Arabidopsis WRKY46, WRKY54, and WRKY70 Transcription Factors Are Involved in Brassinosteroid-Regulated Plant Growth and Drought Responses

Abstract

Plant steroid hormones, brassinosteroids (BRs), play important roles in growth and development. BR signaling controls the activities of BRASSINOSTERIOD INSENSITIVE1-EMS-SUPPRESSOR1/BRASSINAZOLE-RESISTANT1 (BES1/BZR1) family transcription factors. Besides the role in promoting growth, BRs are also implicated in plant responses to drought stress. However, the molecular mechanisms by which BRs regulate drought response have just begun to be revealed. The functions of WRKY transcription factors in BR-regulated plant growth have not been established, although their roles in stress responses are well documented. Here, we found that three Arabidopsis thaliana group III WRKY transcription factors, WRKY46, WRKY54, and WRKY70, are involved in both BR-regulated plant growth and drought response as the wrky46 wrky54 wrky70 triple mutant has defects in BR-regulated growth and is more tolerant to drought stress. RNA-sequencing analysis revealed global roles of WRKY46, WRKY54, and WRKY70 in promoting BR-mediated gene expression and inhibiting drought responsive genes. WRKY54 directly interacts with BES1 to cooperatively regulate the expression of target genes. In addition, WRKY54 is phosphorylated and destabilized by GSK3-like kinase BR-INSENSITIVE2, a negative regulator in the BR pathway. Our results therefore establish WRKY46/54/70 as important signaling components that are positively involved in BR-regulated growth and negatively involved in drought responses.

Figure 1.

WRKY46, WRKY54, and WRKY70 Function Redundantly and Play Positive Roles in the BR Pathway. (A) WRKY46, WRKY54, and WRKY70 mRNA levels were determined in the wild type and bes1-D treated with 1 μM BL or mock control for 2.5 h. The averages and sd were derived from three biological replicates. (B) Top: The growth phenotype of 3-week-old wild type, wrky46, wrky54, wrky70, and wrky46 wrky54 wrky70 triple mutant (abbreviated as w54t in all figures). Bottom: BES1 protein levels were determined by immunoblot and a loading control was shown at the bottom. (C) The measurement of blade lengths, blade widths, and petiole lengths of the sixth leaves. Error bars indicate sd, n = 13 (*P < 0.05, **P < 0.01; Student’s t test). (D) Transgenic complementation of w54t mutant with PWRKY54:WRKY54-FLAG fusion gene and empty vector as the control. Top: Four-week-old wild-type transgenic plants with vector (w54t) or WRKY54 (w54t) are shown. Bottom: WRKY54 protein accumulation was detected in the transgenic plants by immunoblot with anti-FLAG antibody and HERK1 loading control was shown at the bottom. (E) BES1 protein accumulation was determined in 4-week-old w54t leaves soaked in 0.5× liquid MS medium with 1 μM BL or DMSO for 30 min. (F) Hypocotyl lengths of 5-d-old seedlings grown on 0.5× MS medium with 0, 10, and 100 nM BL. Mean was calculated and the sd was also presented. Error bars indicate sd (*P < 0.05, **P < 0.01; Student’s t test).

Figure 2.

WRKY46, WRKY54, and WRKY70 Regulate the Expression of BR Target Genes. (A) Venn diagram showing overlaps among genes up- or downregulated in w54t with those differentially expressed in bes1-D. (B) Clustering analysis of genes differentially expressed in w54t under control conditions within the wild type, w54t, and bes1-D. Values indicate normalized expression levels. (C) The expression of genes downregulated in w54t was examined using 4-week-old plants treated with or without 1 μM BL. The averages and sd are derived from three biological replicates. (D) The expression of genes upregulated in w54t was examined as described in (C).

Figure 3.

WRKY46, WRKY54, and WRKY70 Directly Interact with BES1 Both in Vivo and in Vitro. (A) WRKY46/54/70 interact with BES1 by BiFC assay in vivo. Cotransformation of WRKY46/54/70-YFPC and BES1-YFPN led to the reconstitution of YFP signal, whereas no signal was detected when BES1-YFPN and YFPC or WRKY46/54/70-YFPC and YFPN were coexpressed (Supplemental Figure 7F). The experiments were performed twice with similar results. (B) and (C) Transient expression of LUC driven by the BR-regulated gene promoters of At2g45210 (B) and At1g43910 (C). The mean and sd were derived from three biological repeats.

Figure 4.

WRKY46, WRKY54, and WRKY 70 Play Negative Roles in Drought Response. (A) Phenotypes of wild-type, wrky46, wrky54, wrky70, and w54t plants before drought (top), after drought (middle), and 2 d after rewatering (bottom). (B) The survival rate after recovery was determined. The mean and sd were from three biological repeats. (C) Venn diagram showing comparisons among genes differentially expressed in w54t and genes up- or downregulated by dehydration in the wild type. (D) Clustering of dehydration downregulated genes in the wild type and w54t mutants under control conditions (W) or dehydration (D). (E) Clustering of dehydration upregulated genes in the wild type and w54t mutants under control conditions (W) or dehydration (D). Values indicate normalized expression levels.

Figure 5.

WRKY54 and BES1 Cooperate to Negatively Regulate Dehydration-Induced Genes. (A) The expression of dehydration-inducible genes, ABI5, GLYI7, and RD20, was determined in the wild type and w54t by RT-qPCR under control conditions (W) or dehydration (D). Error bars indicate sd. (B) Schematic diagram of the promoter region of GLYI7. Wild-type W-box and G-box are indicated. mWbox, the mutation of W-box; mGbox, the mutation of G-box; mGWbox, the mutation of both G-box and W-box. (C) Transient expression of GLYI7P-fLUC and mutated W-box or G-box or both of GLYI7P-fLUC was determined in the presence of WRKY54 and/or BES1 in protoplasts. Error bars indicate sd (*P < 0.05, **P < 0.01; Student’s t test).

Figure 6.

BIN2 Kinase Phosphorylates and Destabilizes WRKY54 Protein. (A) WRKY46/54/70 interact with BIN2 by BiFC assay in vivo. Cotransformation of WRKY46/54/70-YFPC and BIN2-YFPN led to the reconstitution of YFP signal, whereas no signal was detected when BIN2-YFPN and YFPC or WRKY46/54/70-YFPC and YFPN were coexpressed (Supplemental Figure 7F). The experiments were performed twice with similar results. (B) In vitro kinase assays show BIN2 phosphorylates WRKY54/46/70 (top). The loading controls of MBP, MBP-WRKY54/46/70, and GST-BIN2 by CBB staining are shown in bottom panel. (C) The phosphorylation of WRKY54 by BIN2 was inhibited with the increasing concentrations of bikinin (top). The loading controls are shown on the bottom. (D) The WRKY54 protein level was detected in indicated BR mutants and wild type with WRKY54 antibody. The w54t mutant was used as a negative control. (E) WRKY54 protein accumulated upon BL treatment. Two-week-old wild-type seedlings were treated with or without 1 μM BL for indicated time and used to prepare protein to detect WRKY54 (top), BES1 (middle), and a control protein (bottom).

Figure 7.

A Working Model of WRKY46/54/70 Function in Plant Growth and Stress Response. (A) WRKY54 and BES1 protein decreased with increasing drought treatment time. The 8d, 9d, and 10d indicate days of drought treatment or controls. (B) A working model of WRKY46/54/70 in BR-regulated growth and drought stress response. WRKY46/54/70 are regulated by BR signaling through BIN2 and BES1, and cooperate with BES1 to promote plant growth and inhibit drought responses. WRKY46/54/70 also slightly promote BR biosynthesis. Under normal growth conditions (left), WRKY46/54/70 and BES1 positively coregulate growth-related genes and negatively control the expression of drought-responsive genes to promote growth. BRs regulate WRKY46/54/70 both transcriptionally and posttranscriptionally through BES1 and BIN2, respectively. Under drought stress conditions (right), WRKY46/54/70 and BES1 protein are destabilized, which leads to repression of growth-related genes and alleviation of WRKY46/54/70’s inhibitory effect on drought-related genes, leading to reduced growth and increased drought tolerance.