The Grape VlWRKY3 Gene Promotes Abiotic and Biotic Stress Tolerance in Transgenic ...

16 Pages·2017·4.68 MB·English

fpls-09-00545 April23,2018 Time:14:59 #1
ORIGINALRESEARCH
published:25April2018
doi:10.3389/fpls.2018.00545
The Grape VlWRKY3 Gene Promotes
Abiotic and Biotic Stress Tolerance in
Transgenic Arabidopsis thaliana
RongrongGuo1,2,3,HengboQiao1,2,JiaoZhao1,2,XianhangWang1,2,MingxingTu1,2,
ChunleiGuo1,2,RanWan1,2,ZhiLi1,2* andXipingWang1,2*
1StateKeyLaboratoryofCropStressBiologyinAridAreas,CollegeofHorticulture,NorthwestA&FUniversity,Xianyang,
China,2KeyLaboratoryofHorticulturalPlantBiologyandGermplasmInnovationinNorthwestChina,MinistryofAgriculture,
NorthwestA&FUniversity,Xianyang,China,3GuangxiAcademyofAgriculturalSciences,Nanning,China
WRKY transcription factors are known to play important roles in plant responses to
various abiotic and biotic stresses. The grape WRKY gene, WRKY3 was previously
reported to respond to salt and drought stress, as well as methyl jasmonate and
ethylene treatments in Vitis labrusca × V. vinifera cv. ‘Kyoho.’ In the current study,
WRKY3 from the ‘Kyoho’ grape cultivar was constitutively expressed in Arabidopsis
thalianaundercontrolofthecauliflowermosaicvirus35Spromoter.The35S::VlWRKY3
transgenic A. thaliana plants showed improved salt and drought stress tolerance
Editedby:
during the germination, seedling and the mature plant stages. Various physiological
VincenzoLionetti,
SapienzaUniversitàdiRoma,Italy traits related to abiotic stress responses were evaluated to gain further insight into
Reviewedby: the role of VlWRKY3, and it was found that abiotic stress caused less damage
BenedettoRuperti,
to the transgenic seedlings than to the wild-type (WT) plants. VlWRKY3 over-
UniversitàdegliStudidiPadova,Italy
PrateekTripathi, expressionalsoresultedinalteredexpressionlevelsofabioticstress-responsivegenes.
TheScrippsResearchInstitute, Moreover, the 35S::VlWRKY3 transgenic A. thaliana lines showed improved resistance
UnitedStates
to Golovinomyces cichoracearum, but increased susceptibility to Botrytis cinerea,
*Correspondence:
compared with the WT plants. Collectively, these results indicate that VlWRKY3 plays
ZhiLi
[email protected] important roles in responses to both abiotic and biotic stress, and modification of its
XipingWang
expressionmayrepresentastrategytoenhancestresstoleranceincrops.
[email protected]
Keywords:abioticstress,bioticstress,grape,transgenic,VlWRKY3
Specialtysection:
Thisarticlewassubmittedto
PlantMicrobeInteractions, INTRODUCTION
asectionofthejournal
FrontiersinPlantScience
Plantsareregularlyexposedtoabroadrangeofabioticandbioticstresses,suchasdrought,high
Received:04December2017
salinity,extremetemperatures,andpathogeninfection(Mishraetal.,2015).Thesestresses,which
Accepted:09April2018
can occur concurrently, affect growth and development, can also alter species distribution (Shi
Published:25April2018
etal.,2014).Plantshaveevolveddiverseadaptivemechanismstoavoidortoleratestress(Schmidt
Citation:
etal.,2010),includingtherecognitionofstresscuesandthetransductionofthesignalsthatregulate
Guo R,Qiao H,Zhao J,Wang X,
theexpressionofstress-relatedgenes(Chinnusamyetal.,2004).Understandingthemolecularbasis
Tu M,Guo C,Wan R,Li Zand
oftoleranceassociatedwithmultiplestresses,andidentifyingcandidategenesthatconfertolerance
Wang X(2018)TheGrapeVlWRKY3
GenePromotesAbioticandBiotic
StressToleranceinTransgenic Abbreviations: ABA,abscisicacid;CAT,catalase;CDS,codingsequence;d,diameter;DAB,diaminobenzidine;dpi,days
Arabidopsisthaliana. postinoculation;ET,ethylene;hpi,hourspostinoculation;MDA,malonyldialdehyde;MeJA,methyljasmonate;NBT,nitro
Front.PlantSci.9:545. bluetetrazolium;ORF,openreadingframe;pad4,Phytoalexindeficient4;POD,peroxidase;qRT-PCR,quantitativereal-time
doi:10.3389/fpls.2018.00545 PCR;ROS,reactiveoxygenspecies;SOD,superoxidedismutase;SOS,saltoverlysensitive;WT,wild-type.
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Guoetal. VlWRKY3PromotesStressTolerance
withoutcompromisingyieldaregoalsinthedevelopmentofcrop (Marchiveetal.,2007,2013),whileover-expressionofVvWRKY2
varieties with enhanced tolerance of deleterious environmental in tobacco (Nicotiana tabacum cv. Xanthi) enhanced broad
factors(AtkinsonandUrwin,2012). resistancetonecrotrophicfungalpathogens.Moreover,theover-
WRKY transcription factors comprise a large family of expression of VvWRKY2 exhibited altered expression of genes
regulatoryproteinsthathavebeenshowntoplayimportantroles involvedinligninbiosynthesispathwayandcellwallformation,
inresponsestobioticandabioticstresses(Chenetal.,2009;Wang suggesting a role of VvWRKY2 in the responses to biotic and
C.etal.,2013).AdefiningcharacteristicofWRKYtranscription abiotic stresses (Mzid et al., 2007; Guillaumie et al., 2010). In
factors is s 60 amino acids WRKY domain, which is composed anotherstudy,theectopicexpressionoftwoWRKYgenesfrom
of a highly conserved WRKYGQK motif at the N-terminus, Erysiphe necator-resistant Chinese wild V. pseudoreticulata W.
aswellaszinc-finger-likemotifs(eitherCX4−5CX22−23HXHor T.Wang‘Baihe-35-1,’VpWRKY1andVpWRKY2inA.thaliana
CX CX HXC)attheC-terminus(Eulgemetal.,2000;Rushton enhanced resistance to powdery mildew, and increased salt
7 23
et al., 2012). The conserved WRKY domain is essential for toleranceand/orcoldtolerance(Lietal.,2010b).AnotherWRKY
recognizing and binding to a DNA cis-element, named the gene, VpWRKY3 (corresponding to WRKY10 in our previous
W-box,inthepromoterregionsoftargetgenes(Sunetal.,2003). study (Guo et al., 2014), also isolated from V. pseudoreticulata
Previous loss- and gain-of-function studies have ‘Baihe-35-1,’wasalsoshowntoparticipatedinabioticandbiotic
demonstrated that WRKY transcription factors act as either stressresponses(Zhuetal.,2012).Inaddition,over-expressionof
positive or negative regulators in response to abiotic or/and VvWRKY11inA.thalianaenhanceddehydrationstresstolerance
biotic stresses (Eulgem et al., 2000; Pandey and Somssich, (Liu et al., 2011), and VvWRKY33 was reported to be involved
2009; Rushton et al., 2010; Chen et al., 2012). Examples of in defense against the oomycete pathogen Plasmopara viticola
biotic stress induced WRKY genes from Arabidopsis thaliana (Merzetal.,2015).
includes WRKY3 and WRKY4, which promote resistance to WepreviouslyfoundthattheexpressionofWRKY3,encoding
necrotrophic pathogens, while WRKY4 has been shown to a putative Group IIc WRKY gene, was up-regulated in
suppress resistance to biotrophic pathogens (Lai et al., 2008). V.labrusca×V.viniferacv.‘Kyoho’followingsaltanddrought
WRKY8hasbeenreportedtodecreaseresistancetothebacterial stress treatments, as well as exogenous application of MeJA
pathogenPseudomonassyringae,buttopromoteresistancetothe and Eth (Guo et al., 2014). In the current study, we examined
necrotrophicfungusBotrytiscinerea(ChenL.G.etal.,2010).In the putative roles of WRKY3 from ‘Kyoho’ grape in resistance
addition, WRKY11 and WRKY17 also suppress basal resistance to abiotic and biotic stresses by over-expressing the gene in
to P. syringae in A. thaliana (Journot-Catalino et al., 2006), A. thaliana and characterizing the responses of the resulting
while constitutive expression of WRKY18 enhances resistance transgenic plants to drought and salt stresses, as well as to
to this pathogen, except when it is coexpressed with WRKY40 inoculationwithGolovinomycescichoracearum,andB.cinerea.
or WRKY60, which results in the transgenic plants being more
susceptible to both P. syringae and B. cinerea (Xu et al., 2006).
MATERIALS AND METHODS
WRKY18, WRKY40 and WRKY60 have also been reported to
beinvolvedinresponsestoABAandabiotic stressbyactingas
Plant Materials and Pathogen Bacteria
eithertranscriptionactivatororrepressor(ChenH.etal.,2010).
WRKY57 and WRKY63 were shown to have important roles in Two year old V. labrusca × V. vinifera cv. ‘Kyoho’ seedlings
responsestodroughtstress(Renetal.,2010;Jiangetal.,2012). were grown in a greenhouse at the Northwest A&F University,
A co-operative interaction between the WRKY70 and WRKY54 Yangling, Shaanxi, China, and used as a source of material to
genes has also been identified in suppression of osmotic stress cloneWRKY3.A.thalianaL.ecotypeCol-0wasusedfortheover-
tolerance (Li et al., 2012). Finally, it has been suggested that expressionexperimentsandtheWTandtransgenicA.thaliana
WRKY30 plays a role in responses to several abiotic or biotic linesweregrownat22◦C,70%relativehumidityandalong-day
stresses(Scarpecietal.,2013). photoperiod(16hlight/8hdark).
WRKY genes from a range of crop species have also been Arabidopsis thaliana powdery mildew (G. cichoracearum
implicated in responses to biotic and abiotic stresses including isolate UCSC1) was cultured on the A. thaliana pad4 mutant
examplesfromrice(Oryzasativa,(Liuetal.,2005,2007;Shimono plantsat22◦C,andaphotoperiodof16hlight/8hdark.B.cinerea
et al., 2007; Qiu et al., 2007; Peng et al., 2008, 2012; Zhang wasmaintainedat22◦ConPotatoGlucoseAgarasdescribedby
et al., 2008; Abbruscato et al., 2012; Chujo et al., 2013; Wang Wanetal.(2015).
H.H. et al., 2013; Yokokani et al., 2013), wheat (Wang C.
VlWRKY3 Cloning and Sequence
et al., 2013), tomato (Bhattarai et al., 2010; Atamian et al.,
Analysis
2012; Liu et al., 2014), and cotton (Yu et al., 2012; Shi et al.,
2014). We have been studying WRKY genes from grape (Vitis Total RNA was extracted from the leaves of ‘Kyoho’ using
vinifera L.), one of the most economically important fruit the E.Z.N.A. Plant RNA Kit (Omega Bio-tek, United States,
crops in many countries (Wang et al., 2014), and in previous R6827-01), following the manufacturer’s protocol. First-strand
study, we identified 59 WRKY genes in the grape genome cDNA was synthesized using PrimeScriptTM RTase (TaKaRa
have been identified (Guo et al., 2014), however, only a few of Biotechnology, Dalian, China) according to the manufacturer’s
these have been functionally characterized to date. VvWRKY1 instructions.AVlWRKY3cDNA(GeneBankaccessionnumber:
was reported to be involved in defense against downy mildew XM_002275540.3) corresponding to the predicted ORF was
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amplified by PCR using the gene-specific primers F1 (5(cid:48)- ATG Seven-day-old transgenic and WT seedlings grown on MS
GAT AGC TTC TCC ACT C-3(cid:48)) and R1 (5(cid:48)-TTA AAA GGA medium plates were transferred to pots filled with humus and
AGC ATA AAC TTG C-3(cid:48)). The PCR product was cloned wateredwellfor3weeks,duringthesubsequentweek,plantswere
into the pGEM(cid:13)R -T Easy vector (Promega, Madison, WI, irrigatedat2daysintervalswith200mMNaClornotwatered,
United States), and the recombinant plasmid was sequenced corresponding to salt and drought treatments, respectively.
and named pGEM(cid:13)R-T Easy-VlWRKY3. Amino acid sequences Plantsthatwerewellwateredwereusedasacontrol.Following
of homologous WRKY3 proteins from other plant species were thedroughttreatment,plantswerere-wateredandsurvivalrates
obtained from the NCBI database1 using BLASTP. A multiple weredetermined24hlater.Allexperimentswererepeatedthree
sequence alignment of the deduced protein sequences and times.
phylogenetic analyses were carried out using the DNAMAN
Measurements of the Chlorophyll
software.Domainpredictionandthelogorepresentationofthe
WRKYdomainwasperformedusingMEME2. Content, Relative Electrolyte Leakage,
Malondialdehyde (MDA) and
Generation of Transgenic A. thaliana
Endogenous ABA Content
Plants Over-Expressing the Grapevine
Seven-day-oldseedlingsgrownonMSmediumweretransferred
WRKY3 Gene tofreshMSmedium,orMSmediumsupplementedwith130mM
The coding sequence of VlWRKY3 (with XbaI and NaClor200mMmannitol,andgrownfor1weekbeforeseedlings
KpnI sites added to its 5(cid:48) and 3(cid:48) ends, respectively) was wereharvested.Allmeasurementswererepeatedthreetimes.
amplified from pGEM-Teasy-VlWRKY3 using gene-specific Forchlorophyllcontentmeasurements,approximately0.05g
primers F2 (5(cid:48)-GCTCTAGA ATGGATAGCTTCTCCACTC- of fresh leaf material was placed in 5 ml of 96% ethanol
3(cid:48); XbaI site underlined) and R2 (5(cid:48)-GGGGTACC and incubated at 4◦C in the dark overnight. The absorbance
TTAAAAGGAAGCATAAACTTGC-3(cid:48); KpnI site underlined), of the extracted pigments was measured at 665 and 649 nm
and inserted immediately downstream of the CaMV 35S usingaspectrophotometer(HitachiLimited,Tokyo,Japan)and
promoter in the plant over-expression vector, pCambia 2300 the chlorophyll content was calculated as previously described
(Cambia, Brisbane, QLD, Australia), termed as 35S::VlWRKY3. (ZhangH.etal.,2012).
Agrobacterium tumefaciens GV3101 harboring 35S::VlWRKY3 Relative electrolyte leakage was measured as previously
constructs was used to transform A. thaliana using the floral described (Cao et al., 2007). Seedlings were vacuum-infiltrated
dip method (Clough and Bent, 1998). T0 seeds were harvested with deionized water for 20 min and after 2 h the conductivity
and sown on MS medium (Murashige and Skoog, 1962) (C1) of the solutions was determined using a conductivity
supplemented with 75 mg L−1 kanamycin. Three lines (L1, L2, detector. Subsequently, the seedlings were boiled for 20 min
andL3)withthebestperformanceaftertreatmentwith130mM in deionized water and cooled to room temperature. The
NaCland200mMmannitolwereselectedfrom59independent conductivity (C2) of the solution was then determined and the
lines,andT3homozygouslinesweregeneratedandusedforall C1 to C2 (C1/C2) ratio was calculated and used as a measure
subsequentexperiments. of the relative electrolyte leakage. MDA content was measured
aspreviouslydescribed(HeathandPacker,1968).ABAcontent
Abiotic Stress Treatments of Transgenic was measured using an enzyme-linked immunosorbent assay
A. thaliana Lines (ELISA),aspreviouslydescribed(Yangetal.,2001).
To test the effects of different abiotic stresses on the seed
Measurement of the Water Loss Rate
germination rates, approximately 100 seeds from each of the
To determine the water loss rate, 10 leaves were detached from
threeselectedT3homozygouslines,aswellasWTplants,were
vernalizedfor3daysat4◦C,surface-sterilizedandsownonMS 4-week-oldtransgenicandWTplantsandimmediatelyweighed.
The samples were then placed on dry filter paper at a relative
medium or MS medium supplemented with 130 mM NaCl or
humidity of 40–45% at room temperature and weighed over a
200mMmannitol.Thegerminationratewascalculatedbasedon
time course. The water loss rate was calculated as previously
thepercentageofseedlingsthathadreachedthecotyledonstage
described(Guoetal.,2015).
10 days after sowing (Saleki et al., 1993). All seeds used for the
germinationanalysiswereharvestedandstoredunderthesame Detection of Reactive Oxygen Species
conditions.
(ROS) and Activity Assays of Antioxidant
To investigate the abiotic stress tolerance, 5-day-old
Enzymes
transgenic and WT seedlings grown on MS medium plates
were transferred to plates of MS medium, or MS medium Ahistochemicalstainingprocedurewasusedtodetectsuperoxide
supplemented with 130 mM NaCl or 200 mM mannitol. Root and hydrogen peroxide in situ, as described in our previously
lengths and lateral roots number were measured when the report (Guo et al., 2015). Rosette leaves from 5-week-old
seedlingswere20daysold. transgenic and WT plants that had either been subjected to
200 mM NaCl or drought treatments for 1 week, as well as the
1http://blast.ncbi.nlm.nih.gov/Blast.cgi correspondingnon-treatedcontrols,wereinfiltratedin1mg/ml
2http://meme.sdsc.edu/meme/intro.html DABsolutionfor8hor6mMNBTfor2h.Then,thechlorophyll
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Guoetal. VlWRKY3PromotesStressTolerance
was cleared at 80◦C in 80% (v/v) ethanol for 2 h and samples constant increase from 60 to 95◦C. A. thaliana Actin 1 (TAIR3:
wereembeddedin10%(v/v)glycerolforobservations(Kotchoni AT2G37620) was used as the reference gene. Primers used
et al., 2006; Kim et al., 2011). The activities of the antioxidant for qRT-PCR are listed in Supplementary Table S1. Relative
enzymesintheleaves,includingsuperoxidedismutase(SOD,EC expressionlevelswereanalyzedwiththeIQ5software(Bio-Rad,
1.15.1.1), catalase (CAT EC 1.11.1.6) and peroxidase (POD EC Hercules, CA, United States) using the Normalized Expression
1.11.1.7), were extracted from 0.5 g leaves from abiotic stress Method.
treatedplantsaswellascontrolplants,andmeasuredasdescribed
byTuetal.(2016). Statistical Analysis
Allexperimentswereperformedusingthreebiologicalreplicates,
Measurement of Stomatal Aperture in with each biological replicate having three technical replicates.
Response to ABA Treatment DataanalysisandplottingwereperformedusingMicrosoftExcel
(MicrosoftCorporation,UnitedStates)andSigmaplot(v.10.0.
Stomatalapertureassayswereperformedessentiallyasdescribed
Systat, Inc., Point Richmond, CA, United States). Significant
by Pei et al. (1997). Briefly, A. thaliana leaves from 4-
differences were assessed through paired t-test using the SPSS
week-old plants were incubated in buffer containing 10 mM
KCl, 50 µM CaCl , and 10 mM MES/KOH (pH 6.15). Statistics 17.0 software (IBM China Company Ltd., Beijing,
2
China).
To induce stomatal opening, the leaves were first incubated
in the light for 2 h and then treated with 1 or 5 µM
ABA. Stomatal apertures were observed after 2 h under a RESULTS
microscope (BX53, Olympus, Japan) and recorded as the ratio
ofstomatalwidthtolength.Allassessmentswerecarriedoutin
VlWRKY3 Cloning and Sequence
triplicate.
Analysis
Inoculation of A. thaliana With BasedontheVvWRKY3(GSVIVT01010525001)cDNAsequence
fromtheGrapeGenomeDatabase(12×)4,theWRKY3ORFfrom
Pathogenic Bacteria
‘Kyoho’wasobtained.TheVlWRKY3codingsequence(CDS)of
Four-week-old T3 transgenic and WT plants were inoculated
570 bp encoded a 190 amino acid protein, and both the CDS
with G. cichoracearum, and visual scoring of disease reaction
sequence and the corresponding deduced amino acid sequence
phenotypesandasporecountofleaveswereperformed120hpi,
shared100%identitytotheV.viniferahomolog.Aphylogenetic
as previously described (Xiao et al., 2005; Li et al., 2010a).
tree was produced through analysis the VlWRKY3 protein
Leaf samples collected 0, 24, 72, and 120 hpi were used to
sequence and its orthologs from a range of plant species,
determine the expression profiles of disease resistance related
and two clades were contained in the phylogenetic tree,
genes.
each of which contained sequences from monocotyledonous
AB. cinereaconidialsuspension(1.5×106 conidia/ml)was
or dicotyledonous species (Supplementary Figure S1A). The
usedtoinoculatetheplants,aspreviouslydescribed(Wanetal.,
Nicotianatomentosiformisprotein,XP_009615508.1,shared54%
2015).Detachedleaveswereusedformorphologicalobservation,
identity to the VlWRKY3 proteins. The sequence identity
lesion diameter analysis, and expression analysis of defense
between VlWRKY3 and the other proteins in the analysis
relatedgenes,aspreviouslydescribed(Guoetal.,2016).Samples
ranged from 39 to 53%. What’s more, we observed significant
usedformorphologicalobservationandlesiondiameteranalysis
sequenceconservationwithintheWRKYdomainregionsamong
werephotographedandmeasuredat72hpi,whileleavesusedto
the sequences, and the sequence logos indicated the level of
analyzetheexpressionofdefenserelatedgeneswerecollectedat
conservation in the WRKY domain of WRKY proteins at each
0,12,24,and48hpi.
residue position (Supplementary Figure S1B). A total of 48 of
theconservedaminoacidswereidenticalamongalltheanalyzed
Gene Expression Analysis by
WRKY proteins, while there was varying levels of conservation
Quantitative Real-Time RT-PCR
amongsttheresidueslocatedinotherpositions(Supplementary
Total RNA was extracted from A. thaliana leaves using an FigureS1B).
RNA prep plant kit (Tiangen Biotech., China) following the
manufacturer’s protocols. First-strand cDNA was synthesized Generation of 35S::VlWRKY3 Transgenic
using PrimeScriptTMRTase (TaKaRa Biotechnology) according A. thaliana Lines
to the manufacturer’s instructions. Quantitative real-time
To investigate the potential role of VlWRKY3 in response
RT-PCR (qRT-PCR) analysis was conducted using SYBR
to abiotic and biotic stresses, VlWRKY3 was introduced into
green (TaKaRa Biotechnology) and an IQ5 real-time RT-PCR
the A. thaliana WT, generating 59 35S::VlWRKY3 lines. Three
instrument (Bio-Rad, Hercules, CA, United States) with the
lines (L1, L2, L3) were selected to assay for stress tolerance.
followingthermalprofile:95◦Cfor30s,40cyclesof95◦Cfor5s,
The transcript levels of VlWRKY3 in the transgenic lines were
and60◦Cfor30s.Eachreactionwasperformedintriplicatefor
evaluatedbyqRT-PCR(SupplementaryFigureS2A).
each of the three biologically replicated sets of cDNA samples.
To perform the melt-curve analysis, the following program 3http://www.arabidopsis.org/
was added after 40 PCR cycles: 95◦C for 15 s, followed by a 4http://www.genoscope.cns.fr
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qRT-PCR was also used to examine the expression 35S::VlWRKY3 Transgenic A. thaliana
profiles of VlWRKY3 in the three transgenic lines post Plants Have Increased Tolerance to Salt
abiotic stress treatments, or G. cichoracearum and B. cinerea
and Drought Stress
inoculation. Results showed that its expression levels were
In order to evaluate the tolerance capacity of salt and drought
increased upon exposure to salt and drought treatments,
stresses, the performance of 5-week-old 35S::VlWRKY3
as well as G. cichoracearum inoculation (Supplementary
transgenic lines and WT that had been subjected to abiotic
Figures S2B,C). And its expression levels increased
stresses for 1 week was also investigated. The 35S::VlWRKY3
at 12 hpi with B. cinerea, and decreased at 48 hpi
transgenic lines and the WT plants under the normal growth
when compared to expression at 0 hpi (Supplementary
conditions exhibited no evident differences (Figure 3A-a).
FigureS2D).
However, most WT leaves became chlorotic after treatment
Effect of Osmotic Stress on Seed with 200 mM NaCl for 7 days, while the transgenic
lines still remained green and phenotypically normal
Germination and Root Growth in
under the same conditions (Figure 3A-b). All WT plants
35S::VlWRKY3 Transgenic A. thaliana
displayed significant wilting and water loss after 7 days of
Lines
withholding water, but the 35S::VlWRKY3 transgenic lines
The effect of VlWRKY3 on osmotic stress tolerance was only showed mild wilting (Figure 3A-c). All of the plants
tested through comparing the seed germination rates and root were re-watered after 7 days of water deprivation, and
growth of 35S::VlWRKY3 transgenic lines and WT. Seeds from almost all of the WT plants died, whereas 46–74% of the
35S::VlWRKY3 transgenic lines and WT were sown on MS 35S::VlWRKY3transgeniclinessurvivedafter1dofre-watering
basal medium containing NaCl and mannitol to evaluate the (Figures3A-d,B).
response to salt stress and drought stress, respectively. Under Four-week-olddetachedrosetteleavesofWTandVlWRKY3
normal conditions, almost all of the seeds from both the transgenic lines were used to assess the water loss rate. Results
35S::VlWRKY3 transgenic lines and WT germinated. However, showed that the water loss rates of leaves from the three
comparison of germination rates revealed that transgenic seeds 35S::VlWRKY3 transgenic lines were lower than the WT plants
exhibited 27–86% higher than that of WT on MS medium atmosttimepoints(Figure3C).
containing NaCl or mannitol (Figures 1A,B). In addition, InordertodeterminewhethertheproductionofROSinthe
the 35S::VlWRKY3 seedlings had shorter roots and higher 35S::VlWRKY3 transgenic plants was affected by abiotic stress,
number of lateral roots per cm of primary root than those theleavesoftransgenicplantsandWTthathadbeensubjectedto
of WT when grown on MS basal medium, and MS medium abioticstresseswerehistochemicallystainedwithNBTandDAB
containing NaCl. And there was no difference between the to determine the levels of O − and H O , respectively. Results
2 2 2
WT and 35S::VlWRKY3 seedlings in the root lengths and showedthattheleavesoftheWTplantsaccumulatedmuchmore
number of lateral roots per cm of primary root when both ROS than those of the 35S::VlWRKY3 transgenic plants under
of them were grown on MS medium containing mannitol abioticstressconditions(Figures4A,B).
(Figures1D,E). Totesttheantioxidantenzymes-mediatedROSdetoxification
in the 35S::VlWRKY3 transgenic plants, we determined the
35S::VlWRKY3 Transgenic Lines Have
enzymaticactivitiesofSOD,CAT,andPODaftersaltanddrought
Improved Physiological Traits Associated stress treatments. The three 35S::VlWRKY3 transgenic plants
With Osmotic Stress Tolerance possessed higher activities of the three antioxidant enzymes
than those corresponding WT with or without abiotic stress
The improved osmotic stress tolerance of 35S::VlWRKY3
treatments(Figures4C–E),whichwasconsistentwiththelower
transgenic seedlings was correlated with changes in several
accumulationofROSinthetransgenicplants.
physiological parameters, such as the MDA content, relative
electrolyte leakage, and the chlorophyll content, as well as
35S::VlWRKY3 Transgenic A. thaliana
the endogenous ABA content. When grown on MS basal
Plants Have Increased Sensitivity of
medium, the MDA contents, as well as the relative electrolyte
Stomata After ABA Treatment
leakage of the 35S::VlWRKY3 lines and the WT were similar.
However, the MDA contents of transgenic lines decreased Water loss from leaves is strongly influenced by stomatal
by 44 and 27% when compared to the WT after NaCl and regulation, which is in turn affected by the concentration
mannitol treatments, respectively (Figure 2A), and the relative of ABA in the leaves (Yan et al., 2014). The largest water
electrolyte leakage of 35S::VlWRKY3 lines were 45 and 26% loss was observed in WT, whereas 35S::VlWRKY3 transgenic
lower than those of the WT, respectively (Figure 2B). It plants exhibited lower water loss (Figure 3C). In addition,
was also observed that the chlorophyll contents of the leaves we compared the stomatal apertures from the leaves of
of the 35S::VlWRKY3 lines were significantly higher than both transgenic and WT plants that has been treated with
those of WT corresponding after treatments of NaCl and different concentrations of exogenous ABA. As shown in
mannitol treatments (Figure 2C), as was also the case for Figure 5, stomatal apertures of the 35S::VlWRKY3 transgenic
endogenous ABA levels 7 days post the same stress treatments lines were significantly lower than that of WT plants when
(Figure2D). ABA was applied, whereas untreated transgenic and WT
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FIGURE1|Effectofosmoticstressonseedgerminationandpost-germinationgrowthofWTandVlWRKY3transgenicArabidopsisthalianalines.(A)WTand
transgenicseedlings10daysafterseedsweresownonMurashigeandSkoog(MS)mediumorMSbasalmediumsupplementedwith130mMNaCland200mM
mannitol.(B)GerminationratesofseedssownonMS,MS+130mMNaCl,orMS+200mMmannitolplates.Eachdatapointisthemeanofthreereplicatesof
100–120seeds.(C)Seedlingsat15daysaftertransfertoMS,MS+130mMNaCl,orMS+200mMmannitolplates.Seedlingswere5-days-oldatthetimeof
transfer.(D,E)Rootlength(D)andlateralrootnumber(E)ofWTandtransgeniclinesafter15daystreatmentwithorwithout130mMNaCland200mMmannitol.
Eachdatapointisthemeanofthreereplicatesof20–30seedlings.TheerrorbarsindicatetheSD.Asterisksindicatestatisticalsignificance(∗0.01<P<0.05,
∗∗P<0.01,Student’sttest)ofdifferencesbetweentransgeniclinesandWTseedlings.
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FIGURE2|PhysiologicalchangesassociatedwithosmoticstressresponseinWTandVlWRKY3transgenicArabidopsisthalianaseedlings.(A,C,D)
Malondialdehyde(MDA)content(A),Chlorophyllcontent(C)andabscisicacid(ABA)content(D)intheleavesofWTandtransgenicseedlingsgrownwithorwithout
osmoticstress,respectively.(B)RelativeelectrolyteleakagefromdetachedleavesofWTandtransgenicseedlingsgrownwithorwithoutexposuretoosmoticstress.
Inallcases,datavaluesrepresentmeans±SDfromthreeindependentexperiments.Asterisksindicatestatisticalsignificance(∗0.01<P<0.05,∗∗P<0.01,
Student’st-test)ofdifferencesbetweentransgeniclinesandWTseedlings.
plants had similar, and relatively high stomatal apertures ABA biosynthesis associated gene, NCED3 (Iuchi et al., 2001),
(Figure5). after abiotic stress treatments, and its expression levels in the
35S::VlWRKY3 transgenic plants were 5-48 fold greater than in
35S::VlWRKY3 Transgenic A. thaliana theWT(Figure6).
Plants Have Altered Expression Levels of
35S::VlWRKY3 Transgenic A. thaliana
Abiotic Stress Responsive Genes
Plants Have Enhanced Susceptibility to
Weexaminedtheexpressionofseveralabioticstress-responsive
B. cinerea
genesin5-week-old35S::VlWRKY3transgenicA.thalianaplants
that had been subjected to salt or drought stress treatments for SincewepreviouslyfoundthattheexpressionlevelsofVlWRKY3
1 week (Figure 6). The expression levels of RD29A, RD29B, was influenced by MeJA and Eth treatments (Guo et al., 2014),
RD22,andADH1appearedmoreabundantinthe35S::VlWRKY3 we hypothesized that it operates via JA and Eth mediated
transgenic plants than those in the WT after salt and drought signaling pathways. It is also generally accepted that JA and
treatments (Figure 6). In contrast, FRY1, which supresses the Ethactinthenecrotrophicpathogen-induceddiseaseresistance
ABA signaling pathway (Xiong et al., 2001), was expressed at response(OliverandSolomon,2004).ThreedpiwithB.cinerea,
lower levels upon exposure to abiotic stress (Figure 6). SOS2 a necrotrophic pathogen, the phenotypes of leaves from the
and SOS3, which involved in the SOS pathway (Zhu, 2002), 35S::VlWRKY3 transgenic lines and WT were examined and
was up-regulated in the 35S::VlWRKY3 transgenic plants after lesiondiametersontheleaveswerescoredandclassifiedintosize
salt treatment (Figure 6A). However, no evident differences categories. The lesions on the 35S::VlWRKY3 transgenic leaves
wereobservedintheexpressionofDREB2AandERD1between were larger than those on the WT leaves (Figures 7A,B), and
the 35S::VlWRKY3 transgenic and WT plants post drought the proportions of medium and large sized lesions were higher
treatment (Figure6B). We also evaluated the expression of the (Figure7C).TheexpressionlevelofPR1waslowerat12hpi,and
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FIGURE3|PerformanceofWTandVlWRKY3transgenicArabidopsisthalianaplantsundernormalgrowthandabioticstressconditions.(A)Representativeimages
of5-week-oldpottedWTandtransgenicplantsundernormalgrowthandabioticstressconditions.(a)WTandtransgenicplantsgrownfor5weeksundernormal
condition.(b)Five-week-oldWTandtransgenicplantsthatweretreatedwith200mMNaClfor7days.(c)Five-week-oldWTandtransgenicplantsthatwere
deprivedofwaterfor7days.(d)WTandtransgenicplantsthatweredeprivedofwaterfor7daysandthenre-watered.(B)SurvivalratesofWTandtransgeniclines
24hafterre-watering.Eachdatapointisthemeanofthreereplicatesof32plants.(C)WaterlossrateofdetachedleavesofWTandtransgenicplants.Eachdata
pointisthemeanofthreereplicatesof10detachedleaves.In(B,C),theerrorbarsindicatetheSD,Asterisksindicatestatisticalsignificance(∗0.01<P<0.05,
∗∗P<0.01,Student’st-test)ofdifferencesbetweentransgeniclinesandWT.
higherat24and48hpiinthe35S::VlWRKY3transgenicplants maximum expression at 72 hpi (Figures 8C,D). PDF1.2 and
comparedtotheWT(Figure7D),whiletheexpressionofPDF1.2 LOX3wasalsoup-regulatedafterG.cichoracearuminoculation,
wasup-regulatedat12and24hpi,anddown-regulatedat48hpi and reached a peak at 120 hpi, but their expression levels in
inthetransgenicplants(Figure7E). the 35S::VlWRKY3 transgenic plants were suppressed when
comparedtotheWT(Figures8E,F).
35S::VlWRKY3 Transgenic A. thaliana
Plants Have Enhanced Resistance to
DISCUSSION
G. cichoracearum
To further investigate the putative function of VlWRKY3 Members of the WRKY transcription factor family play
in biotrophic pathogens-induced defense responses, the importantrolesindiverseplantdevelopmentalandphysiological
35S::VlWRKY3 transgenic and WT plants were inoculated processes,includingembryogenesis(LagaceandMatton,2004),
with G. cichoracearum. Results showed that the 35S::VlWRKY3 seedcoatandtrichomedevelopment(Johnsonetal.,2002),leaf
transgenic plants were more resistant against G. cichoracearum senescence (Miao and Zentgraf, 2007; Zhou et al., 2011), as
at 5 dpi compared to the WT (Figure 8A), and conidia count well as various plant abiotic and biotic stress responses (Guo
on35S::VlWRKY3transgenicleaveswassignificantlylowerthan et al., 2014). In grape, 59 WRKY genes have been identified
in the WT plants (Figure 8B). The expression patterns of PR1 andclassifiedintothreemaingroups(I-III),withtheVlWRKY3
and NPR1 were significantly increased at 24, 72, and 120 hpi genebelongingtoGroupIIc.Inthisstudy,thegenesequenceof
in transgenic lines compared to the WT plants, and reached VlWRKY3 from ‘Kyoho’ was isolated. Phylogenetic analysis of
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FIGURE4|ReactiveoxygenspecieslevelsandactivityassaysofactivatedoxygenscavengingenzymesinWTandVlWRKY3transgenicArabidopsisthalianaplants
undernormalgrowthandabioticstressconditions.(A,B)HistochemicalstainingassayofO2-andH2O2accumulationwithNBT(A)andDAB(B),inWTand
transgenicleavesfollowingnormalgrowthandabioticstressconditions.Theexperimentwasrepeated3times,and5–10leaveswerestainedineachexperiment.
(C–E)ActivityofSOD(C),CAT(D)andPOD(E)intheleavesofWTandtransgenicplantsundernormalgrowthandabioticstressconditions.Datavaluesrepresent
means±SDfromthreeindependentexperiments.Asterisksindicatestatisticalsignificance(∗0.01<P<0.05,∗∗P<0.01,Student’st-test)ofdifferencesbetween
transgeniclinesandWTplants.
the VlWRKY3 sequence, together with orthologs from a range roots contribute to water uptake and facilitate the extraction of
of plant species, revealed a phylogenetic tree with two distinct nutrientsrequiredforgrowthanddevelopment(Zhuetal.,2012).
clades(SupplementaryFigureS1A),consistentwiththeWRKY3 Interestingly, the number of lateral roots per cm of primary
sequenceevolvingsubstantiallyafterthedivergenceofdicotsand root in VlWRKY3 over-expressing seedlings was higher than in
monocots from their last common ancestor (Zhang L.C. et al., WT, and the roots were shorter than in WT under control and
2012). saltstressconditions(Figures1C–E).Inaddition,thetransgenic
In a previously study, the expression of VlWRKY3 was plants were substantially more tolerant of salt and dehydration
induced post salt and drought stress treatments, as well as than WT plants (Figures 3A,B). These findings suggest that
after addition of exogenous MeJA and Eth (Guo et al., 2014), VlWRKY3isinvolvedinregulatingresponsestoosmoticstress,
suggestingaroleinbothabioticandbioticstressresponses.This andmayhavevaluesasatargetforcropimprovement.
hypothesiswastestedthroughaseriesofexperimentsinvolving To investigate the mechanisms by which VlWRKY3 confers
transgenic A. thaliana plants over-expressing VlWRKY3. First abiotic stress tolerance, we performed several experiments
of all, it was found that the expression levels of VlWRKY3 to monitor the physiological changes associated with stress
in transgenic A. thaliana plants were affected by abiotic responses. It has been reported that drought and salt stresses
and biotic stress treatments (Supplementary Figures S2B–D), can be accompanied by the production of MDA (Levine et al.,
which would be caused by the influence of other genes that 1994),whichwasthemarkeroflipidperoxidationandtherefore
involvedinitsinteractionnetwork.Intermsofgeneralosmotic of membrane damage. We observed a lower MDA content in
stress, transgenic seeds exhibited significantly increased levels 35S::VlWRKY3 transgenic seedlings than in WT after exposure
of germination compared to WT seeds (Figures 1A,B). Lateral to osmotic stress (Figure 2A). It is generally accepted that
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FIGURE5|Stomatalclosureinresponsetoabscisicacid(ABA)treatmentinWTandVlWRKY3transgenicArabidopsisthalianaplants.(A)Comparisonofstomatal
aperturesinresponsetodifferentconcentrationsofexogenousABAinWTandtransgenicplants.(B)Stomatalaperturewidthtolengthratiosfollowingtreatment
withdifferentconcentrationsofexogenousABA.Datavaluesrepresentmeans±SDfromthreeindependentexperiments.Asterisksindicatestatisticalsignificance
(∗0.01<P<0.05,∗∗P<0.01,Student’st-test)ofdifferencesbetweentransgeniclinesandWTplants.
maintenance of integrity and stability of cell membranes under (Desikan et al., 2004). In this study, we observed that the
waterstressconditionsisamajorfactorindroughttolerance,and endogenousABAcontentin35S::VlWRKY3transgeniclineswere
thatthedegreeofcellmembraneinjuryinducedbywaterstress muchhigherthaninWTplants(Figure2D),buttheROSlevels
can be assessed through measurements of electrolyte leakage were lower than in WT plants post drought stress treatment
fromthecells(Bajjietal.,2002).Thelevelsofelectrolyteleakage for 7 days (Figures4A,B). A lower width to length ratio in the
in the WT seedlings were significantly higher than in any of transgenicplantsthaninWTfollowingvariousconcentrationsof
thetransgeniclinesundersaltandmannitolstress(Figure2B), exogenous ABA treatment was also observed (Figure 5). It has
indicating that the cell membranes of the WT suffered more been reported that the accumulation of excessive levels of ROS
damage. As a reliable, non-invasive method for monitoring can cause oxidative damage to cells and stimulate the activity
photosynthetic events, chlorophyll fluorescence was used to of antioxidant systems, such as SOD, CAT and POD (Mittler,
reflect the physiological status of plants (Strasser et al., 2002), 2002).AndsignificantlyhigheractivitiesofSOD,CATandPOD
andweobservedthatthechlorophyllcontentof35S::VlWRKY3 were measured in transgenic plants after normal conditions
transgenicseedlingswassignificantlyhigherthanintheWTplant and salt as well as drought stress for 7 days in the present
aftertheosmoticstresstreatments(Figure2C). study(Figures4C–E),suggestingthat35S::VlWRKY3transgenic
Abscisic acid plays a central role in responses to various plants may be able to scavenge excessive ROS under severe
abiotic stresses (Fujita et al., 2006), and significant differences abiotic stress conditions. Thus, it would be speculated that the
were detected in endogenous ABA content between mature increased endogenous ABA content triggered ROS production,
35S::VlWRKY3transgenicandWTplantsthathadbeenexposed and then promoted the closure of stomata in transgenic plants,
to abiotic stress (Figure 2D). This was confirmed by the andexcessiveROSwasscavengedbySOD,CATandPODwhose
observation that the transcript abundance of NCED3, whose activitieswereincreasedintransgenicplantspostdroughtstress
expression is often used as an indicator of ABA biosynthesis, treatment.
was also significantly higher in the transgenic plants than in Unexpectedly, H O and O − accumulation in transgenic
2 2 2
WT following the abiotic stress treatments (Figure 6). What’s lines were higher than in WT plants at 48 hpi with
more, when plants were subjected to water deficit condition, B.cinerea(SupplementaryFigureS3),andthiswouldpromote
the increased endogenous ABA content could trigger ROS the susceptibility of transgenic lines to B. cinerea infection
production to mediate down stream responses in guard cells and colonization (Figures 7A–C). The difference in ROS
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