Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic ...

19 Pages·2010·4.58 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.285,NO.12,pp.9190–9201,March19,2010
©2010byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Overproduction of the Membrane-bound Receptor-like
Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance
in Arabidopsis*□S
Receivedforpublication,August10,2009,andinrevisedform,January19,2010Published,JBCPapersinPress,January20,2010,DOI10.1074/jbc.M109.051938
YurikoOsakabe‡,ShinjiMizuno§¶,HidenoriTanaka‡,KyonoshinMaruyama§,KeishiOsakabe(cid:1),DaisukeTodaka‡,
YasunariFujita§,MasatomoKobayashi**,KazuoShinozaki‡‡,andKazukoYamaguchi-Shinozaki‡§1
Fromthe‡GraduateSchoolofAgriculturalandLifeSciences,UniversityofTokyo,1-1-1Yayoi,Bunkyo-ku,Tokyo113-8657,the
§BiologicalResourcesDivision,JapanInternationalResearchCenterforAgriculturalSciences(JIRCAS),1-1Hogwash,Tsukuba,
Ibaraki305-8686,the¶GraduateSchoolofScienceandTechnology,ChibaUniversity,Inage,Chiba263-8522,the(cid:1)DepartmentofPlant
Biotechnology,NationalInstituteofAgrobiologicalSciences,2-1-2Kannondai,Tsukuba,Ibaraki305-8602,the**Experimental
PlantDivision,RIKENBioresourceCenter,3-1-1Kouya-dai,Tsukuba,Ibaraki305-0074,andthe‡‡GeneDiscoveryResearchGroup,
RIKENPlantScienceCenter,1-7-22Suehiro-cho,Yokohama,Kanagawa230-0045,Japan
RPK1(receptor-likeproteinkinase1)localizestotheplasma tivityandyields.Anincreasedunderstandingoftheregulatory
membraneandfunctionsasaregulatorofabscisicacid(ABA) mechanisms underlying the plant responses to water deficit
signalinginArabidopsis.Inourcurrentstudy,weinvestigated stresswillprovidekeyinsightsforfuturemolecularbreedingof
the effect of RPK1 disruption and overproduction upon plant suchstress-tolerantplants.Understressconditions,theexpres-
responses to drought stress. Transgenic Arabidopsis overex- sionofavarietyofgenesthatfunctionnotonlyinstresstoler-
pressingtheRPK1proteinshowedincreasedABAsensitivityin ancebutalsoinstressresponsesisup-regulated.Theplanthor-
theirrootgrowthandstomatalclosureandalsodisplayedless mone abscisic acid (ABA)2 regulates a range of physiological
transpirationalwaterloss.Incontrast,amutantlackingRPK1 events,includingseedmaturationanddormancy,thecontrolof
function,rpk1-1,wasfoundtoberesistanttoABAduringthese vegetativegrowth,andthetoleranceofplantstovariousabiotic
processesandshowedincreasedwaterloss.RPK1overproduc- stresses,suchasdrought,salinity,andcold(1).ABAisdramat-
tioninthesetransgenicplantsthusincreasedtheirtoleranceto icallyproducedunderwaterdeficitconditionsandregulatesthe
drought stress. We performed microarray analysis of RPK1 expressionofvariousgenesthatalsorespondtowaterdeficit
transgenicplantsandobservedenhancedexpressionofseveral stress.IthasbeenshownthatthebZIP-typetranscriptionfac-
stress-responsivegenes,suchasCor15a,Cor15b,andrd29A,in torsregulatetheactivationoftheABA-responsivegeneexpres-
addition to H O -responsive genes. Consistently, the expres- sioninArabidopsis(2).Otherfamiliesoftranscriptionfactors,
2 2
sion levels of ABA/stress-responsive genes in rpk1-1 had such as MYC, MYB, homeodomain protein, NAC, and AP2/
decreasedcomparedwithwildtype.Theresultssuggestthatthe EREBP, have been shown to play roles in ABA responses in
overproductionofRPK1enhancesboththeABAanddrought plants(2).
stresssignalingpathways.Furthermore,theleavesoftherpk1-1 To further elucidate the ABA signaling mechanisms,
plantsexhibithighersensitivitytooxidativestressuponABA- geneticandphysiologicalanalyseshavebeenperformedpre-
pretreatment,whereastransgenicplantsoverproducingRPK1 viously, and both negative and positive regulators of ABA
manifest increased tolerance to this stress. Our current data signal transduction pathways have been identified (3). A
suggest therefore that RPK1 overproduction controls reactive numberofrecessivemutationsresultingininsensitivityand
oxygenspecieshomeostasisandenhancesbothwaterandoxi- hypersensitivity to ABA have been isolated, and the factors
dativestresstoleranceinArabidopsis. involvedhavebeenshownplayimportantrolesinABAsig-
naltransductionasbothpositiveandnegativeregulators(1,
4). Recently, also novel ABA receptor proteins, the PYR/
The acclimation of plants to ever-changing environmental PYL/RCARs (5, 6) and GTG1/2 (7), have been reported.
conditionsismediatedbyanorchestratedbutcomplexseriesof Interactions between the PYR/PYL/RCARs and the protein
signalingnetworksthatregulatecellularandmolecularevents. phosphatase2CsarefoundtobestimulatedbyABAandto
Waterdeficitconditionsaffectplantgrowthanddevelopment control its signaling by inhibiting protein phosphatase 2C
viavariousbiologicalandphysiologicalprocessesandthereby activity(5,6).ThePYR/PYL/RCARsencodetheSTARTpro-
limitplantproductivity.Improvingthewaterdeficitstresstol- teins and are functionally redundant in terms of ABA per-
eranceofplantsisthusimportantforincreasingcropproduc- ception (5, 6). Previous studies have also indicated that the
relativelylownumberofrecessiveABA-insensitivemutants
*ThisworkwassupportedbyJapanSocietyforthePromotionofScience is due to such genetic redundancy. Furthermore, interac-
Grant-inaidforScientificResearchC21580125(toY.O.)andbytheMinis-
tryofAgriculture,ForestryandFisheries,Japan.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains 2Theabbreviationsusedare:ABA,abscisicacid;ROS,reactiveoxygenspecies;
supplementalFigs.1and2andTables1–3. RLK, receptor-like kinase; RT, reverse transcription; HA, hemagglutinin;
1Towhomcorrespondenceshouldbeaddressed.Tel.:[phone];Fax: ANOVA,analysisofvariance;HSD,honestlysignificantdifference;GM,ger-
81-3-5841-8009;E-mail:[email protected]. minationmedium;RH,relativehumidity.
9190 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
This is an Open Access article under the CC BY license.
AbioticStressToleranceandROSSignalingMediatedbyRPK1
tions among signaling cascades involved in abiotic stress EXPERIMENTALPROCEDURES
responses,suchasthosefordrought,salinity,andcold,and
PlantMaterialsandGrowthConditions—Seedsofwildtype
betweenabioticandtheotherpathways,suchasbiotic,phy-
Arabidopsis thaliana (Columbia; Co ecotype and Was-
tohormone,andsugarresponses,affectthecomplexityofthe
silewskija; WS ecotype), rpk1-1 (24), srk2e (25), and nced3-2
signaling networks and may cause functional redundancy
(26) mutants and transgenic plants were surface-sterilized,
among the components involved. Reactive oxygen species
sown on germination medium (half-strength Murashige and
(ROS) are partially reduced or activated forms of oxygen
Skoogsaltandvitaminmediumsupplementedwith0.8%(w/v)
(O )andincludesuperoxidefreeradicals,suchasH O ,and
2 2 2 agarand1%(w/v)sucrose),andgrowninagrowthchamberat
hydroxyl radicals, all of which are toxic and can cause the
2500 lux at 22°C under a 16-h light/8-h dark photoperiod.
oxidative destruction of cells. It has also been suggested,
TransgenicandcontrolplantsweregrownonGM-agarplates
however, that ROS also function as key regulators in plant
containingkanamycin(20mg/liter).
growth and development, cell cycle, hormone signaling,
RNAAnalyses—TotalRNAswereextractedfromtheleaves
biotic and abiotic stress responses, and programmed cell
orwholeplantsof3-week-oldArabidopsisgrowninsoilpotsor
death(8–10).ROSproductionisinducedbybothabioticand
onGM-agarplatestreatedwithorwithoutABAorH O with
biotic stress insults, such as high light, osmotic stress, and 2 2
TRIzol reagent (Invitrogen). RNA gel blot analysis was per-
pathogen attack. ROS have now been implicated as impor-
formedasdescribedpreviously(27).Forreal-timequantitative
tantsecondmessengersofABAsignalinginguardcells(11–
RT-PCR,reversetranscriptionoftheRNAsampleswascarried
13).TheguardcellsofanArabidopsisdoublemutantforthe
out with SuperScript III RNase H-reverse transcriptase
membrane-bound NADPH oxidase catalytic subunits,
(Invitrogen)withrandomhexamerprimers.Real-timequanti-
atbohD/atbohF,wereshowntobeimpairedinABA-induced
tative PCR was performed using real-time PCR system 7300
H O production and show a partially repressed ABA-in-
2 2 (AppliedBiosystems,FosterCity,CA)andSYBRPremixExTaq
ducedstomatalclosure(12).SRK2E/OST1andABI1,which
(Takara,Shiga,Japan).Theprimerpairsusedforreal-timePCR
catalyze phosphorylation and dephosphorylation during
arelistedinsupplementalTable1.Meltingcurveanalysiscon-
ABAsignaling,controlABA-stimulatedstomatalclosurevia
firmedthatonlyoneproductwasamplified.SpecificcDNAwas
ROS production (11, 14). Furthermore, recent studies have
quantifiedwithastandardcurvebasedonknownamountsof
indicated that the activation of an antioxidant system for
eachamplifiedcDNAfragment.18SrRNAwasalsoamplified
detoxification of ROS increases tolerance to drought stress
for calibration purposes, and three determinations were per-
(15,16).
formedforeachsample.Weperformedthreebiologicalrepli-
Receptor-like kinases (RLKs) belong to a large plant gene
cates for real-time quantitative RT-PCR. Microarray analysis
family(17)andcontainanextracellulardomain,asingletrans-
wasperformedusingtheAgilentTechnologies(PaloAlto,CA)
membrane domain, and a cytosolic Ser/Thr protein kinase
Arabidopsis22Koligonucleotidearray.TotalRNAwasusedfor
domain.TheRLKsareclassifiedonthebasisoftheirputative
the preparation of Cy5- and Cy3-labeled cDNA probes. The
extracellular ligand-binding domains, and the leucine-rich
Arabidopsis2OligoMicroarraykit(AgilentTechnologies)was
repeat receptor-like kinases are the largest group of RLKs
used to compare transcript profiles as described previously
withintheArabidopsisgenome,withmorethan200members
(28).
(18).Currentstudiesindicatethattheyfunctionindiversesig-
Protein Extraction and Protein Gel Blot Analysis—To pre-
naling events in Arabidopsis, such as brassinosteroid percep-
paretotalproteinextracts,3-week-oldtransgenicplantswere
tion by BRI1 and BAK1 (19); perception of bacterial flagellin
fragmentsbyFLS2(20),whichmediatesapathogenresponse; groundinliquidnitrogenandthawedinextractionbuffer(25
peptide plant hormone phytosulfokine perception by PSKR mMTris-HCl,pH7.0,5mMMgCl2,1mMCaCl2,0.25Msucrose,
(21); and recognition of the CLE family peptide hormone, and a protease inhibitor mixture (Roche Applied Science))
CLV3, by CLV1 (22). RPK1 is a leucine-rich repeat receptor- usingamortarandpestle.Theextractswerethencentrifugedat
likekinaseisolatedfromArabidopsis,anditsgeneexpressionis 10,000(cid:1)gfor10minat4°C,andthemicrosomalfractionwas
inducedbyABA,dehydration,highsalt,andlowtemperatures isolatedbycentrifugationofthesupernatantat100,000(cid:1)gfor
(23,24).TodeterminethefunctionofRPK1inABAsignalingin 60minat4°C.Theresultingmicrosomalmembranefraction
plants, we have previously isolated RPK1-knock-out mutants was suspended in extraction buffer containing 0.5% Triton
and constructed antisense-RPK1 transgenic lines. The loss of X-100. The concentration of extracted proteins was deter-
functionofRPK1showsABAinsensitivityinseedgermination, minedusingtheBio-Radproteinassay.Themicrosomalfrac-
plantgrowth,stomatalclosure,andgeneexpressioninArabi- tions were then separated by SDS-PAGE and blotted onto a
dopsis(24).Inourpresentstudy,wegeneratedtransgenicAra- phenylmethylsulfonyl fluoride membrane. The RPK1 protein
bidopsisoverproducingtheRPK1protein.Wethencompared was detected with anti-RPK1 peptide antibody (24), anti-
the effects of RPK1 disruption and overproduction in Arabi- phosphothreonine antibody from Zymed Laboratories
dopsisplantsupontheirsensitivitytowater-deficitconditions (Invitrogen),ormonoclonalanti-hemagglutinin(HA)antibody
andinvestigatedwhetherRPK1overproductionenhancesthe (Sigma).SignalsweredevelopedusingPierceSuperSignalWest
drought stress response pathways in these transgenic plants. Duraextendeddurationsubstrate(ThermoFisherScientific).
WealsoshowthattransgenicplantsoverproducingRPK1have Construction of Transgenic Plants—The full-length cDNA
an increased tolerance to drought stress as well as oxidative fragment of RPK1 was cloned into the pGreenII vector (29),
stress. whichcontainsthemodifiedcauliflowermosaicvirus35Spro-
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 9191
AbioticStressToleranceandROSSignalingMediatedbyRPK1
moterandthe(cid:2)-sequenceofthetobaccomosaicvirus(30).A endogenous ABA contents of rosette leaves of 4-week-old
double HA-His tag sequence was obtained by annealing the wild type, rpk1-1, 35S:RPK1, and control plants were mea-
primers5(cid:3)-ggccgcttacccatacgacgttccagactacgctggttacccatacga- suredaspreviouslydescribed(31).
cgttccagactacgctagatccggtcaccaccaccaccaccactaagagct-3(cid:3) and ResistancetoDroughtStress—Survivalratesassociatedwith
5(cid:3)-cttagtggtggtggtggtggtgaccggatctagcgtagtctggaacgtcgtatggg- droughttolerancelevelsweremeasuredasdescribedpreviously
taaccagcgtagtctggaacgtcgtatgggtaagc-3(cid:3)andligatingintoNotI- (32) with minor modifications. Briefly, 2–3-week-old plants
andSacI-digestedpGreenIIvectortoproduceHA/Hisfusion. weregrownonGM-agarplatesat22°Cundera16-hlight/8-h
The RPK1 sequence was amplified by PCR using the primers darkphotoperiodandthentransferredtofilterpaperondishes.
5(cid:3)-tctttctcttgtgaagcggccgctgaaaaatgaaact-3(cid:3) and 5(cid:3)-ttcttctagc- Theseplantswerethenleftunwateredfor8hin30%RHunder
ggccgcacacaatctagaaggctggat-3(cid:3),digestedwithNotI,andthen continuous white light and then rewatered for 2 days. The
clonedintothecorrespondingsitesinpGreenIItoobtainthe plantswerethenphotographed,andthenumbersofsurviving
expression plasmid 35S:RPK1 for plant transformation. This green plants were calculated. In addition, 4-week-old plants
vector was introduced into Agrobacterium tumefaciens C58 grownonsoilpotsat22°Cundera16-hlight/8-hdarkphoto-
cells,andplantsweretransformedasdescribedpreviously(24). periodin30–40%RHweresubjectedtodehydrationstressby
AsinglemutationwasintroducedintotheRPK1kinasedomain lackofwatering.After2weeksofwithholdingwater,theplants
bysubstitutinglysine289withglutamicacid(K289E)togener- wererewateredfor1weekandthenphotographed.Thesurviv-
atethemutantkinaseprotein(mRPK1).ThemRPK1fragment inggreenplantswerethencounted.Thirtyplantswereusedin
wasclonedintothepGreenIIvectortoexpressthisproteinin each experiment, and all experiments were repeated three
Arabidopsis. To generate transgenic plants containing RPK1 times.
promoterconstructs,weclonedtheRPK1promoterregion(24) Analysesoffreshweightlosswereperformedusingdetached
also into pGreenII containing RPK1-HA (RPK1pro:RPK1) or rosette leaves at the same developmental stage and size as
mRPK1-HA (RPK1pro:mRPK1) inserts. We subsequently 3-week-old plants. Three leaves per plant were excised and
obtained30transgenicT2linesoverexpressingtheRPK1gene maintainedinagrowthchamberin30%RH.Freshweightswere
usingeachfusionvector.Thelinesshowingthehighestexoge- measured at the indicated periods of time, and five plants of
nous RPK1 expression were then selected for further each genotype and transgenic line were analyzed in indepen-
experiments. dentexperimentsrepeatedfivetimes.
Preparation of Microsomal Membranes from Arabidopsis DetectionofReactiveOxygenSpecies—H O wasdetectedin
2 2
and Immunoprecipitation—Three-week-old transgenic plants leavesusing3,3(cid:3)-diaminobenzidinestainingasdescribedprevi-
were ground in liquid nitrogen and homogenized in an ously (33). Briefly, the first fully expanded leaf from both a
extractionbuffercontaining50mMTris-HCl(pH7.5),5mM 5-week-oldwildtypeanda35S:RPK1plantwerevacuum-infil-
MgCl ,0.25Msucrose,1mMEDTAwithproteinaseinhibitor tratedwith3,3(cid:3)-diaminobenzidinesolution(1mg/ml,pH3.8;
2
mixture (Roche Applied Science). The homogenate was fil- Sigma).Theleaveswerethenplacedinaplasticboxunderhigh
teredthroughMiraclothandcentrifugedat10,000(cid:1)gfor20 humidity until a reddish brown precipitate was observed and
min, and the supernatant was then ultracentrifuged at thenfixedwithasolutionof3:1:1ethanol/lacticacid/glycerol
100,000(cid:1)gfor30mintopelletthemicrosomalmembrane. andphotographed.
This pellet was then solubilized with PreserveXTM-QML H O Sensitivity and Superoxide Dismutase Activity Assay—
2 2
(QBI Life Sciences, Madison, WI). RPK1 proteins were Detachedfullyexpandedleavesfromtherosettesof4-week-
immunoprecipitatedfromthesolubilizedmicrosomalmem- oldwildtype,rpk1-1,35S:RPK1,andcontrolplantsgrownon
braneswithHA-agarose(Sigma). soilat22°Cundera16-hlight/8-hdarkphotoperiodin50%
ABA Sensitivity of Arabidopsis Root Growth—Arabidopsis RHwereharvestedandincubatedfor3daysinsolutionwith
seedsofwildtype,rpk1-1,and35S:RPK1plantsweresterilized or without H O under white light. These leaves were also
2 2
and germinated on GM. Five-day-old young seedlings were pretreated with 50 (cid:1)M ABA for 3 h and then transferred to
then transferred to fresh MS medium containing 1% sucrose H O solution. Photochemical efficiency (Fv/Fm ratio)
2 2
andvariousconcentrationsofABA.Therootlengthwasmea- measurements were taken after 3 days of treatment with
suredafter10daysofculture. H O using a PAM chlorophyll fluorometer (Walz, Effel-
2 2
Stomatal Movement Assay and Measurement of ABA trich, Germany), after the leaves had been allowed to dark-
Content—Stomatalmovementassayswereperformedessen- adaptfor60min.Chlorophyllcontentmeasurementsinthe
tially as described previously (8). Briefly, rosette leaves of leaves were performed as described by Arnon (34) by mea-
4-week-old wild type, rpk1-1, 35S:RPK1 and control plants suring the absorbance at 664 and 647 nm of 80% acetone
grownonsoilat22°Cundera16-hlight/8-hdarkphotope- extracts. Twenty leaves of each genotype were treated and
riod in 50% RH were harvested and incubated for 2 h in a measuredforeachcondition.Allexperimentswererepeated
solution containing 10 mM KCl, 0.2 mM CaCl , and 10 mM three times. Superoxide dismutase activity was determined
2
MES-KOH(pH6.15)underwhitelight(8).Theseleaveswere asdescribed(35).
subsequently transferred to a solution containing the same StasticalAnalysis—Statisticalanalyseswereperformedusing
bufferandABAandincubatedforafurther2h.Guardcells either Student’s t test or one-way analysis of variance
were photographed under a color laser three-dimensional (ANOVA),followedbyTurkey’shonestlysignificantdifference
profilemicroscope(Keyence,Tokyo,Japan).Twentystoma- (HSD) test using Statistical Package, version 7.0.1 (Japanese
tal apertures were measured for each leaf experiment. The edition;JMPSoftware).
9192 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
AbioticStressToleranceandROSSignalingMediatedbyRPK1
(At2g29120), Cor15a (At2g42540),
NCED3 (At3g14440), SRK2E
(At4g33950), Kin2 (At5g15970),
RD29A (At5g52310), and AtrbohD
(At5g47910).
RESULTS
RPK1 Overproduction Increases
ABASensitivityinArabidopsis—To
further understand the molecular
mechanismscontrollingtheRPK1-
mediated ABA signaling pathway,
wegeneratedtransgenicArabidop-
sisplants(Coecotype),whichover-
express the RPK1 gene fused to an
HA tag in the expression vector
underthecontrolofthecauliflower
mosaic virus 35S promoter. Thirty
transgenicT2plantsweresubjected
to RPK1 expression analysis by
Northernblotting.Transgeniclines
overexpressing RPK1 (35S:RPK1)
were selected, and their homozy-
gousT3plantswereusedforfurther
analysis.BybothNorthernandpro-
tein blot analyses, we detected the
highestlevelsofRPK1expressionin
transgeniclinesL1andL2,ascom-
paredwithwildtypecontrolplants
transformedwiththeemptyvector
(Fig.1A).Wealsogeneratedtrans-
genic lines overexpressing non-
tagged RPK1 under the control of
the 35S promoter in Arabidopsis
butcouldnotobtainlinesoverpro-
ducing the RPK1 protein (data not
shown). As shown in Fig. 1B, the
35S:RPK1 plants showed weak
growth retardation (supplemental
Fig.1).Previously,wereportedthat
rpk1 mutants manifest impaired
root growth inhibition when
exposedtoABA.Wethusanalyzed
root growth inhibition by ABA in
our current experiments using the
35S:RPK1linesL1andL2andcom-
FIGURE1.ABAsensitivityoftransgenicArabidopsisoverexpressingRPK1.A,expressionlevelsofRPK1in
transgenicplants.TotalRNAswereextractedfromtransgenicplantscarryingthe35Svector(vectorcontrol(VC)) pared these results with those
andfromfourtransgeniclinesof35S:RPK1(L1–L4;Columbiaecotype)andwereblottedandhybridizedwithacDNA obtained for the rpk1 mutant. The
probeforRPK1.Proteinblotanalysiswasperformedwithmicrosomalfractionsof35S:RPK1lines(L1–L4)usingan
negative effects of ABA on root
anti-RPK1antibody(23).B,35S:RPK1(linesL1–L3)andvectorcontrolplantsgrownonGM-agarplatesfor2weeks.
CandD,assayforrootgrowthinhibitionbyABA.Youngseedlingsof35S:RPK1(linesL1andL2),vectorcontrol,wild growthwerefoundtobereducedin
typeArabidopsis(WS),andrpk1-1weretransferredtoABA-containingmediumandincubatedfor10days.The therpk1mutants,whereasthe35S:
relativerootlengthsweremeasuredandareshownasapercentageoftherootlengthsgrownintheabsenceofABA.
Valuesarethemeans(cid:4)S.D.(n(cid:5)50).Theasterisksindicatestatisticallysignificantdifferencesincomparisons RPK1plantsshowedincreasedsen-
betweenWSandrpk1-1,determinedbyStudent’sttest,orincomparisonsbetweenvectorcontroland35S:RPK1, sitivitytoABA(Fig.1,CandD).The
evaluatedbyone-wayANOVAwithTukey’sHSD(*,p(cid:6)0.0001;**,p(cid:6)0.005).Bars,1cm. roots of the rpk1-1 plants grew
faster than those of the wild type
Accession Numbers—Arabidopsis Genome Initiative locus plantsonABA-containingmedia,whereastherootgrowthof
identifiers for the genes described in this article are as 35S:RPK1 plants was more severely inhibited by ABA com-
follows: RPK1 (At1g69270), FRK1 (At2g19190), AtGLR2.7 paredwiththecontrolplants.
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 9193
AbioticStressToleranceandROSSignalingMediatedbyRPK1
thenatureoftheABAsensitivitythatcontrolswaterlossduring
drought stress, we analyzed stomatal movement responses to
ABAinthe35S:RPK1plants(Fig.2,BandC).Comparedwith
thewildtypecontrols,theguardcellsof35S:RPK1plantsexhib-
ited smaller stomatal apertures without ABA treatment,
whereas rpk1-1 mutant plants showed insensitivity in ABA-
inducedstomatalclosure,similartotheresultsofourprevious
study (24). A slightly increased ABA content in rpk1-1 and
decreased ABA content in 35S:RPK1 was observed (supple-
mental Fig. 2), suggesting that the endogenous ABA levels in
theknock-outandtransgenicplantsdidnotaffecttheirpheno-
types. These data thus suggest that RPK1 overproduction
enhancesABAsignalingduringrootgrowthandalsothesto-
matalclosingofguardcellsinthetransgenicplants.
RPK1 Overproduction Increases the Tolerance to Drought
Stress in Arabidopsis—To investigate the physiological func-
tionofRPK1duringdroughtstress,weexaminedwhetherthe
disruptionoroverproductionofRPK1wouldaffectthetoler-
ancetothisconditioninplants(Fig.3).Wildtype,rpk1-1,35S:
RPK1(L1,L2,andL3lines),nced3-2(amutantofakeyenzyme
in ABA biosynthesis during drought stress; 9-cis-epoxicarot-
enoiddioxygenase3),and35S:NCED3(31)plantsgrownfor2
weeksonGM-agarplatesundernormalconditionsweretrans-
ferredontofilterpaperandthenexposedtodroughtstressby
ceasingthewatersupplyin40%RHfor8h.Afterthisdrought
stresstreatment,theplantswerewateredagain,incubatedfora
further2days,andthenanalyzedforsurvivinggreenplants(Fig.
3,BandC).Thesurvivalratesofrpk1-1andnced3-2werelower
thanthoseofthecontrolplantsafterdroughtstress.However,
the survival rates of the 35S:RPK1 plants were significantly
higher((cid:7)80%)thanthoseofthecontrolplantsandweresimilar
tothoseofthe35S:NCED3plants(Fig.3B).Tofurtherexamine
droughtstresstolerance,weselectedtwoadditional35S:RPK1
lines(L5andL6)thatshowedcharacteristicssimilartothoseof
theL1andL2lines(Fig.3B).Fig.3,DandE,showsthesurvival
ratesof35S:RPK1linesL5,L6,andL3grownonsoilpotsunder
longtermdroughtstress.Thetransgenicplantsgrownonpots
were exposed to drought stress by stopping watering in a
FIGURE2.Responseoftherpk1-1mutantandRPK1overexpressionlines
30–40%RHenvironmentfor2weeks.Afterthisdroughtstress
totheregulationoftranspirationunderwaterstressconditions.A,tran-
spirationalwaterlossinwildtype(WS),rpk1-1,RPK1overexpression(35S:RPK1 treatment,theplantswerewateredandculturedforafurther1
linesL1andL2),andcontrolplants(VC)attheindicatedtimepointsafterleaf week and then analyzed for surviving green plants (Fig. 3D).
detachment. Water loss is expressed as a percentage of the initial fresh
weight.Valuesarethemeans(cid:4)S.D.offivesamplesofthreeleaveseach. The 35S:RPK1 plants showed improved stress tolerance to
B,effectsofABAonstomatalclosureinwildtype,rpk1-1,35S:RPK1L1andL2, droughtcomparedwiththewildtypeplants.Theseresultsindi-
andcontrolplants.LeavesweretreatedwithorwithoutABAfor2hafter
cateimportantrolesfortheRPK1-mediatedsignalingpathway
stomatalpreopeningunderlightfor2h,andthestomatalaperturewasmea-
sured.Valuesarethemeans(cid:4)S.D.of20measurements.Theasterisksindicate inbothshorttermandlongtermdroughtstresstolerancesin
statisticallysignificantdifferencesincomparisonsbetweenwildtypeand Arabidopsis.
rpk1-1,determinedbyStudent’sttest,orincomparisonsbetweencontrol
and35S:RPK1,evaluatedbyone-wayANOVAwithTukey’sHSD(*,p(cid:6)0.0001; RPK1OverproductionEnhancestheTranscriptionofStress-
**,p(cid:6)0.0005;***,p(cid:6)0.005).C,guardcellsof35S:RPK1L1andcontrolplants responsive Genes—To further elucidate the effects of RPK1
treatedwithorwithout5(cid:1)MABAfor2h.Bars,30(cid:1)m. overproductiononABAandstresssignalingpathwaysinAra-
bidopsis during drought stress, we performed comparative
ABAsignalingplaysacrucialroleinreducingwaterlossby microarray analysis of wild type and 35S:RPK1 plants grown
regulatingstomatalclosingunderconditionsofdroughtstress. undernormalconditionsusinganAgilentArabidopsis2Oligo
ToinvestigatetheroleofRPK1inthisprocess,wemeasuredthe Microarray (Agilent Technologies), covering (cid:7)21,000 genes.
freshweightlossindetachedleavesfromrpk1-1and35S:RPK1 Two experiments were performed using RNA samples from
plants (Fig. 2A). The disruption of RPK1 resulted in an two independent lines overexpressing the RPK1 protein (L1
increasedwaterlossratewhencomparedwiththewildtypeWS and L2). Each sample was labeled differently (Cy3 or Cy5).
ecotype.Incontrast,theoverproductionofRPK1intransgenic Genes exhibiting an expression ratio (35S:RPK1/wild type)
Arabidopsisledtoadecreaseinwaterloss.Tofurtherexamine higherthan3arelistedinTable1andsupplementalTable2.
9194 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
AbioticStressToleranceandROSSignalingMediatedbyRPK1
shockprotein(HSP)(36),andcalcium-bindingproteins(Table
1). Genes for stress-responsive transcription factors, such as
TEM1,RAV1,andRAV2/TEM2(37),andasenescence-related
transcriptionfactor,WARKY53(38),werealsofoundtobeup-
regulatedintheRPK1transgenicplants.
Fig.4illustratestherelativetranscriptlevelsofseveralstress-
responsivegenes,standardizedusingabsolute18SrRNAtran-
scriptlevels,duringdroughtstressinArabidopsis.Theexpres-
sion levels of the ABA-responsive genes Kin2, Cor15a, and
RD29A (2) were significantly reduced in the rpk1-1 mutant
afterdroughtstress.Incontrast,theexpressionlevelsofthese
samegeneswerestronglyincreasedin35S:RPK1plants,indi-
catingthatRPK1overproductionenhancestheirtranscription.
The expression levels of the putative glutamate receptor,
AtGLR2.7 (AT2g29120) were also found to be affected by
drought stress (i.e. decreased in rpk1-1 and increased in 35S:
RPK1)(Fig.4andsupplementalTable2),suggestingthatRPK1
alsoregulatestheexpressionofdroughtstress-responsiveGLR.
Theexpressionlevelsofareceptor-likekinaseFRK1,whichis
inducedbydroughtstressinwildtypeplants,werefoundtobe
down-regulatedinrpk1-1andup-regulatedin35S:RPK1(Fig.4
and supplemental Table 2). The expression levels of the
NADPHoxidasegene,whichhasrolesinROSproduction,were
alsoup-regulatedbyRPK1overproduction(Fig.4andsupple-
mental Table 2). In contrast, several stress-responsive genes,
such as RD20, DREB, and NAC (2), were shown to be down-
regulatedinthe35S:RPK1plants(supplementalTable3).This
down-regulation is somewhat unexpected but may be caused
FIGURE3.Droughttoleranceofthe35S:RPK1plants.A,proteinblotanaly-
byafeedbackcontrolmechanismwherebytheactivationofthe
sis was performed with microsomal fractions of the 35S:RPK1 transgenic
plants(linesL1,L2,L5,andL6)usingananti-RPK1antibody.NS,nonspecific RPK1-responsivestress-relatedpathwayrepressestheexpres-
proteins.BandC,enhancedtolerancetorapiddroughtstressinthe35S:RPK1 sion of stress-responsive genes regulated by other such path-
(lines L1 and L2) plants. Transgenic plants, including vector control (VC),
ways in the transgenic plants. Further experiments will be
grownonGM-agarplatesundernormalconditionsweretransferredonto
filterpaper,andwaterwaswithheldfor8hunder40%RHconditions.After needed to clarify this phenomenon. Expression of the cytoki-
droughtstresstreatment,theplantswerewateredandincubatedforafur- nin-responsiveARRgenes(27)andGATAtypetranscription
ther2daysandthenphotographed(B).Survivalratesweredeterminedasthe
numberofvisiblegreenplantsafterrehydration(C),and20plantswereused factorgenes(39)wasalsofoundtobedecreasedin35S:RPK1.
ineachexperiment.Valuesarethemeans(cid:4)S.D.calculatedfromtheresultsof RPK1OverproductionEnhancedROSScavengingActivity—
threeindependentexperiments.Bars,1.0cm.DandE,enhancedtoleranceto
ROSisimplicatedinABAsignalingasanessentialsecondmes-
longtermdroughtstressinthe35S:RPK1(linesL5andL6)plants.Thetrans-
genicplantsweregrownonpotsundernormalconditions,andwaterwas senger,andindeedABA-inducedstomatalclosureinvolvesthe
withheldfor2weeksin30–40%RHconditions.Afterthisdroughtstresstreat- ROSproductionthatactivatesCa2(cid:8)channels(12).Inourpres-
ment,theplantswerewateredandincubatedfor1weekandthenphoto-
entmicroarrayanalysis,ROS-responsivegeneswerefoundto
graphed(D).Survivalratesweredeterminedasthenumberofvisiblegreen
plantsafterrehydration(E),and20plantswereusedineachexperiment. beup-regulatedintheRPK1-overproducingplants(Table1and
Valuesarethemeans(cid:4)S.D.calculatedfromtheresultsofthreeindependent
supplementalTable2),suggestingthatROShomeostasismight
experiments.Theasterisksindicatestatisticallysignificantdifferencesincom-
parisonsbetweenWSandrpk1-1,determinedbyStudent’sttest,orincom- beaffected.TofurtherevaluatetheeffectsofRPK1overproduc-
parisons between VC, 35S:RPK1, and 35S:NCED3, evaluated by one-way tioninthetransgenicplantsduringoxidativestress,weinves-
ANOVAwithTukey’sHSD(*,p(cid:6)0.0001;**,p(cid:6)0.005;***,p(cid:6)0.05).
tigatedtheH O sensitivityofrpk1-1and35S:RPK1leavesby
2 2
detectingtheirabilitytoremoveexogenousH O .Inwildtype
2 2
Genesforwhichtheexpressionratio(wildtype/35S:RPK1)was leaves, pretreatment of ABA caused more severe bleaching
lowerthan3areshowninsupplementalTable3.Incomparison whentheywerefloatedon10mMH O underlight(Fig.5A).
2 2
withthevectorcontrolplants,27geneswerefoundtobeup- Leavesoftherpk1-1mutantshowedhighersensitivitytoexog-
regulatedwithanaverageexpressionratiogreaterthan3(Table enousH O thanthoseofwildtype,suggestingthattheRPK1
2 2
1).Amongthese,12geneswereidentifiedaswaterstress/ABA- mutation impairs the ability to scavenge exogenous H O . In
2 2
responsive genes induced by drought, high salinity, cold, contrast,the35S:RPK1leavesremainedgreenduringincuba-
osmoticstress,andABAtreatment.Theseincludedgenessuch tion with H O both with and without pretreatment of ABA,
2 2
asCor15a,Cor15b,RD29A,andGolS3(2),whichwereobserved indicatingthatRPK1overproductionenhancestheresistance
tobeup-regulatedintheRPK1transgenicplants(Table1and tooxidativestress(Fig.5B).
supplementalTable2).Othergeneswerealsofoundtobeup- H O sensitivitywasfurtherassessedbymeasuringofpho-
2 2
regulatednotonlybywaterstressandABAtreatmentbutalso tochemical efficiency of photosystem II and also the chloro-
byH O treatmentandheatstress,includingANAC036,heat phyll contents in the H O -treated leaves of rpk1-1 and 35S:
2 2 2 2
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 9195
AbioticStressToleranceandROSSignalingMediatedbyRPK1
TABLE1
Theup-regulatedgenesinthemicroarraydataofuntreated35S:RPK1(35S:RPK1/wildtype)
-Foldchange
Locus Gene 3w5iSld:RtPypKe1a/ Dryb,c NaClb,c Coldb,c ABAc,d Heatb,c H2O2c
-fold
Stress-responsive
genes
At2g42540.1 Cold-regulatedprotein,Cor15a 5.4 39.8 38.3,7.8 67.4,30.9 8.2,40.4
At5g52310.1 Lowtemperature-responsiveprotein78(LTI78)/rd29A 3.5 13.4 16.7,20.1 17.9,44.4 30.7,25.20
At4g30650.1 Hydrophobicprotein/lowtemperatureandsalt 3.4 41.3,6.2 3.7
responsiveprotein
At2g42530.1 Cold-regulatedprotein,Cor15b 3.2 20.3,2.5 36.9,18.1 6.3,6.5
Metabolism
At1g09350.1 Galactinolsynthase(GolS3) 4.0 53.9,103.4
At1g75900.1 FamilyIIextracellularlipase3EXL3(EXL3) 3.8 2.3 3.0,3.0 2.2
At1g64400.1 Long-chainacyl-CoAsynthetase,putative 3.6
At3g44870.1 S-Adenosyl-L-methionine:carboxylmethyltransferase 3.2 4.0 11.1 63.3
familyprotein
Disease
At2g43620.1 Chitinase 5.1 2.5,2.2 9.0 5.5,11.9 3.0
At3g52430.1 Phytoalexin-deficient4protein(PAD4) 3.4 3.8 2.3
TF
At1g25560.1 AP2domain-containingtranscriptionfactor 4.4 3.2 2.3 4.5
At1g13260.1 DNA-bindingproteinRAV1(RAV1) 3.9 2.0 4.5,6.6 2.0
At1g68840.1 DNA-bindingproteinRAV2(RAV2) 3.9 3.8 3.0 2.0
At4g23810.1 WRKYfamilytranscriptionfactorAR411(WRKY53) 3.9 4.1,12.5 3.1 2.2
At2g17040.1 ANAC036noapicalmeristem(NAM)familyprotein 3.6 2.4,8.4 3.2 51.9
Hsp
At3g09440.1 Heatshockcognate70kDaprotein3(HSC70-3) 3.7 5.9 2.5 8.6,2.0 2.3
At5g52640.1 Heatshockprotein81-1(HSP81-1)/heatshock 3.4 4.3 2.0 32.7,8.9 11.9
protein83(HSP83)
At5g56010.1 Heatshockprotein(HSP81-3) 3.3 4.0 6.9,4.5
At3g12580.1 Heatshockprotein70,putative/HSP70 3.0 31.0 5.2,3.7 147.7,4.1 47.6,5.6 14.3
At5g23240.1 DNAJheatshockN-terminaldomain-containingprotein 3.0 3.7
Calcium
At5g39670.1 Calcium-bindingEF-handfamilyprotein 4.2 6.1 4.5 8.9 39.4 6.7
At5g62070.1 Calmodulin-bindingfamilyprotein 3.5 2.1
At2g41090.1 Calmodulin-likecalcium-bindingprotein,22kDa(CaBP-22) 3.0 2.9 2.5 2.3
Transporter
At5g50200.1 Nitratetransporter,NRT3.1 5.2 4.3 2.4
Other
At2g24600.1 Ankyrinrepeatfamilyprotein 3.4 4.0,6.2 6.5 6.8
Unknown
At2g20142.1 Expressedprotein 4.2 2.8 3.3 61.8 2.4
At4g38560.1 Expressedprotein 3.1 3.0 3.2
aValuesarethemeansandS.D.oftwodifferent35S:RPK1lines(ratio(cid:7)3.0).
bThemicroarraydata(boldfacecharacters)fromMaruyamaetal.(58)(ratio(cid:7)2.0).
cThemicroarraydataareavailableinGenevestigator(availableontheWorldWideWeb)(ratio(cid:7)2.0).
dMicroarraydata(boldfacecharacters)fromY.FujitaandK.Yamaguchi-Shinozaki(unpublisheddata)(ratio(cid:7)2.0).
RPK1. A decrease in photosynthetic activities was detected control, superoxide dismutase activity was induced by ABA
duringincubationoftheseplantsinH O solution,whereasitis treatment,butitsactivitiesinthe35S:RPK1leavesweresignif-
2 2
acceleratedafterpretreatmentwithABAinthewildtypecon- icantlyhigherthanwildtype.WethenevaluatedROSgenera-
trol(Fig.5C).Severedecreasesinphotosyntheticactivitieswere tionin35S:RPK1ArabidopsisleavesaftertreatmentwithABA.
observed in rpk1-1 leaves after pretreatment with ABA, an H O formationinthetransgenicplantswasvisualizedbypo-
2 2
approximate50%decreaseinthesolutionwithoutH O com- lymerizationwith3,3(cid:3)-diaminobenzidine.H O accumulation
2 2 2 2
pared with wild type in the same solution. In contrast, 35S: wasobservedasdarkbrowndepositsintheleavesofwildtype
RPK1leavesshowedsimilarphotosyntheticactivitiesevenafter plants(Fig.5F).Theleavesof35S:RPK1plants,however,didnot
treatment with H O . Similar results were obtained for the accumulateH O ,indicatingthatRPK1overproductionstim-
2 2 2 2
chlorophyllcontent.Thus,therpk1-1leaveswerefoundtobe ulatesscavengingROS.ThesedatathussuggestthatRPK1plays
more sensitive to H O , particularly after pretreatment with an important role in the control of ABA-responsive H O
2 2 2 2
ABA,whereas35S:RPK1leavesexhibitedahigherchlorophyll homeostasis.
contentcomparedwithwildtypecontrols(Fig.5D).Theregu- MutationoftheRPK1PhosphorylationSiteSuppressesRPK1
lationofantioxidantenzymesiscrucialfortheredoxstatesof Function—Tofurtherelucidatetheactivationmechanismsfor
cells.ThetranscriptionlevelsofgenesencodingROSscaveng- the RPK1 receptor-like kinase, we examined the effects of
ingenzymeswerenotseverelyalteredin35S:RPK1(Table1and amino acid substitutions on its activity in vivo (Fig. 6). Previ-
supplementalTable2),andwethusfurtherinvestigatedthese ously, substitution of the conserved lysine within the kinase
enzymeactivitiesinthetransgenicplants.Theactivityofsuper- domainsoftheplantRLKs(toglutamicacid)hasbeenshownto
oxide dismutase was determined in 35S:RPK1 and wild type abolish their activity (40). We thus created an enzymatically
leaveswithorwithoutABAtreatment(Fig.5E).Inthewildtype inactiveformofRPK1inthesameway(Fig.6A;K289Emutant
9196 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
AbioticStressToleranceandROSSignalingMediatedbyRPK1
beentransformedwithmRPK1did
not show an altered size, whereas
35S:RPK1 plants showed growth
retardation(Fig.6C).Wequantified
bothwaterstress-andROS-related
gene expression during drought
stress. As shown in Fig. 6D, the
expression levels of Kin2 and
AtrbohD in the 35S:mRPK1 trans-
genic plants were low, even under
droughtconditions,comparedwith
35S:RPK1.Thecalculatedwaterloss
rates further indicated that muta-
tion of the RPK1 kinase domain
abolishes RPK1 overproduction
phenotypesinthetransgenicplants
(Fig.6E,left).
Given the inherent nonspecific
effects of ectopic gene expression
underthecontrolofthecauliflower
mosaic virus 35S promoter, we
additionally generated transgenic
plantsexpressingRPK1drivenbyits
own promoter (RPK1pro:RPK1)
(24).TheRPK1pro:RPK1plantsdid
not show the growth retardation
(Fig. 6C). We then estimated the
expression levels of stress-respon-
sive genes during drought stress
in the RPK1pro:RPK1 plants. As
showninFig.6D,theexpressionlev-
els of Kin2 and AtrbohD in the
RPK1pro:RPK1 plants were higher
thaninthecontrolplants.Further-
more, in the rpk1-1 mutant, the
introductionofRPK1pro:RPK1res-
cued the water loss phenotype. In
contrast, RPK1pro:mRPK1 did not
functionintherpk1-1mutant(Fig.
6E,right).Takentogether,ourdata
indicate that RPK1 under the con-
trol of its native promoter shows
enhanced function in transgenic
FIGURE4.ExpressionlevelsofRPK1downstreamgenesinrpk1-1and35S:RPK1.RelativemRNAlevelswere Arabidopsis plants and that inacti-
determinedinrpk1-1and35S:RPK1plantsunderdehydrationconditionsbyquantitativeRT-PCR.Similarresults vation of the RPK1 kinase activity
wereobtainedfromthreeindependentexperiments,andatypicalresultisshownwithS.D.valuescalculated
abolishesitsfunction.
fromthreeindependentPCRsfromthesamebiologicalsample.0h,plantsgrownundernormalconditions;3h,
plantssubjectedtodehydrationstressfor3h.VC,vectorcontrol;WS,wildtype.
DISCUSSION
Plantshaveacquiredtheabilityto
RPK1).WethenproducedtransgenicArabidopsisplantsover- surviveinconditionsofscarcewaterbydevelopinghighlysys-
expressing this K289E mutant RPK1 (mRPK1) driven by the tematicsignalingnetworks,inwhichABAisoneoftheimpor-
cauliflowermosaicvirus35Spromoter.Theproteinsimmuno- tantregulatorsofincreasedplanttolerancetowaterstress(1,2).
precipitatedwithanti-HAantibodyfromthemicrosomalfrac- Inourcurrentstudy,weinvestigatedtheeffectsofoverproduc-
tionsof35S:RPK1and35S:mRPK1transgenicplantswerethen tion of a membrane-localized receptor-like kinase, RPK1, in
subjectedtoWesternblotanalysis.ImmunoprecipitatedRPK1- Arabidopsisandfoundthatitinducesvariousstress-responsive
HA, but not mRPK1-HA, cross-reacted with anti-phospho- genes and enhances the physiological responses to drought
threonineantibody(Fig.6B).Wethenevaluatedtheeffectsof stressandABAsensitivity.Ourdatasuggestthatunregulated
kinaseinactivationontheseplantphenotypes.Plantsthathad RPK1 enhances the downstream stress signal transduction
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 9197
AbioticStressToleranceandROSSignalingMediatedbyRPK1
pathways and increases the toler-
ancetodroughtstressintransgenic
Arabidopsis.
In our previous study (24), we
showed that rpk1 knock-out
mutantshaveadecreasedABAsen-
sitivity.Wewerethusinterestedin
what the effects of overexpressed
RPK1proteinwouldbeontheplant
responses to drought stress. In our
currentstudy,wenowshowthatthe
rpk1-1 mutant has decreased ABA
sensitivity with regard to root
growth inhibition, transpirational
water loss, and stomata closure,
whereas RPK1 overproduction
increases the sensitivity of these
processestoABA(Figs.1and2).In
ourcurrenttransgenicplantsover-
expressingRPK1,theexpressionof
many water stress-inducible genes
wasfoundtobeup-regulatedduring
droughtstress,andtheseplantsalso
showed strong tolerance to both
short term and long term drought
stress insults (Table 1 and Fig. 3).
These results additionally suggest
that the RPK1-signaling pathway
positivelycontrolsthetranscription
ofthesegenesandtherebyenhances
drought stress tolerance in plant
cells. Signaling proteins that func-
tion as transducers or mediators
needtobeactivatedinresponseto
cell signals. Receptor proteins per-
ceivesuchextracellularsignalsand
transduce them to the appropriate
downstream pathway. Enhance-
ment of the water stress and ABA
signalingpathwaysbyRPK1overex-
pression may thus be caused by an
increasedfrequencyofextracellular
signal perception by RPK1 or by
amplification of the signaling cas-
cadedownstreamofRPK1.
Ourmicroarrayanalysisindicates
thatvariousstress-responsivegenes
arecontrolledbyRPK1overproduc-
tion.Theexpressionlevelsofwater
stress-responsive genes, such as
Cor15a,Cor15b,RD29A,andGolS,
which encode LEA (late embryo-
genesis-abundant) proteins and a
key enzyme in biosynthesis of
osmoprotectants (raffinose family
oligosaccharides), are up-regulated
in RPK1 transgenic Arabidopsis.
These LEA proteins and osmopro-
9198 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
AbioticStressToleranceandROSSignalingMediatedbyRPK1
FIGURE6.MutationoftheRPK1phosphorylationsitesuppressestheRPK1function.A,alignmentoftheRPK1kinasedomainwithotherplantRLKs.A
conservedlysineinthekinasedomainisindicatedbydots,andasinglemutationofthisresiduewasintroducedbyasubstitutionwithglutamicacid(K289E;
yieldingmRPK1).B,RPK1isphosphorylatedonthreonineresiduesinvivo.Proteinsimmunoprecipitated(IP)withanti-HAantibodyfromthemicrosomal
fractionsof35S:RPK1and35S:mRPK1transgenicplantswereanalyzedbyWesternblot.RPK1-HAandmRPK1-HAproteinswerebothdetectableonimmuno-
blotsusinganti-HAantibody;phosphorylatedRPK1-HAproteinscouldbedetectedwithaphosphothreonineantibody.C,phenotypesofthe35S:RPK1,
35S:mRPK1,andRPK1pro:RPK1transgenicplants.Two-week-oldplantsareshown.Bar,1cm.D,expressionlevelsofRPK1downstreamgenesin35S:RPK1,
35S:mRPK1,andRPK1pro:RPK1transgenicplants.TherelativemRNAlevelsweredeterminedasshowninFig.4.0h,plantsgrownundernormalconditions;3h,
plantssubjectedtodehydrationstressfor3h.E,transpirationalwaterlossinthe35S:RPK1,35S:mRPK1,andRPK1pro:RPK1plantsoftheColumbiaecotype(left).
TheintroductionoftheRPK1pro:mRPK1constructintotherpk1-1mutantplantdidnotrescuethemutantphenotype(right).Thetranspirationalwaterlossrate
wasmeasuredasshowninFig.2.VC,controlplants.
tectantshavebeenshowntofunctionintheprotectionofcells ing drought stress. NADPH oxidases have been suggested to
duringosmoticstress(41,42).Theexpressionlevelofadrought functioninABA-inducedROSproductioninguardcells(8,11,
stress-responsive receptor-like kinase gene, FRK1 was also 12). In the RPK1pro:RPK1 plants, the transcription of water
foundtobeup-regulatedintheRPK1transgenicplants.Fur- stress-andROS-relatedgeneswasalsofoundtohaveincreased,
thermore,theexpressionofROS-responsivegenes,suchasthe althoughgrowthretardation,whichcansometimesresultfrom
HSP genes, is also up-regulated in RPK1 transgenic plants. the strong overexpression of stress-responsive genes (e.g. see
TheseHSPproteinsfunctionaschaperones,whichplayimpor- Ref.28),didnotoccurintheseplants.Ourcurrentresultsthus
tant roles in the acquisition of not only thermotolerance but suggest that RPK1 overproduction causes the increased tran-
also the adaptation to various environmental stresses (36). scription of water stress- and ROS-responsive genes via the
Genes encoding enzymes involved in fatty acid metabolism, enhancementofthesesignalingpathways.
suchasalong-chainacyl-coenzymeAsynthetase,arealsoup- Differenttypesofenvironmentalstresscauseanincreasein
regulated in the RPK1 transgenic plants (Table 1 and oxidative damage to photosynthetic proteins and pigments.
supplemental Table 2). Long-chain acyl-coenzyme A synthe- ROS signaling is controlled and modulated by its production
tase catalyzes free fatty acids to acyl-coenzyme A thioesters, and subsequent scavenging, which is mediated by scavenging
andthisisakeystepinfattyacidmetabolism,includingphos- enzymesandnon-enzymaticantioxidants(14,47).ROShome-
pholipid,triacylglycerol,andjasmonatebiosynthesisandfatty ostasisismaintainedbyalargegenenetworkinArabidopsisto
acid(cid:2)-oxidation(43).(cid:2)-Oxidationrepresentsthedegradation control the steady-state level of these molecules (48). ABA-
stepforfattyacidsinperoxisomesandplaysimportantrolesin induced ROS production has been described in several plant
ROS production and JA biosynthesis in response to environ- species(12,15),andABAisalsothoughttoregulateROSscav-
mentalstresses(44–46).TheexpressionleveloftheNADPH engingenzymes,suchascatalases,superoxidedismutases,glu-
oxidasegeneisalsoup-regulatedbyRPK1overproductiondur- tathione peroxidases, and ascorbate peroxidases (13, 15,
FIGURE5.Therpk1-1mutantshowsincreasedsensitivitytoH O ,whereas35S:RPK1plantsshowenhancedtolerancetoH O .AandB,responseof
2 2 2 2
detachedleavesfromwildtype,rpk1-1,35S:RPK1,andvectorcontrolplants(VC)totreatmentwith10mMH2O2.Theplantsweregrownonsoil,andtheleaves
wereharvestedandincubatedunderwhitelightfor3daysinH O solution.ChloroticlesionswereformedinresponsetoH O treatmentandwereincreased
bypretreatmentwith50(cid:1)MABAfor3h.Bars,1.0cm.CandD,ph2oto2chemicalefficiency(C)andchlorophyllcontents(D)aresh2ow2ninleavestreatedwithvarious
concentrationsofH2O2for3dayswithorwithoutpretreatmentof50(cid:1)MABAfor3h.Valuesarethemeans(cid:4)S.D.ofthreereplicates(n(cid:5)10).Theasterisksfor
wildtype(WS)andvectorcontrolplantdataindicatesignificantdifferencesversusthoseobtainedundercontrolconditions,respectively.Theasterisksfor
rpk1-1and35S:RPK1valuesindicatesignificantdifferencesversusthoseobtainedforthewildtypeandcontrolplantsunderequivalentexperimentalcondi-
tions,respectively,evaluatedbyone-wayANOVAwithTurkey’sHSD(*,p(cid:6)0.0001;**,p(cid:6)0.005).C,valuesofnon-treatedleaves.E,superoxidedimutase
activitiesintheleavesof35S:RPK1linesL5andL6andcontrolplants(vectorcontrol)grownonsoilpotsfor5weeks.Enzymeactivitieswereassayedinthree
independentreplicates,andthevaluesshownarethemeans(cid:4)S.D.Theasterisksindicatestatisticallysignificantdifferencesinacomparisonofvectorcontrol
and35S:RPK1underthesameconditions,determinedbyone-wayANOVAwithTukey’sHSD(*,p(cid:6)0.0001).F,ROSproductioninleavesof35S:RPK1andcontrol
plants.Thedetachedleavesfromtheseplantswerestainedwith3,3(cid:3)-diaminobenzidine(DAB).Thephotographsshownareofrepresentativeleavesfromthree
independentexperiments.Bar,0.25cm.
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 9199