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. 2019 Feb 5;179(4):1844–1860. doi: 10.1104/pp.18.01466

GOLDEN2-LIKE Transcription Factors Regulate WRKY40 Expression in Response to Abscisic Acid1,[OPEN]

Rafiq Ahmad 1,2, Yutong Liu 1,2, Tian-Jing Wang 1,2, Qingxiang Meng 1, Hao Yin 1, Xiao Wang 1, Yifan Wu 1, Nan Nan 1, Bao Liu 1, Zheng-Yi Xu 1,3,4
PMCID: PMC6446771  PMID: 30723180

The GARP (Golden2, ARR-B, Psr1) family transcription factors GOLDEN2-LIKE 1 and -2 (GLK1/2) modulate the ABA response.

Abstract

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 to abscisic acid (ABA) remain unknown. In this study, we used a glk1 glk2 double mutant to examine the role of GLK1/2 in the ABA response. The glk1 glk2 double mutant displayed ABA-hypersensitive phenotypes during seed germination and seedling development and an osmotic stress-resistant phenotype during seedling development. Genome-wide RNA sequencing analysis of the glk1 glk2 double mutant revealed that GLK1/2 regulate several ABA-responsive genes, including WRKY40, in the presence of ABA. Chromatin immunoprecipitation and gel retardation assays showed that GLK1/2 directly associate with the WRKY40 promoter via the recognition of a consensus sequence. Additionally, RNA sequencing analysis of the glk1 glk2 double mutant and wrky40 single mutant revealed that GLK1/2 and WRKY40 control a common set of downstream target genes in response to ABA. Furthermore, results of a genetic interaction test showed that the glk1 glk2 wrky40 triple mutant displayed similar ABA hypersensitivity 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, we propose that the GLK1/2-WRKY40 transcription module plays a negative regulatory role in the ABA response.

The phytohormone abscisic acid (ABA) plays essential roles in the induction of stomatal closure and other adaptive responses under environmental stresses, thus regulating optimal plant growth and development (McAinsh et al., 1990; Leung and Giraudat, 1998; Borsani et al., 2002; Finkelstein et al., 2002; Xiong and Zhu, 2002; Nambara and Marion-Poll, 2005; Zhu, 2016; Singh et al., 2017; Sussmilch et al., 2017). ABA also plays important biological roles in the maintenance of seed dormancy, inhibition of seed germination, acceleration of senescence, and induction of stress tolerance (Zeevaart and 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). Genetic screening of seeds for sensitivity to ABA during germination has led to the identification of several key modulators in the ABA signaling pathway, including ABA INSENSITIVE1 (ABI1), ABI2, ABI3, ABI4, and ABI5 (Koornneef et al., 1984; Finkelstein, 1994; Nakashima and Yamaguchi-Shinozaki, 2013). Among these, ABI1 and ABI2, type 2C proteins with negative regulatory roles in ABA signaling, physically interact with and inhibit downstream targets, such as the Ser/Thr protein kinase OPEN STOMATA1 (OST1; Assmann, 2003; Yoshida et al., 2006; Vlad et al., 2009); ABI3 encodes a transcription factor that shares high homology with maize (Zea mays) viviparous1 (Giraudat et al., 1992); ABI4 is a member of the ERF/AP2 transcription factor family (Finkelstein et al., 1998); and ABI5 is a basic Leu zipper transcription factor that is phosphorylated by SRK2D/SnRK2.2, SRK2E/SnRK2.6/OST1, and SRK2I/SnRK2.3 to regulate the expression 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 factors, including CBF/DREB, WRKY, AP2, RD22BP, MYBs, NAC, and ABF/AREB, have been extensively studied at the transcriptional level (Kizis et al., 2001; Xiong and Zhu, 2002; Yamaguchi-Shinozaki and Shinozaki, 2006; Jiang et al., 2017). Recently, it was shown that transcription factor hierarchy is essential for defining environmental stress and the ABA response network (Song et al., 2016). The WRKY proteins constitute a large family of transcription factors that are evolutionarily conserved in lower and higher plants (Eulgem et al., 2000). WRKY proteins contain a conserved WRKY DNA-binding domain of approximately 60 amino acids, followed by a C2H2 or C2HC zinc-finger motif (Eulgem et al., 2000), and exhibit strong binding affinity toward the W-box motif (C/T)TGAC(T/C; Eulgem et al., 2000; Ülker and Somssich, 2004). The Arabidopsis (Arabidopsis thaliana) genome harbors 74 WRKY genes, and different WRKY transcription factors play positive or negative regulatory roles in abiotic stress and ABA response (Marè et al., 2004; Xie et 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, hormone 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) genes encode GARP nuclear transcription factors, which are B-type Arabidopsis response regulators (ARRs; Riechmann et al., 2000). The GLK genes have been shown to function in chloroplast development in Arabidopsis, 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 also play important roles in photosynthesis, defense response, 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; Leister and Kleine, 2016; Nagatoshi et al., 2016). In Arabidopsis, GLK genes are functionally redundant, as shown by the glk1 glk2 double mutant, which exhibits a perturbed phenotype (Fitter et al., 2002; Yasumura et al., 2005; Waters et al., 2008). The GLK genes contain two highly conserved domains at the C terminus: GCT-box and DNA-binding domain (Rossini et al., 2001). However, the role that GLK transcription factors specifically play in the ABA response and the underlying molecular mechanisms remain unknown.

In this study, we show that the glk1 glk2 double mutant displays an ABA-hypersensitive phenotype, while transgenic plants overexpressing GLK1/2 (Pro-35S:GLK1/2-2xFlag) show an ABA-hyposensitive phenotype, during seed germination and seedling development. Genome-wide transcriptome analysis of the glk1 glk2 double mutant treated with or without ABA revealed that GLK1/2 are required for the regulation of essential ABA-responsive genes. Intriguingly, we found GLK1/2 to specifically activate the expression of WRKY40, which plays an important negative regulatory role in ABA response. Chromatin immunoprecipitation (ChIP) experiments showed that GLK1/2 directly associate with the WRKY40 promoter through the recognition of a consensus sequence, resulting in the activation of gene transcription. Results of genetic analysis showed that the glk1 glk2 wrky40 triple mutant displayed similar ABA hypersensitivity to the wrky40 single mutant and the glk1 glk2 double mutant. By contrast, the glk1 glk2 wrky40 abi5-c (ABI5 CRISPR/Cas9 mutant) quadruple mutant suppressed the ABA-hypersensitive phenotypes of glk1 glk2 wrky40 and displayed similar ABA hyposensitivity to the abi5-7 single mutant. Based on these results, we propose that the GLK1/2-WRKY40 transcription module plays a negative regulatory role in the ABA response.

RESULTS

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

To evaluate the role of GLK1/2 during seed germination, wild-type, glk1 and glk2 single mutant, and glk1 glk2 double mutant seeds were grown in different ABA concentrations (Xu et al., 2013), and the germination greening ratio (calculating cotyledons greening after seed germination) was measured (He et al., 2012; Kong et al., 2015; Wang et al., 2018b). The glk1 and glk2 single mutants did not display a noticeable phenotype compared with the wild type; however, the glk1 glk2 double mutant showed increased ABA sensitivity (Fig. 1, A and B). To further examine the role of GLK1/2 in seedling development, 3-d-old seedlings grown on Murashige and Skoog (MS) medium were transferred to medium containing different concentrations of ABA (containing full MS, 2% [w/v] Suc, 1% [w/v] agar, and 0, 15, or 30 μM ABA) for 7 d. Root growth of the glk1 glk2 mutant was dramatically retarded at different ABA concentrations compared with the wild type and the glk1 and glk2 single mutants (Fig. 1, C and D). To confirm these results, we evaluated the ABA sensitivity of transgenic plants overexpressing GLK1 and GLK2. Ectopic expression of GLK1 or GLK2 was achieved using the strong cassava vein mosaic virus promoter (Supplemental Fig. S1A). Two independent transgenic overexpressing lines of each gene, ProGLK1:GLK1-2xFlag (GLK1OX-1 and GLK1OX-2) and ProGLK2:GLK2-2xFlag (GLK2OX-1 and GLK2OX-2), were used for seed germination assays. All transgenic lines displayed an ABA-hyposensitive phenotype compared with the wild type in terms of seed germination (Supplemental Fig. S1, B and C). We also examined the growth of transgenic GLK1OX and GLK2OX seedlings by transferring 3-d-old seedlings to MS medium containing 2% (w/v) Suc, 1% (w/v) agar, and 15 or 30 μM ABA for 7 d. Transgenic GLK1OX and GLK2OX seedlings showed ABA-hyposensitive phenotypes compared with wild-type seedlings (Supplemental Fig. S1, D and E). Taken together, these results indicate that Arabidopsis GLK1/2 are involved in the response to ABA.

Figure 1.

Figure 1.

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GLK1/2 play negative roles in ABA responses. A and B, Effect of exogenous ABA on seed germination. Seeds of the wild type (WT), glk1, glk2, and glk1 glk2 mutants 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 greening ratio was measured. Error bars indicate sd (n = 3). Statistical analyses were performed between the wild type and glk1 glk2 treated with 0.5 or 1 μM ABA. P1 = 0.00445 and P2 = 0.00393 (Student’s t test). C and D, Effect of exogenous ABA on root growth. The wild type, glk1, glk2, and glk1 glk2 mutants grown on MS plates for 3 d were transferred to MS medium containing 2% (w/v) Suc, 1% (w/v) agar, and 0, 15, or 30 μM ABA for 7 d. C, Images of plants were taken at 7 d after transfer. D, To quantify root growth, primary root length was measured on 7-d-old plants after transfer. Three independent experiments were performed using 20 plants per experiment. Error bars indicate sd (n = 3). Statistical analyses were performed between the wild type and glk1 glk2 treated with 15 or 30 μM ABA. **, P < 0.01 (Student’s t test).

To examine whether GLK1/2 play a regulatory role during seed dormancy, we compared the germination ability of freshly harvested wild-type, glk1, glk2, and glk1 glk2 seeds sown on MS medium; this experiment was conducted under both light and dark and with and without prechilling for 3 d at 4°C (Ding et al., 2014), because the strength of primary dormancy is reflected by the degree of requirement for light and/or chilling to promote germination (Finkelstein, 1994). Results showed that the germination rate of glk1 glk2 seeds was lower than that of wild-type seeds when exposed to light without prechilling, indicating that the double mutant seeds were more dormant than wild-type seeds (Supplemental Fig. S2A). All mutant and wild-type seeds germinated after prechilling treatment under light (Supplemental Fig. S2B). We observed that both wild-type and glk1 glk2 seeds germinated in the dark within a single day following prechilling, whereas glk1 glk2 seeds were more dormant (Supplemental Fig. S2C). However, in the absence of light and prechilling, most seeds failed to germinate (Supplemental Fig. S2D). These results indicate that the glk1 glk2 double mutant showed increased seed dormancy over the wild type and the glk1 and glk2 single mutants.

GLK1/2 Modulate ABA-Mediated Regulation of Gene Expression

To evaluate the effect of GLK1/2 on ABA-mediated transcriptional regulation, we performed RNA sequencing (RNA-seq) analysis of wild-type and glk1 glk2 seedlings treated with or without 10 μM ABA for 1 h. Using stringent statistical and filtering criteria, we identified 1,718 differentially expressed genes (DEGs) in the comparison between glk1 glk2 and wild-type seedlings treated with ABA, of which 877 were up-regulated (URGs) and 841 were down-regulated (DRGs) in the double mutant compared with the wild type. In the comparison between glk1 glk2 and wild-type seedlings without ABA treatment, 1,238 DEGs were identified, of which 394 were URGs and 844 were DRGs in the double mutant compared with the wild type (Fig. 2, A and B; Supplemental Tables S1 and S2). Our data suggest that, under normal conditions, GLKs positively regulate a number of biological processes, including photosynthesis and response to abiotic stress stimuli, such as oxidation, wounding, water deprivation, and hyperosmotic stress (Supplemental Fig. S3; Supplemental Table S3), and negatively regulate other biological processes, such as microtubule-based events, 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 of GLK1/2 by taking advantage of a temporal induction system. Comparison of DRGs with microarray data showing target genes of GLK1/2 revealed that 27.4% of the GLK1 target genes and 52% of the GLK2 target genes overlapped with DRGs (Supplemental Table S4). These overlapping genes mostly function in chlorophyll biosynthesis, light harvesting, and electron transport (Waters et al., 2009). After 1 h of ABA treatment, GLKs showed positive regulation of some biological processes, such as gene transcription and response to oxidation/reduction, hormone stimulus, hormone-mediated signaling, and water deprivation (Supplemental Fig. S3; Supplemental Table S3), and negative regulation of other biological processes, including oxidation/reduction, translation, development, glucan metabolism, DNA replication, and lipid biosynthesis (Supplemental Fig. S3; Supplemental Table S3). To confirm the results of RNA-seq analysis, expression of a few ABA- and other abiotic stress-responsive genes, which were categorized into Gene Ontology (GO) terms such as response to abiotic stimulus and response to water deprivation under ABA 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 induced in wild-type seedlings after ABA treatment but dramatically impaired in glk1 glk2 double mutants (Fig. 2C). Previously, using an inducible system and transcriptome analysis, Waters et al. (2009) showed that COR15A and COR15B 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 network, including ABI5 (Lopez-Molina et al., 2001), PYL11 (Lim and Lee, 2015), ABI3 (Giraudat et al., 1992), and DARK INDUCIBLE11 (DIN11; Fujiki et al., 2001), were negatively regulated by GLK1/2 in response to ABA (Fig. 2C). These results indicate that GLK1/2 control the expression of multiple essential ABA-responsive genes.

Figure 2.

Figure 2.

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GLK1/2 direct the transcriptional networks of ABA response genes. A and B, Bioinformatic analysis of DEGs. A, Venn diagrams showing overlap of DEGs of DRGs and URGs between the wild type (WT) and glk1 glk2 under ABA treatment for 0 and 1 h. B, Hierarchical clustering analyses of DRGs and URGs under ABA treatment for 0 and 1 h. C, RT-qPCR analysis of abiotic stress-responsive genes. GAPDH was used as an internal control. Error bars indicate sd (n = 3).

GLK1/2 Negatively Regulate Osmotic and Dehydration Stress Responses

To gain further insights into the biological role of GLK1/2 in the response to osmotic stress, plants grown on MS plates showing the same root length were transferred to MS medium containing 2% (w/v) Suc, 1% (w/v) agar, and mannitol (0, 100, or 150 mM) or NaCl (0, 100, or 150 mM), and root growth was evaluated. Root growth did not display noticeable variation among wild-type, glk1, glk2, and glk1 glk2 seedlings; however, under osmotic and salt stress conditions, glk1 glk2 showed relatively longer roots than wild-type, glk1, and glk2 seedlings (Fig. 3). We also examined the phenotypes of GLK1OX and GLK2OX transgenic plants. Both GLK1OX and GLK2OX transgenic lines showed hypersensitivity to salt and osmotic stresses (Supplemental Fig. S4). Reactive oxygen species (ROS) are produced in different organelles under abiotic stress conditions because of metabolic imbalance (Skopelitis et 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 accumulation leads 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 enriched among the DRGs (P < 0.01; Supplemental Table S3), we examined salt- and osmotic stress-induced production of superoxide and hydrogen peroxide (H2O2) in wild-type and glk1 glk2 double mutant seedlings by staining with nitroblue tetrazolium (NBT) or 3,3′-diaminobenzidine (DAB). In the absence of osmotic stress, staining with DAB or NBT showed no differences between wild-type and glk1 glk2 seedlings (Supplemental Fig. S5, A and B). However, under osmotic stress, wild-type seedlings showed more intense staining with DAB and NBT than glk1 glk2 seedlings (Supplemental Fig. S5, A and B), indicating that the glk1 glk2 double mutant exhibits higher ROS detoxification capability than the wild type. These data suggest that, in addition to the role of GLK1/2 in ABA response, GLK1/2 also regulate the expression of genes in response to ROS accumulation induced by osmotic stress. We also examined the response of mutant and wild-type seedlings in response to dehydration stress and observed that the survival rate of glk1 glk2 seedlings was significantly higher than that of wild-type, glk1, and glk2 seedlings (Supplemental Fig. S5, C and D). These results indicate that GLK1/2 are redundant and play negative regulatory roles in response to osmotic and dehydration stresses.

Figure 3.

Figure 3.

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GLK1/2 play negative roles in osmotic and salt stress responses. A and B, Effect of osmotic stress on root growth. The wild type (WT), glk1, glk2, and glk1 glk2 mutants grown on MS plates for 3 d were transferred to MS medium containing 2% (w/v) Suc, 1% (w/v) agar, and mannitol (0, 100, or 150 mM) for 7 d. A, Images of plants were taken at 7 d after transfer. B, To quantify root growth, primary root length was measured on 7-d-old plants after transfer. Three independent experiments were performed using 20 plants per experiment. Error bars indicate sd (n = 3). Statistical analyses were performed between the wild type and glk1 glk2 treated with 100 and 150 mM mannitol. *, P < 0.05, **, P < 0.01 (Student’s t test). C and D, Effect of salt stress on root growth. The wild type, glk1, glk2, and glk1 glk2 mutants grown on MS plates for 3 d were transferred to MS medium containing 2% (w/v) Suc, 1% (w/v) agar, and NaCl (0, 100, or 150 mM) for 7 d. C, Images of plants were taken at 7 d after transfer. D, To quantify root growth, primary root length was measured on 7-d-old plants after transfer. Three independent experiments were performed using 20 plants per experiment. Error bars indicate sd (n = 3). Statistical analyses were performed between the wild type and glk1 glk2 treated with 100 or 150 mM NaCl medium. **, P < 0.01 (Student’s t test).

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

To investigate the transcriptional regulatory mode of GLKs, we employed a protoplast transfection system (Wang et al., 2007). In this system, the GUS reporter gene was driven by the minimal 35S promoter (−46 to 0 bp) together with a cis-acting regulatory sequence that could be recognized by the GAL4 DNA-binding domain (named as reporter; Fig. 4A; Wang et al., 2007). In addition, the GAL4 DNA-binding domain was fused to the herpes simplex virus VP16 activation domain (GD-VP16; positive control) or to full-length GLK1 or GLK2 protein (named as effector; Fig. 4A). We cotransfected the reporter construct together with effector constructs encoding GD-VP16, GD-GLK1, or GD-GLK2 fusion proteins. Compared with the negative controls, GD-GLK1 and GD-GLK2 fusion proteins dramatically induced the expression of the GUS reporter gene (Fig. 4B). This result indicates that GLK1 and GLK2 possess transcriptional activation capability.

Figure 4.

Figure 4.

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GLK1/2 activate primary ABA response genes. A and B, GLK1/2 are transcriptional activators. A, Effectors and reporter constructs used in the transfection assays. GD, GAL4 DNA-binding domain; VP-16, herpes simplex virus VP16 activation domain. B, GD, GD-GLK1/2, and GD-VP16 were cotransfected with the reporter GAL4-GUS, and GUS activity was assayed after protoplasts were incubated in darkness for 20 to 23 h. Error bars indicate sd (n = 3). C, Temporal induction of GLK1/2 activates the expression of ABA response genes. β-Estradiol (20 μM) was introduced for 4 h, and expression of WRKY40, RbohB, COR15A, and COR15B was measured using RT-qPCR analysis. Error bars indicate sd (n = 3).

Next, to test the in vivo transcriptional activation capabilities of GLK1 and GLK2, we took advantage of the β-estradiol-inducible system (Schlücking et al., 2013). This system contains successive transcription units in which the G10-90 promoter controls the expression of a chimeric XVE fusion protein (Ishige et al., 1999; Schlücking et al., 2013), which is composed of the DNA-binding domain of the bacterial repressor LexA (X), a transactivating domain of VP16 (V; Dalrymple et al., 1985), and the C-terminal region of the estrogen receptor from human (E; Green et al., 1986). In the presence of β-estradiol, the hormone binds to the receptor domain, which leads to a conformational change, thereby enabling the DNA-binding domain to bind to the LexA operator, which activates the minimal 35S promoter (Benfey and Chua, 1990). Results of RT-qPCR showed that treatment with different concentrations of β-estradiol (0, 1, 10, 20, 50, and 100 μM) for 4 h induced the expression of GLK1, GLK2, and the GFP gene in a dose-dependent manner (Supplemental Fig. S6). Since the induction kinetics of GLK1 and GLK2 were saturated at 20 μm β-estradiol, we examined the expression of stress-responsive genes, including WRKY40, RbohB, COR15A, and COR15B, at this concentration (Fig. 4C). We found that all these genes were rapidly induced upon the temporal induction of GLK1 and GLK2. However, no noticeable alterations in GFP induction were detected, indicating that GLK1/2 specifically induce the expression of these genes.

GLK1/2 Control the Expression of WRKY40 by Directly Binding to the Consensus Sequence Located in Its Promoter Region

Previously, GLK1/2 were shown to specifically recognize a consensus sequence, CCAATC, in the promoter region of target genes (Waters et al., 2009; Guo et al., 2018). Since GLK1/2 are transcriptional activators, we investigated the direct target genes of GLK1/2 in response to ABA. We analyzed the promoter sequences (−500 to 0 bp) of DRGs identified in glk1 glk2 seedlings treated with ABA and categorized under the GO terms response to abiotic stimulus and response to water deprivation. A total of 13 genes, including WRKY40, NRT1.1, RAB18, COR413, and PUB22, harbored the consensus CCAATC motif in the promoter regions (Supplemental Table S5). We further investigated the WRKY40 gene, as it was the only gene among the 13 genes that has been previously reported to negatively regulate ABA response during seed germination and seedling development (Chen et al., 2010) and therefore was the most likely target of GLK1/2. We found that the WRKY40 promoter harbors the CCAATC motif in the region from −500 to 0 bp (Fig. 5A). To examine whether this sequence is required for GLK1/2-mediated transcriptional activation, we generated a chimeric reporter construct by fusing 500 bp of the WRKY40 promoter (−500 to 0 bp) with the luciferase (LUC) reporter gene (ProWRKY40:LUC). Another construct was generated using a variant of the WRKY40 promoter, which carried a mutation in the GLK1/2-binding motif (MProWRKY40:LUC). We cotransfected ProWRKY40:LUC or MProWRKY40:LUC with ProUBQ10:GUS, a chimeric construct of GUS under the control of the UBQ10 promoter, into Arabidopsis protoplasts isolated from transgenic plants expressing ProSUPERR:sXVE-GFP, ProSUPERR:sXVE-GLK1, or ProSUPERR:sXVE-GLK2 using the polyethylene glycol-mediated transformation method (Fig. 5A; Xu et al., 2013). Induction of GLK1 or GLK2 by β-estradiol dramatically activated LUC expression in protoplasts transfected with ProWRKY40:LUC but not in protoplasts transfected with MProWRKY40:LUC (Fig. 5B). This result indicates that the consensus GLK1/2 recognition site is required for GLK1/2-mediated transcriptional activation.

Figure 5.

Figure 5.

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GLK1/2 bind to the promoter region of WRKY40 and activate the expression via recognition of the consensus sequence. A and B, Transcriptional activation of the WRKY40 promoter by GLK1/2 via recognition of the consensus sequence. A, Protoplasts from transgenic plants harboring ProSUPERR:sXVE-GFP, ProSUPERR:sXVE-GLK1, and ProSUPERR:sXVE-GLK2 were cotransformed with reporter containing WRKY40 promoter sequence and normalizing plasmids, incubated for 23 h, and then incubated an additional 4 h with 20 μm β-estradiol. Mutant, Consensus sequence CCAATC located at the WRKY40 promoter region mutated as AAAAAA; WT, original WRKY40 promoter sequence including consensus CCAATC. B, The LUC activity was normalized by GUS activity. Error bars indicate sd (n = 3). **, P < 0.01 (Student’s t test). C and D, GLK1/2 bind to the promoter region of WRKY40. C, Schematic representation of WRKY40 promoter with GLK1/2 binding site (P2) and non-GLK1/2 binding site (P1). D, Transgenic plants expressing genomic DNA of GLK1 (ProGLK1:GLK1-2xFLAG) or GLK2 (ProGLK2:GLK2-2xFLAG) driven by their native promoters were used to perform ChIP-qPCR at different time points under the condition of ABA treatment. ChIP-qPCR was performed with (with Ab) or without (no Ab) FLAG antibody. **, P < 0.01 (Student’s t test). E, Coomassie Blue-stained gel showing levels of recombinant GST proteins used in EMSA. F, EMSA analysis of the binding of recombinant GLK1/2 protein to the promoter of WRKY40. mProbe is the biotin-labeled probe with a mutation from CCAATC to CCGGTC.

Next, to examine whether GLK1/2 bind to the promoter region of WRKY40, we fused GLK1 or GLK2 genomic DNAs (gGLK1 or gGLK2) with two FLAG epitopes (2×FLAG) at the C terminus and cloned gGLK1-2×FLAG and gGLK2-2×FLAG fusions under the control of native promoters (ProGLK1:gGLK1-2×FLAG and ProGLK2::gGLK2-2×FLAG). These constructs were introduced into the glk1 glk2 double mutant separately. Both ProGLK1:gGLK1-2×FLAG and ProGLK2::gGLK2-2×FLAG complemented the phenotype of the glk1 glk2 double mutant (Supplemental Fig. S7, A and B). Moreover, the expression of stress-responsive genes that was increased or reduced in the glk1 glk2 double mutant was recovered to the 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 CCAATC motif was significantly enriched, while the nonconsensus CCACTC sequence showed no enrichment (Fig. 5, C and D). Additionally, to determine the in vitro binding of GLK1/2 to the consensus sequence, we performed electrophoretic mobility shift assays (EMSAs). Full-length GLK1/2 proteins tagged with glutathione S-transferase (GST-GLK1 and GST-GLK2) were capable of binding to probes containing P2 (CCAATC), whereas GST alone did not bind to the probes (Fig. 5, E and F). When a mutant probe (CCGGTC) was used for EMSA, the binding activity of GST-GLK1/2 fusion proteins was diminished (Fig. 5F). Collectively, these results indicate that GLK1/2 bind to the promoter of WRKY40 via the consensus sequence.

GLK1/2 and WRKY40 Regulate Common Target Genes in Response to ABA

To examine whether GLK1/2 and WRKY40 control common target genes in response to ABA, we performed RNA-seq analysis of the glk1 glk2 double mutant and wrky40 single mutant treated with 10 μm ABA for 0, 1, or 3 h. Using stringent statistical and filtering criteria, we identified URGs and DRGs in glk1 glk2 versus the wild type and wrky40 versus the wild type comparisons under different lengths of ABA exposure. A substantial number of URGs and DRGs were shared in each time point with statistical significance (Fig. 6A; Supplemental Tables S6 and S7). Hierarchical clustering analysis showed that GLK1/2 and WRKY40 systematically impact the transcription of common gene targets in response to ABA (Fig. 6B; Supplemental Tables S6 and S7). Furthermore, scatterplot analysis showed positive correlations between URGs and DRGs in glk1 glk2 and wrky40 mutants under ABA treatment for 0, 1, and 3 h (Fig. 6C; Supplemental Tables S6 and S7). We further confirmed RNA-seq data using RT-qPCR. Results of RT-qPCR analysis showed that ABI5 and DIN11 were up-regulated, while RbohB and COR15A were down-regulated, in glk1 glk2 and wrky40 (Fig. 6D). Of note, we detected similar expression patterns in the glk1 glk2 wrky40 triple mutant, suggesting that GLK1/2 and WRKY40 act in the same genetic pathway to regulate the expression of target genes. Taken together, these results suggest that GLK1/2 and WRKY40 share common target genes in response to ABA.

Figure 6.

Figure 6.

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GLK1/2 and WRKY40 direct similar transcriptional networks of ABA response genes. A and B, Bioinformatic analysis of DEGs. A, Venn diagram showing overlap of DEGs between glk1 glk2 and wrky40 under ABA treatment for 0, 1, or 3 h. P values were calculated with two-tailed hypergeometric tests. B, Hierarchical clustering analyses of DRGs and URGs under ABA treatment for 0, 1, or 3 h. C, Scatterplots showing a positive correlation between URGs and DRGs in glk1 glk2 and wrky40 mutants under ABA treatment for 0, 1, and 3 h. WT, Wild type. D, Real-time PCR analysis of abiotic stress-responsive genes. GAPDH was used as an internal control. Error bars indicate sd (n = 3).

Next, we performed RNA-seq analysis of 10-d-old wild-type, glk1 glk2 double mutant, and pyr1 pyl1 pyl2 pyl4 quadruple mutant seedlings treated with 10 μm ABA for 3 h (see “Materials and Methods”). Because glk1 glk2 and pyr1 pyl1 pyl2 pyl4 mutants displayed contrasting phenotypes of seed germination and seedling growth in response to ABA, we compared URGs in glk1 glk2 (523 genes) with DRGs in pyr1 pyl1 pyl2 pyl4 (2,300 genes) and DRGs in glk1 glk2 (874 genes) with URGs in pyr1 pyl1 pyl2 pyl4 (1,833 genes; Supplemental Fig. S8; Supplemental Table S8). A substantial number of genes overlapped, which has statistical significance (Supplemental Fig. S8). Transcript levels of GLK1/2 were increased in the pyr1 pyl1 pyl2 pyl4 receptor mutant (Supplemental Table S8), suggesting that GLK1/2 might act as negative regulators in the PYL/PYR-mediated ABA signaling pathway. This conclusion was further supported by the significant increase in transcript levels of positive regulators of ABA signaling, including ABI5, ABI3, and PYL11, in the glk1 glk2 double mutant under ABA treatment (Fig. 2C).

GLK1/2 and WRKY40 Function in the Same Genetic Pathway

To examine the genetic interaction between GLK1/2 and WRKY40, we crossed glk1, glk2, and glk1 glk2 mutants separately with wrky40, thereby generating glk1 wrky40, glk2 wrky40, and glk1 glk2 wrky40 mutants, respectively. During seed germination, the glk1 glk2 double mutant and wrky40 single mutant displayed ABA-hypersensitive phenotypes compared with the wild type, glk1, and glk2 (Fig. 7, A and B). Additionally, glk1 wrky40, glk2 wrky40, and glk1 glk2 wrky40 displayed similar ABA sensitivity to wrky40, indicating that WRKY40 functions genetically downstream of GLK1/2 during seed germination (Fig. 7, A and B). Next, 3-d-old wild-type and mutant seedlings were transferred to medium containing 15 or 30 μm ABA for 7 d, and relative root lengths were measured. The glk1 glk2 and wrky40 mutants displayed ABA-hypersensitive phenotypes, while glk1 wrky40, glk2 wrky40, and glk1 glk2 wrky40 displayed similar ABA hypersensitivity to wrky40 (Fig. 7, C and D). We also overexpressed WRKY40 in the glk1 glk2 double mutant background, as an alternative approach (Supplemental Fig. S9A). In the resulting transgenic lines, the ABA-hypersensitive phenotype of glk1 glk2 was suppressed, although no effect on chloroplast development was observed (Supplemental Fig. S9, B and C). Taken together, these results indicate that WRKY40 functions genetically downstream of GLK1/2 during both germination and seedling development. Previously, it was reported that WRKY40 directly binds to the promoter of ABI5, which functions genetically downstream of WRKY40, thus suppressing the activity of ABI5 in the presence of ABA (Shang et al., 2010). Our RNA-seq data confirmed that the transcript level of ABI5 was higher in the glk1 glk2 double mutant than in the wild type in the presence of ABA.

Figure 7.

Figure 7.

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GLK1/2 and WRKY40 function in the same genetic pathway. A and B, Effect of exogenous ABA on seed germination. Seeds of the wild type (WT), wrky40, glk1, glk2, glk1 glk2, glk1 wrky40, glk2 wrky40, and glk1 glk2 wrky40 mutants were planted on one-half-strength MS plates supplemented with DMSO or different concentrations of ABA. A, Images were taken after incubation on plates for 7 d. B, The germination greening ratio was measured. Error bars indicate sd (n = 3). Statistical analyses were performed between the wild type and glk1 glk2, the wild type and wrky40, the wild type and glk1 wrky40, the wild type and glk2 wrky40, and the wild type and glk1 glk2 wrky40 treated with 0.5 or 1 μM ABA. **, P < 0.01 (Student’s t test). C, and D, Effect of exogenous ABA on root growth. The wild type, wrky40, glk1, glk2, glk1 glk2, glk1 wrky40, glk2 wrky40, and glk1 glk2 wrky40 mutants 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 or 30 μM ABA 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). Statistical analyses were performed between the wild type and glk1 glk2, the wild type and wrky40, the wild type and glk1 wrky40, the wild type and glk2 wrky40, and the wild type and glk1 glk2 wrky40 treated with 15 and 30 μM ABA. **, P < 0.01 (Student’s t test).

To confirm the genetic interactions among GLK1/2, WRKY40, and ABI5, we tried to cross abi5-7 (Nambara et al., 2002; Tamura et al., 2006) with the glk1 glk2 wrky40 triple mutant. However, since GLK1 and ABI5 are tightly linked genetically, we failed to generate an abi5-7 glk1 glk2 wrky40 quadruple mutant. To overcome this limitation, we took advantage of CRISPR/Cas9 technology (Ossowski et al., 2008; Sablok et al., 2011; Li et al., 2013; Carbonell et al., 2014) and isolated three independent 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 was detected after the ATG of ABI5 (55 bp after the ATG in glk1 glk2 wrky40 abi5-cr-1 and 53 bp after the ATG in glk1 glk2 wrky40 abi5-cr-3), resulting in a frame shift and consequently a premature stop codon (Supplemental Fig. S10A). In glk1 glk2 wrky40 abi5-cr-2, a 1-bp deletion was detected at 55 bp after ATG, causing a frame shift (Supplemental Fig. S10A). To exclude the effect of the Cas9 gene per se, we isolated glk1 glk2 wrky40 abi5-cr lines by screening for nonhygromycin resistance (Supplemental Fig. S10B). Screening for ABA sensitivity among mutants during seed germination and seedling growth revealed that wrky40, glk1 glk2, and glk1 glk2 wrky40 exhibited ABA-hypersensitive phenotypes compared with the wild type, whereas glk1 glk2 wrky40 abi5-cr-1, glk1 glk2 wrky40 abi5-cr-2, and glk1 glk2 wrky40 abi5-cr-3 showed ABA-hyposensitive phenotypes compared with the wild type during seed germination (Fig. 8, A and B) and seedling development (Fig. 8, C and D). These results indicate that ABI5 functions genetically downstream of GLK1/2 and WRKY40. In summary, we propose that GLK1/2 possibly could be activated via core ABA signaling components, PYL/PYRs-PP2Cs-SnRKs, and subsequently induce the expression of WRKY40 by directly binding to the promoter sequence. WRKY40 further directly binds to the ABI5 promoter region to suppress the ABI5 expression (Fig. 8E).

Figure 8.

Figure 8.

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ABI5 functions genetically downstream of GLK1/2 and WRKY40. A and B, Effect of exogenous ABA on seed germination. Seeds of the wild type (WT), abi5-7, glk1 gk2, wrky40, glk1 glk2 wrky40, glk1 glk2 wrky40 abi5-c-1, glk1 glk2 wrky40 abi5-c-2, and glk1 glk2 wrky40 abi5-c-3 were planted on one-half-strength MS plates supplemented with DMSO or different concentrations of ABA. A, Images were taken after incubation on plates for 7 d. B, Germination greening ratio was measured. Error bars indicate sd (n = 3). Statistical analyses were performed between the wild type and abi5-7, the wild type and glk1 glk2 wrky40 abi5-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 μM ABA. *, P < 0.05, **, P < 0.01 (Student’s t test). C and D, Effect of exogenous ABA on root growth. The wild 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-3 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 or 30 μM ABA 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). The wild type and abi5-7, the wild type and glk1 glk2 wrky40 abi5-c-1, the wild type and glk1 glk2 wrky40 abi5-c-2, and the wild type and glk1 glk2 wrky40 abi5-c-3 were treated with 15 and 30 μM ABA. **, P < 0.01 (Student’s t test). E, After ABA perception, ABA receptor PYL/PYRs recruit PP2Cs, thereby activating SnRKs. GLK1/2 possibly could be activated via core ABA signaling components, PYL/PYRs-PP2Cs-SnRKs, and subsequently induce the expression of WRKY40 by directly binding to the promoter sequence. WRKY40 further directly binds to the ABI5 promoter region to suppress the ABI5 expression.

DISCUSSION

Genes encoding plant-specific GARP transcription factors regulate diverse processes, including nutrient sensing, root and shoot development, chloroplast development, and circadian clock oscillation maintenance (Safi et al., 2017). In Arabidopsis, the GARP family comprises 56 genes, including G2-LIKE genes and genes encoding B-type ARR proteins that harbor N-terminal receiver domains (Riechmann et al., 2000). Many decades ago, the golden2 (g2) mutant was described as having a golden color phenotype in maize (Jenkins, 1926). Several orthologous pairs of GLKs have been identified in rice (Oryza sativa), Arabidopsis, tomato (Solanum lycopersicum), and pepper (Capsicum annuum); these proteins exhibit overlapping expression profiles and redundant functions (Fitter et al., 2002; Powell et al., 2012; Brand et al., 2014). In Arabidopsis and maize, the loss of function of GLK results in an abnormal morphology in chloroplast ultrastructure. In addition to the role of GLK genes in chloroplast biogenesis, overexpression of GLK1 confers enhanced resistance to nonhost fungal pathogens and Cucumber mosaic virus (Savitch et al., 2007; Schreiber et al., 2011; Han et al., 2016). Moreover, Murmu et al. (2014) showed that GLKs are involved in jasmonic acid-dependent susceptibility to biotrophic pathogens and jasmonic acid-independent resistance to the necrotrophic pathogen Botrytis cinerea in Arabidopsis. In this study, we found that the glk1 glk2 double mutant showed ABA-hypersensitive response and osmotic stress-resistant phenotypes compared with glk1, glk2, and the wild type. Accordingly, ectopic expression of GLK1 and GLK2 decreased the sensitivity to ABA and increased that to osmotic stress. These results indicate that GLK1 and GLK2 negatively regulate ABA and osmotic stress responses during seed germination and seedling development. In addition to the negative role of GLK1/2 in ABA-mediated inhibition of seed germination and seedling growth, we also found that genes including RbohB, RbohD, and peroxidase-superfamily-gene were down-regulated in glk1 glk2. In accordance with this result, salt- and osmotic stress-induced superoxide and H2O2 accumulation were reduced in the glk1 glk2 double mutant compared with the wild type. These data indicate that GLK1/2 positively impact ROS generation under abiotic stress conditions. Considering the diverse roles 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; Leister and Kleine, 2016; Nagatoshi et al., 2016), we conclude that the function of GLK1/2 in the response to ABA is uncoupled from that in the production of ROS.

Genome-wide RNA-seq analysis of the glk1 glk2 double mutant revealed that GLK1/2 regulate some of the essential ABA-responsive genes. Previously, taking advantage of the inducible expression system, Waters et al. (2009) showed that GLK1/2 regulate chlorophyll biosynthesis, light harvesting, and electron transport at the transcriptional level. Additionally, among the target genes of GLK1/2, Waters et al. (2009) also determined that some of the ABA- and stress-responsive genes, such as COR15A and COR15B, were rapidly and specifically induced. Consistent with these results, we showed that the induction of COR15A and COR15B was dramatically impaired in the glk1 glk2 double mutant in the presence of ABA and confirmed the induction of COR15A and COR15B using the β-estradiol-inducible system. Intriguingly, we identified that GLK1/2 directly affected the expression of WRKY40 in the presence of ABA. Previously, it was reported that the wrky40 mutant shows ABA-hypersensitive phenotypes during seed germination and seedling development (Chen et al., 2010; Shang et al., 2010). Additionally, previous ChIP-qPCR analyses show that WRKY40 directly binds to the W-box motif located in the promoter of ABI5, thus directly repressing ABI5 expression (Chen et al., 2010; Shang et al., 2010). Our genetic interaction analyses showed that the loss of function of ABI5 diminished the ABA-hypersensitive phenotype of glk1 glk2 and wrky40, indicating that ABI5 functions genetically downstream of GLK1/2 and WRKY40. Using ChIP-qPCR analysis, we showed that GLK1/2 bind to the promoter of WRKY40 via a consensus GLK1/2-binding sequence in vivo. Moreover, we demonstrated that GLK1/2 and WRKY40 share common downstream target genes in response to ABA.

In Arabidopsis, WRKY domain-containing proteins constitute a superfamily of up to 100 representative proteins and are widely known to function in plant development 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 germination and seedling development, with the strongest phenotype observed in wrky40 (Shang et al., 2010). Moreover, the wrky40 wrky18 double mutant displays stronger ABA hypersensitivity than wrky40 and wrky40 wrky18 wrky60 mutants, indicating that WRKY40 plays a more important role in ABA response than other WRKYs (Shang et al., 2010). Moreover, in this study, GLK1/2-regulated and PYR/PYL-regulated genes showed the opposite correlation. Because the expression of a subset of ABA-responsive genes was blocked in the glk1 glk2 double mutant, we propose that GLK1/2 act as negative regulators of the PYL/PYR-mediated ABA signaling pathway. However, we did not detect putative phosphorylation target sites of SnRKs, which are a core component of the PYR/PYL-PP2C-SnRK signaling module in GLK1/2. In addition to the typical SnRK2 phosphorylation sites, SnRK2s also recognize atypical phosphorylation sites (Furihata et al., 2006; Sirichandra et al., 2010; Umezawa et al., 2013; Wang et al., 2013; Peirats-Llobet et al., 2016). Thus, it is possible that GLK1/2 might be the direct targets of SnRK2 via atypical sites. Considering this possibility, GLK1/2 may act as the upstream target of ABI5, which could be phosphorylated by SnRK2s. The PYR/PYL-PP2C-SnRK signaling module possibly activates GLK1/2, thereby inducing the expression of WRKY40, a negative regulator, to prevent the overinduction of ABA-responsive genes. Another possibility is that calcium-dependent protein kinases or mitogen-activated protein kinases, which are essential regulators of ABA signaling (Asano et al., 2012; de Zelicourt et al., 2016), directly phosphorylate GLK1/2 to regulate the activity of GLK1/2. However, to confirm this possibility, further investigation is needed. In conclusion, we predict that this molecular mechanism helps maintain plant fitness under changing environmental conditions.

MATERIALS AND METHODS

Plant Growth Conditions and Genotyping

Arabidopsis (Arabidopsis thaliana) Columbia-0 (Col-0) plants were grown in the greenhouse at 23°C, maintaining 60% relative humidity condition with 160 μmol m−2 s−1 under a 16/8-h light/dark photoperiod for physiological experiments (Moore et al., 2003). Col-0 single-knockout lines glk1 (At2g20570) and glk2 (At5g44190) and the glk1 glk2 double knockout line, N9805, N9806, and N9807, respectively, were obtained from the Nottingham Arabidopsis Stock Centre 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 mutants 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, and WRKY40-F/R, respectively. All PCR primers used for genotyping are listed in Supplemental Table S9.

Construction of Plasmids and Generation of Transgenic Plants

Gene-specific primers GLK1-C-F/R or GLK2-C-F/R were used to isolate GLK1 and GLK2 cDNA from a cDNA library by PCR. To generate the Pro-35s:GLK1-2×FLAG and Pro-35s:GLK2-2×FLAG constructs, full-length GLK1 or GLK2 was amplified and cloned into pCsV1300 vector with a 2xFLAG tag using XbaI and BamHI sites. To generate the Pro-35s:WRKY40-2Xflag construct, full-length WRKY40 cDNA was amplified by PCR using primers WRKY40-C-F/R and cloned into pCsV1300 vector with a 2xFLAG tag using XbaI and BamHI sites (Xu et al., 2012). To generate GD-GLK1 and GD-GLK2 constructs, full-length GLK1 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 using NdeI and SacI sites (Wang et al., 2007). To generate ProSUPERR:sXVE:GLK1 and ProSUPERR:sXVE:GLK2 constructs, full-length GLK1 or GLK2 was amplified by PCR using primers GLK1-sXVE-F/R or GLK2-sXVE-F/R and inserted into the sXVE vector using XbaI and BamHI sites. To generate a ProWRKY40:LUC construct, an intact or mutated 0.5-kb WRKY40 promoter sequence carrying AAAAAA substitution was amplified using XhoI and SpeI and inserted into the LUC reporter gene. To generate GLK1-GST and GLK2-GST constructs, full-length GLK1 or GLK2 was amplified by PCR using primers GLK1-GST-F/R or GLK2-GST-F/R and inserted into the pGEX-4T1 vector using BamHI and EcoRI sites. To generate ProGLK1:GLK1-2xFLAG and ProGLK2:GLK2-2xFLAG constructs, the genomic sequences of GLK1 or GLK2 containing promoter regions were amplified using GLK1-G-F/R (2xFLAG/XbaI/PstI) and GLK2-G-F/R (2xFLAG/XbaI/PstI) primers. Subsequently, the constructs were inserted into the binary vector pCAMBIA1302 (Invitrogen). To knock out both ABI5 gene copies, three abi5-cr constructs were designed using the AtU6-26-sgRNA-SK and pCAMBIA1300-pYAO:Cas9 plasmids according to the method described previously (Yan et al., 2015). A link to a Web tool for automated design of the target sequence(s) is available at http://crispr.mit.edu/. The constructs were transformed into wild-type or glk1 glk2 wrky40 triple mutant plants. PCR and Sanger sequencing were used to gain the mutation forms. T3 seeds were screened with hygromycin, and non-hygromycin-resistant seeds were used for the following experiments (Jia et al., 2016). The Pro-35s:WRKY40-2Xflag constructs were transformed into the glk1 glk2 double mutant. All primers are listed in Supplemental Table S9. Transgenic plants were grown on B5 plates treated with 50 mg L−1 hygromycin (Clough and Bent, 1998).

Assays of Sensitivity to ABA and Osmotic Stress

For tests of the ABA effect on germination, seeds were incubated at 4°C for 3 d to break dormancy prior to germination, and then sterilized seeds were grown on one-half-strength MS medium with or without ABA for 7 d in a greenhouse. Germination ratios were calculated according to the percentages of cotyledon greening emergencies after seed germination (He et al., 2012; Kong et al., 2015; Wang et al., 2018a). For testing root elongation under ABA or osmotic stress treatments, 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, or mannitol in the incubator at 23°C, maintaining 60% relative humidity condition with 60 μmol m−2 s−1 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 MS medium treated with 10 μM ABA for 0, 1, or 3 h with Trizol (Invitrogen). The growing condition was 160 μmol m−2 s−1 light intensity under a 16/8-h light/dark photoperiod (Moore et al., 2003). Materials were collected from three independent biological replicates from Col-0, glk1 glk2, wrky40, and pyr1 pyl1 pyl2 pyl4 mutants under different lengths of ABA exposure. At least 3 μg of RNA was 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 more than 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 was performed in paired-end mode with 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 clean reads were mapped to the Arabidopsis reference genome (TAIR10) using TopHat v.2.1.0 (Trapnell et al., 2009) with TAIR10 gene annotation as the transcript index. 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. The DEGs were filtered according 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 GO grouping of DEGs was performed by hypergeometric distribution in R (version 3.1.0; https://www.r-project.org/; Lucent Technologies), with an adjusted P < 0.05 as a cutoff to determine significantly enriched GO terms. Venn diagrams were generated 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-day conditions were used for isolation of protoplasts (Jin et al., 2001; Hyunjong et 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 incubated under darkness for 20 to 23 h. GUS activities were measured using a Fluoroskan Finstruments microplate reader (MTX Lab Systems; Tiwari et al., 2003; Wang et al., 2005).

Induced Transgene Expression in Stably Transformed Arabidopsis Plants

To induce transgene expression in stably transformed seedlings, selected plants transformed with ProSUPERR:sXVE:GLK1, ProSUPERR:sXVE:GLK2, or ProSUPERR:sXVE:GFP constructs were grown in one-half-strength MS liquid medium and then supplemented with 20 μm β-estradiol and induced for 4 h (Schlücking et al., 2013).

Transient Dual-Luciferase Reporter System

To examine reporter gene expression, ProWRKY40:LUC or MProRKY40:LUC was cotransformed with effector gene ProSUPERR:sXVE:GLK1 or ProSUPERR:sXVE:GLK2 protoplasts and incubated for 23 h followed by 4-h treatment with 20 μM β-estradiol (Schlücking et al., 2013). Transformed protoplasts were pelleted by low-speed centrifugation (500 rpm). Total RNA was prepared using an RNA extraction kit (Ambion) and used for RT-qPCR analysis by the comparative cycle threshold method, in which LUC transcript levels were normalized using the GUS transcript. The specific primers used for real-time PCR are listed in Supplemental Table S9.

ChIP Assays

The ChIP assay was performed according to the method described previously (Haring et al., 2007) with a slight modification (Liu et al., 2018a, 2018b). Two-week-old transgenic 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, which was 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 complexes were isolated by binding to protein A beads. The DNA fragments in the immunoprecipitated complexes were released by reversing the cross-linking at 65°C for 8 h and then extracted with phenol/chloroform, precipitated in ethanol, and resuspended in water. The specific primers used for real-time PCR are listed in Supplemental Table S9. ACT7 was 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 onto glutathione beads. The beads were then washed three times (Xu et al., 2013). The purified protein was confirmed by SDS-PAGE and prepared for EMSA. The probes were synthesized and labeled with biotin by Sangon Biotechnology. Double-strand probe (50 fmol) was incubated with protein in binding buffer for 10 min. The mixture was then separated by nondenatured 6% native polyacrylamide gel with 0.5× Tris-borate/EDTA for 30 min, and the protein was transferred to a nylon membrane via wet transfer and detected according to the instructions 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 method described previously with a slight modification (Nguyen et al., 2017). Five-day-old seedlings grown in liquid MS medium were treated with or without 100 mM NaCl or mannitol for 12 h. To detect O2−, treated plants were vacuumed infiltrated for 3 min and then stained for 4 h with 0.05% (w/v) NBT and 10 mM NaN3 in 10 mm potassium phosphate (pH 7.8). To detect H2O2, plants were 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, plants were grown on soil in a greenhouse with 160 μmol m−2 s−1 under a 16/8-h light/dark photoperiod (23°C, 60% relative humidity) 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 (rehydration; Kang et al., 2002; Xu et al., 2012).

Protein Extraction and Western-Blot Assay

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

Accession Numbers

Data generated in this study are deposited in the National Center for Biotechnology Information Sequence Read Archive (accession nos. PRJNA513154 and PRJNA513157).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

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

Footnotes

1

This work was supported by the National Natural Science Foundation of China (31601311 and 31771352 to Z.-Y.X.), the Fundamental Research 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.).

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