Sucrose Transporter AtSUC9 Mediated by a Low Sucrose Level is Involved in Arabidopsis Abiotic ...

14 Pages·2015·1.23 MB·English

Sucrose Transporter AtSUC9 Mediated by a Low Sucrose Level
is Involved in Arabidopsis Abiotic Stress Resistance by
Regulating Sucrose Distribution and ABA Accumulation
Wanqiu Jia1,4,5, Lijun Zhang1,4,5, Di Wu1,4, Shan Liu1, Xue Gong1, Zhenhai Cui1, Na Cui1, Huiying Cao1,
R Longbing Rao2 and Che Wang1,3,*
e
g 1CollegeofBiologicalScienceandTechnology,ShenyangAgriculturalUniversity,Shenyang,China
u
l 2ResearchInstituteofSubtropicalForestry,ChineseAcademyofForestry,Hangzhou,China
a 3KeyLaboratoryofNortheastRiceBiologyandBreeding,MinistryofAgriculture,andKeyLaboratoryofNorthernJaponicaRiceGeneticsandBreeding,
r
MinistryofEducation,RiceResearchInstitute,ShenyangAgriculturalUniversity,Shenyang,China
P 4Theseauthorscontributedequallytothiswork. Do
ap 5Presentaddress:CollegeofBiologicalScienceandTechnology,ShenyangAgriculturalUniversity,Shenyang110866,China. wnlo
e *Correspondingauthor:E-mail,[email protected];Fax,[phone]. ad
r (Received July 20, 2014; Accepted May 29, 2015) e
d
fro
m
Sucrose (Suc) transporters (SUCs or SUTs) are important Introduction h
regulators in plant growth and stress tolerance. However, ttp
s
the mechanism of SUCs in plant abiotic stress resistance Abioticstresses,suchassalt,coldanddrought,areenvironmen- ://a
remains to be dietermined. Here, we found that AtSUC9 tal factors that greatly limit crop production and plant distri- ca
d
expression was induced by abiotic stress, including salt, os- butionworldwide(Chinnusamyetal.2005,FujiiandZhu2012), em
motic and cold stress conditions. Disruption of AtSUC9 led and the cellular, molecular and physiological responses of ic.o
to sensitive responses to abiotic stress during seed germin- plants to thesestresses havebeen analyzedintensively (Xiong up
etal.2002,Zhu2002).ThehormoneABAisanimportantfactor .c
ation and seedling growth. Further analyses indicated that o
m
the sensitivity phenotype of Atsuc9 mutants resulted from inmediatingcold,saltandwaterstressresponses(Chinnusamy /p
higherSuccontentinshootsandlowerSuccontentinroots, et al. 2005). Many ABA-regulated genes have been identified, cp/a
ascomparedwiththatinwild-type(WT)plants.Inaddition, severalofwhichareactuallypositiveABAsignalingregulators, rtic
we found that the expression of AtSUC9 is induced in par- including ABA-insensitive 3 (ABI3), ABI4 and ABI5 (Lopez- le-a
ticularbylowlevelsofexogenousandendogenousSuc,and Molina et al. 2001, Finkelstein and Ginson 2002, Nambara bs
deletion of AtSUC9 affected the expression of the low Suc et al. 2002), as well as the ABA response element-binding tra
c
level-responsive genes. AtSUC9 also showed an obvious re- factorgene(ABF)(Choietal.2000).Severalnegativeregulators t/5
6
sponsetotreatmentswithlowconcentrationsofexogenous of ABA responses have also been identified, including ROP10 /8
/1
Suc during seed germination, seedling growth and Suc dis- (theRho-likesmallGprotein)(Zhengetal.2002),thecalcium 57
tribution, and Atsuc9 mutants hardly grew in abiotic stress sensor calcineurin B-like protein (CBL9) (Pandey et al. 2004), 4/1
treatments without exogenous Suc. Moreover, our results and ABI1 and ABI2 (Gosti et al. 1999, Merlot et al. 2001). In 84
6
illustrated not only that deletion of AtSUC9 blocks abiotic addition,thereareseveralabioticstress-inducedABA-regulated 21
6
stress-inducibleABAaccumulationbutalsothatAtsuc9mu- genes, including RD29A (Yamaguchi-Shinozaki and Shinozaki b
y
tantshadalowercontentofendogenousABAinstresscon- 1994), and the kinases KIN1 and KIN2 (Kurkela and Borg- gu
ditions than in normal conditions. Deletion of AtSUC9 also Franck 1992). Studies on mutation in genes in both the ABA es
andsucrose(Suc)signalingpathways,suchasthoseleadingto t o
inhibited the expression of many ABA-inducible genes n
(SnRk2.2/3/6, ABF2/3/4, ABI1/3/4, RD29A, KIN1 and KIN2). impaired function of Suc-induction 4 (isi4) (Laby et al. 2000, 12
These results indicate that AtSUC9 is induced in particular Rooketal.2001)andABI4(Rooketal.2006),haveestablisheda Ap
bylowSuclevelsthenmediatesthebalanceofSucdistribu- relationship between ABA signaling and Suc responses at the ril 2
0
tion and promotes ABA accumulation to enhance molecularlevel. 19
Arabidopsis abioticstress resistance. Suc,whichisproducedduringphotosynthesis,isasourceof
carbon and energy. Suc plays an important role in regulating
Keywords: ABA (cid:2) Abiotic stress (cid:2) AtSUC9 (cid:2) Low levels of plant growth and plant stress tolerance; however, stress can
Suc (cid:2) Suc transporter. inhibit photosynthesis, thereby affecting Suc supply and
decreasing the plant’s tolerance to environmental stresses
Abbreviations: ABI, abscisic-acid insensitive; MS, Murashige (for reviews, see Yu 1999, Gupta and Kaur 2005, Ruan et al.
and Skoog; qRT-PCR, quantitative real-time PCR; RT–PCR, 2010). Thus, Suc transport and distribution are key steps in
reverse transcriptase–PCR; Suc, sucrose; SUCs/SUTs, sucrose plant stress resistance (Roitsch 1999, Lemoine et al. 2013).
transporters; WT, wild type. Suc transporters (SUCs or SUTs) are important exporters of
PlantCellPhysiol.56(8):1574–1587 (2015) doi:10.1093/pcp/pcv082,AdvanceAccesspublicationon10June2015,
availableonlineatwww.pcp.oxfordjournals.org
!TheAuthor2015.PublishedbyOxfordUniversityPressonbehalfofJapaneseSocietyofPlantPhysiologists.
Allrightsreserved.Forpermissions,pleaseemail:[email protected]
PlantCellPhysiol.56(8):1574–1587(2015)doi:10.1093/pcp/pcv082
Sucfromhigherplantsourceleavestosinktissues.Todate,>80 Results
SUCgeneshavebeencloned(Sunetal.2008,Hiroseetal.2010,
Identification of T-DNA insertion mutants of
Ku¨hn and Grof 2010). Phylogenetic analysis has shown five
distinct subfamilies, called the type SUT1–SUT5 clade (Ku¨hn AtSUC9
and Grof 2010). There are some reports on the role of SUC ToexaminethefunctionoftheAtSUC9gene,weisolatedthree
duringabioticstress,includingsalt,droughtandlowtempera- T-DNA insertion alleles of this gene, AtSUC9-1 (SALK_049523),
tures.Whencelery(ApiumgraveolensL.)wasplacedin300mM AtSUC9-4 (CS329542) and AtSUC9-5 (SALK_013812), from the
NaCl,expressionofAgSUT1,aH+/Suctransporterwithahigh collectionofT-DNA-transformedArabidopsislines(Arabidopsis
affinityforSuc,decreasedinallorgans,withthelargestdecrease BiologicalResourceCenter).Sequenceanalysisindicatedthatthe
in the roots, suggesting that Suc transporters are associated insertionsiteislocatedinthepromoterat578bpbeforeATGin
withsalttolerance(Noiraudetal.2000).Analysisoftheexpres- theAtSUC9-5allele,inthefirstexonat425bpafterATGinthe
sionlevelsofSuctransportersunderdroughtandsalinitystres- AtSUC9-4alleleandinthethirdexonat1,812bpafterATGinthe D
o
sesinriceshowedthatonlyOsSUT2ofthefivericeSUCgenes AtSUC9-1allele(SupplementaryFig.S1a).Indeed,reversetran- w
n
was up-regulated during drought and salinity treatments scription–PCR(RT–PCR)analysisfailedtodetectanyfull-length loa
(Ibraheem et al. 2011). Under low temperature stress, AtSUC9 transcript in the mutants (Supplementary Fig. S1b). de
d
Arabidopsis thaliana was found to up-regulate the expression TheabnormaltranscriptthatresultedfromtheT-DNAinsertion, fro
of AtSUC1, and to a lesser extent AtSUC2 (Lundmark et al. if stably expressed, is unlikely to be functional. Therefore, the m h
2006). Our previous findings showed that AtSUC2 and AtSUC9mutantsarelikelytobenull. ttp
s
AsttrSeUssCes4aanrediAmBpAortdaunrtinrgegsueleadtogrserimninthateiorne,spsoenedselintgogarboiwottihc AtSUC9 mutants are more sensitive to salt, ://ac
a
and Suc distribution. AtSUC2 and AtSUC4 mutants have osmotic stress and low temperature de
m
higher Suc content in shoots and lower Suc content in roots To gain insights into the possible functions of AtSUC9 during ic.o
than the wild-type (WT) plants under abiotic stress, so we u
abiotic stress, we examined the germination rates in the WT p
speculate that the mutants would grow better in abiotic .c
andAtsuc9mutantsinabioticstressconditions.Undercontrol o
m
stress in exogenous Suc media. Unexpectedly, we found the conditions, the germination rates of the mutant seeds were /p
opposite result. The AtSUC2 and AtSUC4 mutants are much lower than those of WT seeds at 1d, but the germination of cp/a
moresensitivetoabioticstresstreatmentswithexogenousSuc mutant and WT seeds was identical at 5d (Fig. 1). However, rtic
than without it, suggesting the complicated mechanism of le
additionof50or100mMNaClsignificantlyinhibitedtheger- -a
SUCs and the role of other SUCs might play in abiotic stress. b
minationofthemutantseedscomparedwiththatoftheWT s
However, the physiological functions of other SUCs at the seeds,from1to5d(Fig.1).SimilartotheresponsestoNaCl, trac
whole-plant level in plant abiotic stress tolerance are poorly exposuretoosmoticstress(viamediacontainingdifferentcon- t/5
6
known. centrations of mannitol) and low temperature, the mutant /8/1
InArabidopsis,nineSUCgeneshavebeenidentifiedinthree 5
plants germinated significantly less than the WT (Fig. 1). We 7
distinct subfamilies: the SUT1, SUT2 and SUT4 clades (Aoki further investigated the effect of abiotic stress on seedling 4/1
et al. 2003). AtSUC1, AtSUC2, AtSUC5, AtSUC6, AtSUC7, growth by examining the length of roots and the area of true 846
AtSUC8 and AtSUC9 are members of the high-affinity dicot 2
leaves under control and stress conditions. There was no sig- 1
6
SUT1 clade (Ku¨hn and Grof 2010); AtSUC3/SUT2 belongs to nificant difference observed in growth status among WT and by
theSUT2clade(Meyeretal.2000);andthereisoneSUT4clade g
mutant seedlings under control conditions (Fig. 2). However, u
SSUucCstrhaansspaocrltoesre, rAetlaStUioCn4sh(Wipetiosetheetirasl.pe2c0i0a0l)f.uTnchteiolnosca(tSitoandloerf underabioticstress,themutantseedlingshadshorterrootsand est o
smallerleavesthanWTseedlings(Fig.2). n
etal.1999).Forexample,AtSUC2isakeyproteinrequiredfor 12
phloem loading of Suc in source leave (Gottwald et al. 2000, Expression of AtSUC9 is induced by salt, osmotic Ap
Chandran et al. 2003). AtSUC1 is highly expressed in pollen stress and low temperature ril 2
0
(Stadler et al. 1999), AtSUC5 in seeds (Baud et al. 2005) and 1
Asthenextstepinfunctionalanalysis,weexaminedthechange 9
AtSUC8inflowers(Saueretal.2004,Sivitzetal.2007).AtSUC6
in AtSUC9 expression in shootsand roots underabiotic stress
and AtSUC7 encode aberrant proteins (Sauer et al. 2004).
by quantitative RT–PCR (qRT–PCR). These analyses revealed
Compared with other SUCs, AtSUC9 is located in almost all
thattheexpressionofAtSUC9ishighlyinducedinshootsand
tissue.Inaddition,AtSUC9showslowsubstratespecificityand
roots by several stress conditions, including salt, osmotic and
transports a wide range of glucosides, including helicin,
cold at nearly all treatment times (Fig. 3a), indicating that
salicin, arbutin, maltose, fraxin, esculin, turanose and a
AtSUC9isanimportantabioticstress-inducibleprotein.
methyl-D-glucose(Sivitzetal.2007).However,thephysiological
functions of AtSUC9 at the whole-plant level are largely un-
AtSUC9 mutants alter Suc distribution in shoots
known, except that AtSUC9 regulates early flowering under
and roots in response to salt, osmotic stress and
short-dayconditions(Sivitzetal.2007).Here,wedemonstrate
low temperature
a new physiological role for the Suc transporter AtSUC9 in
plant response to abiotic stress mediated by ABA and low Nullmutations inAtSUC9 mightcause a change inSuc distri-
levelsofSuc. bution in shoots and roots. Therefore, we examined Suc
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W.Jiaetal.|RoleofAtSUC9inabioticstressresistance
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Fig.1 SeedgerminationofAtSUC9mutantsunderabioticstress.Thegerminationrateswererecordedin1/2MSmediawithorwithoutNaCl, nlo
mannitol or cold treatments during a period from 1 to 5d after stratification for the WT, Atsuc9-1, Atsuc9-4 and Atsuc9-5 (n=3; *P<0.05; ad
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**P<0.01;Student’st-test). d
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Fig.2 SeedlinggrowthofAtSUC9mutantsunderabioticstress.(a)Seedlinggrowthin1/2MSmediawithorwithoutNaCl,mannitolorcold A
p
treatmentsat15dafter5dgrowthon1/2MSmedium.EachimageshowstheWT,Atsuc9-1,Atsuc9-4andAtsuc9-5,oneseedlingeach,fromleft ril 2
toright.Scalebar=1cm.(b)Twenty-day-oldseedlingstreatedornotwithNaCl,mannitolorcoldwerechosenforinvestigationoftheleafarea 0
1
9
andprimaryrootlength.Asterisksindicatestatisticallysignificantdifferencesbetweennormalconditionsandstressconditionsattheindicated
timesaccordingtoStudent’st-test(*P<0.05;**P<0.01).
contents of AtSUC9 mutants during abiotic stress. The results control treatment, Suc contents in WT and mutant roots
indicatedthatunderthecontrolconditions,therewaslessSuc showed an obvious increase in the presence of abiotic stress,
intherootsofthemutantplantsthaninthoseofWTplants, suggestingthataccumulation ofSucinrootsmaybeveryim-
but therewasnodifference in Succontents oftheshootsbe- portantforplantstresstolerance.However,Succontentinthe
tween the WT and mutant plants (Fig. 3b). Under abiotic roots of AtSUC9 mutants is much less than that in WT roots,
stress, the Suc content in the shoots of AtSUC9 mutants is whichmayaffectthemutants’stresstolerance.Takentogether,
more than that ofthe WT, which mayaffect plant growth by thesedatashowthatduringstress,Sucdistributionisalteredin
inducingnegativefeedbackonphotosynthesisandcarbonme- AtSUC9 mutants, and this may explain why the growth of
tabolism (Lemoine et al. 2013). In addition, compared with AtSUC9mutantsisinhibitedunderstressconditions.
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Fig.3 ChangesinSuccontentoftheWT,Atsuc9-1,Atsuc9-4andAtsuc9-5,andchangesoftheexpressionofAtSUC9withstresstreatments.(a) 2 A
ChangesintheexpressionofAtSUC9withstresstreatments(200mMNaCl,300mMmannitoland10(cid:3)C).Dataarethemean±SD(n=3).The p
expressionofAtSUC9intheuntreatedWTwasusedascontrol.TherelativeexpressionofAtSUC9inthetreatmentgroupswasnormalizedbythe ril 2
0
controlgroup.(b)Theseedlingswerecollectedafter20dofabioticstresstreatments,andSuccontentinshootsandrootsoftheWTandAtsuc9 1
9
mutantswasmeasured(n=3;*P<0.05;**P<0.01;Student’st-test).
AtSUC9 shows an obvious response to low levels
WeexaminedtheexpressionofthephotosyntheticgenesCAB
of endogenous Suc
and RBCS in mutant and WT plants under abiotic stress. The
Manyreportshaveindicated thatunderabiotic stress,photo- resultsshowedthattheexpressionofCABandRBCSisinhibited
synthesis, which is the main source of Suc production, is in- inboththemutantandtheWT,suggestingthatphotosynthesis
hibited(forreviews,seeYu1999,Ruanetal.2010).AtSUC9isan isinhibitedduringstress(SupplementaryFig.S2).Theinhib-
ultrahigh affinity protein, i.e. it is capable of transporting Suc itionofphotosynthesisindicatedthattheremightbeaverylow
evenwhenSuclevelsareverylow(Sivitzetal.2007).Therefore, supplyofSucforsinktissuesduringstresses.
we hypothesized that AtSUC9 plays a role in decreasing Suc To determine whether AtSUC9 responds to low levels of
levelsafterabioticstress-inducedinhibitionofphotosynthesis. endogenous Suc, we analyzed the expression of AtSUC9,
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W.Jiaetal.|RoleofAtSUC9inabioticstressresistance
AtSUC2andAtSUC4intwotreatmentswhichleadtoalowlevel suggestingthatAtsuc9mutantsshowedamoreobvioussensi-
of endogenous Suc (Caspar et al. 1985, Caspar et al. 1989, tivity response to abiotic stress without exogenous Suc than
Thimmetal.2004).Thetreatmentswereimplementedatthe withit.TheseresultsfurtherprovedthatAtSUC9playsarolein
endofthenightandafteranadditional6hofdarkness(Thimm responsetoabioticstresses,whichiscloselylinkedtothelevel
etal.2004),orinthemiddleofnight(Casparetal.1985,Caspar ofSuc.
etal.1989).TheresultsshowedthatbothlowSuctreatments
Deletion of AtSUC9 blocks abiotic stress-inducible
leadtosimilarchangesintheexpressionofAtSUC2,AtSUC4and
ABA accumulation, and even results in a lower
AtSUC9,i.e.adecreaseofAtSUC4,amildincreaseofAtSUC2and
anobviousincreaseofAtSUC9inresponsetolowSuc(Fig.4a), ABA content in abiotic stress compared with
illustratingthatAtSUC9inparticularisinducedbylowlevelsof normal conditions
endogenous Suc. Additionally, we analyzed the expression of
ABAisawell-knownphytohormonethatcanenhanceabiotic
some low Suc level-responsive genes in the WT and Atsuc9 stress tolerance, including tolerance to salinity, cold and D
o
mutants treated during the day and in the middle of the w
drought, and ABAisclosely relatedtotheSuc signaling path- n
night. By analyzing the expression change of some genes in lo
way (Zhu 2002). Thus, the ABA content was examined in the a
themiddleofthenight,comparedwithtreatmentduringthe shootsandrootsoftheWTandAtSUC9mutantsundernormal ded
day, in the WT (data not shown) and referring to previous conditions and abiotic stress. The results showed a very high fro
studies (Lam et al. 1994, Thimm et al. 2004), we screened m
ABA content in both shoots and roots in AtSUC9 mutants h
some low Suc level-responsive genes, including low Suc- compared with that in the WT in normal conditions (Fig. 6), ttp
s
induced genes,asparagine synthetase1(ASN1)andPSII 10kDa suggestingthatdeletionofAtSUC9mightrequireagreaterABA ://a
phosphoprotein (PSBH); low Suc-inhibited genes, ribosomal c
contenttomaintainnormalgrowth.Further,theABAcontent a
d
protein S8 (RPS8) and ATPase beta-subunit (ATPB); and non- e
inrootsandshootsoftheWTwashigherinabioticstressthan m
responsive genes, ribosomal protein S11 (RPS11) and Cyt B6 innormalconditions(Fig.6),whichisanimportantresponsein ic.o
(PETB). The results showed that deletion of AtSUC9 resulted u
plantabioticstresstolerance(forreviews,seeXiongetal.2002, p
in obvious expression changes of low Suc-induced genes and .c
Zhu2002,Chinnusamyetal.2005).However,theABAcontent o
m
low Suc-inhibited genes, and no obvious changes in non-re- inbothshootsandrootsofAtSUC9mutantswasmuchlowerin /p
sponsive gene expression in both day and night treatments; abioticstressthaninnormalconditions(Fig.6),suggestingthat cp/a
more obvious changes can been seen in the night treatment, deletionofAtSUC9notonlyinhibitedtheabioticstress-induced rtic
especiallyfullyinhibitedexpressionoflowSuc-inducedgenesin ABA content, but also led to a lower ABA content compared le-a
Atsuc9-1inthenighttreatment(SupplementaryFig.S3),illus- b
withthatinnormalgrowthconditions. s
tratingthatAtSUC9regulatessomelowSuc-responsivegenes, tra
c
further indicating that AtSUC9 is involved in response to low Deletion of AtSUC9 inhibited the expression of t/5
6
levelsofendogenousSuc. ABA- and stress-inducible genes /8
/1
5
AtSUC9 shows an obvious response to low levels WenexttestedtheexpressionofthefollowingABA-inducible 7
4
of exogenous Suc and stress-responsive genes in AtSUC9 mutants: SnRK2.2, /1
8
4
SnRK2.3 and SnRK2.6 (Boudsocq et al. 2004); ABF genes 6
To verify further the role of AtSUC9 in low levels of Suc, we 2
(ABF2/AREB1, ABF3 and ABF4) (Choi et al. 2000, Uno et al. 16
examinedtheeffectoflowlevelsofexogenousSuc(0–3%)on b
2000); ABI1 (Leung et al. 1994); ABI3 (Giraudat et al. 1992); y
theexpressionofAtSUC9.WefoundthatAtSUC9expressionis g
ABI4 (Finkelstein et al. 1998); RD29A (Yamaguchi-Shinozaki u
up-regulatedinbothshootsandrootsinresponsetolowlevels e
s
ofexogenousSuc(Fig.4b),andtheexpressionincreasesfurther and Shinozaki 1994); and KIN1 and KIN2 (Kurkela and Borg- t o
following a decrease in the concentration of exogenous Suc. Franck 1992). Our results showed that in the absence of ABA n 1
2
Then, we examined seed germination, seedling growth and treatment, the expression of almost all the genes in mutants A
p
Suc distribution in the WT and Atsuc9 mutants in 0% or 3% Hinocwreeavseedr, stlhigehtelyxp(r<es0s.i5o-nfolodf),aclolmgepnaeresdinwimthutthanetWs Tde(cFriega.s7ed). ril 20
Suc treatments. The results showed that AtSUC9 mutants are 1
approximately1-foldto2.5-foldinresponsetoABAtreatment, 9
moresensitivethanWTplantsto0%Suctreatmentintermsof
comparedwiththatintheWT(Fig.7),indicatingthatdeletion
seed germination (Fig. 4c) and seedling growth (Fig. 4d, e).
of AtSUC9 blocks the increase of ABA-inducible gene expres-
Moreover,thedisruptionofAtSUC9greatlyaffectedSucdistri-
sion. Additionally, we examined the expression of these ABA-
butioninthesourceandsinkinthe0%Suctreatment,similar
and stress-related genes in WT and AtSUC9 mutants in salt
totheresultsduringabioticstress(Fig.4f).
stress.TheresultsalsoshowedthatdeletionofAtSUC9blocks
Toanalyzefurthertherelationshipbetweenthefunctionof
theincreaseofABA-induciblegeneexpressionundersaltstress
AtSUC9 and a low Suc level in abiotic stress, we investigated
treatments(SupplementaryFig.S6).
seedlinggrowthofthemutantsunderstressconditionswithout
exogenous Suc. Compared with the control condition, Atsuc9
AtSUC9 mutants are more sensitive to ABA
mutantsexhibitedasmallerleafshapeandamuchshorterroot
treatments
system than the WT (Fig. 5) under abiotic stress without ex-
ogenousSuc.Inparticular,AtSUC9mutantsbarelygrewunder Additionally,weexaminedthegerminationratesandseedling
relativelyhigherconcentrationsofosmoticandsalttreatments, growthintheWTandAtsuc9mutantsunderABAtreatments.
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Fig.4 AtSUC9responsestolowlevelsofSuc.(a)AtSUC9responsestolowlevelsofendogenousSuc.TheexpressionofAtSUC9,AtSUC2and 19
AtSUC4duringtheday,inthemiddleofthenightorafteranadditional6hafternighttreatment.Dataarethemean±SD(n=3).Theexpression
ofgenesinthedaytreatmentwassetto1.(b)ChangesintheexpressionofAtSUC9withdifferentconcentrationsofexogenousSuc.Dataarethe
mean±SD(n=3).Theexpressionofgenesin3%Suc-treatedWTwassetto1.(c)ChangesinseedgerminationofAtsuc9mutantswithno
exogenousSuctreatment.Thegerminationrateswererecordedin1/2MSmediasupplementedwith3or0%Sucduring1–4dafterstratification
for the WT, Atsuc9-1, Atsuc9-4 and Atsuc9-5 (n=3; *P<0.05; **P<0.01; Student’s t-test). (d and e) Changes in seedling growth of Atsuc9
mutantswithnoexogenousSuctreatment.Eachimage(d)shows20-d-day-oldWT,Atsuc9-1,Atsuc9-4andAtsuc9-5at3or0%Suctreatment,
twoseedlingseach,fromlefttoright.Scalebar=1cm.Twenty-day-oldseedlingswerechosentobeinvestigatedintermsofleafareaandprimary
rootlength(e).(f)ChangesofSuccontentinAtsuc9mutantswithnoexogenousSuctreatment.Theseedsweregerminatedandgrownon1/2
MS media with 3 or 0% Suc. The seedlings were collected at 20d and the Suc content in shoots and roots was measured (n=3; *P<0.05;
**P<0.01;Student’st-test).
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Fig.5 AtSUC9mutantsshowhypersensitivityinresponsetoabioticstresstreatmentswithoutexogenousSuc.(a)Seedlinggrowthin1/2MS em
mediawithoutexogenousSucwithNaCl,mannitolorcoldat20dafterstratification.EachimageshowstheWT,Atsuc9-1,Atsuc9-4andAtsuc9-5, ic
.o
oneseedlingeach,fromlefttoright.Scalebar=1cm.(b)SeedlinggrowthwithabioticstresstreatmentswithoutexogenousSuc.Twenty-day-old u
p
seedlingswerechosentobeinvestigatedintermsoftheleafareaandprimaryrootlength.Asterisksindicatestatisticallysignificantdifferences .c
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amongNaCl,mannitolorcoldtreatmentattheindicatedtimesaccordingtoStudent’st-test(*P<0.05;**P<0.01). m
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Fig.6 DeletionofAtSUC9inhibitsabioticstress-inducibleABAaccumulation.The20-day-oldseedlingswerecollectedafter6htreatmentunder 0
1
abiotic stress (200mM NaCl, 350mM mannitol and 4(cid:3)C), and the endogenous ABA content in shoots and roots of the WT, Atsuc9-1 and 9
Atsuc9-4wasmeasured.
Theresultsshowedthatundercontrolconditions,thegermin- mutant seedlings had shorter roots and smaller leaves
ationratesofthemutantseedswerelowerthanthoseofWT than WT seedlings (Fig. 8). These results illustrated that
seedsat1d,butthegerminationofmutantandWTseedswas AtSUC9 mutants are more sensitive in response to ABA
identical at 5d (Fig. 8). However, addition of 50 or 100mM treatments.
NaCl significantly inhibited the germination of the mutant
Atsuc9-1/Atabi1-1 double mutants show increased
seedscomparedwiththatofWTseeds,from1to5d(Fig.8).
plant abiotic stress tolerance
In addition, there was no significant difference observed in
growthstatusamongWTandmutantseedlingsundercontrol ToanalyzefurthertheroleofAtSUC9inABAsignalingunder
conditions (Fig. 8). However, under ABA treatment, the abioticstress,wegeneratedAtsuc9-1/Atabi1-1doublemutants,
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Fig.7 DeletionofAtSUC9inhibitstheexpressionofABA-induciblegenes.ExpressionofABA-induciblegeneswasassayedbyqRT–PCRforthe ril 2
0
WT, Atsuc9-1, Atsuc9-4 and Atsuc9-5. –ABA, no ABA treatment; +ABA, treatment with 40mM ABA. Data are the mean±SD (n=3). The 1
9
expressionofgenesintheWTwithnoABAtreatmentwassetto1.
then planted the parent mutants and double mutants in that the deletion of both AtSUC9 and AtABI1 is beneficial to
normal and abiotic stress conditions. The results showed that plantabioticstresstolerance.
innormalconditions,Atsuc9-1grewtoslightlysmallersizethan
theWT(ColandLer),andAtabi1-1andtheAtsuc9-1/Atabi1-1
double mutantdid not differ fromthe WT(Fig. 9). Inabiotic
Discussion
stress,Atsuc9-1washypersensitive,similartowhatisshownin
Fig.2,andthegrowthofAtabi1-1wasnotdifferentfromthatof Suc is produced in the leaves of plants during photosynthesis
the WT (Fig. 9); however, Atsuc9-1/Atabi1-1 double mutants andisthentransportedthroughouttheplantbySUCsinorder
grewtoalargersizecomparedwiththeWT(Fig.9),indicating to support plant growth. Thus, it is accepted that SUCs are
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W.Jiaetal.|RoleofAtSUC9inabioticstressresistance
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Fig.8 SeedgerminationandseedlinggrowthofAtSUC9mutantsunderABAtreatment.(a)Thegerminationrateswererecordedin1/2MS .co
m
mediawithorwithoutABAtreatmentsduringaperiodfrom1to5dafterstratificationfortheWT,Atsuc9-1,Atsuc9-4andAtsuc9-5.(b)Fifteen-
/p
day-oldseedlingsafter5dgrowthon1/2MSmediumtreatedornotwithABAwerechosentobeinvestigatedintermsoftheprimaryroot cp
laecncgotrhdianngdtloeaSftaurdeean.At’sstte-rtiesskts(in*Pd<ica0t.e05st;a*t*iPst<ica0ll.y01s)ig.nificantdifferencesbetweennormalconditionsandstressconditionsattheindicatedtimes /article
-a
b
s
tra
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importantforplantgrowthandstresstolerance(Rollandetal. 2005,Sivitzetal.2007),apreviousstudyfoundnodifferencesin t/5
6
2002). AtSUC2, AtSUC4 (in A. thaliana) and PtaSUT4 (in seedling growth on media containing Suc (Sivitz et al. 2007), /8
/1
Populus tremula) have been proved to be involved in plant and we also found that the growth of three AtSUC9 mutants 5
7
abiotic stress tolerance at the whole-plant level (Frost et al. didnotdifferfromthegrowthoftheWTinnormalMurashige 4/1
2012, Gong et al. 2015), but almost all other results have and Skoog (MS) medium (containing 3% exogenous Suc) 84
6
been determined strictly through gene expression changes (Fig.4).Aprevious studyshowedthatmutationofAtsuc9al- 2
1
6
during abiotic stress. The cross-talk between SUCs and plant lelesleadstoadelayinfloraltransitionundershort-daycondi- b
y
stress resistance at the whole-plant level has not been fully tions(Sivitzetal.2007).Thus,itispossiblethatenvironmental g
u
investigated. In the current study, we found that AtSUC9, a signalsmaybeakeyfactorininducingthebiologicalfunctionof es
high-affinitySuctransporter,isinvolvedinabioticstresstoler- AtSUC9. t o
n
anceinseedgermination,seedlinggrowth,Sucdistributionand Inourstudy,wefoundthatexpressionofAtSUC9inshoots 12
ABA accumulation regulated by low levels of Suc in androotsisstronglyinducedbysalt,droughtandlowtempera- Ap
Arabidopsis. ture (Fig. 3a). Three AtSUC9 mutants were more sensitive to ril 2
0
salt, drought and low temperature than the WT (Fig. 2). It is 1
AtSUC9 plays an important role under abiotic 9
well known that source–sink Suc distribution is beneficial for
stresses
plantgrowthandstresstolerance(Roitsch1999).Waterstress
Ingeneral,mosttypesofplantscontainafamilyofSUCscom- induceslargealterationsinsource–sinkrelationshipsforregu-
posedofseveralmembers.Forexample,fivemembersmakeup lationofgrowthpriorities(Bermanetal.1996).Here,deletionof
a SUC family in rice and nine members make up a family in AtSUC9 fully breaks the Suc balance in the shoots and roots
Arabidopsis (Ku¨hn and Grof 2010). These proteins can play underabioticstresses,includingosmotic,saltandcoldstresses
rolesindifferent partsoftheplantorduringdifferent growth (Fig.3a),whichmaybethemainreasonfortheabioticstress-
stages,includingplantstressresistance(GuptaandKaur2005, inducedsensitivityofAtsuc9mutants.Uptonow,allresultson
Ku¨hnandGrof2010).AtSUC9belongstotheSUTIclade,which the role of SUCs at the whole-plant level in abiotic stress
is a group of high-affinity Suc transporters (Sauer et al. 2004). showedthattheSucimbalanceinsinkandsourcewasinduced
AtSUC9islocalizedtotheplasma membranebytransient ex- by a defect of SUC functions, including those of AtSUC2,
pression in the onion (Allium cepa) epidermis. Although AtSUC4andPtaSUT4(Frostetal.2012,Gongetal.2015),sug-
AtSUC9 is expressed in nearly the entire plant (Schmid et al. gesting that SUCs might be involved in plant abiotic stress
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PlantCellPhysiol.56(8):1574–1587(2015)doi:10.1093/pcp/pcv082
Fig.9 TheAtabi1-1mutantrecoversthehypersensitivephenotypeofAtSUC9mutationunderabioticstress.Fifteen-day-oldseedlingsoftheWT D
o
(ColorLer),Atsuc9-1,Atabi1-1andAtsuc9-1/Atabi1-1after5dgrowthon1/2MSmediumtreatedornotwithNaCl,mannitolorcoldwere w
n
chosentobeinvestigatedintermsoftheprimaryrootlengthandleafarea.Asterisksindicatestatisticallysignificantdifferencesbetweennormal lo
a
conditionsandstressconditionsattheindicatedtimesaccordingtoStudent’st-test(*P<0.05;**P<0.01). d
e
d
fro
m
h
tolerance largely dependent on regulating the Suc content in that AtSUC9 might be required when low levels of Suc need ttps
thesourceandsink.Sucaccumulationinshootsshouldoccur to be transported in plant abiotic stress tolerance. Here, we ://a
c
by interfering with phloem loading of Suc in source organs. found that AtSUC9 was particularly and highly induced by ad
e
AtSUC9 belongs to the SUT1 clade whose members have low levels of exogenous and endogenous Suc (Fig. 4a, b). m
ic
been proved to play a role in Suc loading in the leaf. For ex- Atsuc9mutantshadhypersensitivitytolowlevelsofexogenous .o
u
ample AtSUC2 has been confirmed as the main SUC respon- Suc treatments in seed germination and seedling growth, and p
.c
sible for phloem loading at the whole-plant level in normal Suc distribution was affected in the source and sink (Fig. 4), om
conditions (Gottwald et al. 2000, Srivastava et al. 2008). A similar to the results of Atsuc9 mutants in abiotic stress. In /p
c
p
lower Suc content in the AtSUC9 mutant roots could be pro- addition,deletionofAtSUC9obviouslyaffectedtheexpression /a
duced bya decreaseofSuc supply fromsourcesand/or byan ofmanygenesthatwereresponsivetolowlevelsofendogenous rtic
le
effectonSucdistributionactivities.Todate,aroleforSUCsin Suc(SupplementaryFig.S3).AtSUC9mutantshardlygrewin -a
b
phloemunloadingofSucinrootshasnotbeenestablished.A abioticstressconditionswithoutexogenousSuc(Fig.5).These s
tra
recent studyshowedthatSuc unloadingin plantaisprobably results are not found in AtSUC2 and AtSUC4 (Fig. 6a; Gong c
regulated by SWEET proteins rather than SUCs (Chen et al. et al. 2015), suggesting that AtSUC9 functions in particular in t/56
/8
2012). When SUCs are located in sinks, they probably play a lowSuclevels.ThespecialroleofAtSUC9maybeacontribu- /1
5
roleinretrievalofescapedSucbackintothephloemtoregulate tiontofurtherunderstandingtwopreviousfindings.Oneisthe 7
4
Suc distribution in the sink (Truernit and Sauer 1995). physiological phenomenon of Atsuc9 mutants in short days, /1
8
4
Therefore, how AtSUC9 regulates the root Suc content is still becauseAtSUC9playsagreaterroleinlongperiodsofdarkness 6
2
anopenquestion.ApreviousstudyfoundthatAtSUC9shows which produce a low Suc level in vivo. It also explains why 16
b
lowsubstratespecificityandtransportsawiderangeofgluco- Atsuc2 and Atsuc4 mutants have better growth in stress con- y
g
sides (Sivitz et al. 2007), which may also be involved in plant ditionswithoutexogenousSucthanwithit.Thereasonisthat u
e
s
stresstolerance. AtSUC9istriggeredandplaysagreaterrolethanAtSUC2and t o
AtSUC4 in the stresses without exogenous Suc, because the n 1
Functional specificity of AtSUC9 in response to condition requires the plant to transport Suc even at a very 2 A
p
low Suc levels lowlevel. ril 2
0
Manyreportsillustratedthatunderabioticstress,photosynthe- 1
AtSUC9 functions in ABA accumulation and 9
sisisinhibited(forreviews,seeYu1999,Ruanetal.2010).With
SnRK2s signaling transduction in abiotic stress
regardtotheeffectofwaterdeficiencyonwholeplantsunder
abiotic stress, source limitations such as a reduced photosyn- ABAisaphytohormonethatmediatesavarietyofgrowthand
thetic capacity, resulting in a decreased export of assimilates, developmental processes, as well as environmental responses,
seemtoberesponsiblefordecreasedcropload(Bermanetal. suchasdrought,saltandcoldstressresponses(Chenetal.2006,
1996). In our study, we found decreased expression of photo- Seo et al. 2012). It is confirmed that cold, salt and osmotic
synthesis genes in both WT and mutant plants under abiotic stresses induce production of endogenous ABA in plants (for
stresses, further proving that abiotic stress inhibits photosyn- reviews, see Xiong et al. 2002, Zhu 2002, Chinnusamy et al.
thesis(SupplementaryFig.S2).Theinhibitionofphotosynthe- 2005), Here, high ABA accumulation in roots in response to
sis might lead to low levels of Suc transported to sink tissue. abioticstresswasalsoobserved(Fig.6).Additionally,theresults
Compared with the other Arabidopsis SUCs, AtSUC9 had an demonstrated that the disruption of AtSUC9 leads to a high
ultrahigh affinity for Suc (with an apparent K value= ABAaccumulationinnormalconditions(Fig.6),whichmight
m
0.07±0.02mM Suc) (Sivitz et al. 2007); thus, we speculate be in order to activate other AtSUCs to recover the loss of
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