Dolichol Biosynthesis and Its Effects on the Unfolded Protein Response and Abiotic Stress ...

21 Pages·2008·3.24 MB·English

ThePlantCell,Vol.20:1879–1898,July2008,www.plantcell.orgã2008AmericanSocietyofPlantBiologists
Dolichol Biosynthesis and Its Effects on the Unfolded Protein
Response and Abiotic Stress Resistance in Arabidopsis W OA
HairongZhang,aKiyoshiOhyama,b,1JulieBoudet,c,1ZhizhongChen,aJilaiYang,aMinZhang,aToshiyaMuranaka,b,d
ChristopheMaurel,cJian-KangZhu,e,fandZhizhongGonga,f,g,2
aState Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University,
Beijing100193,China
bRIKENPlantScienceCenter,Tsurumi-ku,Yokohama,Kanagawa230-0045,Japan
cBiochimieetPhysiologieMole´culairedesPlantes,InstitutdeBiologieInte´grativedesPlantes,Unite´ MixtedeRecherche5004,
CentreNationaldelaRechercheScientifique/Unite´ MixtedeRecherche0386,InstitutNationaldelaRechercheAgronomique/
MontpellierSupAgro/Universite´ Montpellier2,F-34060MontpellierCedex1,France
dKiharaInstituteforBiologicalResearch,Yokohama CityUniversity,Yokohama,Kanagawa244-0813,Japan
eDepartment of Botany and Plant Sciences, Institute for Integrative Genome Biology, University of California, Riverside,
California92521
fChina Agricultural University–University of California-Riverside Center for Biological Sciences and Biotechnology, Beijing
100193,China
gNationalCenterforPlantGeneResearch,Beijing100193,China
Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of
sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological
functionsofdolicholsinplantsarelargelyunknown.WeisolatedanArabidopsisthalianamutant,lew1(forleafwilting1),that
showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when
expressedinEscherichiacolicatalyzedtheformationofdolicholwithachainlengtharoundC inaninvitroassay.Thelew1
80
mutationreducedthetotalplantcontentofmaindolicholsby;85%andcausedproteinglycosylationdefects.Themutation
also impairedplasmamembrane integrity,causing electrolyteleakage,lower turgor,reduced stomatalconductance, and
increased drought resistance. Interestingly, drought stressinthe lew1 mutantinduced higher expression oftheunfolded
proteinresponsepathwaygenesBINDINGPROTEINandBASICDOMAIN/LEUCINEZIPPER60aswellasearlierexpression
of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark
sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that
dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in
Arabidopsis.
INTRODUCTION Dallner,1988;SwiezewskaaandDanikiewiczb,2005).Thebio-
synthesis of dolichols takes place mainly in the endoplasmic
Dolichols are long-chain unsaturated polyisoprenoids present
reticulum(ER)andtoalesserextentinperoxisomes(Chojnacki
in all eukaryotic cells; they consist of 15 to 23 isoprenic
andDallner,1988).Thisbiosynthesisisinitiatedbytheformation
units depending on the species (Chojnacki and Dallner, 1988;
offarnesyldiphosphate(FPP),primarilythroughthemevalonate
SwiezewskaaandDanikiewiczb,2005).Inanimals,dolicholsare
pathway (Chojnacki and Dallner, 1988; Swiezewskaa and
broadly distributed in different cells. They are localized within
Danikiewiczb, 2005). The formation of longer chain polyprenyl
thetwohalvesofthephospholipidbilayer,withhighcontentsin
diphosphateiscatalyzedbyenzymescalledcis-prenyltransfer-
theplasmamembranes,peroxisomes,Golgivesicles,andlyso-
ases, which sequentially add isopentenyl diphosphates (IPPs)
somes.Dolicholsexistasfreealcoholsorproductsderivedby
onto FPP (Grabinska and Palamarczyk, 2002). The number of
phosphorylation,esterification,orglycosylation(Chojnackiand
IPPs added is species-dependent. Polyprenyl diphosphate is
furtherconvertedtodolicholanddolichylphosphatebyaseries
ofreactions(GrabinskaandPalamarczyk,2002).Dolichylphos-
1Theseauthorscontributedequallytothiswork.
phate serves as a glycosyl carrier lipid in the biosyntheses of
[email protected].
The author responsible for distribution of materials integral to the proteinC-andO-mannosylation,inglycosylphosphatidylinositol
findingspresentedinthisarticleinaccordancewiththepolicydescribed anchors,andisoneoftherate-limitingfactorsinN-linkedprotein
in the Instructions for Authors (www.plantcell.org) is: Zhizhong Gong glycosylation in yeast and mammalian cells (Burda and Aebi,
([email protected]).
1999;Jonesetal.,2005).
WOnlineversioncontainsWeb-onlydata.
OAOpenAccessarticlescanbeviewedonlinewithoutasubscription. The first eukaryotic cis-prenyltransferase–encoding gene,
www.plantcell.org/cgi/doi/10.1105/tpc.108.061150 RER2, which is responsible for dolichol synthesis, was cloned
1880 ThePlantCell
from the yeast mutant rer2 (Sato et al., 1999). The multicopy sion profiling of soybean leaves shows that some genes are
suppressorofrer2,STR1,encodesaproteinsimilartoRER2,but inducedbybothosmoticandUPRstresses,suggestingthatthe
withdifferentphysiologicalrolesduringcellgrowth(Satoetal., twopathwaysmayconvergeinplants(Irsigleretal.,2007).These
2001).rer2showsmanyphenotypes,includingtemperatureand results suggest complex crosstalk between drought stress re-
hygromycinsensitivity,slowgrowth,defectsinN-andO-glyco- sponsesandtheUPRpathway(Alvimetal.,2001;Koiwaetal.,
sylation, and abnormal accumulation of ER and Golgi mem- 2003;Irsigleretal.,2007).
branes(Satoetal.,1999).Bycontrast,thestr1mutantdoesnot Throughageneticscreen,weisolatedtheArabidopsismutant
have noticeable phenotypes (Sato et al., 2001). Through com- lew1(forleafwilting1).Here,weshowthatlew1isaltered ina
parisonofcis-prenyltransferasesequenceswithplantgenomic gene encoding a cis-prenyltransferase for dolichol synthesis.
sequences,ahomologousgene(AT2G23410)wasclonedfrom lew1 mutant plants showed a leaf-wilting phenotype under
Arabidopsis thaliana (Cunillera et al., 2000; Oh et al., 2000). normalgrowthconditionsduetoimpairedmembraneintegrity.
Expressionofthisgeneinyeastrer2complementsitsthermo- lew1mutantplantswerehypersensitivetotunicamycinandhad
sensitive phenotype (Cunillera et al., 2000). A recombinant reducedlevelsofproteinglycosylation.Droughtstressinduced
protein produced from AT2G23410 shows dehydrodolichyl higher expression of the UPR genes BiP and bZIP60 as well
diphosphate synthase activity, and dolichols could be synthe- as earlier expression of the abiotic stress–responsive genes
sizedinaninvitroassay(Cunilleraetal.,2000;Ohetal.,2000).It COR47andRD29Ainlew1thaninthewildtype.Furthermore,
shouldbenotedthatEscherichiacolicis-prenyltransferasesyn- drought treatment suppressed dark-induced senescence in
thesizesundecaprenyldiphosphate(C ),whichfunctionsasa lew1.ThesedataimplicateLEW1ashavingcrucialrolesindoli-
55
carrier lipid in cell wall polysaccharide synthesis (Kato et al., cholsynthesis,theUPRpathway,andtheabioticstressresponse
1999). However, no dolichol-deficient mutants have been iso- inArabidopsis.
latedfromanymulticellularorganisms,includingplants,andthe
physiologicalrolesofdolicholsinplantsarelargelyunknown.
N-Glycosylation of secreted or membrane proteins occurs, RESULTS
shortly after synthesis, in the lumen of the ER. Inhibition of
N-glycosylation often reduces the folding efficiency, enhances
lew1MutantPlantsExhibitaLeaf-WiltingPhenotypeunder
incorrectfolding,andacceleratesthedegradationofthehypo-
NormalGrowthConditionsDespiteIncreasedHydraulic
glycoprotein. Chaperone proteins such as BiP (for BINDING
ConductivityinRoots
PROTEIN)playcrucialrolesinassistingproteinfoldingduringER
stress, which can result from N-glycosylation defects (Wilson, Thelew1mutantwasisolatedduringageneticscreenforleaf-
2002).Increasesinsecretoryactivityandintheaccumulationof wilting phenotypes (Figure 1A) in an ethyl methanesulfonate–
unfoldedproteinsinducethetranscriptsofchaperonesthrough mutagenizedArabidopsisM2population(Chenetal.,2005).We
the unfolded protein response (UPR) pathway (Wilson, 2002). found that lew1 plants showed a clear leaf-wilting phenotype
Recently, two BASIC DOMAIN/LEUCINE ZIPPER (bZIP) tran- evenunderwell-waterednormalgrowthconditions.Thewiltingin
scriptionfactorgenes,bZIP60andbZIP28,wereisolated,bothof lew1wasobservedmainlyinthemarginsofoldleavesbutnotin
which have been shown to activate BiP expression, probably youngleaves.Thisdiffersfromleafwiltingnormallycausedby
throughERstressresponseelement–likesequences(Iwataand water deficit, in that it was not seen in all leaves and was not
Koizumi,2005;Liuetal.,2007a).bZIP60andbZIP28aremem- associated with changed water status of the seedlings. The
brane proteins that are anchored to the ER membrane under overallsizeoflew1matureplantswassmallerthanthatofwild-
normalconditions.UponERstress,bZIP28,andprobablyalso type plants (Figure 1A). There was no apparent difference in
bZIP60, is cleaved and the bZIP domain translocates to the floweringtimebetweenlew1andwild-typeplants(Figure2A).
nucleus to activate the expression of BiP (Iwata and Koizumi, Inprinciple,leafwiltinginplantsmaybecausedby(1)reduced
2005;Liuetal.,2007a). wateruptakebyroots,(2)lowwatertransportthroughthexylem,
Drought stress is one of the most damaging environmental (3)hightranspiration,(4)lowosmoticpotentialinleafcells,or(5)
conditionsthatlimitplantgrowthanddistribution.Droughtstress impairedcellormembraneintegrity.Weobservedthatwild-type
induces the accumulation of the phytohormone abscisic acid, andlew1plantsshowedsimilarroot:shootratios(Figure1B).This
which,asanendogenoussignal,playscrucialrolesinmediating suggestedthatnomajorimbalancebetweenwateruptakeand
plant responses in order to adjust the water deficit (Xiong water loss was created by gross morphological differences
etal.,2002;Christmannetal.,2006;ShinozakiandYamaguchi- between the two genotypes. The intrinsic capacity of roots to
Shinozaki,2007).Duringdroughtstress,manygenesareupre- takeupwater(i.e.,theirhydraulicconductivity[Lp])wasdeter-
r
gulated. Expression of some of these genes is mediated by mined from measurements of pressure-dependent sap flow in
abscisicacid,whileothersareabscisicacid–independent(Xiong excisedrootsystems(Boursiacetal.,2005).AsshowninFigure
etal.,2002;Christmannetal.,2006;ShinozakiandYamaguchi- 1C,lew1plantsexhibitedasignificantlygreater(+64%)Lp than
r
Shinozaki,2007).TheexpressionofBiPwasfoundtobeinduced wild-type plants. Previous studies have shown that aquaporin
bywaterstressinsoybean(Glycinemax)(Cascardoetal.,2000), activitycontributestomostoftheLp inArabidopsis(Tournaire-
r
and overexpression of BiP in transgenic tobacco (Nicotiana Rouxetal.,2003).Inordertounderstandwhethertheenhanced
tabacum)improvesplantdroughttolerance(Alvimetal.,2001). Lp in lew1 was due to enhanced root aquaporin activity or to
r
However, the upregulation of BiP is not necessarily related to enhancedflowthroughthecellwalls,wetestedtheeffectsoftwo
increaseddroughttolerance(Koiwaetal.,2003).Geneexpres- independentaquaporininhibitors,mercuryandazide.Mercuryis
Dolichol,ERStress,andAbioticStress 1881
Figure1. ThePhenotypesofthelew1Mutant.
(A)Wild-typeandlew1mutantplantsgrowninsoil.Left,lew1(arrowspointtowiltedpartsofleaves);right,thewildtype.
(B)Comparisonofroot:shootratio.Dataaremeans6SE(n=41).
(C)Lprofwild-typeandlew1roots.Thenumbersinparenthesesindicatethenumbersofplantsanalyzed.Dataaremeans6SE.
(D)and(E)EffectsofaquaporininhibitorsonLp ofwild-typeandlew1plants:azideat1mM,30min(D)andmercuryat50mM,60min(E).Dataare
r
means6SE.Thenumbersinparenthesesindicatethenumbersofplantsanalyzed.
(F)Structureofxyleminleaves(toprow)andstems(bottomrow)ofwild-typeandlew1plants.Arrowspointtoxylem.Bars=5mm.
(G)Graftingexperiments.Left,lew1shootwithwild-typeroot;right,wild-typeshootwithlew1root.
the mostcommonlydescribed aquaporinblocker, butazideis In conclusion, the leaf-wilting phenotype of lew1 was not
even more potent, as it can reversibly block Lp of wild-type causedbydefectsinrootwateruptakeorxylemtransport.
r
plants byup to 80% (Tournaire-Roux etal.,2003). The results
showed that wild-type and lew1 plants had similar levels of
Thelew1MutationImpairsCellTurgorinLeavesby
inhibition,particularlyinthecaseofazide(Figures1Dand1E),
CausingIncreasedElectrolyteLeakageandReduces
which meant that most of the increase in Lp shown by lew1
r LeafTranspiration
(+64%)couldbeaccountedforbyenhancedaquaporinactivity.
Theseresultsdisproveapossiblehydrauliclimitationofrootsin In contrast to the increased Lp, increased transpiration could
r
the leaf-wilting phenotype. They further suggest that the lew1 causeleafwilting.Surprisingly,wefoundthatstomatalconduc-
mutationincreasestheefficiencyofwatertransportacrossroot tance,asmeasuredusingaporometer,wasreducedby;75%in
cells. lew1(Figure2B).Consistentwiththis,spontaneouswaterloss
We previously showed that leaf wilting in lew2 is caused by fromdetachedleaveswasslowerinlew1thaninthewildtype
impededwatertransportduetoacollapsedxylem(Chenetal., (Figure 2C). Importantly, lew1 plants grown in soil were more
2005). To investigate a possible xylem defect, we examined resistanttodroughtstressthanwerewild-typeplants(Figure2A).
cross sections of vascular bundles in the xylem of stems and A closer inspection of leaf epidermis showed that, under
leaves and found no difference between lew1 and wild-type normal growth conditions, 60% of stomata were closed in the
plants (Figure 1F). We also performed grafting experiments to wilted parts of lew1 leaves but only ;15% of stomata were
testwhetherleafwiltinginlew1mighthavebeenduetoanoverall closed in wild-type leaves (Figures 2D and 2E). Even under
defect of water supply to the shoot. When lew1 shoots were conditions(e.g.,highlight)thatpromotestomatalopening,when
graftedontowild-typeroots,leafwiltingstilloccurred.Bycon- allstomataofwild-typeleaveswerefullyopen,moststomatain
trast, when grafted onto lew1 roots, wild-type shoots did not thewiltedpartsoflew1leavesremainedpartlyclosed(Figure2F).
showanywilting(Figure1G). By contrast, no apparent difference in stomatal opening was
1882 ThePlantCell
Figure2. AnalysisofDroughtResistanceandRelatedParametersinlew1.
(A) Comparison of wild-type and lew1 plant growth under normal (left) and drought stress (right) conditions. For drought treatment, 3-week-old
seedlingsweresubjectedtowaterwithholdingfor2weeks.
(B)Stomatalconductanceinthewildtypeandlew1.Dataaremeans6SE.Thenumbersinparenthesesindicatethenumbersofanalyzedplants.Twoto
fourleaveswereusedperplantforthewildtype,andonetotwoleaveswereusedperplantforlew1.
(C)Waterlossfromdetachedleaves.Threeindependentexperimentswereperformed,and15leavespergenotypewereusedineachexperiment.Data
aremeans6SE(Student’sttest;**statisticallysignificantdifference[P<0.01]).
(D)Stomataclosureratesofthewildtypeandlew1.Forlew1plants,stomatafromwiltingpartsweremeasured.About40stomataweremeasuredin
eachofthreeexperimentsforboththewildtypeandlew1.Dataaremeans6SE(Student’sttest;**statisticallysignificantdifference[P<0.05]).
(E)Stomatacomparisonbetweenthewildtypeandlew1.Thestomataonthewiltingpartofaleafareshown.Bars=20mm.
(F)Comparisonofstomatainthewildtypeandlew1underconditionsthatpromotefullstomatalopening.Thestomatafromthewiltingpartofalew1leaf
werenotopenunderconditionswhenallstomatainthewild-typeleafwereopen.Bars=20mm.
(G)Comparisonofstomatainthewildtypeandlew1.Stomataonnonwiltingpartsintheleafbaseoflew1areshown.Bars=20mm.
(H)Osmoticpotentialinthewildtypeandlew1undernormalgrowthconditions.Valuesaremeans6SE,n=3(Student’sttest;*statisticallysignificant
difference[P<0.05]).
(I)Comparisonofleafelectrolyteleakagebetweenlew1andthewildtype.Valuesaremeans6SE,n=3(Student’sttest;*statisticallysignificant
difference[P<0.05]).
(J)Typicalrecordingtraceshowingmeasurementsofturgorinepidermalcellsofawild-typeleaf.Followingsuccessfulimpalementofacellwiththe
pressure probe, the intracellular recording was continued until a stationary turgor was reached. The trace shows successive recordings in three
individualcells.
(K)Mean(6SE)epidermalcellturgorinareasofleavesofwild-typeandlew1plantsshowingnoobvioussignofwilting(wildtype,n=22cells;lew1,n=
21cells).Thereisastatisticallysignificantdifference(P=0.0005)betweenthetwogenotypes.
Dolichol,ERStress,andAbioticStress 1883
foundintheunwiltedpartsoflew1andwild-typeleaves(Figure center.OurfailuretoobtainhomozygousT-DNAinsertionplants
2G). These results suggested that, in the wilted parts of lew1 suggested that complete disruption of LEW1 is lethal. Plants
leaves,guardcellsandpossiblysurroundingcellswerenotable heterozygousfortheT-DNAinsertionwerecrossedwiththelew1
toestablishanormalturgor.Toassessfurtherthebasisofthe mutant.TheF2progenysegregatedintothreedifferentpheno-
apparent leaf-wilting phenotype of lew1, leaf cell turgor was types, including typical wild-type and lew1 phenotypes, as
investigatedusingthecellpressureprobetechnique.Because expected. We also observed a third class of plants that were
lew1 leaves could show signs of early senescence, the mea- smaller,showedearliersenescence,andproducedfewerseeds
surementswereperformedinyoung,growingleavesthatdidnot than lew1 or wild-type plants (Figure 3Ba). Genetic and DNA
showanyofthesesymptoms.Yet,localzonesofwilting,mostly sequenceanalysisshowedtheseplantstobeheterozygousfor
inthemostdistalpartoftheblade,couldbeobservedinlew1. boththeT-DNAinsertionandthelew1mutation.RT-PCRanal-
Amongallleafcelltypesinvestigated,thelargefusiformepider- ysis indicated that LEW1 transcripts were present in these
malcellslocatedattheedgeofthebladeyieldedthemoststable heterozygousplants,perhapsatreducedlevels(Figure3Bb).
micropipette impalements and turgor recordings. In wild-type
plants,cellturgormeasurementscouldbeperformedallalong
TheLEW1GeneEncodesaNovel
the leaf edge, yielding a mean stationary turgor value of P =
0 cis-PrenyltransferaseProtein
4.7060.21bars(n=22cells)(Figure2J).Bycontrast,impale-
mentofthewiltedareainlew1leavesdidnotallowanystable TheLEW1geneencodesapolypeptideof254aminoacidswitha
turgorrecording.Measurementsperformedinotherareasofthe predicted molecular mass of 28.52 kD. Because the genes
leafprovidedameanturgorvalueofP =3.3460.29bars(n=21 encoding cis-prenyltransferases form a large family with an
0
cells),whichwassignificantlylower(P=0.0005)thanthevaluein overalllowlevelofsequencesimilarity,weselectedforcompar-
wild-typeleaves.Thus,leafcellsoflew1showedasignificantly isononlythosecandidatesthatwerethemostcloselyrelatedto
reducedturgor,eveninareasshowingnoclearsignofwilting. LEW1 (Figure 3C). The previously identified AT2G23410 and
Undernormalgrowthconditions,theosmoticpotentialoflew1 yeastRER2wereincludedinthisanalysis.Figure3Dshowsthat
leaveswasslightlyhigherthanthatofwild-typeleaves(Figure2H). inArabidopsis,LEW1(AT1G11755)hasonlyonecopy,whereas
Thiswasprobablyduetoaslightosmoticcompensationinthe inrice(Oryzasativa),therearetwohomologs(Os03G0197000
wiltedlew1cells.Wethenexaminedcellmembraneintegrityby andOs02G0197700).TheproductofAT2G23410,RER2,anda
measuringelectrolyteleakage.AsshowninFigure2I,lew1leaves RER2homologinE.coli(P60472)belongtoanotherhomology
had a higher electrolyte ion leakage than did wild-type leaves, subgroup(Satoetal.,1999;Cunilleraetal.,2000;Ohetal.,2000).
suggestingthatthelew1mutationimpairedmembraneintegrity. The analysisindicates thatLEW1isanew member of the cis-
Inconclusion,theseresultssuggestthattheleaf-wiltingphe- prenyltransferasefamily.
notypeoflew1isduetoincreasedsoluteleakagecausedbycell In order to confirm that LEW1 encodes a functional cis-
membranelesions.Thereducedcellturgorwouldleadtopartial prenyltransferase, we expressed LEW1 in E. coli (Figure 4A)
stomatalclosureandlesstranspirationinlew1. andanalyzeditsenzymeactivityinvitro.Theproteinsextracted
fromE.colicarryingLEW1oranemptyvectorwereincubated
with14C-labeledIPPandFPP,andthereactionproductswere
Mapping-BasedCloningoftheLEW1Gene
analyzed by normal-phase thin layer chromatography (TLC)
To clone the LEW1 gene, lew1 plants in the Columbia gl1 (Figure4B).TheauthenticsolanesolswithchainlengthsofC
45
accession were crossed with wild-type Landsberg plants, and andC wereusedasstandardcompounds(Figure4B).Because
90
F2 progeny was grown in soil under normal conditions. lew1 E.coliisabletocatalyzethesynthesisofdolicholwithachain
plants with a leaf-wilting phenotype were selected for genetic lengthofC ,thelabelingofC productswasequallydetected
55 55
mapping. LEW1 was initially mapped to the upper arm of inbothreactions,asexpected.However,largerreactionprod-
chromosome1.Fine-mappingnarrowedthelew1mutationtoa uctsweredetectedintheextractfromE.coliexpressingLEW1.
regioncoveredbyfourBACclones,T23J18,F25C20,F12F1,and Theseproducts migrated slightlyfasterthanthe C solanesol
90
T28K15(Figure3A).Furtherfine-mappingdelimitedlew1onBAC standardsandthereforemustbesomewhatsmaller(Figure4B).
clonesF25C20andF12F1.Wesequencedalloftheopenreading TheseresultsindicatethatLEW1possessescis-prenyltransfer-
frames from the lew1 mutant in this region and found a single aseactivityfordolicholsynthesisinvitro.
G-to-A mutation in the open reading frame of AT1G11755, ApreviousstudyindicatedthatArabidopsisAT2G23410was
whichcontainssevenexons(Figure3A).Themutationoccurred abletocomplementthetemperature-dependentgrowthpheno-
inthesecondexonandchangedGGT(encodingGly-21)toGAT typeofyeastmutantrer2(Cunilleraetal.,2000).Weexpressed
(encodingAsp-21)inlew1(Figure3A). LEW1andAT2G23410(asapositivecontrol)intherer2mutant
ThecDNAofAT1G11755wasamplifiedandoverexpressedin andcomparedgrowthat23,30,and378C.AsshowninFigure
lew1 and wild-type plants under the control of the cauliflower 4C,allofthetestedyeaststrainsgrewverywellat238Cinuracil
mosaicvirus35Spromoter.Weobtained25independenttrans- (URA)-containing medium. By contrast, the rer2 mutant, either
geniclew1linesandfoundthattheleaf-wiltingphenotypewas nativeorexpressingtheemptyvector,wasnotabletogrowat
complemented in 21 of these lines (Figure 3B shows a repre- temperatures higher than 308C. However, when transformed
sentativeplantoflew1+LEW1).Inparalleltothis,anArabidopsis with a plasmid carrying the LEW1 cDNA under control of the
line (SALK_032276) carrying a T-DNA insertion in the second glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter,
exon of AT1G11755 was obtained from the Arabidopsis stock rer2 was able to grow, albeit more slowly than the rer2 strain
1884 ThePlantCell
Figure3. PositionCloningofLEW1andSequenceAnalysisofLong-Chaincis-Prenyltransferases.
Dolichol,ERStress,andAbioticStress 1885
Figure4. BiochemicalCharacterizationofLEW1andComplementationoftheYeastrer2Mutant.
(A)EctopicexpressionofLEW1inE.coli.ProteinsextractedfromE.colicellswereanalyzedon12%SDS-PAGEgelsandstainedwithCoomassieblue.
ThearrowpointstotheexpressedLEW1proteininE.coli.M,marker;Con,emptyvectorcontrol;LEW1,isopropylthio-b-galactoside–inducedLEW1.
(B)TLCanalysisofLEW1reactionproducts.[14C]IPPand[14C]FPPwereincubatedwithtotalproteinsisolatedfromE.colistrainstransformedwith
eithertheemptyvector(Con)orLEW1.Atthecompletionofthereaction,lipidproductswereextractedandsubjectedtoTLCanalysis.SolanesolsC
45
andC wereusedasstandards.AC band(representingaproductthatcanbesynthesizedbythenativecis-prenyltransferaseinE.coli)wasequally
90 55
detectedinE.colitransformedwitheitheremptyvectororLEW1,indicatingthatthereactionsweresuccessful.Ori,originalspot.
(C)LEW1partiallycomplementedthetemperature-sensitivephenotypeoftheyeastrer2mutant.rer2cellstransformedwithanemptyvector,LEW1,orDPS
(AT2G23410)weregrownovernight.Serialdecimaldilutionswerespottedontoplatesofsyntheticdropoutmediumwithallaminoacids(URA+)orexcluding
URA(URA(cid:1)).URAisaselectionmarkerofthetransformedclone.Plateswereincubatedattheindicatedtemperaturesandphotographedafter4d.
expressing AT2G23410. These results indicate that LEW1 can 2004). We performed lipid analysis to compare the dolichol
partiallycomplementthehightemperature–sensitivephenotype contentsamongwild-type,lew1mutant,andtransgenicplants
oftherer2mutant. overexpressingLEW1(linesOE14andOE34).Figure5Fshows
that LEW1 transcripts increased in the two overexpression
lines. The dolichols in Arabidopsis exist as free lipids, lipids
DolicholContentsAreReducedinlew1MutantPlantsbut
covalently bound to proteins, or lipid carriers linked to oligo-
AreIncreasedinTransgenicPlantsOverexpressingLEW1
saccharidesforproteinglycosylation.Thetotaldolicholswere
DolicholswithchainlengthsofC andC havebeenreported extractedandanalyzedbyliquidchromatography–massspec-
75 80
tobethemostabundantformsinArabidopsis(Gutkowskaetal., trometry (LC-MS) following previously established methods
Figure3. (continued).
(A)PositionalcloningofLEW1.TheLEW1locuswasmappedtobetweentwopolymorphicmarkersonBACclonesT23J18(recombinants,11of1638)
andT28K15(recombinants,17of1638).FurthermappingdelimitedtheLEW1locustoaregionwithinBACsF12F1(recombinants,3of1638)and
T28K15.Allcandidateopenreadingframesweresequencedinthisregion,andonlyonemutation(G159A)wasfoundinAT1G11755.
(B)(a)Complementationofthelew1mutant.Shownarethewildtype(gl1),thelew1mutant,thelew1mutanttransformedwith35S-LEW1(cDNA),the
wildtype(Columbia[Col]),andaheterozygousplantoflew1crossedwithalineinwhichtheLEW1genewasdisruptedbyaT-DNAinsertion.TheT-DNA
wasinsertedatposition205countingfromthefirstputativeATGofAT1G11755.(b)RT-PCRanalysisofLEW1transcriptsinthewildtype,lew1,anda
heterozygousplantoflew1crossedwiththeT-DNAline.rRNAisshownasacontrolforloading.
(C)Alignmentoflong-chaincis-prenyltransferasesfromdifferentorganisms:rice,Os02g0197700andOs03g0197000;human,NP_612468;mouse,
BAE39779;Xenopus,NP_001016090;fruitfly,AAF51407;E.coli,P60472;Micrococcusluteus,BAA31993;Saccharomycescerevisiae,BAA36577
(RER2);Arabidopsis,AT2G23410andAT1G11755(LEW1).
(D)PhylogenetictreeofLEW1andrelatedproteinsfromdifferentorganisms:Arabidopsis,At2g23400,At5g58780,At5g58782,At5g58784,At5g58770,
At5g60510,At5g60500,andAt2g17570;rice,Os01g0857200,Os06g0167400,andOs07g0607700;monkey,XP_001110733;dog,XP_541219;Aedes
aegypti,EAT38464;rat,XP_001058252;S.cerevisiae,NP_013819/SRT1andNP_010088;theothersarethesameasin(C).Thephylogenetictreewas
constructedonthealignmentusingMEGA(version4.0)(seeSupplementalDataSet1online).Bootstrapvalueswerecalculatedfrom1000trialsandare
shownateachnode.Theextentofdivergenceaccordingtothescale(relativeunits)isshownatbottom.
1886 ThePlantCell
(Skorupinska-Tudek et al., 2003; Gutkowska et al., 2004). lew1MutantsAreHypersensitivetotheGlycosylation
Dol-C (amixtureofdolicholsC toC )waspurchased InhibitorTunicamycin
65-105 65 105
andusedasastandardcompound.Becausewedidnothave
TunicamycinisaspecificinhibitorofUDP-N-acetylglucosamine-
purestandardsamplesforeachdolicholtoquantifytheexact
dolichyl-phosphateN-acetylglucosaminephosphotransferase(EC
amounts, here we only show the relative dolichol contents in
2.7.8.15), the first enzyme in the biosynthesis of dolichol-linked
eachsample.AsindicatedinFigure5A,wedetectedDol-C
75
oligosaccharidesforproteinglycosylationmodification(Lehrman,
andDol-C atdifferentretentiontimes(Dol-C ,25min,mass
80 75
1991;Koizumietal.,1999).Blockingglycosylationbytunicamycin
analysis of m/z 1063 to 1065; Dol-C , 26 min, m/z 1131 to
80
inducesUPR,causingenhancedexpressionofsomeUPRpath-
1133).Basedonthisinformation,wecomparedthepeakareas
waygenes(Koizumietal.,1999;Lukowitzetal.,2001;Martinezand
of Dol-C and Dol-C of wild-type (Figure 5B), lew1 (Figure
75 80
Chrispeels, 2003). Because the lew1 mutation impairs protein
5C),OE14(Figure5D),andOE34(Figure5E)plants.Compared
glycosylation,wetestedthesensitivityoflew1totunicamycin.As
with wild-type levels, the relative contents of Dol-C and
75
showninFigure6C,lew1plantsweremoresensitivethanwild-type
Dol-C were 13 and 15% in lew1, 109 and 103% in line
80 plantstoexposureto0.1mg/mLtunicamycin.Overexpressionof
OE14,and111and109%inlineOE34,respectively.Thesere-
LEW1inthewildtypedidnotobviouslyincreasetheresistanceof
sultsstronglysuggestthatLEW1catalyzesthebiosynthesisof
transgenicplantstotunicamycin,suggestingthatLEW1isnota
Dol-C andDol-C inArabidopsis.
75 80
limitingfactorforproteinglycosylation.
Thelew1MutationImpairsProteinN-Glycosylation
Thelew1MutantIsHypersensitivetoDarkStress
Dolicholactsasalipidcarrierduringproteinglycosylation,and
Defects in protein glycosylation greatly affect the biogenesis of
mutationsintheyeastRER2leadtodefectsinproteinglycosyl-
membraneandsecretedproteinsbyinterferingwiththeirfolding,
ation(Satoetal.,1999).First,weusedperiodicacidSchiff(PAS)
trafficking,degradation,andtargetingtotheirfinalsitesinthecell;
stainingtoexaminetheglycoproteinlevels(Nanjoetal.,2006)in
therefore, defects in protein glycosylation generally lead to ER
wild-typeandlew1plants.Intotalproteinextractsseparatedby
stress. We observed that mutant plants with one copy of the
SDS-PAGE and stained by PAS (Figure 6A), several relatively
T-DNAinsertionalleleoflew1andtheothercopyasthelew1point
smallbandswerefoundtobemuchlessabundantinlew1thanin
mutation allele showed an early-senescence phenotype (Figure
the wild type, indicating that protein glycosylation levels were
3B). We further tested whether lew1 exhibits a more severe
reducedbythemutation.
membrane-damage phenotype in the dark, because prolonged
We then used concanavalin A–Sepharose, which binds to
dark treatment induces membrane damage and senescence
high-mannose-typeN-glycansofglycoproteins,toenrichglyco-
(Fujikietal.,2000).lew1andwild-typeplantsgrownunderwell-
proteinsfromthetotalproteinextracts(Koiwaetal.,2003).The
wateredconditionsfor2weeksweretransferredtodarknessat
enriched glycoproteins were separated by SDS-PAGE. As 228Cfor10d.Allofthelew1plantsbecameyellow,butwild-type
showninFigure6B,morebandsweredetectedinthewildtype
plants did not. lew1 plants transformed with the LEW1 over-
than in lew1. It is also noticeable that the peptide profile from
expressionconstruct(35S-LEW1)exhibitedsimilarphenotypesas
lew1 was smeared, indicating that proteins in lew1 may be
wild-type plants (Figure 7A). Electrolyte leakage from leaves of
abnormally glycosylated. We further treated the enriched pro-
lew1plantswasgreatlyincreasedandreached35%after10din
teinswithendoglycosidaseHtocleaveN-linkedglycans,andthe thedark,whereaswild-typeleavesshowedonly;10%leakage
productswerecomparedbySDS-PAGE.AsshowninFigure6B,
underthesameconditions(Figure7B).Theseresultsshowthat
similar bands were detected in both the wild type and lew1,
moreseveremembranedamageoccurredinlew1thaninthewild
suggestingthatthedifferenceswereduetochangesinprotein
typeunderprolongeddarktreatment.
glycosylation.
Because tunicamycin inhibits protein glycosylation, we also
Third, we recovered from SDS-PAGE two bands that were
investigatedtheeffectsofthiscompoundonplantsensitivityto
present in the total protein extracts from the wildtype but not
prolongeddarkness.Sprayingtunicamycinonwild-typeleaves
fromlew1(Figure6B,arrows).Trypticpeptideswereresolvedby
acceleratedthedark-inducedsenescenceandthereforepheno-
matrix-assistedlaser-desorptionionizationtimeofflight(MALDI-
copied to some extent the dark-sensitive phenotype of lew1
TOF)MS(AutoflexIITOF/TOF;Bruker)andanalyzedbyMascot
(Figure 7C). In addition, tunicamycin treatment of lew1 leaves
atwww.Matrixscience.com.Thesmallerbandcorrespondedto
renderedtheirsenescenceinthedarkmorepronounced(Figure
the glycoprotein b-thioglucoside glucohydrolase encoded by
7C).Thedataoverallsuggestthatthelew1phenotypeinthedark
TGG1 in Arabidopsis (Xue et al., 1995; Husebye et al., 2002).
isrelatedtoaglycosylationdefect.
TGG1islocalizedinthevacuole,andunderglycosylatedTGG1
wasalsofoundinthestt3amutantofArabidopsisinaprevious
DarknessStress–ResponsiveGenesAreHyperinducedin
study (Koiwa et al., 2003). The partial sequence of the larger
lew1byDarkTreatment
product was identical to that of a glycoprotein named SKU5
(Sedbrook et al., 2002). SKU5 is localized to both the plasma Weselectedtwolatedark-induciblemarkergenes,DIN2(fordark-
membrane and the cell wall and is involved in directional root inducible2)andDIN9,tocomparetheirexpressionpatternsinlew1
growth in Arabidopsis (Sedbrook et al., 2002). These results and wild-typeplants. DIN2 encodes a b-glucosidase,and DIN9
further demonstrate that LEW1 plays critical roles in protein encodesamannose-6-phosphateisomerase(Fujikietal.,2000).
glycosylation. We also examined a leaf senescence marker gene, YLS4 (for
Figure5. AnalysisoftheRelativeDolicholContentsinWild-Type,lew1,andLEW1OverexpressionPlantsbyLC-MS.
Thepeakareainthewildtypewastakenas100%,andtherelativecontentsinotherplantswerecomparedwiththatinthewildtype.
(A)Thedolicholstandards(left,Dol-C ;right,Dol-C ).Arrowsindicatethepeaks.
75 80
(B)Thewildtype.
(C)lew1mutant.
(D)Overexpressionline14(OE14).
(E)Overexpressionline34(OE34).
(F)RNAgelblotanalysisofLEW1expressioninOE14andOE34.Duetoitslowexpression,LEW1transcriptcouldnotbedetectedinthewildtypeand
thelew1mutantbyRNAgelblotundertheconditionsused.OE15andOE21,twoothertransgeniclinesnotusedinthisstudy,arealsoincluded.The
bottompanelshowsanRNAgelasaloadingcontrol.
1888 ThePlantCell
Figure6. lew1ImpairsProteinN-Glycosylation,andlew1PlantsAreHypersensitivetoTunicamycin.
(A)Comparisonofproteinglycosylationpatternsbetweenthewildtypeandlew1byPASstaining.Thirtymicrogramsoftotalproteinwereresolvedby
SDS-PAGEanddetectedbyPASstaining.Thearrowpointstothebandsmissingfromlew1.
(B)ConcanavalinA–SepharosebindingassayforN-glycosylatedproteins.Totalproteinsextractedfromthewildtypeandlew1wereincubatedwith
concanavalinA–Sepharose,andtheboundproteinswereeluted,treatedwith(+)orwithout((cid:1))endoglycosidaseH(EndoH),resolvedbySDS-PAGE,
andstainedwithCoomassieblue.Thearrowsatleftpointtothetwoproteinbandsfoundinthewildtypebutnotinlew1.Thesetwobandswere
recovered and analyzed by MALDI-TOF MS. The top one corresponds to SKU5, and the bottom one corresponds to TTG1. Endoglycosidase H
treatmentresolvedtheproteinsfoundinboththewildtypeandlew1.M,marker.
(C)lew1seedlingsaresensitivetotunicamycin(TM).SeedlingsweregerminatedonMSmediumorMSmediumcontaining0.1mg/mLtunicamycinand
grownfor10dinagrowthchamber.WT,wildtype(gl1);lew1,lew1mutant;35S-LEW1,awild-typelineoverexpressingLEW1underthecontrolofthe
35Spromoter(line14);lew+35S-LEW1,alew1mutantlineoverexpressingLEW1underthecontrolofthe35Spromoter(line5).
YELLOW-LEAF-SPECIFICGENE4),whichisinducedinoldleaves day-oldseedlingsgrowninthelightwerepretreatedbywithhold-
bydarkness(Fujikietal.,2005).Two-week-oldseedlingswerekept ingwaterfor7dpriortothedarktreatment,whichproducedonlya
underdarknessfor24and48h.RNAgelblotanalysisrevealed modestdroughtstress.Controlplantswerewateredeverythird
DIN2,DIN9,andYLS4transcriptstobesubstantiallyinducedin day,andbothcontrolanddrought-treatedplantswerekeptinthe
lew1at24and48hinthedark,whereasthetranscriptswerenot darkorlightforvarioustimes.Afteranadditional15dofdrought
yet or less detected in the wild type and two transgenic lines treatment,wild-typeplantsgrowninthelightwereseriouslywilted
overexpressingLEW1atthesetimepointsinthedark(Figure7D). (Figure 8A, 15 d in light) and did not recover growth after
rewatering. By contrast, lew1 plants, which developed clear
drought-tolerant phenotypes in light, were able to regrow after
DroughtorOsmoticStressSuppressestheDarkness
watering (see Supplemental Figure 1 online). These results are
Sensitivityoflew1Plants,andlew1SeedlingsAreMore
consistentwithpreviousdata(Figure2A)andindicatethatlew1is
ToleranttoOsmoticStress
toleranttodroughtstressatdifferentdevelopmentalstages.
Becauselew1plantsaremoretoleranttodroughtstress,wenext lew1plantsgrowingunderwell-wateredconditionsinthedark
analyzedtheresponseoflew1todroughtstressinthedark.Ten- for15dbecameyellowishandwereseriouslydamaged(Figure