THE MOLECULAR BASIS OF DEHYDRATION TOLERANCE IN …arquivo.ufv.br/DBV/PGFVG/BVE684/htms/pdfs_revisao/e... · 2002-04-17 · stresses in plants, possibly reflecting increased energy

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Annu.Rev. Plant Physiol. Plant Mol. Biol. 1996. 47:377–403Copyright© 1996by AnnualReviewsInc. All rightsreserved

THE MOLECULAR BASISOFDEHYDRATION TOLERANCE INPLANTS

J. IngramandD. Bartels

Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829 Köln,Germany

KEY WORDS: dehydration stress,desiccation tolerance, late-embryogenesis-abundant (LEA)proteins,osmolytes,ABA responsiveness

ABSTRACT

Molecular studies of drought stressin plants usea variety of strategies andinclude different species subjected to a wide range of water deficits. Initialresearchhasbynecessity been largely descriptive, andrelevant geneshavebeenidentifiedeither byreferencetophysiologicalevidenceorbydifferential screen-ing.A largenumberof geneswithapotential rolein droughttolerancehavebeendescribed, and major themes in themolecular response have been established.Particular areas of importance are sugar metabolism andlate-embryogenesis-abundant (LEA) proteins. Studies have begun to examine mechanisms thatcontrol thegeneexpression,andputative regulatory pathwayshavebeenestab-lished. Recent attempts to understand gene function have utilized transgenicplants. Theseeffortsareof clear agronomic importance.

CONTENTSINTRODUCTION..................................................................................................................... 378RESEARCH STRATEGIES..................................................................................................... 378

TolerantSystems.................................................................................................................. 379Genetic ModelSystems........................................................................................................ 380CropPlants.......................................................................................................................... 380

GENESWITH UPREGULATED EXPRESSIONIN RESPONSETO DEHYDRATION .... 380Metabolism.......................................................................................................................... 382Osmotic Adjustment............................................................................................................. 382Structural Adjustment.......................................................................................................... 384

Degradation and Repair ...................................................................................................... 384Removal of Toxins............................................................................................................... 385Late-Embryogenesis-Abundant Proteins............................................................................. 385

SUGARS................................................................................................................................... 388REGULATION OFGENEEXPRESSIONDURING DEHYDRATION............................... 389

Promoter Studies................................................................................................................. 390Second Messengers and Signaling Molecules..................................................................... 393Posttranscriptional Control ................................................................................................. 394Downregulationof Genes.................................................................................................... 395

TRANSGENICPLANTSASSESSING GENEFUNCTION.................................................. 395FUTURE PERSPECTIVES...................................................................................................... 396

INTRODUCTION

This review considersmolecularmechanisms involved in dehydrationtoler-ancein plants.Most plantsencounterat leasttransientdecreasesin relativewatercontentat somestageof their life, andmanyalsoproducehighly desic-cation-tolerantstructuressuchasseeds,spores,or pollen.Indeed,physiologi-cal droughtalsooccursduring cold andsalt stresses,whenthe main damagecausedto theliving cell canberelatedto waterdeficit (84,124).Althoughwearestill far from a completeunderstanding of thedamagecausedby drought,or theplant’s tolerancemechanisms, muchmolecular datahasbeencollectedoverthepastfew years.Currentknowledgeof theregulatorynetworkgovern-ing thedrought-stressresponsesis alsofragmentary,with almostno informa-tion on signalperception.However,signaltransduction, via ABA at least,andthe promotermodules of several responsegenes, are startingto be elucidated.

Someof the most recentefforts to understandgenefunction have usedtransgenic plants,and thesestudies have significant implications for cropdevelopment.Plantbreedinghasalreadyprovidedanenormousimprovementin the drought toleranceof crop plants (1), with selectionoften allowingdesiredtraitsto betransferredfrom closewild relatives.However,mostof thetraits are complex,and their molecularbasis is frequently not understood.With our rapidly expandingknowledgeof theunderlyingmolecularprocessesinvolved in dehydrationtolerance,togetherwith the technology of genema-nipulation, crop improvementcan nowalso be basedon geneticmaterialtransferred from any organismand usedin a directedmanner.

RESEARCHSTRATEGIES

Dehydrationtolerancehasbeeninvestigatedusing threemain approachesinplants:(a) examiningtolerantsystems,suchasseedsandresurrectionplants;(b) analyzingmutantsfrom geneticmodelspecies;and(c) analyzingthe ef-fects of stresson agriculturally relevant plants.

378 INGRAM & BARTELS

Tolerant Systems

Oneapproachof physiological researchin dehydrationtolerancehasbeentousespecificstructuresor speciesthat canwithstandseveredesiccation.Mostprominentin this categoryarecertainseeds(73, 82), but desiccation-tolerantspeciessuch as resurrectionplants (angiosperms)(8), mosses(particularlyTortula ruralis), andferns(98) arealsoincluded.Both seedsandtheresurrec-tion plantCraterostigmaplantagineumsurviveseveredehydration; therefore,the detailed molecular analysesof thesesystemsshould reveal expressedgenes thatcontainthe geneticinformation for desiccationtolerance.

SEEDS Thefinal maturationstageof thedevelopmentof seedsis characterizedby desiccation,andasmuchas90%of theoriginalwateris removedin attaininga stateof dormancywith unmeasurablemetabolism (73).This desiccatedstateallowssurvival underextremeenvironmentalconditions andfavorswide dis-persal.Theembryocannotwithstanddesiccationat all developmentalstages;toleranceis usually acquiredwell beforematurationdrying but is lost asgerminationprogresses.Theseedsof manyspecieshavebeenusedto isolatethemRNAandproteinsrelatedto thedesiccation-toleranceresponse,including,in particular,thoseof Arabidopsisthaliana (100)andof cropspeciessuchascotton(Gossypiumspp.)(6), barley(Hordeumvulgare) (9), maize(Zeamays)(99), andrice (Oryza sativa) (91). However,a significantcomplicationwiththesestudiesis thedifficulty of separatingthepathwaysleadingto desiccationtolerance fromthose involved withother aspectsof development.

Themainachievementof molecularstudieswith seedshasbeentheidenti-fication andcharacterizationof the late-embryogenesis-abundant(LEA) pro-teins.LEA-protein mRNAs first appearat the onsetof desiccation,dominatethe mRNA populationin dehydratedtissues(111),andgraduallyfall severalhoursafterembryosbeginto imbibewater(seesectionon Late-Embryogene-sis-AbundantProteins).

RESURRECTIONPLANTS Resurrectionplantsareuniqueamongangiospermsintheir ability to survive during drought,whenprotoplastic desiccationcanleave<2% relative water contentin the leaves(8). When water is withheld frommatureindividualsof C.plantagineum,changesrapidlyoccuratthemRNA andproteinlevels(8),eventuallyleadingto thetolerantstate.A particularadvantageof theseplantsin studiesat themolecularlevel is thatdesiccationtolerancecanbeinvestigated in bothwholeplantsandundifferentiatedcalluscultures(Tol-erantcallusof C. plantagineumis obtainedby pretreatmentwith ABA) (8). Inthecallustissue,andto a certainextentin whole C. plantagineumplants,thetransitionto thetolerantstateis largelyfreeof thecomplicationsof developmentor otheradjustmentsinherentin seedsor otherplantsystems.Oneof themost

DEHYDRATION TOLERANCE 379

strikingfeaturesof thedesiccation-inducedgenescharacterizedfromvegetativetissuesof C. plantagineumhasbeentheir similarity to thegenesexpressedinseeds of otherspecies.

Genetic Model Systems

Geneticmodel systemsare a secondmajor approachto the examination ofdehydrationtolerance.Thesesystems takeadvantageof detailedgeneticinfor-mation,a widerange ofmutants,and thefeasibility of positional genecloning.Progressin understandingthe role of ABA in desiccationtolerancehasbeenachievedby characterizingmutants,suchastheABA-deficientmutantsflacca(tomato,Lycopersiconesculentum) (22) anddroopy(potato,Solanumtubero-sum) (108).A numberof mutations related toABA action arealso available inA. thaliana,andtheir analysishasprovidedmanyinsightsinto ABA-mediateddroughtresponses.A. thaliana lines that are lesssensitive to ABA than thewild-type havemutations at the abi loci [43; seealso the maizevp1 mutant(82)]. Thedetailedgeneticinformationavailablefor A. thaliana facilitatedtheisolationof theABI1 andABI3 genesby positional cloning(42,74,86).ABI3is specificallyexpressedin seedsandprobablyencodesa transcriptionfactorable to activatelea-type genes(100), andABI1 encodesa calcium-regulatedphosphatase.

Crop Plants

A third approachin researchingdehydrationtolerancehasbeento usespeciesimportantto agriculture toanalyze theplantresponseafterdrought stress.Thistype of study is usefulbecause,throughintensive breedingor in vitro selec-tion, lines areavailablewith differing degreesof tolerance.Thus,correlativeevidencecanbesoughtfor genesputativelyinvolvedin thedroughtresponse.The transientandmoderatedroughtstressrepresentedin studiesof crop spe-ciesprobablydescribesthemostcommonform of dehydrationthatmostplantsarelikely to encounter.Theintensity of researchhasthusenabledamuchmorecompletepicture of the possible factors involved in drought tolerancetoemerge.

GENES WITH UPREGULATED EXPRESSION INRESPONSE TO DEHYDRATION

To establish the basicresponsesof plantsto drought,two of the approachesalready outlined—examination of tolerant systemsand crop plants—havebeenmostproductive.Onetypeof analysisinvolvestargetinggenesthoughttobeimportant,suchasthosefor the manyenzymesin drought-induced metabo-lic pathways.A secondapproachusesdifferentialscreeningto isolateupregu-latedgenes.Theseexperiments havebeensuccessfulin describingmanygenes


encodingproteinsof known function associatedwith desiccation(Table 1).Differential screeninghas also revealedmany genesof unknown function,which areincludedin Tables1 and2; the largestgroupis the arrayof LEA-protein-relatedgenes(Table3). Someof thegenesmaybeinvolvedin secon-dary problemsof drought-stressedplants,suchas increasedsusceptibility topathogens,e.g.pcht28(encodingan acidic endochitinase)(Table1; 17) andSC514(encodinglipoxygenase)(Table 1; 10). Genesinvolved in signaling

Table 1 Genesupregulatedby drought stressa andencodingpolypeptidesof knownfunction

cDNA Source Encodedpolypeptide Ref

GapC-Crat Craterostigmaplantagineum Cytosolic glyceraldehyde 3-phosphatedehydrogenase

129

pSPS1 C. plantagineum Sucrose-phosphate synthase b

pSS1; pSS2 C. plantagineum Sucrosesynthases 36

pPPC1 Mesembryanthemumcrystallinum

Phosphoenolpyruvatecarboxylase 130

pBAD Hordeumvulgare (barley) Betaine aldehydedehydrogenase 54

cAtP5CS Arabidopsisthaliana δ1-pyrroline-5-carboxylatesynthetase

145

RD28 A. thaliana Waterchannel 141

SAM1; SAM3 Lycopersiconesculentum(tomato)

S-adenosyl-L-methionine synthetases37

rd19A; rd21A A. thaliana Cysteineproteases 67

UBQ1 A. thaliana Ubiquitin extension protein 66

pMBM1 Triticum aestivum (wheat) L-isoaspartyl methyltransferase 90

SC514 Glycinemax(soybean) Lipoxygenase 10

cATCDPK1;cATCDPK2

A. thaliana Ca2+-dependent, calmodulin-independent protein kinases

127

PKABA1 T. aestivum Protein kinase 4

cAtPLC1 A. thaliana Phosphatidylinositol-specificphospholipase C

53

Apx1gene Pisumsativum(pea) Cytosolic ascorbateperoxidase 89

Sod 2 gene P. sativum Cytosolic copper/zinc superoxidedismutase

135

P31 L. esculentum Cytosolic copper/zinc superoxidedismutase

102

pcht28 L. chilense Acidic endochitinase 17

Atmyb2 A. thaliana MYB-protein-relatedtranscriptionfactor

128

ERD11; ERD13 A. thaliana GlutathioneS-transferases 63

cAtsEH A. thaliana Soluble epoxidehydrolase 61aThe best-characterizedplantgenesfrom which cDNA cloneshave beendemonstratedto show

increasedmRNAexpression levelsin responsetodroughtstresshavebeenincluded. Droughtstresshasbeentakento includequitediversetreatments, ideallywherewaterhasbeenwithheld from theplant, but alsofor example by applying osmotic stresswithmannitol solutionsorby detachingplantorgans.

bIngram& Bartels,unpublisheddata.


andcontrolprocessesare consideredin thesectionon Second MessengersandSignalingMolecules.

Metabolism

Changesin primarymetabolismarea generalresponseto stressin plants.Forexample,a cDNA-encodingglyceraldehyde-3-phosphate dehydrogenase,iso-lated from the resurrectionplant C. plantagineum(Table1; 129), showsin-creasedexpressionduring droughtand upon ABA treatment.However, in-creasedlevels of the enzymeare also associatedwith other environmentalstressesin plants,possibly reflectingincreasedenergydemand.Proteasesmayalsobe an importantfeatureof stressmetabolism,dispensingwith redundantproteinsanddepolymerizing vacuolarstoragepolypeptides,therebyreleasingaminoacids for themassivesynthesisof new proteins(Tables1 and 2;50).

Enzymesof sugarmetabolism areprobably criticalin desiccationtolerance.It has beendemonstratedthat certainsugars may becentral tothe protection ofa wide rangeof organismsagainstdrought (seesectionon Sugars).In C.plantagineum,theoverall transcriptlevelsof sucrose-phosphatesynthaseandsucrosesynthaseincreaseimmediatelyin responseto drought(36; J Ingram&D Bartels,unpublisheddata).Theexpressionpatternis complexif the kineticsof individual transcripttypesarefollowed over the entirecourseof dehydra-tion.

Enzymesinvolved in the synthesis of other compoundsthat can act ascompatiblesolutes—andwhosetranscriptlevelsareclearly upregulateddur-ing drought—includeδ∆1-pyrroline-5-carboxylatesynthetase(prolinebiosyn-thesis)(Table 1; 145) and betainealdehydedehydrogenase(glycine betainebiosynthesis) (Table 1;54).

Theinduction of themRNA encodingphosphoenolpyruvatecarboxylaseinMesembryanthemumcrystallinum (Table1; 130)highlightstheimportanceofCrassulaceanacidmetabolismin enablingcarbonfixation with minimal waterloss.Suchmetabolism is amajorresponsein awidevarietyof plantsto growthin dry conditions(139).

OsmoticAdjustment

Total waterpotential canbe maintainedduring mild droughtby osmoticad-justment, which involves utili zing sugarsor other compatible solutes (12).Both ion andwaterchannelsarelikely to beimportantin regulatingwaterflux,and the relevanceof thesechannelsto drought-stresshasbeensupportedbythe isolation of channelproteingenesexpressedin responseto waterdeficit.The7acDNA from pea(Pisumsativum) (Table2; 50) encodesa polypeptidewith characteristicfeaturesof ion channels,while the RD28 cDNA (A.thaliana) (Table1; 141)andprobablyalsotheH2-5 cDNA (C. plantagineum)


(J-B Mariaux & D Bartels,unpublisheddata)encodeputativewater-channelproteins(28).

Table 2 Genesupregulatedby drought stressa but encodingpolypeptides ofunknownfunction

cDNA Source Featuresof encodedpolypeptide Ref

26g Pisumsativum(pea) Some similarity to aldehydedehydrogenase

50

7a P. sativum Similar tochannelproteins 50

kin2 Arabidopsisthaliana Similarity to animalantifreezeproteins

69

pcC37-31 Craterostigmaplantagineum Similar toearly-light-inducibleproteins

7

TSW12 Lycopersiconesculentum(tomato)

A lipid transferprotein 125

pLE16 L. esculentum Similar tolipid transferproteins 107

15a P. sativum Similarity to proteases 50

pA1494 A. thaliana Similarity to proteases 136

ERD1 A. thaliana Similar toa Clp ATP-dependentproteasesubunit

64

Ha hsp17.6; Hahsp17.9

Helianthusannuus(sunflower)

Low-molecular-weight heat-shockproteins

21

Athsp70-1 A. thaliana Similar tothe HSP70 heat-shock-protein family

66

Athsp81-2 A. thaliana Similar tothe HSP81 heat-shock-protein family

66

BLT4 Hordeumvulgare (barley) Similar toproteaseinhibitors 32

P22 Raphanussativus(radish) Similar toproteaseinhibitors 77

BnD22 Brassicanapus (rape) Similar toproteaseinhibitors 31

pMAH9 Zeamays(maize) Similar toRNA-bindingproteins 47

MsaciA Medicagosativa (alfalfa) Similar topUM90-1 andpSM2075polypeptides

70

pUM90-1 M. sativa Similar toMsaciAandpSM2075polypeptides

80

pSM2075 M. sativa Similar toMsaciAandpUM90-1polypeptides

79

pBN115 B. napus Similar topolypeptidesencodedbypBN19 andpBN26(B. napus) ,andCOR15 (A. thaliana)

134

RD22 A. thaliana Similar toanunidentifi edseedprotein from Vicia faba

56

salT Oryzasativa (rice) 18

lti65 gene; lti78gene

A. thaliana 95

pcC13-62 C. plantagineum 104aSeeFootnote ain Table 1.


Structural Adjustment

Droughtstresshasbeenshownto causealterationsin the chemicalcomposi-tion andphysicalpropertiesof thecell wall (e.g.wall extensibility), andsuchchangesmay involve the genesencodingS-adenosylmethionine synthetase(Table 1;37). Under nonstressfulconditions, increased expression ofS-adeno-syl-L-methionine synthetasegenescorrelateswith areaswherelignification isoccurring (101).Thus, the increased expression in drought-stressedtissuecould thusalsobe dueto lignification in the cell wall. Cell elongationstopsunderprolongeddroughtstress,andthenlignificationprocesses seem tobegin(94a).Esparteroetal (37)alsonotedthatfungalelicitors causethecoinductionof S-adenosyl-L-methionine synthetase transcriptwith thoseof otherenzymes,e.g.S-adenosyl-L-homocystein hydrolaseor a methyltransferase,requiredforcell wall formation.

The C. plantagineum pcC37-31cDNA (Table 2; 7) encodesthe dsp-22protein, whosemRNA levels increasein responseto various stresses.ThecDNA shows significant homology to early light-inducible protein (ELIP)genes(1a).Light is involved in theregulationof thegeneexpression,andtheencodeddsp-22proteinis chloroplastic.ELIPsmayplayarole in theassemblyof the photosystem (1a). During desiccation,C. plantagineumchloroplastsundergomorphological changes,andthusthe dsp-22proteincould bind pig-mentsor help maintainassembledphotosyntheticstructuresessentialfor re-sumingactivephotosynthesisduringresurrection.

Degradation andRepair

Genesencodingproteinswith sequencesimilarity to proteases,andwhich areinducedby drought,havebeenisolatedfrom both pea(Table2; 50) and A.thaliana (Tables1 and2; 64,67,136).Oneof thefunctionsof theseenzymescould be to degradeproteinsirreparablydamagedby the effectsof drought(50). During early drought in A. thaliana, there is an increasein levels ofmRNA encodingubiquitin extensionprotein(66),a fusion proteinfrom whichactive ubiquitin is derivedby proteolytic processing.This increasemay besignificant in terms of protein degradation,becauseubiquitin has a role intaggingproteinsfor destruction.During droughtstress,proteinresiduesmaybe modified by chemicalprocessessuch as deamination, isomerization, oroxidation,andit is thus likelythat enzymeswith functions inproteinrepairareupregulatedin responseto drought. Indeed,the responseto desiccationinmossesmaylargelyberepairbased(98).An exampleof suchrepairprocessesis theobservationthatL-isoaspartylmethyltransferasesmayconvertmodifiedL-isoaspartylresiduesin damagedproteinsbackto L-aspartylresidues(Table1; 90).


Mudgett& Clarke(90) havearguedthatsuchrepairmechanismscouldbeparticularlyimportantduringdesiccation,whenproteinturnoverratesare low.Although Escherichiacoli mutants lacking the enzymegrow normally in thelogarithmic phasewhenthereis high proteinturnover,theysurvivepoorly inthe stationary phase when turnoveris much lower(75).

Theproductsof two drought-inducedgenesisolatedby differentialscreen-ing havesequencesimilarity to heat-shockproteins(Table2; 66). Theseen-coded proteinsare probablychaperonins,involvedin proteinrepair byhelpingotherproteinsto recovertheir nativeconformationafterdenaturationor mis-folding during water stress.The low-molecular-weightheat-shockproteins(Table2; 21) may alsobe chaperonins.This function hasbeendemonstratedfor a mammalian low-molecular-weightheat-shockprotein (58). An alterna-tive functionmaybein the sequestrationof specific mRNAsin cellssubjectedto drought(96).

Removalof Toxins

Enzymesconcernedwith removingtoxic intermediatesproducedduringoxy-genic metabolism, suchas glutathione reductaseand superoxidedismutase,increasein responseto drought stressand are probably very important intolerance(89).Decreasingleaf watercontentandconsequentstomatalclosureresultin reducedCO2 availability andtheproductionof activeoxygenspeciessuchassuperoxideradicals(117). Increasedphotorespiratory activity duringdroughtis alsoaccompaniedby elevatedlevelsof glycolate-oxidaseactivity,resulting in H2O2 production(89). This could explain why genesencodingenzymesthat detoxify active oxygen speciessuch as ascorbateperoxidase(Table1; 89) andsuperoxidedismutase(Table1; 102,135) havebeenfoundupregulatedin responseto drought.

Late-Embryogenesis-Abundant Proteins

The genesencodinglate-embryogenesis-abundant(LEA) proteinsareconsis-tently representedin differential screensfor transcriptswith increasedlevelsduring drought.LEA proteinswere first describedfrom researchinto genesabundantlyexpressedduring the final desiccationstageof seeddevelopment(see above).Circumstantial evidencefor their involvement in dehydrationtoleranceis strong: Thegenesare similar to manyof those expressedinvegetativetissuesof drought-stressedplants(Table3), anddesiccationtreat-mentscanoften induceprecociousexpressionin seeds.ABA canalsoinducethe leagenes inseeds and vegetativetissues.

GENERAL FEATURES Groupingsfor dividing theLEA proteinsoriginatefroma dot matrix analysiswith proteinsfrom cotton.A groupwasassignedon thebasisof onecotton LEAprotein showingregions ofsignificanthomologywith


atleastoneproteinfromanotherspecies(33).The“type” of cottonproteinsusedfor thesegroupingswereLEA D19(Group1),LEA D11[Group2 (alsotermeddehydrins)],andLEA D7 (Group3). ThecottonproteinsLEA D113(34, 35)and LEAD95 (40) nowdefine twoadditionalclasses. This systemwill remainuseful untilclear functionscan be assigned.

LEA proteins appearto be located in many cell types andat variableconcentrations(19,34,35,45),andwithin thecell theyappearto bepredomi-nantly—butnot exclusively—cytosolic (19, 45, 91, 114).The concentrationsin the cell are characteristicallyvery high. For example,in maturecottonembryocells, the D7 LEA proteinsrepresentabout4% of nonorganellar cy-tosolicprotein(about0.34mM) (111).

A generalstructuralfeatureof theLEA proteinsis their biasedaminoacidcomposition,which resultsin highly hydrophilic polypeptides,with just a fewresidues providing 20–30%of their total complement. Forexample, a deduced

Table 3 Genesupregulatedby drought stressa thatencodepolypeptidesrelatedto late-embryo-genesis-abundantLEA proteins

cDNA Source Relationshipof encodedpolypeptideto LEA proteins

Ref

Ha ds10 Helianthusannuus(sunflower)

D19-LEA-protein related 3

Em Triticum aestivum (wheat) D19-LEA-protein related 76

B19.1; B19.3;B19.4

Hordeumvulgare (barley) D19-LEA-protein related 39

pLE25 Lycopersiconesculentum(tomato)

D113-LEA-proteinrelated 23

Ha ds11 H. annuus D113-LEA-proteinrelated 3

pRABAT1 Arabidopsisthaliana D11-LEA-protein related 72

pcC27-04 Craterostigmaplantagineum D11-LEA-protein related 104

M3 (RAB-17) Zeamays(maize) D11-LEA-protein related 20

B8; B9; B17 H. vulgare D11-LEA-protein related 20

pLE4 L. esculentum D11-LEA-protein related 23

pcC6-19 C. plantagineum D11-LEA-protein related 104

TAS14 L. esculentum D11-LEA-protein related 46

pLC30-15 L. chilense D11-LEA-protein related 16

H26 Stellaria longipes D11-LEA-protein related 110

pRAB 16A Oryzasativa (rice) D11-LEA-protein related 91

pcECP40 Daucuscarota (carrot) D11-LEA-protein related 62

ERD10; ERD14 A. thaliana D11-LEA-protein related 65

pMA2005 T. aestivum D7-LEA-proteinrelated 26

pMA1949 T. aestivum D7-LEA-proteinrelated 27

pcC3-06 C. plantagineum D7-LEA-proteinrelated 104

pcC27-45 C. plantagineum D95-LEA-protein-related 104aSeeFootnote ain Table 1.


D19 protein from cotton contains13% glycine and 11% glutamic acid (6).Furthermore, mostLEA proteinslackcysteine andtryptophanresidues.

ROLES We awaitdirectexperimentalevidencethatLEA proteinscanprotectspecificcellularstructuresor amelioratetheeffectsof droughtstress.Becausetheyarehighlyhydrophilic, it appearsunlikelythattheyoccurin specificcellularstructures.Also, their high concentrationsin the cell and biasedamino acidcompositions suggestthat theydo not functionas enzymes (6).

The randomlycoiled moietiesof someLEA proteinsareconsistentwith arole in binding water.Total desiccationis probablylethal,andthereforesuchproteins could help maintain the minimum cellular water requirement.McCubbin& Kay (83) havefoundthat theEm protein(D19-group)(Table3;76) from wheatis considerablymorehydratedthanmostglobularpolypeptidesbecauseit is over 70% randomcoil in normalphysiological conditions.Therandomcoil tailsof theD113proteinscouldalsobindconsiderableamountsofwater,although thelong N-terminalhelicaldomainwould not sharethis prop-erty (34,35).

A majorproblemunderseveredehydrationis thatthelossof waterleadstocrystallizationof cellularcomponents, whichin consequencedamages cellularstructures.This may be counteractedby LEA proteins,andsomeof theLEAproteinscould essentiallybe consideredcompatible solutes,which supportsthe likely role of sugarsin maintaining the structureof the cytoplasmin theabsenceof water.Bakeret al (6) havesuggestedthat LEA proteinsD11 andD113couldbeinvolvedin the“solvation” of cytosolic structures.The randomcoiling would permit their shapeto conform to that of other structuresandprovidea cohesivelayerwith possiblygreaterstability thanwould beformedby sugars.Their hydroxylatedgroupswould solvatestructuralsurfaces.Fur-thermore,theycouldbesuperiorto sucroseasprotectantsin beinglesslikelyto crystallize.However,for theD11-relatedproteinRAB-17,a regulatoryrolehas been postulated (see below).

Bakeret al (6) havehypothesizedthat the 11-amino-acidmotif (T/A A/TQ/E A/T A/T K/R Q/ED K/R A/T X ED/Q) (34) of LEA proteinD-29 (whichis alsopresentin D7 LEA proteins)couldcounteracttheirreversiblydamagingeffects of increasingionic strengthin the cytosol during desiccation.Suchproblemscouldbemitigatedby theformationof saltbridgeswith aminoacidresiduesof highly chargedproteins.The repeatingelementsmostlikely existas amphiphilic helices(34), which meansthat hydrophobicand hydrophilicaminoacidsarecontainedin particularsectorsof thehelix. Thehelicesprob-ably form intramolecularbundles,which would presenta surfacecapableofbinding both anionsandcations.Furtheranalysesof theD7-groupmoleculeshaveallowedprecisestructuralpredictionsto bemade:Theintersurfaceedges


of the interactinghelical regionsof the (putative)dimer revealperiodicallyspaced bindingsitesfor suitablycharged ions.

SUGARS

The involvementof soluble sugarsin desiccationtolerancein plantsis sug-gestedby studiesin which the presenceof particularsoluble sugarscan becorrelated withthe acquisition of desiccation tolerance (73).Suchstudieshavefollowed work with animals,fungi, yeast,andbacteria,in which a high levelof the disaccharidetrehalosehasbeenestablishedas important in survivingdesiccation.Trehaloseis the mosteffectiveosmoprotectantsugarin termsofminimum concentrationrequired(25). Whereastrehaloseis extremelyrareinplants,sucrose—togetherwith other sugars—appearsable to substitute. Al -though sugaraccumulation is not the only way in which plants deal withdesiccation(12), it is considered animportantfactor intolerance.

Many studieswith seedshavedemonstratedthe accumulation of solublesugarsduringtheacquisitionof desiccation tolerance(73);similar results havebeendemonstratedin resurrectionplants. A common themehas emerged.Various solublecarbohydratesmay be presentin fully hydratedtissues,butsucroseusuallyaccumulatesin thedriedstate.For example,desiccationin theleavesof C. plantagineum is accompaniedby conversionof the C8-sugar2-octulose(90% of thetotal sugar inhydratedleaves) intosucrose,which thencomprisesabout40% of thedry weight(11).

Total water potentialcan be maintained during mild droughtby osmoticadjustment. Sugarsmay serveascompatible solutespermitting suchosmoticadjustment, although many other compounds usuallyassociatedwith saltstressare also active, such as proline, glycine betaine,and pinitol (54, 84,145). Increasingsucrosesynthesisandsucrose-phosphatesynthaseactivity isnot only a drought-responseof desiccation-tolerantplantssuchasC. plantagi-neum(36) but also of plantsthat cannotwithstandextremedrying, suchasspinach(109).

Oneway sugarsmay protectthecell during severedesiccationis by glassformation:Ratherthansolutescrystallizing,throughthe presenceof sugarsasupersaturatedliquid is producedwith the mechanicalpropertiesof a solid(68). Glassformation hasbeendemonstratedin viable maizeseedsand hasbeenassociatedwith their viability (137). Differential scanningcalorimetryhasbeenusedto examinethe effect of termperatureon glassformation bysugarmixtures;only sugarmixturesequivalentin concentrationandcomposi-tion to thosein desiccation-tolerantembryosareableto form glassat ambienttemperatures(68). It seemslikely that sugarcomposition, rather than justconcentration,is relatedto glassformation.During desiccation,glasswouldfill space,thus preventingcellular collapse,and in restrictingthe molecular


diffusion requiredby chemicalreactionswould permita stablequiescentstate(68).

Phosphofructokinaseis a tetramericenzymethat usually dissociates irre-versibly into inactivedimersduring dehydration(14). However,it wasfoundthat in vitro the disaccharidessucrose,maltose,and trehalosestabilize theactivity of theenzyme duringdrying.

Croweet al (24) haveshownthat, in vitro, drying andrehydrationof themodel-membranesarcoplasmicreticulumusuallyresultsin the fusion of ves-icles and loss of the ability to transportcalcium.However,when the sugartrehalosewaspresentat concentrationsequivalentto thosein desiccation-tol-erantorganisms, functionalvesicleswerepreserved.Many otherstudiesshowthatsugarscanprotectmembranesin vitro (25); it is suggestedthatsugarsalterphysical propertiesof dry membranesso that they resemblethoseof fullyhydrated biomolecules.

Themechanismby which proteinsarestabilizedby sugarsis betterunder-stoodthanthesituationwith membranes.Infraredspectroscopyhasshownthattrehaloseprobably forms hydrogenbondsbetweenits hydroxyl groupsandpolarresiduesin proteins(25).Hydrogenbonding betweenthehydroxylgroupof trehaloseand the phosphateheadgroup of phospholipids can be inferredfrom comparisonsof changesin theinfraredspectrumof themoleculesduringdehydration.Strauss& Hauser(120)usedthecationEu3+, which is knowntoform a specific ionic bridge to the phosphateof phospholipids, to showthatsucroseis probablyboundbetweenphosphatesitesin dry membranes.Thiswasinferredfrom experimentsin which Eu3+ ionswereaddedto preparationsof sucroseandphosphatidylcholinevesicles;thestabilizationof liposomesbysucroseduring freezedrying decreasedas the Eu3+ ions wereadded,whichsuggestscompetitive bindingof sucroseandEu3+ at thephosphatesitesof thephospholipids.

REGULATION OF GENEEXPRESSIONDURINGDEHYDRATION

Themachineryleadingto theexpressionof drought-stressgenesconformstothegeneralcellularmodel,with acomplexsignaltransductioncascadethatcanbe divided into the following basic steps:(a) perception ofstimulus; (b)processing,including amplification and integration of the signal; and (c) aresponsereactionin the form of denovogeneexpression.No moleculardataareavailableon the perceptionof droughtstress,althoughturgor changehasbeensuggestedas a possible physical signal. An attractivemodel for theactivationof a transductionpathwayby a stresssignalhasbeenderivedfromstudyingtheheat-shockresponsein yeast(60).Kamadaet al (60) suggestthatheat-inducedactivation of a particular pathwayis in responseto increased


membranefluidity in the cell wall. The cell detectsthis weaknessin the cellwall by sensingstretchin theplasmamembrane.Examplessuchasthis fromsimple systems may provide the conceptualframeworkfor devisingexperi-mentsin plants.

The drought-activatedsignal transmission processhas begunto be dis-sected at the molecular level, mostly on the basis of studies of isolateddrought-responsive genes.Endogenous ABAlevels havebeen reportedtoincreaseasa resultof waterdeficit in manyphysiological studies,andthere-fore ABA is thoughtto be involved in thesignaltransduction(15, 43). Manyof thedrought-related genescanbeinducedby exogenousABA; however,thisdoesnot necessarilyimply that all thesegenesarealsoregulatedby ABA invivo.

We now discusspromoter studies,signalingmolecules,andbothposttran-scriptionalandposttranslationalmodificationsin thecontextof drought-regu-lated geneexpression.

Promoter Studies

CIS- AND TRANS-ACTING ELEMENTS Many of the changesin mRNA levelsobservedduringdroughtreflecttranscriptionalactivation.Treatmentwith ABAcanalsoinducethesechanges,andthis treatmenthasbeenutili zedfor settingup experimentalsystemsto define cis- and trans-acting elements.cis- andtrans-acting elementsinvolved in ABA-induced geneexpressionhavebeenanalyzed extensively(Tables 4 and5; 43).

Table 4 cis-actingpromoterelements relevantto ABA or drought

Gene Element Sequencea Ref

Rab16A(Oryzasativa)

ABRE (Motif I) GTACGTGGCGC 119

EM(Triticumaestivum)

Em1A GGACACGTGGC 51

Hex3(synthetic tetramer)(derivedfrom Nicotianatabacum)

GGTGACGTGGC 71

rab28(Zeamays)

ABRE CCACGTGG 106

Cat1(Zeamays)

CCAAGAAGTC-CACGTGGAGGTGGAAGAG

138

HVA22(Hordeumvulgare)

ABRE3 andCE1 GCCACGTACA andTGCCACCGG

118

CDeT27-45(Craterostigmaplantagineum)

AAGCCCAAATTTCA-CAGCCCGATAACCG

93

rd29(Arabidopsisthaliana)

DRE TACCGACAT 144

aThe G-box coreelementsACGT are initalic.


The best-characterizedcis-elementin the contextof droughtstressis theABA-responsive element (ABRE), which contains the palindromic motifCACGTG with the G-box ACGT core element(44). ACGT elementshavebeenobservedin a multitude of plant genesregulatedby diverseenviron-mental and physiological factors. Systematic DNA-binding studies haveshown that nucleotidesflanking the ACGT core specify the DNA-proteininteractionsandsubsequentgeneactivation(57). G-box-relatedABREs havebeenobservedin manyABA-responsivegenes,althoughtheir functionshavenot alwaysbeenprovenexperimentally.The best-studied examplesof theseABRE promoter elementsareEm1afrom wheatandMotif I from therice rab16Agene(Table4; 81, 92). Multiplecopiesof theelements fusedto aminimal35S promoterconfer an ABA responseto a reportergene(51, 119), whichsupportsthe hypothesisthat ABREs are critical for the ABA induction ofrelevantgenes(althoughit is difficult to explain why single copiesare not

Table 5 Characterizationof promotersin transgenic plants

Gene Nativegene activity Reportergene activity Ref

Rab16B Embryos ofOryzasativa Nicotiana tabacumembryos 142

Em Embryos ofTriticumaestivum Nicotiana tabacumembryos 81

Rab17 Embryos ofZeamays The embryos andendosperm ofArabidopsis thaliana

131

Hex3(synthetic tetramer)(derivedfrom Nicotiana tabacum)

Matureseeds ofN. tabacum;inducible in seedlingsby desicca-tion, salt,andABA

71

Rd22 DehydratedA. thalianaplants Constitutive inflowers andstemsof A. thaliana; inducible in N.tabacumby ABA or dehydration

56

Rd29A DehydratedA. thalianaplants Inducible by dehydration in mostvegetativeparts ofA. thaliana;inducible in N. tabacumby cold,ABA, andsalt

143,144

CDeT27-45 C. plantagineumdehydratedor ABA-treatedvegetativetissues

In embryos andmaturepollenofboth A. thalianaandN. tabacum

39a,88


In developingembryos andmaturepollenof both A. thalianaandN.tabacumalsoinducible in theirleavesandguardcells

87,39a,123


Embryosof both A. thalianaandN.tabacum; inducible in A. thalianaleavesby dehydration

a

DC8 Embryos ofDaucuscarota D. carotaseedtissues 49

DC3 Embryos ofDaucus carota N. tabacumseedlings;alsoinduc-ible in the leaves byeitherdryingor ABA treatment

132

aR Velasco, F Salamini & D Bartels,unpublisheddata.


sufficient for this response).TheABA effecton transcription wasorientationindependentin both the wheatand rice elements,which suggeststhat theypossiblyfunctionasenhancerelementsin their nativegenes.Electrophoretic-mobility-shift assaysand methylation-interferencefootprinting have shownthatbothEm1aandMotif1 interactwith nuclearproteins;theseDNA-bindingproteinsareconstitutively expressedin anABA-independentmanner(51,92).cDNAs encodingABRE-bindingproteins(wheatEMBP-1andtobaccoTAF-1) havebeenclonedandshownto containa basicregion adjacentto a leucine-zippermotif that is characteristicof transcriptionfactors(51, 97). Despitethefact that both proteinsexhibit specific and distinct binding properties,theirroles in vivo arenot understood.It seemspossiblethat they arenot directlyinvolved in ABA-responsivegeneexpressionbut that they cooperatewithother regulatoryfactors.

Recently,two differentelementshavebeendescribedthatmustbepresentto allow a single copy of the ABRE to mediatetranscriptionalactivationinresponseto ABA, and thus define an ABA responsecomplex.An ABREelementin the barleyAmy32bα-amylasepromoterhasbeenshownto allowABA-stimulated transcription toincreaseonly in the presenceof an O2Selementthat interactswith the ABRE within tight positional constraints. Asecondcouplingelementhasbeenidentified during promoteranalysisof theABA-inducedbarleyHVA22promoter(118).Thecoupling element(CE1)actstogether with aG-box-type ABRE(GCCACGTACA) in conferringhigh ABAinduction,whereastheABRE aloneis not sufficientfor transcriptional activa-tion. CE1-like elementshavebeenfound in manyotherABA-regulatedpro-moters,but their function remainsto be demonstrated(118). The specificsequence of a couplingelement may profoundly affectthe specificity ofABA-driven geneexpressionand may explain differencesbetweenfunctional andnonfunctionalABREs.

In promoterssuchasCDeT27–45or CDeT6–19,isolatedfrom C. plantagi-neum,G-box-relatedABREs do not appearto be major determinants of theABA or drought response(87, 88). The CDeT27–45promotercontainsanelementthatspecificallybindsnuclearproteinsfrom ABA-treatedtissue;thispromoterfragmentis essentialbut not sufficient for conferringa responsetoABA on a reporter gene (93).

Besidesthe ABA-mediatedgeneexpression,the investigation of drought-induced genes in A. thaliana has also revealed ABA-independent signaltransductionpathways(144). The A. thaliana genesrd29A and rd29B aredifferentially inducedunderconditionsof dehydration, saltor cold stress,andABA treatment.The rd29A genehasat leasttwo cis-actingelements.1. The9-bp direct repeatsequence,TACCGACAT, termedthe dehydration-respon-sive element (DRE), functions in the initial rapid response of rd29A todrought,salt,or low temperature(144).2. TheslowerABA responseis medi-


atedby another fragmentthat containsanABRE (143).It will be interesting toseewhetherthesamecis elementsfunctionin otherA. thalianagenesthatareinducedduringprogressivedrought;besidesABA, at leasttwo otherdifferentsignalsareinvolved in this induction (48). The existenceof ABA-dependentand-independentpathwaysis corroboratedby studieson theaccumulation ofthreedistinct Lea transcriptsin barleyembryos.Selectedtranscriptsincreasedin responseto osmoticstresswithout requiring ABA, whereasinduction bysaltdid requireABA (38).

A different class ofpotentialtranscription factors withrelevance to droughtstressis representedby the A. thaliana geneAtmyb2.This geneencodesanMYB-relatedproteinandis inducedby dehydrationor saltstressandby ABA(128).Plantmyb-relatedgenescomprisea largefamily thatmayplay variousrolesin generegulation.TheATMYB2 proteinexpressedin E. coli hasbeenshownto bind theMYB-recognition sequence,PyAACTG,which supportsitsrole as a DNA-binding protein.Another A. thaliana droughtstress–inducedgene,rd22 (56), hasa promoterwith no ABRE but with two recognitionsitesfor the transcription factors MYCand MYB.Binding of theATMYB2 proteinappears likelybuthas notbeen provenexperimentally.

ASSESSMENTOFPROMOTERSIN TRANSGENIC PLANTS Promoteranalysisusingtransientexpressionassayshasresultedin thecharacterizationof severaldistinctcis-actingelementsandthecloning of relatedtranscriptionfactors.However,tests with a rangeof promoters derived fromdrought- or ABA-induciblestructuralgenesin transgenicplantshaveshownthat the promoteractivitiesdefinedin transientassaysarenotalwayscorrelatedwith theexpressionpatternsof their corresponding structural genes.A summaryof resultsis givenin Table5. A problemwith theapproach couldbe theuse of heterologous plantexpres-sion systems.Although the genesare alwaysactive in seeds,expressioninvegetativetissuesis notalwaysinducedupondroughtorABA treatment,whichpointsto anincompleteactivationof thetranscriptionalmachinery.It is inter-estingto notethat ectopic expression of theotherwise seed-specificabi-3geneproduct(42) allows the ABA-mediatedactivationof Lea genesin vegetativetissuesof A. thaliana (100). Similarly, the CDeT27–45promoter from C.plantagineumwasonly fully responsiveto ABA in A. thaliana in the presenceof the ABI3 product (39a). Theseexperimentssuggestthat the ABI3 geneproductcan functionallyinteractwith different promoters.

SecondMessengersandSignalingMolecules

Proteinphosphorylation and dephosphorylation (via kinasesand phosphory-lases,respectively)aremajor mechanisms of signal integration in eukaryoticcells.Two A. thalianagenesencodingcalcium-dependentkinasesareinducedby dehydration(Table 1; 127), which suggeststhat they may participatein


phosphorylation processesoccurringin responseto drought.A serine-threon-ine-typeproteinkinasehasalsobeenisolatedfrom wheatandshowsaccumu-lation in ABA-treatedembryosandin dehydratedshoots(Table1; 4). How-ever,thephosphorylation targetsof thesekinasesarenot yet known,andtheirexact roles are obscure.

A role for protein phosphorylation in the drought-stressresponseis alsosuggestedon the basisof functional studiesof the ABA-responsive RAB17proteinfrom maize(45). This proteinis highly phosphorylatedin vivo, prob-ably viacatalysisby casein kinase2. The RAB17 proteinhas been foundto bedistributedbetweenthecytoplasmandthenucleusof maizeembryos,in differ-ent statesof phosphorylation(5, 45).Biochemicalstudies showed that RAB17binds peptides with nuclearlocalization signals andthatthebinding isdepend-enton phosphorylation.It hasbeensuggestedthatRAB17 mediatesthetrans-port ofspecific nuclear-targeted proteinsduringstress (45).

Cytoplasmiccalciumactsasasecondmessengerin manycellularprocessesandmayalsobe involved in thesignalingpathwaysmediatingtheexpressionof drought-relatedgenes(13). Stomatalclosureis an early plant responsetodrought,andincreasesin thecytosolicconcentrationof freecalcium,togetherwith pH changes,areconsideredto be primary eventsin the ABA-mediatedreductionof stomatalturgor(115).However,it is likely thatcalcium,togetherwith phosphorylationprocesses,playsa moregeneralrole in themechanismsassociatedwith drought-stressperception.For example,the A. thaliana ABI1geneproductis thought to bea calcium-activatedphosphoprotein phosphatase(74, 86). Furthermore,a transcriptencodinga phosphatidylinositol-specificphospholipaseC, an enzymeinvolved in catalyzingthe synthesisof inositol1,4,5-triphosphate, increases during dehydration (Table 1; 53); inositol-triphosphatestimulatesthe release of Ca2+ from intracellularstores.

PosttranscriptionalControl

Much of the effort to understandgeneregulationduring drought has beendevotedto transcriptionalmechanisms,but it has becomeclear that otherpotentialcontrolpointsincludemRNA processing,transcriptstability, transla-tion efficiency, and protein modification or turnover.Generalposttranscrip-tional mechanismsin plantshaverecentlybeenreviewed(41, 121).Evidenceis emergingthat thesemechanisms alsoplay a role duringstressresponses.InC. plantagineum,droughtstressinducessomeproteinsthataresynthesizedina light-dependentmanner(seeabove);for someof theseproteinsthelevelsofthemRNA do not parallelthoseof theproteins,which suggestsposttranscrip-tional regulation(2). A more detailedanalysisof alfalfa (Medicagosativa)suggeststhat increasedmRNA stability is involvedin theaccumulationof theMsPRP2transcript(30). ThemaizepMAH9cDNA cloneencodesa transcriptthat is upregulatedby drought.The correspondingproteinhasRNA-binding


characteristics,which suggeststhat itmayplayarole in the selective stabiliza-tion of mRNAs(Table2; 78).

A secondmajorcontrolpoint appearsto betheposttranslationalmodifica-tion of proteins,in which phosphorylation is a key mechanism.For example,phosphorylation is involved in the modification of the fructose-1,6-bisphos-phatasein drought-stressedleavesof sugarbeet(Betavulgaris) (52).Someofthe proteasesinducedby drought stress(Tables1 and 2) may also haveafunction in posttranslational modification. Schaffer& Fischer(113) havehy-pothesizedthat a thiol protease,the mRNA of which is cold-inducedin to-mato, could proteolytically activatecertainproteins.This mechanismcouldalso operateduring drought stress.It hasalso beensuggestedthat putativeproteaseinhibitorsinducedduringdrought(Table2) havea role in controllingthe activityof endogenousproteases(31).

Downregulation of Genes

Until now,mostresearchhasfocusedonunderstanding howrelevantgenesareupregulatedduring droughtstress.However, the responseto drought alsoinvolves the downregulation of severalgenes.For example,studiesof C.plantagineumhave revealed thattranscriptsencodingproteinsrelevantto pho-tosynthesis aredownregulatedduring thedehydrationprocessandthuspossi-bly reducephotooxidative stress(C Bockel & D Bartels,unpublished data).Jianget al (59) havealsoshownthat the promoter regionsof storageproteingenescontainthe information for their downregulation during seeddesicca-tion. Furthermore,it has recentlybeenreportedthat histoneH1 transcriptsaccumulatein responseto droughtstressin vegetativetissuesof tomato,and itwas suggestedthat H1 histonesare implicatedin the repressionof geneex-pression(E Bray,personal communication).

TRANSGENICPLANTS ASSESSINGGENEFUNCTION

Transgenicplantsallow the targetedexpressionof drought-relatedgenesinvivo andarethereforeanexcellentsystemto assessthefunctionandtoleranceconferredby theencodedproteins.With ectopicexpressionof genesinvolvedin controlling ABA biosynthesis,it shouldalsobepossibleto alterthehormo-nalbalancein vivo andthusto clarify theroleof ABA in thedroughtresponse.Anotherpurposefor usingtransgenicplantsis to improvedroughttolerance inagronomicallyvaluable plants.However, despiteextensive research,examplesof transgenicplantswith improvedstresstolerancearescarce(seealso12).Areasonfor this is thatstresstoleranceis likely to involvetheexpressionof geneproductsfrom several pathways.

The accumulationof low–molecularweight metabolites that act asosmo-protectantsis a widespreadadaptationto dry, saline, and low-temperature


conditionsin manyorganisms.In engineeringplantsthatsynthesizeprotectiveosmolytes,microorganismsappearto beusefulsourcesfor genes.Transgenictobacco plantsthatsynthesizeandaccumulatethe sugar alcoholmannitolhavebeenobtainedby introducing a bacterialgenethat encodesmannitol1-phos-phatedehydrogenase.Plantsproducingmannitol showedincreasedsalt toler-ance(122).Similarly, a freshwatercyanobacteriumthatwastransformedwithE. coli bet genesproducedsignificantamountsof glycine betaine;this stabi-lized photosynthetic activity in the presenceof sodium chloride, allowingimprovedgrowth(94).Tobaccoplantsthataccumulatethepolyfructosemole-cule fructanhavebeenengineeredusingmicrobial (Bacillussubtilis or Strep-tococcusmutans) fructosyltransferasegenes.Theseplantsshowedimprovedgrowth under polyethylene-mediateddrought stress(105), with a positivecorrelationobservedbetweenthe level of accumulatedfructansanddegreeoftolerance.The mechanismby which fructansconfer toleranceis not known,althougha mere osmotic effect seems unlikely.

Oneconsequenceof droughtandmanyotherstressesis the production ofactivatedoxygen molecules that causecellular injury, and thereforeplantswith increasedconcentrationsof oxygenscavengersshouldshow improvedperformancesunder nonlethal stress conditions.WhentobaccoMn-superoxidedismutase wasoverexpressedin alfalfa,theplantsshowedanincreasedgrowthrate after freezing stress (85).

Although Lea-relatedgenesareupregulatedabundantlyin mostplantsdur-ing all typesof osmotic stress,separateectopicexpressionof threedifferentrepresentatives in tobaccodid not yield an obvious drought-tolerant phenotype(55). However,this result is perhapslesssurprisingconsideringthat droughtstressdoesinducean array of different LEA-relatedproteinsin plants.It isalsolikely thatotherfactorsarerequiredfor theexpressionof tolerancewhereLEA-typeproteinsareinvolved.

FUTUREPERSPECTIVES

Despitethemanygenesthathavebeenidentified in associationwith droughtstress,muchof thedatais descriptive,with thefunctionsof only a few of theencodedproteinsestablished.The productionof mutantsusingan antisense-RNA approachis a powerful techniquethat should continue to elucidatecertainaspectsof stresstolerance,but it hasbeenmostsuccessfulonly withwell-characterizedareasof plant metabolism. It is also difficult to devisescreeningproceduresfor usefuldehydration-tolerancemutants, becauseof thearray of processessimultaneouslyaffectedby drought.Resurrectionplantswould be an excellentsourcefor mutantswith decreasedtolerance,but C.plantagineum, as well as many other resurrection-plantspecies,has apolyploid genomeandis thusunsuitable.Mutantanalysessofar exploitedfor


droughtstresshavebeenwith ABA-relatedmutations, and the powerof theapproachis shownin the cloning of Abi1 andAbi3 (43), which hasprovidednewperspectives.Anothervaluableapproachmaybeto identify thosemetabo-lic stepsthat aremostsensitiveto droughtstress(a techniqueusedto geneti-cally dissectsaltstressin yeast)(116).Suchanapproachcanat leastbegintoelucidatewhich gene productsare of primaryimportance.

The plant hormoneABA regulatesdifferent aspectsof the drought-stressresponse, andthus the synthesisof pureactive ABA analogues (103)may helpin the development of probesfor ABA-binding proteins,which could thenshedsomelight on primary signals.In contrastwith thesituation with signalperception,someinformation is availableon cis- andtrans-regulatoryfactors.Severalelementsin a promoterneedto cooperatewith multiple DNA-bindingproteins to mediate geneexpression.Therecentlydescribedcoupling elements(118) are probablyonly a beginningin resolvingtheregulatory network. Littleprogress hasbeen madewith the cloning and analysis of drought-relatedtranscriptionfactors,althougha biochemical approachanduseof therecentlyestablished yeastone- and two-hybrid systems(133) should producenewinsights. Regulation atstagesbeyondtranscription mustalsobefurtherconsid-ered,becausethiscouldmakeamajorcontribution to thefinal geneexpressionpattern.

Thecomplexity of droughttoleranceapparentthroughoutthis reviewpointsto control by multiple genes,andthus the identificationof quantitative-trait-loci (QTLs) for droughtresistancemay well be an effectiveanalyticaltool.The approachhas just begunto be applied to the environmental-stressre-sponsesof plants(126)andis particularlypromisingconsideringthatsaturatedDNA–markermapsarenow availablefor bothgeneticmodelplantsandcropplants.

Themolecularanalysisof the drought response hasarrived atastage whereresearchcanbuild upona largecollectionof characterizedgenes.The useofnovel approachescombining genetic,biochemical,andmolecular techniquesshouldprovideexcitingresultsin the near future.

ACKNOWLEDGMENTS

Theauthors wish to thank F.Salaminifor hissupport andfor comments onthemanuscript,M. Pasemannfor help in preparingthe manuscript,and the EUPTP project for financial support.We apologizeto all colleagueswho havecontributedto this researchareawhosework hasnot beencited becauseoflimited space.

Any Annual Reviewchapter, aswell asany arti clecited in an Annual Reviewchapter,may bepurchased fromthe Annual ReviewsPreprints and Reprints service.

1-800-347-8007; 415-259-5017; email: [email protected]


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