GOLDEN2-LIKE Transcription Factors Regulate WRKY40 · GOLDEN2-LIKE Transcription Factors RegulateWRKY40 Expression in Response to Abscisic Acid1[OPEN] RaﬁqAhmad,2 Yutong Liu,2 Tian-Jing

GOLDEN2-LIKE Transcription Factors Regulate WRKY40Expression in Response to Abscisic Acid1[OPEN]

Rafiq Ahmad,2 Yutong Liu,2 Tian-Jing Wang,2 Qingxiang Meng, Hao Yin, Xiao Wang, Yifan Wu,Nan Nan, Bao Liu, and Zheng-Yi Xu3,4

Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University,Changchun 130024, People’s Republic of China

ORCID IDs: 0000-0001-5481-1675 (B.L.); 0000-0001-9820-2329 (Z.X.).

Arabidopsis (Arabidopsis thaliana) GARP (Golden2, ARR-B, Psr1) family transcription factors, GOLDEN2-LIKE1 and -2 (GLK1/2), function in different biological processes; however, whether and how these transcription factors modulate the response toabscisic acid (ABA) remain unknown. In this study, we used a glk1 glk2 double mutant to examine the role of GLK1/2 in theABA response. The glk1 glk2 double mutant displayed ABA-hypersensitive phenotypes during seed germination and seedlingdevelopment and an osmotic stress-resistant phenotype during seedling development. Genome-wide RNA sequencing analysisof the glk1 glk2 double mutant revealed that GLK1/2 regulate several ABA-responsive genes, includingWRKY40, in the presenceof ABA. Chromatin immunoprecipitation and gel retardation assays showed that GLK1/2 directly associate with the WRKY40promoter via the recognition of a consensus sequence. Additionally, RNA sequencing analysis of the glk1 glk2 double mutantand wrky40 single mutant revealed that GLK1/2 and WRKY40 control a common set of downstream target genes in response toABA. Furthermore, results of a genetic interaction test showed that the glk1 glk2 wrky40 triple mutant displayed similar ABAhypersensitivity to the wrky40 single mutant and the glk1 glk2 double mutant, while the glk1 glk2 wrky40 abi5-c (ABI5 CRISPR/Cas9 mutant) quadruple mutant displayed similar ABA hyposensitivity to the abi5-7 single mutant. Based on these results, wepropose that the GLK1/2-WRKY40 transcription module plays a negative regulatory role in the ABA response.

The phytohormone abscisic acid (ABA) plays essen-tial roles in the induction of stomatal closure and otheradaptive responses under environmental stresses, thusregulating optimal plant growth and development(McAinsh et al., 1990; Leung and Giraudat, 1998;Borsani et al., 2002; Finkelstein et al., 2002; Xiong andZhu, 2002; Nambara and Marion-Poll, 2005; Zhu, 2016;Singh et al., 2017; Sussmilch et al., 2017). ABA also playsimportant biological roles in the maintenance of seed

dormancy, inhibition of seed germination, accelerationof senescence, and induction of stress tolerance (Zeevaartand Creelman, 1988; Borsani et al., 2002; Finkelstein et al.,2002; Xiong and Zhu, 2002; Nambara and Marion-Poll,2005; Park et al., 2015; Zhu, 2016; Li et al., 2018). Geneticscreening of seeds for sensitivity to ABA during germi-nation has led to the identification of several key modu-lators in the ABA signaling pathway, including ABAINSENSITIVE1 (ABI1), ABI2, ABI3, ABI4, and ABI5(Koornneef et al., 1984; Finkelstein, 1994; Nakashima andYamaguchi-Shinozaki, 2013). Among these, ABI1 andABI2, type 2C proteins with negative regulatory roles inABA signaling, physically interact with and inhibitdownstream targets, such as the Ser/Thr protein kinaseOPENSTOMATA1 (OST1;Assmann, 2003; Yoshida et al.,2006; Vlad et al., 2009);ABI3 encodes a transcription factorthat shares high homology with maize (Zea mays) vivip-arous1 (Giraudat et al., 1992); ABI4 is a member of theERF/AP2 transcription factor family (Finkelstein et al.,1998); and ABI5 is a basic Leu zipper transcription fac-tor that is phosphorylated by SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1, and SRK2I/SnRK2.3 to regulate the ex-pression of stress-responsive genes (Piskurewicz et al.,2008; Nakashima et al., 2009; Guo et al., 2011; Fan et al.,2018; Wang et al., 2018a; Wu et al., 2018).

Various types of stress-responsive transcription fac-tors, including CBF/DREB, WRKY, AP2, RD22BP,MYBs, NAC, and ABF/AREB, have been extensivelystudied at the transcriptional level (Kizis et al., 2001;Xiong and Zhu, 2002; Yamaguchi-Shinozaki and

1This work was supported by the National Natural Science Foun-dation of China (31601311 and 31771352 to Z.-Y.X.), the FundamentalResearch Funds for the Central Universities (#2412018BJ002 to Z.-Y.X.),and the Natural Science Foundation of Jilin Province of China(20180101233JC to Z.-Y.X.).

2These authors contributed equally to the article.3Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Zheng-Yi Xu ([email protected]).

Z.-Y.X. devised the project; Z.-Y.X. and B.L. supervised the project;R.A. performed the physiological analysis in response to ABA andabiotic stress; Y.L. performed the RNA sequencing experiment andanalyzed data; T.-J.W. performed cell biological assays andChIP-qPCR analysis; Q.M., H.Y., Y.W., N.N., and X.W. performedmolecular cloning and physiological analyses; Z.-Y.X., and B.L. wrotethe article; all authors reviewed, revised, and approved the article.

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Shinozaki, 2006; Jiang et al., 2017). Recently, it wasshown that transcription factor hierarchy is essential fordefining environmental stress and the ABA responsenetwork (Song et al., 2016). The WRKY proteins con-stitute a large family of transcription factors that areevolutionarily conserved in lower and higher plants(Eulgem et al., 2000). WRKY proteins contain a con-served WRKY DNA-binding domain of approximately60 amino acids, followed by a C2H2 or C2HC zinc-fingermotif (Eulgem et al., 2000), and exhibit strong bindingaffinity toward the W-box motif (C/T)TGAC(T/C;Eulgem et al., 2000; Ülker and Somssich, 2004). TheArabidopsis (Arabidopsis thaliana) genome harbors74 WRKY genes, and different WRKY transcriptionfactors play positive or negative regulatory roles inabiotic stress and ABA response (Marè et al., 2004; Xieet al., 2005; Miller et al., 2008; Jiang and Deyholos, 2009;Wu et al., 2009; Zhang et al., 2009b; Ren et al., 2010).The GARP (Golden2, ARR-B, Psr1) transcription

factors play essential roles in plant development, hor-mone signaling, organogenesis, pathogen resistance,nutrient sensing, and circadian rhythm maintenance(Kerstetter et al., 2001; Fitter et al., 2002; Tajima et al.,2004; Onai and Ishiura, 2005; Savitch et al., 2007;Schreiber et al., 2011; Canales et al., 2014; Huang et al.,2014; Han et al., 2016). GOLDEN2-LIKE (GLK) genesencode GARP nuclear transcription factors, whichare B-type Arabidopsis response regulators (ARRs;Riechmann et al., 2000). The GLK genes have beenshown to function in chloroplast development in Ara-bidopsis, maize, and the moss Physcomitrella patens(Rossini et al., 2001; Fitter et al., 2002; Yasumura et al.,2005). Other studies have shown that GLK genes alsoplay important roles in photosynthesis, defense re-sponse, fruit development, and ozone tolerance(Savitch et al., 2007; Kakizaki et al., 2009; Waters et al.,2009; Kobayashi et al., 2013; Murmu et al., 2014; Leisterand Kleine, 2016; Nagatoshi et al., 2016). In Arabi-dopsis, GLK genes are functionally redundant, asshown by the glk1 glk2 double mutant, which exhibits aperturbed phenotype (Fitter et al., 2002; Yasumuraet al., 2005; Waters et al., 2008). The GLK genes con-tain two highly conserved domains at the C terminus:GCT-box and DNA-binding domain (Rossini et al.,2001). However, the role that GLK transcription fac-tors specifically play in the ABA response and the un-derlying molecular mechanisms remain unknown.In this study, we show that the glk1 glk2 double

mutant displays an ABA-hypersensitive phenotype,while transgenic plants overexpressingGLK1/2 (Pro-35S:GLK1/2-2xFlag) show an ABA-hyposensitive phenotype,during seed germination and seedling development.Genome-wide transcriptome analysis of the glk1 glk2double mutant treated with or without ABA revealedthat GLK1/2 are required for the regulation of essentialABA-responsive genes. Intriguingly, we found GLK1/2to specifically activate the expression ofWRKY40, whichplays an important negative regulatory role in ABA re-sponse. Chromatin immunoprecipitation (ChIP) experi-ments showed that GLK1/2 directly associate with the

WRKY40 promoter through the recognition of a con-sensus sequence, resulting in the activation of genetranscription. Results of genetic analysis showed thatthe glk1 glk2 wrky40 triplemutant displayed similar ABAhypersensitivity to the wrky40 single mutant andthe glk1 glk2 double mutant. By contrast, the glk1 glk2wrky40 abi5-c (ABI5 CRISPR/Cas9 mutant) quadruplemutant suppressed the ABA-hypersensitive phenotypesof glk1 glk2 wrky40 and displayed similar ABA hypo-sensitivity to the abi5-7 single mutant. Based on theseresults, we propose that the GLK1/2-WRKY40 tran-scription module plays a negative regulatory role in theABA response.

RESULTS

The glk1 glk2 Double Mutant Displays Increased ABASensitivity during Seed Germination, Seedling Growth,and Induced Seed Dormancy

To evaluate the role of GLK1/2 during seed germi-nation, wild-type, glk1 and glk2 single mutant, and glk1glk2 double mutant seeds were grown in different ABAconcentrations (Xu et al., 2013), and the germinationgreening ratio (calculating cotyledons greening afterseed germination) was measured (He et al., 2012; Konget al., 2015; Wang et al., 2018b). The glk1 and glk2 singlemutants did not display a noticeable phenotype com-pared with the wild type; however, the glk1 glk2 doublemutant showed increased ABA sensitivity (Fig. 1, A andB). To further examine the role of GLK1/2 in seedlingdevelopment, 3-d-old seedlings grown on Murashigeand Skoog (MS) medium were transferred to mediumcontaining different concentrations of ABA (containingfull MS, 2% [w/v] Suc, 1% [w/v] agar, and 0, 15, or30mMABA) for 7 d. Root growth of the glk1 glk2mutantwas dramatically retarded at different ABA concentra-tions compared with the wild type and the glk1 and glk2singlemutants (Fig. 1, C andD). To confirm these results,we evaluated the ABA sensitivity of transgenic plantsoverexpressing GLK1 and GLK2. Ectopic expression ofGLK1 or GLK2 was achieved using the strong cassavavein mosaic virus promoter (Supplemental Fig. S1A).Two independent transgenic overexpressing lines ofeach gene, ProGLK1:GLK1-2xFlag (GLK1OX-1 andGLK1OX-2) and ProGLK2:GLK2-2xFlag (GLK2OX-1 andGLK2OX-2), were used for seed germination assays. Alltransgenic lines displayed an ABA-hyposensitive phe-notype compared with the wild type in terms of seedgermination (Supplemental Fig. S1, B and C). We alsoexamined the growth of transgenic GLK1OX andGLK2OX seedlings by transferring 3-d-old seedlings toMS medium containing 2% (w/v) Suc, 1% (w/v) agar,and 15 or 30 mM ABA for 7 d. Transgenic GLK1OX andGLK2OX seedlings showed ABA-hyposensitive pheno-types comparedwithwild-type seedlings (SupplementalFig. S1, D and E). Taken together, these results indicatethat Arabidopsis GLK1/2 are involved in the responseto ABA.

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GLK1/2 Modulate the ABA Response


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To examine whether GLK1/2 play a regulatory roleduring seed dormancy, we compared the germinationability of freshly harvested wild-type, glk1, glk2, andglk1 glk2 seeds sown on MS medium; this experimentwas conducted under both light and dark and with andwithout prechilling for 3 d at 4°C (Ding et al., 2014),because the strength of primary dormancy is reflectedby the degree of requirement for light and/or chillingto promote germination (Finkelstein, 1994). Resultsshowed that the germination rate of glk1 glk2 seeds waslower than that of wild-type seeds when exposed tolight without prechilling, indicating that the doublemutant seeds were more dormant than wild-type seeds(Supplemental Fig. S2A). All mutant and wild-typeseeds germinated after prechilling treatment underlight (Supplemental Fig. S2B). We observed that bothwild-type and glk1 glk2 seeds germinated in the darkwithin a single day following prechilling, whereas glk1

glk2 seedsweremore dormant (Supplemental Fig. S2C).However, in the absence of light and prechilling, mostseeds failed to germinate (Supplemental Fig. S2D).These results indicate that the glk1 glk2 double mutantshowed increased seed dormancy over the wild typeand the glk1 and glk2 single mutants.

GLK1/2 Modulate ABA-Mediated Regulation ofGene Expression

To evaluate the effect of GLK1/2 on ABA-mediatedtranscriptional regulation, we performed RNA se-quencing (RNA-seq) analysis of wild-type and glk1 glk2seedlings treated with or without 10 mM ABA for 1 h.Using stringent statistical and filtering criteria, weidentified 1,718 differentially expressed genes (DEGs)in the comparison between glk1 glk2 and wild-type

Figure 1. GLK1/2 play negative roles in ABA responses. A and B, Effect of exogenous ABA on seed germination. Seeds of the wildtype (WT), glk1, glk2, and glk1 glk2mutants were planted on one-half-strength MS plates supplemented with dimethyl sulfoxide(DMSO) or different concentrations of ABA. A, Images were taken after incubation on plates for 7 d. B, The germination greeningratio was measured. Error bars indicate SD (n = 3). Statistical analyses were performed between the wild type and glk1 glk2 treatedwith 0.5 or 1 mM ABA. P1 = 0.00445 and P2 = 0.00393 (Student’s t test). C and D, Effect of exogenous ABA on root growth. Thewild type, glk1, glk2, and glk1 glk2mutants grown onMS plates for 3 d were transferred to MSmedium containing 2% (w/v) Suc,1% (w/v) agar, and 0, 15, or 30 mM ABA for 7 d. C, Images of plants were taken at 7 d after transfer. D, To quantify root growth,primary root lengthwasmeasured on 7-d-old plants after transfer. Three independent experimentswere performed using 20 plantsper experiment. Error bars indicate SD (n = 3). Statistical analyseswere performed between thewild type and glk1 glk2 treatedwith15 or 30 mM ABA. **, P , 0.01 (Student’s t test).

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seedlings treated with ABA, of which 877 were up-regulated (URGs) and 841 were down-regulated(DRGs) in the double mutant compared with the wildtype. In the comparison between glk1 glk2 and wild-type seedlings without ABA treatment, 1,238 DEGswere identified, of which 394 were URGs and 844 wereDRGs in the double mutant compared with the wildtype (Fig. 2, A and B; Supplemental Tables S1 and S2).Our data suggest that, under normal conditions, GLKspositively regulate a number of biological processes,including photosynthesis and response to abiotic stressstimuli, such as oxidation, wounding, water depriva-tion, and hyperosmotic stress (Supplemental Fig. S3;Supplemental Table S3), and negatively regulateother biological processes, such as microtubule-basedevents, cell cycle processes, cytoskeleton organization,meiosis, and spindle organization (Supplemental Fig.S3; Supplemental Table S3). Previously, Waters et al.(2009) identified potential direct target genes ofGLK1/2 by taking advantage of a temporal induction

system. Comparison of DRGs with microarray datashowing target genes of GLK1/2 revealed that 27.4% ofthe GLK1 target genes and 52% of the GLK2 targetgenes overlapped with DRGs (Supplemental Table S4).These overlapping genesmostly function in chlorophyllbiosynthesis, light harvesting, and electron transport(Waters et al., 2009). After 1 h of ABA treatment, GLKsshowed positive regulation of some biological processes,such as gene transcription and response to oxidation/reduction, hormone stimulus, hormone-mediated sig-naling, and water deprivation (Supplemental Fig. S3;Supplemental Table S3), and negative regulation of otherbiological processes, including oxidation/reduction,translation, development, glucan metabolism, DNA rep-lication, and lipid biosynthesis (Supplemental Fig. S3;Supplemental Table S3). To confirm the results of RNA-seq analysis, expression of a few ABA- and other abioticstress-responsive genes, which were categorized intoGene Ontology (GO) terms such as response to abioticstimulus and response to water deprivation under ABA

Figure 2. GLK1/2 direct the transcrip-tional networks of ABA response genes.A and B, Bioinformatic analysis ofDEGs. A, Venn diagrams showing over-lap of DEGs of DRGs and URGs be-tween the wild type (WT) and glk1 glk2under ABA treatment for 0 and 1 h. B,Hierarchical clustering analyses of DRGsandURGs under ABA treatment for 0 and1 h. C, RT-qPCR analysis of abiotic stress-responsive genes. GAPDH was usedas an internal control. Error bars indicateSD (n = 3).
















treatment, was examined by quantitative real-time PCR(RT-qPCR; Fig. 2C). The expression of several stress-responsive genes such as WRKY40 (Chen et al., 2010),RbohB (Zhang et al., 2009a), and COR15A and COR15B(Yamaguchi-Shinozaki and Shinozaki, 2005) was inducedin wild-type seedlings after ABA treatment but dramati-cally impaired in glk1 glk2 double mutants (Fig. 2C).Previously, using an inducible system and transcriptomeanalysis, Waters et al. (2009) showed that COR15A andCOR15B are the primary transcriptional targets of GLK1/2; these results are consistent with our data. In addition,some of the essential genes in the ABA regulatory net-work, including ABI5 (Lopez-Molina et al., 2001), PYL11(Lim and Lee, 2015), ABI3 (Giraudat et al., 1992), andDARK INDUCIBLE11 (DIN11; Fujiki et al., 2001), werenegatively regulated by GLK1/2 in response to ABA(Fig. 2C). These results indicate that GLK1/2 control theexpression of multiple essential ABA-responsive genes.

GLK1/2 Negatively Regulate Osmotic and DehydrationStress Responses

To gain further insights into the biological role ofGLK1/2 in the response to osmotic stress, plants grownon MS plates showing the same root length weretransferred to MS medium containing 2% (w/v) Suc,1% (w/v) agar, and mannitol (0, 100, or 150 mM) orNaCl (0, 100, or 150 mM), and root growth was evalu-ated. Root growth did not display noticeable variationamong wild-type, glk1, glk2, and glk1 glk2 seedlings;however, under osmotic and salt stress conditions, glk1glk2 showed relatively longer roots than wild-type,glk1, and glk2 seedlings (Fig. 3). We also examined thephenotypes of GLK1OX and GLK2OX transgenicplants. Both GLK1OX and GLK2OX transgenic linesshowed hypersensitivity to salt and osmotic stresses(Supplemental Fig. S4). Reactive oxygen species (ROS)are produced in different organelles under abiotic stressconditions because of metabolic imbalance (Skopelitiset al., 2006; Suzuki et al., 2012; Ivanchenko et al., 2013;Khan and Khan, 2017; Singh et al., 2017; Qi et al., 2018;Yang and Guo, 2018), and excess ROS accumulationleads to injury or cell death (Nakagami et al., 2004;Queval et al., 2007; Cha et al., 2015; Zhao et al., 2018).Since the GO term oxidation reduction was enrichedamong the DRGs (P, 0.01; Supplemental Table S3), weexamined salt- and osmotic stress-induced productionof superoxide and hydrogen peroxide (H2O2) in wild-type and glk1 glk2 double mutant seedlings by stainingwith nitroblue tetrazolium (NBT) or 3,39-diaminobenzidine(DAB). In the absence of osmotic stress, staining withDAB or NBT showed no differences between wild-typeand glk1 glk2 seedlings (Supplemental Fig. S5, A and B).However, under osmotic stress, wild-type seedlingsshowed more intense staining with DAB and NBT thanglk1 glk2 seedlings (Supplemental Fig. S5, A and B),indicating that the glk1 glk2 double mutant exhibitshigher ROS detoxification capability than thewild type.These data suggest that, in addition to the role of

GLK1/2 in ABA response, GLK1/2 also regulate theexpression of genes in response to ROS accumulationinduced by osmotic stress. We also examined the re-sponse of mutant and wild-type seedlings in responseto dehydration stress and observed that the survivalrate of glk1 glk2 seedlings was significantly higher thanthat of wild-type, glk1, and glk2 seedlings (SupplementalFig. S5, C and D). These results indicate that GLK1/2 areredundant and play negative regulatory roles in re-sponse to osmotic and dehydration stresses.

GLK1/2 Specifically Activate the Expression of SomeEssential ABA-Responsive Genes

To investigate the transcriptional regulatory mode ofGLKs, we employed a protoplast transfection system(Wang et al., 2007). In this system, the GUS reportergene was driven by the minimal 35S promoter (246 to0 bp) together with a cis-acting regulatory sequencethat could be recognized by the GAL4 DNA-bindingdomain (named as reporter; Fig. 4A; Wang et al.,2007). In addition, the GAL4 DNA-binding domainwas fused to the herpes simplex virus VP16 activationdomain (GD-VP16; positive control) or to full-lengthGLK1 or GLK2 protein (named as effector; Fig. 4A).We cotransfected the reporter construct together witheffector constructs encoding GD-VP16, GD-GLK1, orGD-GLK2 fusion proteins. Comparedwith the negativecontrols, GD-GLK1 and GD-GLK2 fusion proteinsdramatically induced the expression of the GUS re-porter gene (Fig. 4B). This result indicates that GLK1and GLK2 possess transcriptional activation capability.

Next, to test the in vivo transcriptional activationcapabilities of GLK1 and GLK2, we took advantage ofthe b-estradiol-inducible system (Schlücking et al.,2013). This system contains successive transcriptionunits in which the G10-90 promoter controls the ex-pression of a chimeric XVE fusion protein (Ishige et al.,1999; Schlücking et al., 2013), which is composed of theDNA-binding domain of the bacterial repressor LexA(X), a transactivating domain of VP16 (V; Dalrympleet al., 1985), and the C-terminal region of the estrogenreceptor from human (E; Green et al., 1986). In thepresence of b-estradiol, the hormone binds to the re-ceptor domain, which leads to a conformationalchange, thereby enabling the DNA-binding domain tobind to the LexA operator, which activates the minimal35S promoter (Benfey and Chua, 1990). Results of RT-qPCR showed that treatment with different concentra-tions of b-estradiol (0, 1, 10, 20, 50, and 100 mM) for 4 hinduced the expression of GLK1, GLK2, and the GFPgene in a dose-dependent manner (Supplemental Fig.S6). Since the induction kinetics of GLK1 and GLK2were saturated at 20 mM b-estradiol, we examined theexpression of stress-responsive genes, includingWRKY40, RbohB, COR15A, and COR15B, at this con-centration (Fig. 4C). We found that all these genes wererapidly induced upon the temporal induction of GLK1and GLK2. However, no noticeable alterations in GFP


Ahmad et al.











induction were detected, indicating that GLK1/2 spe-cifically induce the expression of these genes.

GLK1/2 Control the Expression of WRKY40 by DirectlyBinding to the Consensus Sequence Located in ItsPromoter Region

Previously, GLK1/2 were shown to specifically rec-ognize a consensus sequence, CCAATC, in the pro-moter region of target genes (Waters et al., 2009; Guoet al., 2018). Since GLK1/2 are transcriptional activa-tors, we investigated the direct target genes of GLK1/2

in response to ABA. We analyzed the promoter se-quences (2500 to 0 bp) of DRGs identified in glk1 glk2seedlings treated with ABA and categorized under theGO terms response to abiotic stimulus and response towater deprivation. A total of 13 genes, includingWRKY40, NRT1.1, RAB18, COR413, and PUB22, har-bored the consensus CCAATC motif in the promoterregions (Supplemental Table S5). We further investi-gated theWRKY40 gene, as it was the only gene amongthe 13 genes that has been previously reported to neg-atively regulate ABA response during seed germinationand seedling development (Chen et al., 2010) andtherefore was the most likely target of GLK1/2. We

Figure 3. GLK1/2 play negative roles inosmotic and salt stress responses. A andB, Effect of osmotic stress on rootgrowth. The wild type (WT), glk1, glk2,and glk1 glk2 mutants grown on MSplates for 3 d were transferred to MSmedium containing 2% (w/v) Suc, 1%(w/v) agar, and mannitol (0, 100, or150mM) for 7 d. A, Images of plants weretaken at 7 d after transfer. B, To quantifyroot growth, primary root length wasmeasured on 7-d-old plants after transfer.Three independent experiments wereperformed using 20 plants per experi-ment. Error bars indicate SD (n = 3). Sta-tistical analyses were performed betweenthe wild type and glk1 glk2 treated with100 and 150 mM mannitol. *, P , 0.05,**, P , 0.01 (Student’s t test). C and D,Effect of salt stress on root growth. Thewild type, glk1, glk2, and glk1 glk2 mu-tants grown on MS plates for 3 d weretransferred to MS medium containing2% (w/v) Suc, 1% (w/v) agar, and NaCl(0, 100, or 150 mM) for 7 d. C, Images ofplants were taken at 7 d after transfer. D,To quantify root growth, primary rootlength was measured on 7-d-old plantsafter transfer. Three independent experi-ments were performed using 20 plantsper experiment. Error bars indicate SD

(n = 3). Statistical analyses were per-formed between the wild type and glk1glk2 treated with 100 or 150 mM NaClmedium. **, P , 0.01 (Student’s t test).






found that theWRKY40 promoter harbors the CCAATCmotif in the region from 2500 to 0 bp (Fig. 5A). To ex-amine whether this sequence is required for GLK1/2-mediated transcriptional activation, we generated achimeric reporter construct by fusing 500 bp of theWRKY40 promoter (2500 to 0 bp) with the luciferase(LUC) reporter gene (ProWRKY40:LUC). Another con-struct was generated using a variant of the WRKY40promoter, which carried a mutation in the GLK1/2-binding motif (MProWRKY40:LUC). We cotransfectedProWRKY40:LUC orMProWRKY40:LUCwith ProUBQ10:GUS, a chimeric construct ofGUS under the control of theUBQ10 promoter, into Arabidopsis protoplasts isolatedfrom transgenic plants expressingProSUPERR:sXVE-GFP,ProSUPERR:sXVE-GLK1, or ProSUPERR:sXVE-GLK2 us-ing the polyethylene glycol-mediated transformationmethod (Fig. 5A; Xu et al., 2013). Induction of GLK1 orGLK2 by b-estradiol dramatically activated LUC expres-sion in protoplasts transfected with ProWRKY40:LUC butnot in protoplasts transfected with MProWRKY40:LUC(Fig. 5B). This result indicates that the consensus GLK1/2recognition site is required for GLK1/2-mediated tran-scriptional activation.

Next, to examine whether GLK1/2 bind to the pro-moter region of WRKY40, we fused GLK1 or GLK2 ge-nomicDNAs (gGLK1 or gGLK2)with two FLAG epitopes(23FLAG) at the C terminus and cloned gGLK1-23FLAGand gGLK2-23FLAG fusions under the control of nativepromoters (ProGLK1:gGLK1-23FLAG and ProGLK2::gGLK2-23FLAG). These constructs were introduced intothe glk1 glk2 double mutant separately. Both ProGLK1:gGLK1-23FLAG and ProGLK2::gGLK2-23FLAG com-plemented the phenotype of the glk1 glk2 double mutant(Supplemental Fig. S7, A and B). Moreover, the expres-sion of stress-responsive genes that was increased orreduced in the glk1 glk2 double mutant was recovered tothe level of the wild type in the complementation lines(Supplemental Fig. S7C). Next, we performed ChIP

coupled with qPCR (ChIP-qPCR) analysis using an anti-FLAG antibody. We found that the consensus CCAATCmotif was significantly enriched, while the nonconsensusCCACTC sequence showed no enrichment (Fig. 5, C andD). Additionally, to determine the in vitro binding ofGLK1/2 to the consensus sequence, we performed elec-trophoretic mobility shift assays (EMSAs). Full-lengthGLK1/2 proteins tagged with glutathione S-transferase(GST-GLK1 and GST-GLK2) were capable of binding toprobes containing P2 (CCAATC), whereas GST alone didnot bind to the probes (Fig. 5, E and F). When a mutantprobe (CCGGTC) was used for EMSA, the binding ac-tivity of GST-GLK1/2 fusion proteins was diminished(Fig. 5F). Collectively, these results indicate that GLK1/2bind to the promoter of WRKY40 via the consensussequence.

GLK1/2 and WRKY40 Regulate Common Target Genes inResponse to ABA

To examine whether GLK1/2 and WRKY40 controlcommon target genes in response to ABA, we per-formed RNA-seq analysis of the glk1 glk2 double mu-tant and wrky40 single mutant treated with 10 mM ABAfor 0, 1, or 3 h. Using stringent statistical and filteringcriteria, we identified URGs and DRGs in glk1 glk2versus the wild type and wrky40 versus the wild typecomparisons under different lengths of ABA exposure.A substantial number of URGs and DRGs were sharedin each time point with statistical significance (Fig. 6A;Supplemental Tables S6 and S7). Hierarchical clusteringanalysis showed that GLK1/2 and WRKY40 system-atically impact the transcription of common gene tar-gets in response to ABA (Fig. 6B; Supplemental TablesS6 and S7). Furthermore, scatterplot analysis showedpositive correlations between URGs and DRGs in glk1glk2 and wrky40mutants under ABA treatment for 0, 1,

Figure 4. GLK1/2 activate primary ABAresponse genes. A and B, GLK1/2 aretranscriptional activators. A, Effectorsand reporter constructs used in thetransfection assays. GD, GAL4 DNA-binding domain; VP-16, herpes sim-plex virus VP16 activation domain. B,GD, GD-GLK1/2, and GD-VP16 werecotransfected with the reporter GAL4-GUS, and GUS activity was assayed af-ter protoplasts were incubated in dark-ness for 20 to 23 h. Error bars indicate SD

(n = 3). C, Temporal induction of GLK1/2 activates the expression of ABA re-sponse genes. b-Estradiol (20 mM) wasintroduced for 4 h, and expression ofWRKY40,RbohB,COR15A, andCOR15Bwas measured using RT-qPCR analysis.Error bars indicate SD (n = 3).


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and 3 h (Fig. 6C; Supplemental Tables S6 and S7). Wefurther confirmed RNA-seq data using RT-qPCR. Re-sults of RT-qPCR analysis showed thatABI5 andDIN11were up-regulated, while RbohB and COR15A weredown-regulated, in glk1 glk2 and wrky40 (Fig. 6D). Ofnote, we detected similar expression patterns in the glk1glk2 wrky40 triple mutant, suggesting that GLK1/2 andWRKY40 act in the same genetic pathway to regulatethe expression of target genes. Taken together, theseresults suggest that GLK1/2 and WRKY40 share com-mon target genes in response to ABA.

Next, we performed RNA-seq analysis of 10-d-oldwild-type, glk1 glk2 double mutant, and pyr1 pyl1 pyl2pyl4 quadruple mutant seedlings treated with 10 mM

ABA for 3 h (see “Materials and Methods”). Becauseglk1 glk2 and pyr1 pyl1 pyl2 pyl4 mutants displayedcontrasting phenotypes of seed germination and seed-ling growth in response to ABA, we compared URGs inglk1 glk2 (523 genes) with DRGs in pyr1 pyl1 pyl2 pyl4(2,300 genes) and DRGs in glk1 glk2 (874 genes) withURGs in pyr1 pyl1 pyl2 pyl4 (1,833 genes; SupplementalFig. S8; Supplemental Table S8). A substantial number

Figure 5. GLK1/2 bind to the promoter region ofWRKY40 and activate the expression via recognition of the consensus sequence.A and B, Transcriptional activation of theWRKY40 promoter byGLK1/2 via recognition of the consensus sequence. A, Protoplastsfrom transgenic plants harboring ProSUPERR:sXVE-GFP, ProSUPERR:sXVE-GLK1, and ProSUPERR:sXVE-GLK2 were cotrans-formedwith reporter containingWRKY40 promoter sequence and normalizing plasmids, incubated for 23 h, and then incubatedan additional 4 hwith 20mM b-estradiol. Mutant, Consensus sequence CCAATC located at theWRKY40 promoter regionmutatedas AAAAAA; WT, original WRKY40 promoter sequence including consensus CCAATC. B, The LUC activity was normalized byGUS activity. Error bars indicate SD (n= 3). **, P, 0.01 (Student’s t test). C andD,GLK1/2 bind to the promoter region ofWRKY40.C, Schematic representation ofWRKY40 promoter withGLK1/2 binding site (P2) and non-GLK1/2 binding site (P1). D, Transgenicplants expressing genomic DNA of GLK1 (ProGLK1:GLK1-2xFLAG) or GLK2 (ProGLK2:GLK2-2xFLAG) driven by their nativepromoters were used to perform ChIP-qPCR at different time points under the condition of ABA treatment. ChIP-qPCR wasperformedwith (with Ab) or without (no Ab) FLAG antibody. **, P, 0.01 (Student’s t test). E, Coomassie Blue-stained gel showinglevels of recombinantGST proteins used in EMSA. F, EMSA analysis of the binding of recombinantGLK1/2 protein to the promoterof WRKY40. mProbe is the biotin-labeled probe with a mutation from CCAATC to CCGGTC.









of genes overlapped, which has statistical significance(Supplemental Fig. S8). Transcript levels of GLK1/2were increased in the pyr1 pyl1 pyl2 pyl4 receptor mu-tant (Supplemental Table S8), suggesting that GLK1/2might act as negative regulators in the PYL/PYR-mediated ABA signaling pathway. This conclusion wasfurther supported by the significant increase in tran-script levels of positive regulators of ABA signaling,including ABI5, ABI3, and PYL11, in the glk1 glk2double mutant under ABA treatment (Fig. 2C).

GLK1/2 and WRKY40 Function in the SameGenetic Pathway

To examine the genetic interaction between GLK1/2and WRKY40, we crossed glk1, glk2, and glk1 glk2 mu-tants separately with wrky40, thereby generating glk1wrky40, glk2 wrky40, and glk1 glk2 wrky40 mutants, re-spectively. During seed germination, the glk1 glk2double mutant and wrky40 single mutant displayed

ABA-hypersensitive phenotypes compared with thewild type, glk1, and glk2 (Fig. 7, A and B). Additionally,glk1 wrky40, glk2 wrky40, and glk1 glk2 wrky40 displayedsimilar ABA sensitivity to wrky40, indicating thatWRKY40 functions genetically downstream of GLK1/2during seed germination (Fig. 7, A and B). Next, 3-d-oldwild-type and mutant seedlings were transferred tomedium containing 15 or 30 mM ABA for 7 d, and rel-ative root lengths were measured. The glk1 glk2 andwrky40 mutants displayed ABA-hypersensitive phe-notypes, while glk1 wrky40, glk2 wrky40, and glk1 glk2wrky40 displayed similar ABA hypersensitivity towrky40 (Fig. 7, C and D). We also overexpressedWRKY40 in the glk1 glk2 double mutant background, asan alternative approach (Supplemental Fig. S9A). In theresulting transgenic lines, the ABA-hypersensitivephenotype of glk1 glk2 was suppressed, although noeffect on chloroplast development was observed(Supplemental Fig. S9, B and C). Taken together, theseresults indicate that WRKY40 functions geneticallydownstream of GLK1/2 during both germination and

Figure 6. GLK1/2 and WRKY40 directsimilar transcriptional networks of ABAresponse genes. A and B, Bioinformaticanalysis of DEGs. A, Venn diagramshowing overlap of DEGs between glk1glk2 and wrky40 under ABA treatmentfor 0, 1, or 3 h. P values were calculatedwith two-tailed hypergeometric tests. B,Hierarchical clustering analyses ofDRGs and URGs under ABA treatmentfor 0, 1, or 3 h. C, Scatterplots showing apositive correlation between URGs andDRGs in glk1 glk2 and wrky40 mutantsunder ABA treatment for 0, 1, and 3 h.WT, Wild type. D, Real-time PCR anal-ysis of abiotic stress-responsive genes.GAPDHwas used as an internal control.Error bars indicate SD (n = 3).


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seedling development. Previously, it was reported thatWRKY40 directly binds to the promoter of ABI5, whichfunctions genetically downstream of WRKY40, thussuppressing the activity of ABI5 in the presence of ABA(Shang et al., 2010). Our RNA-seq data confirmed thatthe transcript level of ABI5 was higher in the glk1 glk2double mutant than in the wild type in the presenceof ABA.To confirm the genetic interactions among GLK1/2,

WRKY40, and ABI5, we tried to cross abi5-7 (Nambaraet al., 2002; Tamura et al., 2006) with the glk1 glk2wrky40 triple mutant. However, since GLK1 and ABI5are tightly linked genetically, we failed to generate anabi5-7 glk1 glk2 wrky40 quadruple mutant. To overcomethis limitation, we took advantage of CRISPR/Cas9technology (Ossowski et al., 2008; Sablok et al., 2011; Liet al., 2013; Carbonell et al., 2014) and isolated threeindependent glk1 glk2 wrky40 abi5-crispr-cas9 (abi5-cr)

lines (Supplemental Fig. S10). In glk1 glk2 wrky40 abi5-cr-1 and glk1 glk2 wrky40 abi5-cr-3, a 1-bp insertion wasdetected after the ATG of ABI5 (55 bp after the ATG inglk1 glk2 wrky40 abi5-cr-1 and 53 bp after the ATG in glk1glk2 wrky40 abi5-cr-3), resulting in a frame shift andconsequently a premature stop codon (SupplementalFig. S10A). In glk1 glk2 wrky40 abi5-cr-2, a 1-bp deletionwas detected at 55 bp after ATG, causing a frame shift(Supplemental Fig. S10A). To exclude the effect of theCas9 gene per se, we isolated glk1 glk2 wrky40 abi5-crlines by screening for nonhygromycin resistance(Supplemental Fig. S10B). Screening for ABA sensitivityamong mutants during seed germination and seedlinggrowth revealed that wrky40, glk1 glk2, and glk1 glk2wrky40 exhibited ABA-hypersensitive phenotypes com-paredwith thewild type, whereas glk1 glk2 wrky40 abi5-cr-1, glk1 glk2 wrky40 abi5-cr-2, and glk1 glk2 wrky40 abi5-cr-3showed ABA-hyposensitive phenotypes compared with

Figure 7. GLK1/2 and WRKY40 function in the same genetic pathway. A and B, Effect of exogenous ABA on seed germination.Seeds of thewild type (WT),wrky40, glk1, glk2, glk1 glk2, glk1wrky40, glk2wrky40, and glk1 glk2wrky40mutantswere plantedon one-half-strength MS plates supplemented with DMSO or different concentrations of ABA. A, Images were taken after in-cubation on plates for 7 d. B, The germination greening ratio was measured. Error bars indicate SD (n = 3). Statistical analyseswereperformed between the wild type and glk1 glk2, the wild type andwrky40, the wild type and glk1 wrky40, the wild type and glk2wrky40, and the wild type and glk1 glk2 wrky40 treated with 0.5 or 1 mMABA. **, P, 0.01 (Student’s t test). C, and D, Effect ofexogenous ABA on root growth. The wild type, wrky40, glk1, glk2, glk1 glk2, glk1 wrky40, glk2 wrky40, and glk1 glk2 wrky40mutants were planted on MS plates for 3 d and transferred to MS medium containing 2% (w/v) Suc and 1% (w/v) agar with 15 or30 mMABA for 7 d. C, Images were taken after incubation on plates for 7 d. D, The relative root growth was measured. Error barsindicate SD (n = 3). Statistical analyses were performed between the wild type and glk1 glk2, the wild type and wrky40, the wildtype and glk1wrky40, thewild type and glk2wrky40, and thewild type and glk1 glk2wrky40 treatedwith 15 and 30mMABA. **,P , 0.01 (Student’s t test).










the wild type during seed germination (Fig. 8, A and B)and seedling development (Fig. 8, C and D). These resultsindicate that ABI5 functions genetically downstream ofGLK1/2 and WRKY40. In summary, we propose thatGLK1/2 possibly could be activated via core ABA sig-naling components, PYL/PYRs-PP2Cs-SnRKs, and sub-sequently induce the expression of WRKY40 by directlybinding to the promoter sequence. WRKY40 further di-rectly binds to the ABI5 promoter region to suppress theABI5 expression (Fig. 8E).

DISCUSSION

Genes encoding plant-specific GARP transcriptionfactors regulate diverse processes, including nutrientsensing, root and shoot development, chloroplast de-velopment, and circadian clock oscillation maintenance(Safi et al., 2017). In Arabidopsis, the GARP familycomprises 56 genes, includingG2-LIKE genes and genesencoding B-type ARR proteins that harbor N-terminalreceiver domains (Riechmann et al., 2000). Many de-cades ago, the golden2 (g2) mutant was described as

Figure 8. ABI5 functions genetically downstream of GLK1/2 and WRKY40. A and B, Effect of exogenous ABA on seed germi-nation. Seeds of the wild type (WT), abi5-7, glk1 gk2, wrky40, glk1 glk2 wrky40, glk1 glk2 wrky40 abi5-c-1, glk1 glk2 wrky40abi5-c-2, and glk1 glk2 wrky40 abi5-c-3 were planted on one-half-strength MS plates supplemented with DMSO or differentconcentrations of ABA. A, Imageswere taken after incubation on plates for 7 d. B, Germination greening ratiowasmeasured. Errorbars indicate SD (n = 3). Statistical analyses were performed between thewild type and abi5-7, the wild type and glk1 glk2wrky40abi5-c-1, the wild type and glk1 glk2 wrky40 abi5-c-2, and the wild type and glk1 glk2 wrky40 abi5-c-3 treated with 0.5 or 3 mMABA. *, P, 0.05, **, P, 0.01 (Student’s t test). C andD, Effect of exogenous ABA on root growth. Thewild type, abi5-7, glk1 gk2,wrky40, glk-1 glk-2 wrky40, glk1 glk2 wrky40 abi5-c-1, glk1 glk2 wrky40 abi5-c-2, and glk1 glk2 wrky40 abi5-c-3were plantedon MS plates for 3 d and transferred to MS medium containing 2% (w/v) Suc and 1% (w/v) agar with 15 or 30 mMABA for 7 d. C,Images were taken after incubation on plates for 7 d. D, The relative root growth was measured. Error bars indicate SD (n = 3). Thewild type and abi5-7, thewild type and glk1 glk2wrky40 abi5-c-1, thewild type and glk1 glk2wrky40 abi5-c-2, and thewild typeand glk1 glk2 wrky40 abi5-c-3were treatedwith 15 and 30 mMABA. **, P, 0.01 (Student’s t test). E, After ABA perception, ABAreceptor PYL/PYRs recruit PP2Cs, thereby activating SnRKs. GLK1/2 possibly could be activated via core ABA signaling com-ponents, PYL/PYRs-PP2Cs-SnRKs, and subsequently induce the expression of WRKY40 by directly binding to the promoter se-quence. WRKY40 further directly binds to the ABI5 promoter region to suppress the ABI5 expression.


Ahmad et al.



having a golden color phenotype in maize (Jenkins,1926). Several orthologous pairs of GLKs have beenidentified in rice (Oryza sativa), Arabidopsis, tomato(Solanum lycopersicum), and pepper (Capsicum annuum);these proteins exhibit overlapping expression profilesand redundant functions (Fitter et al., 2002; Powellet al., 2012; Brand et al., 2014). In Arabidopsis andmaize, the loss of function of GLK results in an abnor-mal morphology in chloroplast ultrastructure. In ad-dition to the role ofGLK genes in chloroplast biogenesis,overexpression of GLK1 confers enhanced resistance tononhost fungal pathogens and Cucumber mosaic virus(Savitch et al., 2007; Schreiber et al., 2011; Han et al.,2016). Moreover, Murmu et al. (2014) showed thatGLKs are involved in jasmonic acid-dependent sus-ceptibility to biotrophic pathogens and jasmonic acid-independent resistance to the necrotrophic pathogenBotrytis cinerea in Arabidopsis. In this study, we foundthat the glk1 glk2 double mutant showed ABA-hypersensitive response and osmotic stress-resistantphenotypes compared with glk1, glk2, and the wildtype. Accordingly, ectopic expression of GLK1 andGLK2 decreased the sensitivity to ABA and increasedthat to osmotic stress. These results indicate that GLK1and GLK2 negatively regulate ABA and osmotic stressresponses during seed germination and seedling de-velopment. In addition to the negative role of GLK1/2in ABA-mediated inhibition of seed germination andseedling growth, we also found that genes includingRbohB, RbohD, and peroxidase-superfamily-gene weredown-regulated in glk1 glk2. In accordance with thisresult, salt- and osmotic stress-induced superoxide andH2O2 accumulation were reduced in the glk1 glk2 dou-ble mutant compared with the wild type. These dataindicate that GLK1/2 positively impact ROS generationunder abiotic stress conditions. Considering the diverseroles of GLK1/2 in different biological processes(Savitch et al., 2007; Kakizaki et al., 2009; Waters et al.,2009; Kobayashi et al., 2013; Murmu et al., 2014; Leisterand Kleine, 2016; Nagatoshi et al., 2016), we concludethat the function of GLK1/2 in the response to ABA isuncoupled from that in the production of ROS.Genome-wide RNA-seq analysis of the glk1 glk2

double mutant revealed that GLK1/2 regulate some ofthe essential ABA-responsive genes. Previously, takingadvantage of the inducible expression system, Waterset al. (2009) showed that GLK1/2 regulate chlorophyllbiosynthesis, light harvesting, and electron transport atthe transcriptional level. Additionally, among the targetgenes of GLK1/2, Waters et al. (2009) also determinedthat some of the ABA- and stress-responsive genes,such as COR15A and COR15B, were rapidly and spe-cifically induced. Consistent with these results, weshowed that the induction of COR15A and COR15Bwas dramatically impaired in the glk1 glk2 doublemutant in the presence of ABA and confirmed the in-duction of COR15A and COR15B using the b-estradiol-inducible system. Intriguingly, we identified thatGLK1/2 directly affected the expression of WRKY40 inthe presence of ABA. Previously, it was reported that

the wrky40 mutant shows ABA-hypersensitive pheno-types during seed germination and seedling develop-ment (Chen et al., 2010; Shang et al., 2010). Additionally,previous ChIP-qPCR analyses show that WRKY40 di-rectly binds to the W-box motif located in the promoterof ABI5, thus directly repressing ABI5 expression (Chenet al., 2010; Shang et al., 2010). Our genetic interactionanalyses showed that the loss of function of ABI5 di-minished theABA-hypersensitive phenotype of glk1 glk2and wrky40, indicating that ABI5 functions geneticallydownstream ofGLK1/2 andWRKY40. Using ChIP-qPCRanalysis, we showed that GLK1/2 bind to the promoterof WRKY40 via a consensus GLK1/2-binding sequencein vivo. Moreover, we demonstrated that GLK1/2 andWRKY40 share common downstream target genes inresponse to ABA.In Arabidopsis, WRKY domain-containing proteins

constitute a superfamily of up to 100 representativeproteins and are widely known to function in plantdevelopment and defense response (Eulgem et al., 2000;Ülker and Somssich, 2004; Pandey and Somssich, 2009).Among these proteins, WRKY2, WRKY40, WRKY18,and WRKY60 function in ABA response (Jiang and Yu,2009; Chen et al., 2010; Shang et al., 2010). Strong ABA-hypersensitive phenotypes are observed in wrky40,wrky18, and wrky60 single mutants during seed ger-mination and seedling development, with the strongestphenotype observed in wrky40 (Shang et al., 2010).Moreover, the wrky40 wrky18 double mutant displaysstronger ABA hypersensitivity than wrky40 and wrky40wrky18 wrky60 mutants, indicating that WRKY40 playsa more important role in ABA response than otherWRKYs (Shang et al., 2010). Moreover, in this study,GLK1/2-regulated and PYR/PYL-regulated genesshowed the opposite correlation. Because the expres-sion of a subset of ABA-responsive genes was blockedin the glk1 glk2 double mutant, we propose that GLK1/2act as negative regulators of the PYL/PYR-mediatedABA signaling pathway. However, we did not detectputative phosphorylation target sites of SnRKs, whichare a core component of the PYR/PYL-PP2C-SnRKsignaling module in GLK1/2. In addition to the typicalSnRK2 phosphorylation sites, SnRK2s also recognizeatypical phosphorylation sites (Furihata et al., 2006;Sirichandra et al., 2010; Umezawa et al., 2013; Wanget al., 2013; Peirats-Llobet et al., 2016). Thus, it is pos-sible that GLK1/2 might be the direct targets of SnRK2via atypical sites. Considering this possibility, GLK1/2 may act as the upstream target of ABI5, whichcould be phosphorylated by SnRK2s. The PYR/PYL-PP2C-SnRK signaling module possibly activatesGLK1/2, thereby inducing the expression of WRKY40,a negative regulator, to prevent the overinductionof ABA-responsive genes. Another possibility isthat calcium-dependent protein kinases or mitogen-activated protein kinases, which are essential regula-tors of ABA signaling (Asano et al., 2012; de Zelicourtet al., 2016), directly phosphorylate GLK1/2 to regulatethe activity of GLK1/2. However, to confirm this pos-sibility, further investigation is needed. In conclusion,





we predict that this molecular mechanism helps main-tain plant fitness under changing environmentalconditions.

MATERIALS AND METHODS

Plant Growth Conditions and Genotyping

Arabidopsis (Arabidopsis thaliana) Columbia-0 (Col-0) plants were grown inthe greenhouse at 23°C, maintaining 60% relative humidity condition with160 mmol m22 s21 under a 16/8-h light/dark photoperiod for physiologicalexperiments (Moore et al., 2003). Col-0 single-knockout lines glk1 (At2g20570)and glk2 (At5g44190) and the glk1 glk2 double knockout line, N9805, N9806, andN9807, respectively, were obtained from the Nottingham Arabidopsis StockCentre and verified using GLK1-F/R and GLK2-F/R PCR primers by PCR-based genotyping. To generate the glk1 wrky40 and glk2 wrky40 double mu-tants and the glk1 glk2 wrky40 triple mutant, glk1, glk2, and glk1 glk2 mutants,respectively, were crossed with the wrky40 mutant (Xu et al., 2006; Liu et al.,2012), and mutants were screened using primers GLK1-F/R, GLK2-F/R, andWRKY40-F/R, respectively. All PCR primers used for genotyping are listed inSupplemental Table S9.

Construction of Plasmids and Generation ofTransgenic Plants

Gene-specific primers GLK1-C-F/R or GLK2-C-F/R were used to isolateGLK1 and GLK2 cDNA from a cDNA library by PCR. To generate the Pro-35s:GLK1-23FLAG and Pro-35s:GLK2-23FLAG constructs, full-length GLK1 orGLK2was amplified and cloned into pCsV1300 vector with a 2xFLAG tag usingXbaI and BamHI sites. To generate the Pro-35s:WRKY40-2Xflag construct, full-length WRKY40 cDNA was amplified by PCR using primers WRKY40-C-F/Rand cloned into pCsV1300 vector with a 2xFLAG tag usingXbaI and BamHI sites(Xu et al., 2012). To generate GD-GLK1 and GD-GLK2 constructs, full-lengthGLK1 or GLK2 was amplified by PCR using primers GLK1-GD-F/R or GLK2-GD-F/R and inserted into the pUC19 vector with an N-terminal GD tag usingNdeI and SacI sites (Wang et al., 2007). To generate ProSUPERR:sXVE:GLK1 andProSUPERR:sXVE:GLK2 constructs, full-lengthGLK1 orGLK2was amplified byPCR using primers GLK1-sXVE-F/R or GLK2-sXVE-F/R and inserted into thesXVE vector using XbaI and BamHI sites. To generate a ProWRKY40:LUCconstruct, an intact or mutated 0.5-kb WRKY40 promoter sequence carryingAAAAAA substitution was amplified usingXhoI and SpeI and inserted into theLUC reporter gene. To generate GLK1-GST and GLK2-GST constructs, full-length GLK1 or GLK2 was amplified by PCR using primers GLK1-GST-F/Ror GLK2-GST-F/R and inserted into the pGEX-4T1 vector using BamHI andEcoRI sites. To generate ProGLK1:GLK1-2xFLAG and ProGLK2:GLK2-2xFLAGconstructs, the genomic sequences ofGLK1 or GLK2 containing promoter regionswere amplified using GLK1-G-F/R (2xFLAG/XbaI/PstI) and GLK2-G-F/R(2xFLAG/XbaI/PstI) primers. Subsequently, the constructs were inserted into thebinary vector pCAMBIA1302 (Invitrogen). To knock out both ABI5 gene copies,three abi5-cr constructs were designed using the AtU6-26-sgRNA-SK andpCAMBIA1300-pYAO:Cas9 plasmids according to the method described previ-ously (Yan et al., 2015). A link to a Web tool for automated design of the targetsequence(s) is available at http://crispr.mit.edu/. The constructs were trans-formed into wild-type or glk1 glk2 wrky40 triple mutant plants. PCR and Sangersequencing were used to gain the mutation forms. T3 seeds were screened withhygromycin, and non-hygromycin-resistant seeds were used for the followingexperiments (Jia et al., 2016). The Pro-35s:WRKY40-2Xflag constructs were trans-formed into the glk1 glk2 double mutant. All primers are listed in SupplementalTable S9. Transgenic plants were grown on B5 plates treated with 50 mg L21

hygromycin (Clough and Bent, 1998).

Assays of Sensitivity to ABA and Osmotic Stress

For tests of theABAeffect ongermination, seedswere incubated at 4°C for 3dto break dormancy prior to germination, and then sterilized seeds were grownon one-half-strength MS medium with or without ABA for 7 d in a greenhouse.Germination ratios were calculated according to the percentages of cotyledongreening emergencies after seed germination (He et al., 2012; Kong et al., 2015;Wang et al., 2018a). For testing root elongation under ABA or osmotic stresstreatments, 3-d-old seedlings were transferred to MS medium containing 2%

(w/v) Suc and 1% (w/v) agar with different concentrations of ABA, NaCl, ormannitol in the incubator at 23°C, maintaining 60% relative humidity conditionwith 60 mmol m22 s21 under a 22/2-h light/dark photoperiod (He et al., 2012).

RNA Isolation and RNA-Seq Library Preparation

Total RNA was isolated from 10-d-old plants that were grown in liquid MSmedium treated with 10 mM ABA for 0, 1, or 3 h with Trizol (Invitrogen). Thegrowing condition was 160 mmol m22 s21 light intensity under a 16/8-h light/dark photoperiod (Moore et al., 2003). Materials were collected from three in-dependent biological replicates from Col-0, glk1 glk2, wrky40, and pyr1 pyl1 pyl2pyl4 mutants under different lengths of ABA exposure. At least 3 mg of RNAwas generated from each material for the next RNA-seq.

Bioinformatics Analyses of RNA-Seq Data

The RNAs were sequenced on the Illumina Hi-Seq platform, yielding morethan 10 million high‐quality, 150‐base, single‐end sequence reads(Supplemental Table S10). The Agilent 2100 Bioanalyzer (Agilent Technologies)was used to determine the quality and concentration of RNA. Sequencing wasperformed in paired-endmodewith a read length of 150 nucleotides. Next, low-quality (less than Q20) reads were excluded from raw data using the FASTX-Toolkit (version 0.0.13; http://hannonlab.cshl.edu/fastx_toolkit/). The cleanreads were mapped to the Arabidopsis reference genome (TAIR10) usingTopHat v.2.1.0 (Trapnell et al., 2009) with TAIR10 gene annotation as the transcriptindex. Gene quantification was performed using Cufflinks (http://ccb.jhu.edu/software/tophat/index.shtml and http://cole-trapnell-lab.github.io/cufflinks/)with genomic annotation from the TAIR10 genome release. TheDEGswerefilteredaccording to the fold change (|log2FC| . 1) and an adjusted P value (P , 0.05),calculated with Cuffdiff (a subpackage of Cufflinks; Yu et al., 2016, 2018). The GOgrouping of DEGs was performed by hypergeometric distribution in R (version3.1.0; https://www.r-project.org/; Lucent Technologies), with an adjusted P ,0.05 as a cutoff to determine significantly enrichedGO terms. Venn diagramsweregenerated using BioVenn (http://www.biovenn.nl/index.php).

Protoplast Isolation, Transfection, and GUS Activity Assay

Rosette leaves of 4-week-old Arabidopsis plants grown under short-dayconditions were used for isolation of protoplasts (Jin et al., 2001; Hyunjonget al., 2006). For testing the GUS activity, effector plasmids encoding the full-length protein of GLK1 or GLK2 fused in frame with GD or transactivator GD-VP16 were cotransfected with reporter Gal4-35S:GUS into protoplasts and in-cubated under darkness for 20 to 23 h. GUS activities were measured using aFluoroskan Finstruments microplate reader (MTX Lab Systems; Tiwari et al.,2003; Wang et al., 2005).

Induced Transgene Expression in Stably TransformedArabidopsis Plants

To induce transgene expression in stably transformed seedlings, selectedplants transformed with ProSUPERR:sXVE:GLK1, ProSUPERR:sXVE:GLK2, orProSUPERR:sXVE:GFP constructs were grown in one-half-strength MS liquidmedium and then supplemented with 20 mM b-estradiol and induced for 4 h(Schlücking et al., 2013).

Transient Dual-Luciferase Reporter System

To examine reporter gene expression, ProWRKY40:LUC orMProRKY40:LUCwas cotransformed with effector gene ProSUPERR:sXVE:GLK1 or ProSUPERR:sXVE:GLK2 protoplasts and incubated for 23 h followed by 4-h treatment with20 mM b-estradiol (Schlücking et al., 2013). Transformed protoplasts werepelleted by low-speed centrifugation (500 rpm). Total RNAwas prepared usingan RNA extraction kit (Ambion) and used for RT-qPCR analysis by the com-parative cycle threshold method, in which LUC transcript levels were nor-malized using the GUS transcript. The specific primers used for real-time PCRare listed in Supplemental Table S9.


Ahmad et al.



http://crispr.mit.edu/




http://hannonlab.cshl.edu/fastx_toolkit/

http://ccb.jhu.edu/software/tophat/index.shtml

http://ccb.jhu.edu/software/tophat/index.shtml

http://cole-trapnell-lab.github.io/cufflinks/

https://www.r-project.org/

http://www.biovenn.nl/index.php



ChIP Assays

The ChIP assay was performed according to the method described previously(Haringet al., 2007)with a slightmodification (Liu et al., 2018a, 2018b). Two-week-oldtransgenic plants under the treatment of ABA for 0 and 6 h were selected for ChIP-qPCR. The antibody anti-FLAG (F1804; Sigma) was added to the chromatin, whichwas isolated and sheared to 200 to 1,200 bp with an FB120 Sonic Dismembrator(Fisher Scientific) for an overnight incubation at 4°C. The antibody-protein complexeswere isolated by binding to protein A beads. The DNA fragments in the immuno-precipitated complexes were released by reversing the cross-linking at 65°C for 8 hand then extracted with phenol/chloroform, precipitated in ethanol, and resus-pended in water. The specific primers used for real-time PCR are listed inSupplemental Table S9. ACT7was used as a negative control.

Pull-Down Assay and EMSA

For the protein pull-down experiment, GST alone or GST-GLK1/2 (3 mg)was induced via the Escherichia coli BL21 (DE3) cell line and immobilized ontoglutathione beads. The beads were then washed three times (Xu et al., 2013).The purified proteinwas confirmed by SDS-PAGE and prepared for EMSA. Theprobes were synthesized and labeled with biotin by Sangon Biotechnology.Double-strand probe (50 fmol) was incubated with protein in binding buffer for10 min. The mixture was then separated by nondenatured 6% native polyac-rylamide gel with 0.53 Tris-borate/EDTA for 30 min, and the protein wastransferred to a nylon membrane via wet transfer and detected according to theinstructions provided with the EMSA kit (Beyotime; Guo et al., 2017).

DAB and NBT Staining Assay

The DAB and NBT staining assay was performed according to the methoddescribed previously with a slight modification (Nguyen et al., 2017). Five-day-old seedlings grown in liquid MS medium were treated with or without100 mM NaCl or mannitol for 12 h. To detect O2

2, treated plants were vac-uumed infiltrated for 3 min and then stained for 4 h with 0.05% (w/v) NBT and10 mM NaN3 in 10 mM potassium phosphate (pH 7.8). To detect H2O2, plantswere vacuumed for 3 min and then stained for 12 h with 0.1% DAB (pH 5.8).After staining, the seedlings were destained by boiling in ethanol:lactic acid:glycerol (3:1:1) solution until colorless (Nguyen et al., 2017).

Drought Tolerance Assay

To test for drought tolerance, plantswere grown on soil in a greenhousewith160 mmol m22 s21 under a 16/8-h light/dark photoperiod (23°C, 60% relativehumidity) for 2 weeks, the water was withheld from 14-d-old plants for 14 d,and the survival rates of plants were determined 2 d after rewatering (rehy-dration; Kang et al., 2002; Xu et al., 2012).

Protein Extraction and Western-Blot Assay

Preparation of protein extracts and western blots were performed accordingto the method described previously with a slight modification (Xu et al., 2013).Briefly, proteins from transgenic plants, the wild type, and mutants wereextracted in a lysis buffer and then separated on 10% SDS-polyacrylamide gels.The proteins on gels were transferred to a polyvinylidene difluoride membraneby semidry electroblotting. The membrane was blocked and incubated with theFLAG or ACTIN antibody and detected with an enhanced chemiluminescencedetection kit.

Accession Numbers

Data generated in this study are deposited in the National Center for Bio-technology Information Sequence Read Archive (accession nos. PRJNA513154and PRJNA513157).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. GLK1/2 overexpression transgenic plants showedABA-hyposensitive phenotype.

Supplemental Figure S2. Lack of GLK1/2 reduced primary seeddormancy.

Supplemental Figure S3. GO analysis.

Supplemental Figure S4. GLK1/2 overexpression transgenic plants showedhypersensitivity to salt and osmotic stresses.

Supplemental Figure S5. GLK1/2 play negative redundant roles underosmotic and dehydration stress conditions.

Supplemental Figure S6. Generation of GLK1/2 transient inductionsystem.

Supplemental Figure S7. Generation of GLK1/2 complementation lines.

Supplemental Figure S8. GLK1/2 transcript levels are regulated by PYL/PYR ABA receptors in response to ABA.

Supplemental Figure S9. Isolating WRKY40 overexpression lines.

Supplemental Figure S10. Generation of CRISPR/Cas9 (abi5-cr) lines.

Supplemental Table S1. DEGs between the wild type and glk1 glk2 undernormal conditions.

Supplemental Table S2. DEGs between the wild type and glk1 glk2 underABA treatment for 1 h.

Supplemental Table S3. GLK1/2-regulated genes stratified into differentbiological processes.

Supplemental Table S4. Overlap genes between DRGs in glk1 glk2 andURGs in GLK1/2 target genes.

Supplemental Table S5. Promoter sequence information of genes thatwere stratified into response to abiotic stimulus and response to waterdeprivation GO terms.

Supplemental Table S6. DEGs between the wild type and glk1 glk2 underABA treatment for 0, 1, or 3 h.

Supplemental Table S7. DEGs between the wild type and wrky40 underABA treatment for 0, 1, or 3 h.

Supplemental Table S8. DEGs between the wild type and pyr1 pyl1 pyl2pyl4 under ABA treatment for 3 h.

Supplemental Table S9. Primer sequences used in different experiments.

Supplemental Table S10. Read numbers and data size of RNA-seq data indetail.

ACKNOWLEDGMENTS

We thank Dr. Da-Peng Zhang (Systems Biology and Bioinformatics Labo-ratory of the Ministry of Education, School of Life Sciences, Tsinghua Univer-sity) for generously providing the wrky40 mutants and Dr. Jian-Kang Zhu(Shanghai Center for Plant Stress Biology and Chinese Academy of SciencesCenter of Excellence inMolecular Plant Sciences, Chinese Academy of Sciences)for generously providing the pyr1 pyl1 pyl2 pyl4 mutants.

Received November 27, 2018; accepted January 29, 2019; published February 5,2019.

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GOLDEN2-LIKE Transcription Factors Regulate WRKY40 · GOLDEN2-LIKE Transcription Factors RegulateWRKY40 Expression in Response to Abscisic Acid1[OPEN] RaﬁqAhmad,2 Yutong Liu,2 Tian-Jing