WRKY71 accelerates

WRKY71 accelerates
WRKY71 accelerates

WRKY71accelerates ?owering via the direct activation of FLOWERING LOCUS T and LEAFY in Arabidopsis thaliana

Yanchong Yu 1,?,Zhenhua Liu 1,?,Long Wang 1,?,Sang-Gyu Kim 2,Pil J.Seo 2,?,Meng Qiao 1,Nan Wang 1,Shuo Li 1,Xiaofeng Cao 3,Chung-Mo Park 2and Fengning Xiang 1,*1

The Key Laboratory of Plant Cell Engineering and Germplasm Innovation,School of Life Sciences,Shandong University,Jinan 250100,China,2

Molecular Signaling Laboratory,Department of Chemistry,Seoul National University,Seoul 151-742,Korea,and 3

State Key Laboratory of Plant Genomics and National Center for Plant Gene Research,Institute of Genetics and Develop-mental Biology,Chinese Academy of Sciences,Beijing 100101,China

Received 30October 2015;revised 19November 2015;accepted 23November 2015;published online 8December 2015.*For correspondence (e-mail xfn0990@https://www.360docs.net/doc/9a4849216.html,).?These authors contributed equally to this work.?

Present address:Department of Bioactive Material Sciences and Research Center of Bioactive Materials,Chonbuk National University,Jeonju 561-756,Korea.

SUMMARY

Flowering is crucial for achieving reproductive success.A large number of well-delineated factors affecting ?owering are involved in complex genetic networks in Arabidopsis thaliana .However,the underlying part played by the WRKY transcription factors in this process is not yet clear.Here,we report that WRKY71is able to accelerate ?owering in Arabidopsis.An activation-tagged mutant WRKY71-1D and a constitutive over-expresser of WRKY71both ?owered earlier than the wild type (WT).In contrast,both the RNA interfer-ence-based multiple WRKY knock-out mutant (w71w8+28RNAi )and the dominant repression line (W71-SRDX )?owered later.Gene expression analysis showed that the transcript abundance of the ?owering time integrator gene FLOWERING LOCUS T (FT )and the ?oral meristem identity genes LEAFY (LFY ),APETALA1(AP1)and FRUITFULL (FUL )were greater in WRKY71-1D than in the WT,but lower in w71w8+28RNAi and W71-SRDX .Further,WRKY71was shown to bind to the W-boxes in the FT and LFY promoters in vitro and in vivo .The suggestion is that WRKY71activity hastens ?owering via the direct activation of FT and LFY .Keywords:WRKY71,transcription factor,?owering,FLOWERING LOCUS T ,LEAFY ,Arabidopsis thaliana .

INTRODUCTION

Flowering needs to be timed appropriately to achieve reproductive success (Gu et al.,2013;Hu et al.,2014;Blu-mel et al.,2015).In Arabidopsis thaliana ,the molecular and genetic basis of the transition from vegetative to reproductive growth has been described in great detail (Andres and Coupland,2012;Dally et al.,2014;Blumel et al.,2015).Four major pathways have been identi?ed,referred to as the photoperiod,vernalization,autonomous and gibberellic acid (GA)pathways (Andres and Coupland,2012).These pathways mediate various signals,such as light,age,circadian clock,photoperiod,temperature,abi-otic stresses and hormones,and then converge to modu-late the expression of a set of ?owering time integrators,notably FLOWERING LOCUS T (FT ),LEAFY (LFY )and SUP-PRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1)(Han et al.,2008;Kumar et al.,2012;Riboni et al.,2013;Hu et al.,2014;Wang,2014),which provide the appropriate signal to the ?oral meristem identity (FMI)genes,LFY ,

APETALA1(AP1),CAULIFLOWER (CAL )and FRUITFULL (FUL ),thereby triggering the switch from the vegetative to the reproductive phase (Han et al.,2008;Davis,2009;Wang,2014).

Photoperiod is a key trigger of FT expression in Ara-bidopsis (Song et al.,2013;Nakamura et al.,2014;Wick-land and Hanzawa,2015).The FT protein,the primary target of CONSTANS (CO )which integrates photoperiod signal,is synthesized in leaf vascular tissue,from where it is translocated to the shoot apical meristem (SAM)(Corbe-sier et al.,2007).In the meristem,FT interacts with the bZIP transcription factor (TF)FD to form a complex which then induces the expression of AP1and FUL to initiate ?owering (Abe et al.,2005).In addition to the photoperiod,FT can also mediate signals of the vernalization and autonomous pathways (Han et al.,2008;Blumel et al.,2015).FLOWER-ING LOCUS C (FLC )is a key gene acting at the conver-gence point of the vernalization and autonomous

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pathways and inhibits?owering by repressing FT expres-sion(Han et al.,2008).The bHLH TF PHYTOCHROME INTERACTING FACTOR4(PIF4)mediates thermal signals to directly activate FT expression to promote?owering (Kumar et al.,2012).LFY plays a crucial role in determining FMI.During the vegetative stage,LFY expression is largely con?ned to leaf primordia;upon?oral induction,gene expression is rapidly increased.During the reproductive phase,it reaches its expression maximum in the?oral meristem(FM)and initiates?oral transition(Parcy,2005). LFY encodes a TF which interacts with a set of?owering time regulators,such as TERMINAL FLOWER1,CO,AP1, CAL and SQUAMOSA PROMOTER BINDING LIKE(SPL) (Liljegren et al.,1999;Ratcliffe et al.,1999;Wagner et al., 1999;Samach et al.,2000;William et al.,2004;Moyroud et al.,2010,2011;Torti et al.,2012).

The WRKY proteins comprise a large family of plant TFs, de?ned by the presence of the heptapeptide WRKYGQK at their N-terminus.The WRKY proteins operate by binding to the W-box(TTGACC/T)present in their target promoters and are involved in a variety of developmental and physio-logical processes(Eulgem et al.,2000;Rushton et al.,2010; Chen et al.,2012).The Arabidopsis genome includes74 WRKY genes,most of which are demonstrated to be involved in defence against pathogens(Rushton et al., 2010).For instance,WRKY33is demonstrated to help Ara-bidopsis defend against infection by the fungal pathogens Alternaria brassicicola and Botrytis cinerea(Zheng et al., 2006;Birkenbihl et al.,2012).Other than pathogen defence, a number of WRKY genes,such as WRKY8,WRKY25, WRKY33and WRKY63,are revealed to be involved in the response to abiotic stress(Jiang and Deyholos,2009;Ren et al.,2010;Li et al.,2011;Hu et al.,2013).In addition,the WRKY genes are also reported to play roles in develop-mental processes,including seed development,trichome morphogenesis and leaf senescence(Johnson et al.,2002; Robatzek and Somssich,2002;Luo et al.,2005;Jiang et al., 2014).It has been demonstrated that AtWRKY6, AtWRKY53,GsWRKY20,MlWRKY12and OsWRKY11are all involved in the determination of?owering time(Robatzek and Somssich,2002;Miao et al.,2004;Luo et al.,2013;Yu et al.,2013;Cai et al.,2014).However,the underlying ways in which WRKY TFs are involved this process are not clear yet.The present paper describes the use of mutant and transgenic materials to demonstrate the involvement of WRKY71in?owering.It has been further demonstrated to directly promote FT and LFY to accelerate?owering.

RESULTS

The WRKY71-1D activation tagged line?owers precociously

A screen of several thousand activation tagging lines selected WRKY71-1D,on the basis of its earlier?owering than Col-0under long-day(LD)conditions(Figure1a,b). The mature WRKY71-1D plants were short,and formed lar-ger?oral organs than did the wild-type(WT)plants(Fig-ure S1a,b).The site of the T-DNA insertion in WRKY71-1D was about1kb from the WRKY71transcription start codon (Figure1c),inducing the over-expression of this gene(but not of any others in the genomic vicinity)(Figure1d).Con-?rmation that the early?owering phenotype of WRKY71-1D was caused by an excess of WRKY71was obtained by showing that transgenic lines constitutively expressing the TF(35S::WRKY71)also?owered precociously(Figures1e and S1c).WRKY71is a282-residue polypeptide harbouring one WRKYGQK domain and one C2H2zinc-?nger motif (Figure S1d),and belongs to group IIc of the WRKY family. The site of WRKY71deposition,as determined from the expression pro?le of a35S::WRKY71-GFP transgenic line(it exhibited an early?owering phenotype resembling35S:: WRKY71,indicating that the tagged WRKY71was func-tional),was limited to the nucleus(Figure S1e).In AH109 yeast cells expressing a GAL4DNA-binding domain–WRKY71fusion protein,a His and LacZ reporter gene driven by the GAL4upstream activation sequence was acti-vated,indicating that WRKY71possesses transcriptional activation activity(Figure S1f).

WRKY71accelerates formation of the FM

Under LD conditions,WRKY71transcript abundance rose steadily in the aerial part of seedlings between8and 16days after germination(DAG)(Figure2a).It was detect-able in the in?orescence meristem,FM and leaf vein(Fig-ure2b).In order to investigate the function of WRKY71in ?owering,the loss-of-function mutant wrky71-1was explored(Figure S2a,b).However,it showed an indistin-guishable phenotype from that of the WT(Figure S2c,d). Analysis of SAM sections showed that in all WRKY71-1D plants sampled,the FM had formed by10DAG,while by this time only18.5?2.2%of wrky71-1mutant plants had reached this stage of development(rising to37.9?2.8% by11DAG).In the WT,the equivalent proportions were 51.2?4.7%(10DAG)and85.2?6.7%(11DAG)(Fig-ure2c).To con?rm this observation,scanning electron microscopy was used to view their SAMs,and the same results were obtained(Figure2d).From these results,we see that WRKY71plays a role in the formation of the FM.

Flowering is delayed in the w71w8+28RNAi and W71-SRDX transgenic plants

Phylogenetic analysis has shown that the closest homo-logues to WRKY71were WRKY8and WRKY28(Wu et al., 2005),suggesting that these two TFs may also act to pro-mote?owering.The two transgenic lines35S::WRKY8and 35S::WRKY28both?owered before the WT,but not by as early as WRKY71-1D(Figure S3a,b),which implied a mea-sure of redundancy between WRKY71,WRKY8and

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WRKY28.The availability of T-DNA insertion mutants for both WRKY8and WRKY28(Figure 3a)allowed for the con-struction of double and triple mutants.The WRKY28tran-script was as abundant in the mutants as in the WT (Figure S3c),indicating that SALK_007497was not the wrky28mutant.Like wrky71-1,none of these mutants had a non-WT ?owering phenotype (Figure S3d,e).Therefore,RNA interference (RNAi)was used to silence WRKY28

in

Figure 1.The WRKY71-1D line ?owers precociously.(a)The ?owering phenotype of 25-day-old plants.(b)The ?owering time of plants grown under long-day (LD)conditions.At least 15plants per line were measured in each replicate,error bars represent SD (n =3).TLN,total leaf number;DAG,days after ger-mination.Statistically signi?cant differences in the measurements were determined based on Student’s t -test (**P <0.01).

(c)The T-DNA insertion site in WRKY71-1D .Black arrows denote genes,and the black triangles identify the 35S enhancer copies present.(d)Transcript levels of related genes.

(e)The phenotype of 25-day-old plants.OE1and OE2,WRKY71over-expression lines;TLN,total leaf number.Values represent mean SD (n =3).

(a)

(b)i

ii

iii

(d)

(c)

Figure 2.WRKY71accelerates the initiation of the ?oral meristem (FM).

(a)Expression in aerial part of the wild-type (WT)seedlings.D,days after germination.Error bars rep-resent SD (n =3).

(b)In situ hybridization of a WRKY71antisense probe to the shoot apex of 10-day-old (i)and 14-day-old (ii)WT plant,a control hybridization of a WRKY71sense probe is shown in the right column (iii).Arrows indicate the hybridization signals.im,in?orescence meristem;fm,?oral meristem;lv,leaf vein.Scale bar =50l m.

(c)Shoot apical meristem sections of Col-0,WRKY71-1D and wrky71-1.Scale bar =50l m.

(d)The rate of FM formation.Values are derived from the mean of 100individuals per line,error bars represent SD (n =3).Statistically signi?cant differences of the measurements were determined based on Student’s t -test (**P <0.01).

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w71w8SALK_007497,which was designated as w71w8+28RNAi (Figure 3b).In parallel,chimeric repres-sor gene-silencing technology (CRES-T)(Hiratsu et al.,2003)was employed by fusing WRKY71with SRDX driven by the CaMV 35S promoter (Figure 3c).Both the w71w8+28RNAi and W71-SRDX lines ?owered signi?-cantly later than the WT (Figure 3d,e).

WRKY71accelerates ?owering in a partially FT -dependent manner

Col-0,WRKY71-1D ,w71w8+28RNAi and W71-SRDX all ?owered rather later when exposed to short-day (SD)as opposed to LD conditions.The WRKY71-1D line reached ?owering before the WT,while w71w8+28RNAi ?owered later,suggesting that the mutant was able to respond nor-mally to a photoperiod cue.All four lines responded simi-larly to both prolonged vernalization treatment and the application of GA (Table 1).The transcript abundance of major ?owering regulators was unaltered (Figure S4),as was that of WRKY71in any of the sampled loss-of-function mutants associated with the four pathways (Figure 4a).Furthermore,WRKY71-1D was crossed with each of the mutants ?c-3,gai-t6and ft-10,to obtain a set of double mutants.WRKY71-1D ?c-3?owered much earlier than

either WRKY71-1D or ?c-3;?nally,WRKY71-1D gai-t6?ow-ered earlier than WRKY71-1D but later than gai-t6(Fig-ure 4b).However,WRKY71-1D ft-10?owered earlier than ft-10,and this occurred much later than for WRKY71-1D ,indicating that the precocity of WRKY71-1D was reversed in a ft-10background (Figure 4b).The implication was that WRKY71accelerated ?owering in a partially FT -dependent manner.

WRKY71positively promotes ?owering integrator and FMI genes

The ?owering integrator and FMI genes FT ,LFY ,AP1,CAL and FUL were all increased in WRKY71-1D ,while FT ,LFY ,AP1and FUL were decreased in w71w8+28RNAi and W71-SRDX (Figures 5a and S5).For the stages checked,WRKY71transcripts were maintained at higher levels in WRKY71-1D and W71-SRDX throughout,and were unde-tectable in w71w8+28RNAi ,while in the WT the gene was gradually increased from 8to 16days (Figure 5b).The transcript levels of FT and LFY in the WT began to increase after 10DAG,as was also the case in WRKY71-1D .How-ever,the increase in WRKY71-1D occurred rapidly,and expression of FT and LFY was higher in WRKY71-1D than in the WT at each time point (Figure 5b).

In

Figure 3.The phenotype of Col-0,WRKY71-1D ,w71w8+28RNAi and W71-SRDX .(a)T-DNA insertion sites in the SALK_107668and SALK_007497lines.

(b)Transcript levels of related genes in Col-0and w71w8+28RNAi .Lower-case letters above columns indicate signi?cant differences in each group (one-way ANOVA ,P <0.01).

(c)The 35S:WRKY71-SRDX transgene.CaMV 35S represents the CaMV 35S promoter of Tobacco mosaic virus .SRDX is the 12-residue repression domain.(d)The ?owering phenotype of 30-day-old plants.

(e)The ?owering time of plants grown under long-day conditions.At least 15plants per line were measured in each replicate,error bars represent SD (n =3).RLN/CLN,rosette/cauline leaf number.Lower-case letters above columns indicate signi?cant differences (one-way ANOVA ,P <0.01).

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w71w8+28RNAi or W71-SRDX,their increase only became apparent by14DAG,which occurred later than in the WT(Figure5b).These data,which were fully consistent with the lines’?owering behaviour(Figure3d,e),implied that WRKY71accelerated?owering mainly via the activa-tion of genes associated with the transition from vegetative to reproductive growth.

WRKY71promotes?owering of Arabidopsis mainly via direct activation of FT and LFY

The promoter sequences of FT,LFY,AP1and CAL(but not FUL)harbour W-boxes(TT TGAC T/C):three are present in the FT promoter,six in the LFY promoter,?ve in the AP1 promoter and one in the CAL promoter(Figures6a and S6a).Since the W-box is the target of WRKY TFs,the W-boxes were labelled with digoxigenin-ddUTP to perform an electrophoretic mobility shift assay(EMSA).WRKY71 strongly bound to the last two,but not the?rst,FT pro-moter W-boxes,and to the?rst?ve,but not the sixth,LFY promoter W-boxes;when200-fold competitors(unlabelled W-box sequences)were added,the binding signals decreased remarkably.However,when200-fold mutant competitors(unlabelled mutated W-box sequences)were added,the binding signals remained strongly(Figure6b). It also bound weakly to the CAL W-box,but not to any of the AP1W-boxes(Figure S6b).A chromatin immunopre-cipitation(ChIP)assay based on the35S::WRKY71-GFP transgenic line indicated that WRKY71interacted in vivo with the FT and LFY promoters but not with the CAL one (Figures6c and S6c).When a dual-luciferase reporter assay was used to examine whether WRKY71was able to acti-vate FT and LFY expression,LUC activity proved to be greater when driven by the native FT and LFY promoters than by the same promoters carrying mutated W-boxes (Figure6d,e).Furthermore,we crossed WRKY71-1D with lfy-2.Since the hybrid line was unable to set?ower,a count was made of the number of rosette leaves at bolting. The?owering time of WRKY71-1D lfy-2was delayed (Figure6f),as was also the case for WRKY71-1D ft-10 (Figure4b),suggesting that the?owering of WRKY71-1D depended on the presence of functional copies of FT and LFY.The overall conclusion was that WRKY71promoted ?owering via the direct modulation of FT and LFY expres-sion(Figure6g).

DISCUSSION

Substantial progress has been achieved over the past20 years in de?ning the roles of many of the WRKY TFs(Rush-ton et al.,2010).The effect of the ectopic expression of AtWRKY6,AtWRKY53,GsWRKY20,MlWRKY12and OsWRKY11suggests that they all are involved in the deter-mination of?owering time(Robatzek and Somssich,2002; Miao et al.,2004;Luo et al.,2013;Yu et al.,2013;Cai et al., 2014).Knock-outs of the?rst four of these genes produce a phenotype similar to that of the WT or are not shown.The phenotype of the RNAi knock-down of OsWRKY11is dia-metrically opposite to that generated by its constitutive expression(Cai et al.,2014).The present?ndings add WRKY71to this list,and unlike for the other genes the underlying molecular mechanisms have been clari?ed in some detail.WRKY71transcript was gradually increased in the green part of seedlings over the period8–16DAG,and thereafter decreased(Figure2a).This was in line with pub-licly available AtGenExpress data which showed that

Conditions Lines RLN CLN TLN DAG

LD Col-012.7?0.5b 3.1?0.6a15.8?0.8b26.7?1.2b WRKY71-1D8.9?0.7a 2.9?0.3a11.8?0.9a20.5?1.4a

w71w8+28RNAi15.9?0.7c 3.2?0.4a19.1?0.9c32.7?1.3c

W71-SRDX15.7?0.8c 3.2?0.4a18.9?0.8c32.8?1.3c SD Col-058.0?4.3b8.5?0.7b66.5?4.4b95.5?3.5b WRKY71-1D49.1?4.5a 5.8?1.0a54.9?4.3a81.0?3.7a

w71w8+28RNAi69.4?7.1c9.1?0.9bc78.5?7.6c102.4?6.9c

W71-SRDX69.3?6.4c9.1?0.8bc77.3?6.5c102.0?5.6c Ver Col-09.4?0.5b 3.4?0.5ab12.8?0.7b23.0?1.3b WRKY71-1D 6.3?0.7a 3.0?0.0a9.3?0.7a16.0?1.1a

w71w8+28RNAi12.9?0.9c 4.0?0.5b16.8?1.2c29.2?1.8c

W71-SRDX13.8?0.5c 3.6?0.5ab16.5?0.9c27.1?3.1c GA Col-010.8?0.8b 3.3?0.5a14.1?0.6b23.3?1.3b WRKY71-1D7.1?0.6a 3.0?0.0a10.1?0.6a16.3?1.4a

w71w8+28RNAi13.7?1.2c 3.9?0.3ab17.2?1.5c29.0?1.7c

W71-SRDX13.9?0.6c 3.6?0.5a16.8?1.1c27.6?4.3c The data are presented as mean?SD(n=3).

LD/SD,long/short-day conditions;Ver,vernalization treatment given;GA,exogenous gib-berellic acid applied;RLN/CLN/TLN,rosette/cauline/total leaf number;DAG,days after germination.

Different lower-case letters indicate signi?cant differences in each group of different condi-tions(one-way ANOVA,P<0.01).Table1Flowering times of plants grown under various conditions

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WRKY71transcript in the green parts of seedlings increased at 8days and ?nally decreased at 21days (https://www.360docs.net/doc/9a4849216.html,/expviz/expviz.jsp).The manner of tem-poral expression of WRKY71is similar to that of the ?ower-ing regulator genes AGAMOUS-LIKE17(AGL17)and SOC1(Han et al.,2008),in which transcript levels of three genes increase steadily during the ?oral transition.In addition,WRKY71transcript can be found in the in?orescence meristem,FM and leaf vein (Figure 2b).Therefore,WRKY71might play a role in determining ?owering.The FM formation in wrky71-1plants was completed later than in the WT,while the WRKY71-1D plants had formed their FM before this stage was reached by the WT,indicating that WRKY71plays a role in formation of the FM (Fig-ure 2c,d).However,the ?owering time of wrky71-1was similar to that of the WT (Figure S2c,d),implying that other WRKY genes must also be involved.The simultaneous silencing of WRKY71and its close homologues WRKY8and WRKY28induced a substantial delay in ?owering,as was also the case for the W71-SRDX line (Figure 3d,e).The conclusion is that WRKY71,WRKY8and WRKY28are func-tionally redundant with respect to the determination of ?owering time.

As a ?owering pathway integrator,FT expression is affected by a large number of factors,some of which act as repressors and others as positive regulators.The former class includes several MADS box TFs (Hartmann et al.,2000;Searle et al.,2006;Hu et al.,2014),AP2-like proteins (Aukerman and Sakai,2003;Schmid et al.,2003;Castillejo and Pelaz,2008)and B-box proteins (Cheng and Wang,2005;Wang et al.,2014),while the latter class includes CO (Wenkel et al.,2006;Cao et al.,2014;Wang et al.,2014),NUCLEAR FACTOR Y (Cao et al.,2014)and GIGANTEA (GI)(Sawa and Kay,2011).The signals are ?nally conveyed to AP1and FUL in order to effect the switch from vegetative to reproductive growth (Wickland and Hanzawa,2015).In our study FT was decreased in w71w8+28RNAi and W71-SRDX and increased in WRKY71-1D (Figure 5),as was also the case for its targets AP1and FUL (Figure S5).This expression model is exactly consistent with the ?owering time behaviour of these lines (Figure 3d,e).This observa-tion was taken to demonstrate that WRKY71is able to posi-tively regulate FT .In the ft-10background,the early ?owering of WRKY71-1D was reversed (Figure 4b).Thus,the conclusion was that WRKY71acts to accelerate ?ower-ing in at least a partially FT -dependent manner.

Besides acting in ?ower development,LFY has also been proved to be a ?owering time gene.The genes CO ,GI ,FCA and FVE have been demonstrated to affect ?owering either by activating LFY expression or by modulating the response to LFY (Nilsson et al.,1998).Any reduction in LFY transcript abundance induces a delay in ?owering,while its enhancement accelerates it (Blazquez et al.,1997).The delay in ?owering shown by the agl17mutant is likely to be a consequence of the decrease of LFY (Han et al.,2008).Application of GA reverses the delayed ?owering of the BBX19over-expression line by inducing LFY (Wang et al.,2014).SPL3directly upregulates LFY to promote ?owering (Yamaguchi et al.,2009;Teotia and Tang,2015).Consistently,in the present study,less LFY transcript was present in w71w8+28RNAi and W71-SRDX than in the WT,and more in WRKY71-1D (Figure 5),consistent

with

Figure 4.WRKY71accelerates ?owering in a partially FT -dependent manner.

(a)Transcript levels of WRKY71in ?owering mutants.Ten-day-old plants were used to determinate the transcript levels of WRKY71.Error bars represent SD (n =3).

(b)The ?owering time of ?owering mutants and WRKY71-1D 9mutant hybrids grown under long-day conditions.At least 15plants per line were measured in each replicate,error bars represent SD (n =3).Statistically signi?cant differences in the measurements were determined based on Student’s t -test (*P <0.05;**P <0.01).

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their altered ?owering times (Figure 3d,e).In addition,the ?owering of WRKY71-1D was attenuated in the lfy-2back-ground (Figure 6f).These results suggest that WRKY71is also probably able to accelerate ?owering of Arabidopsis by regulating LFY expression.

It was possible to demonstrate that WRKY71is capable of interacting in vitro and in vivo with the FT and LFY pro-moter W-boxes (Figure 6b,c),thereby supporting the notion that WRKY71directly activates transcription of FT and LFY .Further supporting evidence was that LUC activity was higher when driven by a native FT or LFY promoter rather than by a mutated FT or LFY promoter,respectively (Figure 6d,e).In summary,we have proved that WRKY71acts as a promoter of ?owering,and triggers ?owering by directly promoting expression of FT and LFY (Figure 6g).EXPERIMENTAL PROCEDURES Plant materials and growth conditions

The Arabidopsis lines employed in this study were in the Col-0genetic background unless otherwise indicated.Seeds were sown

in soil,held at 4°C for 72h and then at 20–22°C,at 65%relative humidity.Two alternative photoperiods were imposed:LD (16h/8h,light/dark)and SD (8h/16h,light/dark),using 110l mol pho-tons m à2sec à1of white light.Seeds were ?rst surface-sterilized and then grown on 1/2MS (Murashige and Skoog)agar medium [Sigma,https://www.360docs.net/doc/9a4849216.html,/; 2.2g L à1MS salinity,0.05%(w/v)MES,0.7%(w/v)agar,pH 5.70],held at 4°C for 72h and then at 20–22°C.GABI_651G03,SALK_107668,SALK_007497and lfy-2(N6229)were obtained from the Nottingham Arabidopsis Stock Centre (NASC).To over-express WRKY71,the full-length WRKY71coding sequence (CDS)was inserted into pB2GW7,and then transformed into Col-0plants using the ?oral dip method (Clough and Bent,1998).Transgenic regenerants were recognized by a screen based on Basta resistance.To construct the w71w8+28RNAi transgenic plants,a fragment of WRKY28cDNA was inserted into pK7GWIWG2(I),then introduced into the w71w8SALK_007497mutants.

Selection of the WRKY71-1D activation tagging mutant

The pSKI015vector containing the CaMV 35S enhancer element was transformed into Col-0plants (Weigel et al.,2000).T 0seeds were grown in soil,and screened by spraying twice a week over 3weeks with Basta.The T-DNA insertion event in WRKY71-1D was validated using Southern blot analysis.The T-DNA

insertion

Figure 5.WRKY71upregulates various ?oral integrator and ?oral meristem identity genes.

(a)WRKY71,FT and LFY transcript levels in the wild type (WT),WRKY71-1D ,w71w8+28RNAi and W71-SRDX plants 10days after germination.Error bars repre-sent SD (n =3).Lower-case letters above columns indicate signi?cant differences in each group (one-way ANOVA ,P <0.01).(b)Temporal variation in WRKY71,FT and LFY transcription.Error bars represent SD (n =3).D,days after germination.

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102Yanchong Yu et al.

(b)(d)

(e)

(f)

(g)

I II

III IV

V VI

VII

IX

VIII

Figure 6.WRKY71binds to the promoters of FT and LFY .

(a)The FT and LFY promoter structure and the fragments used for the chromatin immunoprecipitation (ChIP)assay.I –IX represent W-boxes;the short lines indi-cate the ChIP assay fragments;CK,control fragment lacking a W-box.(b)The electrophoretic mobility shift assay.Probes,GST,GST:WRKY71,competitors and mutated competitors at a 509and 2009molar excess were present (+)or absent (à)in each reaction.I –IX indicate the reaction of individual FT and LFY pro-moter W-boxes.DNA –protein complexes are arrowed.(c)The ChIP assay.AB and Mock represent ChIP signals in the presence and absence of the GFP antibody.–IX are as in part (b)and CK as in (a).Error bars represent SD (n =3).Statistically signi?cant differences were determined based on Student’s t -test (*P <0.05;**P <0.01).(d)The constructs used for the transient transcriptional activity assays in Arabidopsis leaf protoplast.(FT )and (LFY ),mutated W-boxes.(e)The transient transcriptional activity assay shows the activation of the native and mutated FT or LFY promoters.The Luc/Ren ratio indicates the pFT:LUC and mpFT:LUC ,pLFY:LUC and mpLFY:LUC activities relative to the internal control (REN driven by the 35S promoter),respectively.W71,WRKY71;Con,control.Error bars represent SD (n =3).Lower-case letters above columns indicate signi?cant differences in each group (one-way ANOVA ,P <0.01).(f)The ?owering time of plants grown under long-day conditions.At least 15plants per line were measured in each replicate.Error bars represent SD (n =3).Statistically signi?cant differences of the measurements were determined based on Student’s t -test (*P <0.05;**P <0.01).(g)A proposed model of how WRKY71regulates ?owering.WRKY71accelerates ?owering via direct activation of FT and LFY in Arabidopsis.

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WRKY71promotes ?owering 103

site was determined by thermal asymmetric interlaced PCR(Liu et al.,1995)and sequencing.

Measurement of?owering time

The number of rosette and cauline leaves and the total number of leaves at the moment when the?rst?ower became visible were taken as predictors of?owering time.In some cases,the day when the?rst visible?ower appeared was used to estimate the?owering time.Vernalization was provided by holding plants at4°C for6weeks under SD,after which they were grown under LD conditions.Exoge-nous GA was given by spraying plants grown under LD with20l M GA twice a week from7DAG to the appearance of the?rst?ower. Tissue sectioning and in situ hybridization

Soil-grown plants were harvested at various time points,and pre-pared for sectioning according to a previous study(Yu et al., 2013).The sectioning and staining was performed as per our pre-vious study(Qiao et al.,2012).Data were based on the inspection of at least100plants.

The in situ hybridization procedure followed the protocol described previously(Mayer et al.,1998)and used soil-grown WT plants.The201-bp30end of the WRKY71cDNA was inserted into pGEMa-T easy,and then the probes were generated by using a digoxigenin RNA labelling kit(Roche,https://www.360docs.net/doc/9a4849216.html,/).The probes were applied at a concentration of5l g mlà1in50%(v/v) formamide and stored atà80°C.

Transcription analysis

For the transcription pro?ling of WRKY71,RNA was isolated from the aerial part of soil-grown WT plants harvested at various time points.For the transcription pro?ling of?owering-related genes, RNA was extracted from the aerial parts of soil-grown plants (2595098cm3plastic plates with32small holes,6969 7cm3for each hole)at zeitgeber time16(ZT16).Both semi-quanti-tative RT-PCR and quantitative RT-PCR(qRT-PCR)were used to estimate transcript levels.The procedures were as described in our previous study(Qiao et al.,2012).Gene-speci?c primer sequences are listed in Table S1,and TUB2was used as a reference.Each qRT-PCR was represented by three biological replicates. Subcellular localization and transcriptional activation analysis

The full-length WRKY71CDS(up to but excluding the stop codon) was inserted into pB7FWG2,and introduced into Col-0as described above.The roots of transgenic lines were used for the determina-tion of WRKY71subcellular localization.For the transcriptional acti-vation analysis,the same sequence was inserted into pGBKT7,and this was then introduced into AH109containing the His3and LacZ reporter genes.The transformed AH109was grown on yeast nitro-gen base medium with or without His,and was used to perform b-galactosidase assay as described previously(Ren et al.,2010). The EMSA and ChIP assays

The full length of the WRKY71CDS was inserted into pGEX-4T-2, and transformed into Escherichia coli BL21(DE3)cells.The GST: WRKY71transgene was induced by1m M isopropyl b-D-1-thioga-lactopyranoside for3h,and the transgene product was puri?ed using a MagneGST Protein Puri?cation System(Promega,http:// https://www.360docs.net/doc/9a4849216.html,/).Probes were generated using a Dig Gel Shift kit(Roche).The relevant oligonucleotide sequences are listed in Table S1.

The ChIP assay followed the protocol of a previous study (Haring et al.,2007).Ten-DAG transgenic seedlings used in the subcellular localization assay were?xed with1%(v/v)formalde-hyde,and then the chromatins were sheared by sonication. DNA–protein complexes were immunoprecipitated from the sheared chromatins using a GFP antibody(Millipore,http:// https://www.360docs.net/doc/9a4849216.html,/).The DNA was then isolated from these complexes and ampli?ed by qRT-PCR(primer sequences are listed in Table S1).

Transcription dual-luciferase assay

Genomic DNA segments of2.2and2.3kb including either the native FT and LFY promoters or W-box-mutated FT and LFY pro-moters,respectively,were inserted into pGreenII0800-LUC(Hel-lens et al.,2005)to active the LUC reporter gene.The renilla luciferase gene REN driven by the35S promoter was used as the internal control.The full-length WRKY71CDS was inserted into pB2GW7to serve as an effector.All primers are listed in Tabl S1. Arabidopsis mesophyll protoplasts were prepared and transfected according to a previous study(Yoo et al.,2007).The LUC/REN activity was tested by using the Dual-Luciferase Reporter Assay System(Promega).

ACKNOWLEDGEMENTS

This research was?nancially supported by the National Special Science Research Program of China(grant no.2013CB967300), National High Technology Research and Development Program ‘863’(grant no.2013AA102602-4),National Transgenic Project of China(grant no.2013ZX08010002-002),National Natural Science Foundation(grant nos30970243,31270328,31471515,31201269, 31210103901and31500232),China Postdoctoral Science Founda-tion funded project(grant nos2014M551893and2015M572014), Science and Technology Plan of Shandong Province(grant no. 2013GNC11010),Promotive Research Fund for Excellent Young and Middle-aged Scientists of Shandong Province(BS2013SW027), Research Program for International S&T Cooperation Projects of Shandong Province(grant no.2011176),Shandong Province Natu-ral Science Foundation(ZR2014CP002)and a State Key Laboratory of Plant Genomics grant(no.2011B0525).We thank Dr Robert Koebner(UK)for English editing of the manuscript.

CONFLICT OF INTEREST

The authors declare no con?icts of interest. SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.

Figure S1.The ectopic expression of WRKY71results in a variant phenotype.

Figure S2.The?owering phenotype of wrky71-1mutant.

Figure S3.WRKY8,WRKY28and WRKY71are redundant.

Figure S4.Transcript levels of?owering related genes in Col-0, WRKY71-1D,w71w8+28RNAi and W71-SRDX.

Figure S5.Transcript levels of the?oral meristem identity genes in four lines.

Figure S6.The interaction of WRKY71with the AP1and CAL promoters.

Table S1.Primers used in this study.

REFERENCES

Abe,M.,Kobayashi,Y.,Yamamoto,S.,Daimon,Y.,Yamaguchi,A.,Ikeda, Y.,Ichinoki,H.,Notaguchi,M.,Goto,K.and Araki,T.(2005)FD,a bZIP

?2015The Authors

The Plant Journal?2015John Wiley&Sons Ltd,The Plant Journal,(2016),85,96–106 104Yanchong Yu et al.

protein mediating signals from the?oral pathway integrator FT at the shoot apex.Science,309,1052–1056.

Andres, F.and Coupland,G.(2012)The genetic basis of?owering responses to seasonal cues.Nat.Rev.Genet.13,627–639.

Aukerman,M.J.and Sakai,H.(2003)Regulation of?owering time and?oral organ identity by a microRNA and its APETALA2-like target genes.Plant Cell,15,2730–2741.

Birkenbihl,R.P.,Diezel,C.and Somssich,I.E.(2012)Arabidopsis WRKY33Is

a Key Transcriptional Regulator of Hormonal and Metabolic Responses

toward Botrytis cinerea Infection.Plant Physiol.159,266–285. Blazquez,M.A.,Soowal,L.N.,Lee,I.and Weigel,D.(1997)LEAFY expres-sion and?ower initiation in Arabidopsis.Development,124,3835–3844. Blumel,M.,Dally,N.and Jung,C.(2015)Flowering time regulation in crops -what did we learn from Arabidopsis?Curr.Opin.Biotechnol.32,121–129.

Cai,Y.H.,Chen,X.J.,Xie,K.,Xing,Q.K.,Wu,Y.W.,Li,J.,Du,C.H.,Sun,Z.X.

and Guo,Z.J.(2014)Dlf1,a WRKY transcription factor,is involved in the control of?owering time and plant height in rice.PLoS ONE,9,e102529. Cao,S.,Kumimoto,R.W.,Gnesutta,N.,Calogero,A.M.,Mantovani,R.and Holt,B.F.(2014)A distal CCAAT/NUCLEAR FACTOR Y complex promotes chromatin looping at the FLOWERING LOCUS T promoter and regulates the timing of?owering in Arabidopsis.Plant Cell,26,1009–1017. Castillejo,C.and Pelaz,S.(2008)The balance between CONSTANS and TEMPRANILLO activities determines FT expression to trigger?owering.

Curr.Biol.18,1338–1343.

Chen,L.G.,Song,Y.,Li,S.J.,Zhang,L.P.,Zou,C.S.and Yu,D.Q.(2012)The role of WRKY transcription factors in plant abiotic stresses.BBA-Gene Regul.Mech.1819,120–128.

Cheng,X.F.and Wang,Z.Y.(2005)Overexpression of COL9,a CONSTANS-LIKE gene,delays?owering by reducing expression of CO and FT in Ara-bidopsis thaliana.Plant J.43,758–768.

Clough,S.J.and Bent, A.F.(1998)Floral dip:a simpli?ed method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J.

16,735–743.

Corbesier,L.,Vincent,C.,Jang,S.et al.(2007)FT protein movement con-tributes to long-distance signaling in?oral induction of Arabidopsis.

Science,316,1030–1033.

Dally,N.,Xiao,K.,Holtgrawe,D.and Jung,C.(2014)The B2?owering time locus of beet encodes a zinc?nger transcription factor.Proc.Natl Acad.

Sci.U.S.A.111,10365–10370.

Davis,S.J.(2009)Integrating hormones into the?oral-transition pathway of Arabidopsis thaliana.Plant,Cell Environ.32,1201–1210.

Eulgem,T.,Rushton,P.J.,Robatzek,S.and Somssich,I.E.(2000)The WRKY superfamily of plant transcription factors.Trends Plant Sci.5,199–206. Gu,X.,Le,C.,Wang,Y.,Li,Z.,Jiang,D.,Wang,Y.and He,Y.(2013)Ara-bidopsis FLC clade members form?owering-repressor complexes coor-dinating responses to endogenous and environmental cues.Nat.

Commun.4,1947.

Han,P.,Garcia-Ponce,B.,Fonseca-Salazar,G.,Alvarez-Buylla,E.R.and Yu,

H.(2008)AGAMOUS-LIKE17,a novel?owering promoter,acts in a FT-

independent photoperiod pathway.Plant J.55,253–265.

Haring,M.,Offermann,S.,Danker,T.,Horst,I.,Peterhansel,C.and Stam, M.(2007)Chromatin immunoprecipitation:optimization,quantitative analysis and data normalization.Plant Methods,3,11.

Hartmann,U.,Hohmann,S.,Nettesheim,K.,Wisman,E.,Saedler,H.and Huijser,P.(2000)Molecular cloning of SVP:a negative regulator of the ?oral transition in Arabidopsis.Plant J.21,351–360.

Hellens,R.P.,Allan,A.C.,Friel,E.N.,Bolitho,K.,Grafton,K.,Templeton, M.D.,Karunairetnam,S.,Gleave,A.P.and Laing,W.A.(2005)Transient expression vectors for functional genomics,quanti?cation of promoter activity and RNA silencing in plants.Plant Methods,1,13.

Hiratsu,K.,Matsui,K.,Koyama,T.and Ohme-Takagi,M.(2003)Dominant repression of target genes by chimeric repressors that include the EAR motif,a repression domain,in Arabidopsis.Plant J.34,733–739.

Hu,Y.R.,Chen,L.G.,Wang,H.P.,Zhang,L.P.,Wang,F.and Yu,D.Q.(2013) Arabidopsis transcription factor WRKY8functions antagonistically with its interacting partner VQ9to modulate salinity stress tolerance.Plant J.

74,730–745.

Hu,J.Y.,Zhou,Y.,He,F.,Dong,X.,Liu,L.Y.,Coupland,G.,Turck,F.and de Meaux,J.(2014)miR824-regulated AGAMOUS-LIKE16contributes to ?owering time repression in Arabidopsis.Plant Cell,26,2024–2037.Jiang,Y.Q.and Deyholos,M.K.(2009)Functional characterization of Ara-bidopsis NaCl-inducible WRKY25and WRKY33transcription factors in abiotic stresses.Plant Mol.Biol.69,91–105.

Jiang,Y.J.,Liang,G.,Yang,S.Z.and Yu,D.Q.(2014)Arabidopsis WRKY57 functions as a node of convergence for jasmonic acid-and auxin-mediated signaling in jasmonic acid-induced leaf senescence.Plant Cell, 26,230–245.

Johnson,C.S.,Kolevski,B.and Smyth,D.R.(2002)TRANSPARENT TESTA GLABRA2,a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor.Plant Cell,14,1359–1375. Kumar,S.V.,Lucyshyn,D.,Jaeger,K.E.,Alos,E.,Alvey,E.,Harberd,N.P.

and Wigge,P.A.(2012)Transcription factor PIF4controls the thermosen-sory activation of?owering.Nature,484,242–245.

Li,S.,Fu,Q.,Chen,L.,Huang,W.and Yu,D.(2011)Arabidopsis thaliana WRKY25,WRKY26,and WRKY33coordinate induction of plant thermo-tolerance.Planta,233,1237–1252.

Liljegren,S.J.,Gustafson-Brown,C.,Pinyopich,A.,Ditta,G.S.and Yanof-sky,M.F.(1999)Interactions among APETALA1,LEAFY,and TERMINAL FLOWER1specify meristem fate.Plant Cell,11,1007–1018.

Liu,Y.G.,Mitsukawa,N.,Oosumi,T.and Whittier,R.F.(1995)Ef?cient isola-tion and mapping of Arabidopsis thaliana T-DNA insert junctions by ther-mal asymmetric interlaced PCR.Plant J.8,457–463.

Luo,M.,Dennis,E.S.,Berger,F.,Peacock,W.J.and Chaudhury,A.(2005) MINISEED3(MINI3),a WRKY family gene,and HAIKU2(IKU2),a leucine-rich repeat(LRR)KINASE gene,are regulators of seed size in Arabidop-sis.Proc.Natl Acad.Sci.U.S.A.102,17531–17536.

Luo,X.,Sun,X.L.,Liu,B.H.et al.(2013)Ectopic expression of a WRKY homolog from Glycine soja alters?owering time in Arabidopsis.PLoS ONE,8,e73295.

Mayer,K.F.,Schoof,H.,Haecker,A.,Lenhard,M.,Jurgens,G.and Laux,T.

(1998)Role of WUSCHEL in regulating stem cell fate in the Arabidopsis shoot meristem.Cell,95,805–815.

Miao,Y.,Laun,T.,Zimmermann,P.and Zentgraf,U.(2004)Targets of the WRKY53transcription factor and its role during leaf senescence in Ara-bidopsis.Plant Mol.Biol.55,853–867.

Moyroud,E.,Kusters,E.,Monniaux,M.,Koes,R.and Parcy,F.(2010)LEAFY blossoms.Trends Plant Sci.15,346–352.

Moyroud,E.,Minguet,E.G.,Ott,F.et al.(2011)Prediction of Regulatory Interactions from Genome Sequences Using a Biophysical Model for the Arabidopsis LEAFY Transcription Factor.Plant Cell,23,1293–1306. Nakamura,Y.,Andres,F.,Kanehara,K.,Liu,Y.C.,Dormann,P.and Coup-land,G.(2014)Arabidopsis?origen FT binds to diurnally oscillating phospholipids that accelerate?https://www.360docs.net/doc/9a4849216.html,mun.5,3553. Nilsson,O.,Lee,I.,Blazquez,M.A.and Weigel,D.(1998)Flowering-time genes modulate the response to LEAFY activity.Genetics,150,403–410. Parcy,F.(2005)Flowering:a time for integration.Int.J.Dev.Biol.49,585–593.

Qiao,M.,Zhao,Z.J.,Song,Y.G.,Liu,Z.H.,Cao,L.X.,Yu,Y.C.,Li,S.and Xiang,F.N.(2012)Proper regeneration from in vitro cultured Arabidopsis thaliana requires the microRNA-directed action of an auxin response fac-tor.Plant J.71,14–22.

Ratcliffe,O.J.,Bradley,D.J.and Coen,E.S.(1999)Separation of shoot and ?oral identity in Arabidopsis.Development,126,1109–1120.

Ren,X.,Chen,Z.,Liu,Y.,Zhang,H.,Zhang,M.,Liu,Q.,Hong,X.,Zhu,J.K.

and Gong,Z.(2010)ABO3,a WRKY transcription factor,mediates plant responses to abscisic acid and drought tolerance in Arabidopsis.Plant J.

63,417–429.

Riboni,M.,Galbiati,M.,Tonelli,C.and Conti,L.(2013)GIGANTEA enables drought escape response via abscisic acid-dependent activation of the ?origens and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1.

Plant Physiol.162,1706–1719.

Robatzek,S.and Somssich,I.E.(2002)Targets of At WRKY6regulation dur-ing plant senescence and pathogen defense.Genes Dev.16,1139–1149. Rushton,P.J.,Somssich,I.E.,Ringler,P.and Shen,Q.J.(2010)WRKY tran-scription factors.Trends Plant Sci.15,247–258.

Samach, A.,Onouchi,H.,Gold,S.E.,Ditta,G.S.,Schwarz-Sommer,Z., Yanofsky,M.F.and Coupland,G.(2000)Distinct roles of CONSTANS tar-get genes in reproductive development of Arabidopsis.Science,288, 1613–1616.

Sawa,M.and Kay,S.A.(2011)GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana.Proc.Natl Acad.Sci.U.S.A.108,11698–11703.

?2015The Authors

The Plant Journal?2015John Wiley&Sons Ltd,The Plant Journal,(2016),85,96–106

WRKY71promotes?owering105

Schmid,M.,Uhlenhaut,N.H.,Godard,F.,Demar,M.,Bressan,R.,Weigel,D.

and Lohmann,J.U.(2003)Dissection of?oral induction pathways using global expression analysis.Development,130,6001–6012.

Searle,I.,He,Y.H.,Turck,F.,Vincent,C.,Fornara,F.,Krober,S.,Amasino, R.A.and Coupland,G.(2006)The transcription factor FLC confers a?ow-ering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis.Genes Dev.20,898–912.

Song,Y.H.,Ito,S.and Imaizumi,T.(2013)Flowering time regulation:pho-toperiod-and temperature-sensing in leaves.Trends Plant Sci.18,575–583.

Teotia,S.and Tang,G.L.(2015)To bloom or not to bloom:role of micro-RNAs in plant?owering.Mol.Plant,8,359–377.

Torti,S.,Fornara,F.,Vincent,C.,Andres,F.,Nordstrom,K.,Gobel,U., Knoll,D.,Schoof,H.and Coupland,G.(2012)Analysis of the Arabidopsis shoot meristem transcriptome during?oral transition identi?es distinct regulatory patterns and a leucine-rich repeat protein that promotes?ow-ering.Plant Cell,24,444–462.

Wagner,D.,Sablowski,R.W.M.and Meyerowitz,E.M.(1999)Transcriptional activation of APETALA1by LEAFY.Science,285,582–584.

Wang,J.W.(2014)Regulation of?owering time by the miR156-mediated age pathway.J.Exp.Bot.65,4723–4730.

Wang,C.Q.,Guthrie,C.,Sarmast,M.K.and Dehesh,K.(2014)BBX19inter-acts with CONSTANS to repress FLOWERING LOCUS T transcription, de?ning a?owering time checkpoint in Arabidopsis.Plant Cell,26,3589–3602.

Weigel,D.,Ahn,J.H.,Blazquez,M.A.et al.(2000)Activation tagging in Ara-bidopsis.Plant Physiol.122,1003–1013.Wenkel,S.,Turck,F.,Singer,K.,Gissot,L.,Le Gourrierec,J.,Samach,A.

and Coupland,G.(2006)CONSTANS and the CCAAT box binding com-plex share a functionally important domain and interact to regulate?ow-ering of Arabidopsis.Plant Cell,18,2971–2984.

Wickland,D.P.and Hanzawa,Y.(2015)The FLOWERING LOCUS T/TERM-INAL FLOWER1gene family:functional evolution and molecular mecha-nisms.Mol.Plant,8,1–15.

William,D.A.,Su,Y.H.,Smith,M.R.,Lu,M.,Baldwin,D.A.and Wagner,D.

(2004)Genomic identi?cation of direct target genes of LEAFY.Proc.Natl Acad.Sci.U.S.A.101,1775–1780.

Wu,K.L.,Guo,Z.J.,Wang,H.H.and Li,J.(2005)The WRKY family of tran-scription factors in rice and Arabidopsis and their origins.DNA Res.12, 9–26.

Yamaguchi,A.,Wu,M.F.,Yang,L.,Wu,G.,Poethig,R.S.and Wagner,D.

(2009)The MicroRNA-Regulated SBP-Box Transcription Factor SPL3Is a Direct Upstream Activator of LEAFY,FRUITFULL,and APETALA1.Dev.

Cell,17,268–278.

Yoo,S.D.,Cho,Y.H.and Sheen,J.(2007)Arabidopsis mesophyll proto-plasts:a versatile cell system for transient gene expression analysis.Nat.

Protoc.2,1565–1572.

Yu,Y.C.,Hu,R.B.,Wang,H.M.,Cao,Y.P.,He,G.,Fu,C.X.and Zhou,G.K.

(2013)MlWRKY12,a novel Miscanthus transcription factor,participates in pith secondary cell wall formation and promotes?owering.Plant Sci.

212,1–9.

Zheng,Z.Y.,Abu Qamar,S.,Chen,Z.X.and Mengiste,T.(2006)Arabidopsis WRKY33transcription factor is required for resistance to necrotrophic fungal pathogens.Plant J.48,592–605.

?2015The Authors

The Plant Journal?2015John Wiley&Sons Ltd,The Plant Journal,(2016),85,96–106 106Yanchong Yu et al.

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