositol polyphosphate-1-phosphatase. The authors demonstrated thatthe fry1 mutation results in elevated levels of inositol-1,4,5-triphosphate inboth control and ABA-treated plants, super-induction of ABA- and stress-inducible genes in response to ABA, salt and dehydration, and plants thatare less tolerant to freezing, drought and salt stress. The authors pro-posed that FRY1 is a negative regulator of ABA and stress signaling andprovide genetic evidence that phosphoinositols have an important role inABA and stress signal transduction in plants.

33.�

Lee H, Xiong L, Gong Z, Ishitani M, Stevenson B, Zhu JK:The Arabidopsis HOS1 gene negatively regulates cold signaltransduction and encodes a RING finger protein that displayscold-regulated nucleo-cytoplasmic partitioning. Genes Dev2001, 15:912-924.

Map-based cloning of HOS1 and further phenotypic characterization ofthe hos1 mutant are described. The DREB1/CBF genes are super-induced by cold in hos1 mutant plants, implying that HOS1 functionsupstream of DREB1/CBF transcription factors. Mutant hos1 plantsshowed reduced expression of FLC (Flowering Locus C), which is anegative regulator of vernalization. HOS1 encodes a variant RING-fingerprotein that exhibits nucleo-cytoplasmic partitioning in response to coldtreatment.

34. Guo Y, Xiong L, Ishitani M, Zhu JK: An Arabidopsis mutation intranslation elongation factor 2 causes superinduction of CBF/

DREB1 transcription factor genes but blocks the induction oftheir downstream targets under low temperatures. Proc NatlAcad Sci USA 2002, 99:7786-7791.

35. Lee H, Guo Y, Ohta M, Xiong L, Stevenson B, Zhu JK: LOS2, agenetic locus required for cold-responsive genetranscription encodes a bi-functional enolase. EMBO J 2002,21:2692-2702.

36.�

Xiong L, Ishitani M, Lee H, Zhu JK: The Arabidopsis LOS5/ABA3locus encodes a molybdenum cofactor sulfurase andmodulates cold stress- and osmotic stress-responsive geneexpression. Plant Cell 2001, 13:2063-2083.

This report describes the map-based cloning of LOS5/ABA3, whichencodes a molybdenum cofactor sulfurase. The authors demonstratedthat LOS5/ABA3 is a key regulator of ABA biosynthesis, stress-respon-sive gene expression, and stress tolerance.

37. Xiong L, Lee H, Ishitani M, Zhu JK: Regulation of osmotic stress-responsive gene expression by the LOS6/ABA1 locus inArabidopsis. J Biol Chem 2002, 277:8588-8596.

38.�

Xiong L, Gong Z, Rock CD, Subramanian S, Guo Y, Xu W, GalbraithD, Zhu JK: Modulation of abscisic acid signal transduction andbiosynthesis by an Sm-like protein in Arabidopsis.Dev Cell 2001, 1:771-781.

The authors demonstrate that a sad1 mutant allele results in increasedplant sensitivity to drought and ABA and impairment in ABA biosynthesisin response to drought. The SAD1 gene encodes a polypeptide similar tomultifunctional Sm-like snRNP proteins that are required for mRNAsplicing, export, and degradation. These results suggested a critical rolefor mRNA metabolism in the control of ABA signaling as well as in theregulation of ABA homeostasis.

39. Liu J, Zhu JK: A calcium sensor homolog required for plant salttolerance. Science 1998, 280:1943-1945.

40.�

Liu J, Ishitani M, Halfter U, Kim CS, Zhu JK: The Arabidopsisthaliana SOS2 gene encodes a protein kinase that is requiredfor salt tolerance. Proc Natl Acad Sci USA 2000, 97:3730-3734.

The positional cloning of SOS2 is described. SOS2 encodes a proteinkinase with an N-terminal catalytic domain similar to that of the SNF1/AMPK kinases. The regulatory domain of SOS2 is novel and is required forthe function of this protein in Arabidopsis.

41.�

Shi H, Ishitani M, Kim C, Zhu JK: The Arabidopsis thaliana salttolerance gene SOS1 encodes a putative Naþ/Hþ antiporter.Proc Natl Acad Sci USA 2000, 97:6896-6901.

The positional cloning of SOS1 is reported. SOS1 encodes a putativeplasma membrane Naþ/Hþ antiporter. SOS1 expression is upregulatedby salt stress and this upregulation is diminished in sos2 and sos3 mutantplants.

42. Shi H, Xiong L, Stevenson B, Lu T, Zhu JK: The Arabidopsis saltoverly sensitive 4 mutants uncover a critical role for vitamin B6

in plant salt tolerance. Plant Cell 2002, 14:575-588.

43.��

Qui QS, Guo Y, Dietrich MA, Schumaker KS, Zhu JK: Regulation ofSOS1, a plasma membrane Naþ/Hþ exchanger in Arabidopsisthaliana, by SOS2 and SOS3. Proc Natl Acad Sci USA 2002,99:8436-8441.

The results demonstrate that SOS1 contributes to plasma membraneNaþ/Hþ exchange. SOS2 and SOS3 were shown to activate SOS1 activityby phosphorylation of the Naþ/Hþ exchanger; SOS3 does not regulateSOS1 directly but operates through SOS2.

44. Quintero FJ, Ohta M, Shi H, Zhu JK, Pardo JM: Reconstitution inyeast of the Arabidopsis SOS signaling pathway for Naþ

homeostasis. Proc Natl Acad Sci USA 2002, 99:9061-9066.

45. Zhu JK: Genetic analysis of plant salt tolerance usingArabidopsis. Plant Physiol 2000, 124:941-948.

46.�

Rus A, Yokoi S, Sharkhuu A, Reddy M, Lee BH, Matsumoto TK,Koiwa H, Zhu JK, Bressan RA, Hasegawa PM: AtHKT1 is a salttolerance determinant that controls Naþ entry into plant roots.Proc Natl Acad Sci USA 2001, 98:14150-14155.

The authors identified two T-DNA insertion mutations in AtHKT1 (hkt1-1and hkt1-2) that functionally disrupt expression and suppress the Naþ

hypersensitive phenotype of the sos3 mutant. The results indicated thatAtHKT1 is a salt tolerance determinant that controls Naþ entry and high-affinity Kþ uptake.

47. Apse MP, Blumwald E: Engineering salt tolerance in plants.Curr Opin Biotechnol 2002, 13:146-150.

198 Plant biotechnology

Current Opinion in Biotechnology 2003, 14:194–199 www.current-opinion.com

48. Iuchi S, Kobayashi M, Taji T, Naramoto M, Seki M, Kato T, Tabata S,Kakubari Y, Yamaguchi-Shinozaki K, Shinozaki K: Regulation ofdrought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acidbiosynthesis in Arabidopsis. Plant J 2001, 27:325-333.

49. Gong D, Zhang C, Chen X, Gong Z, Zhu JK: Constitutiveactivation and transgenic evaluation of the function of anArabidopsis PKS protein kinase. J Biol Chem 2002,277:42088-42096.

50. Guo Y, Xiong L, Song CP, Gong D, Halfter U, Zhu JK: A calciumsensor and its interacting protein kinase are global regulatorsof abscisic acid signaling in Arabidopsis. Dev Cell 2002,3:233-244.

51.��

Seki M, Narusaka M, Kamiya A, Ishida J, Satou M, Sakurai T,Nakajima M, Enju A, Akiyama K, Oono Y et al.: Functionalannotation of a full-length Arabidopsis cDNA collection.Science 2002, 296:141-145.

The authors isolated 14 668 nonredundant cDNA groups, equivalent to�60% of all predicted genes in Arabidopsis. The authors also obtained 50-expressed sequence tags from 14 034 nonredundant cDNAs and con-structed a promoter database. The database is useful for promoteranalysis and for the correct annotation of predicted transcription unitsand gene products. The RIKEN Arabidopsis full-length (RAFL) cDNAs area useful resource for analyses of the expression profiles, functions, andstructures of plant proteins. The RAFL cDNA clones are available fromthe RIKEN Bioresource Center (http://www.brc.riken.go.jp/lab/epd/Eng/index.html).

Plant molecular responses to stress Seki et al. 199

www.current-opinion.com Current Opinion in Biotechnology 2003, 14:194–199