BZR1 Positively Regulates Freezing Tolerance via CBF-Dependent and CBF- Independent Pathways in Arabidopsis  Hui Li, Keyi Ye, Yiting Shi, Jinkui Cheng, Xiaoyan Zhang, Shuhua Yang  Molecular Plant  Volume 10, Issue 4, Pages 545-559 (April 2017) DOI: 10.1016/j.molp.2017.01.004 Copyright © 2017 The Author Terms and Conditions

Figure 1 Freezing Phenotypes of BIN2-Overexpressing Plants and bin2-3 bil1 bil2 Mutant. (A and C) Freezing phenotypes of BIN2-overexpressing (A) and the bin2-3 bil1 bil2 mutant (C) under non-acclimated and acclimated conditions. The wild-type (WT), transgenic plants overexpressing BIN2-Myc driven by super promoter (BIN2-OE1 and BIN2-OE15), and bin2-3 bil1 bil2 mutant were grown on 1/2 MS plates at 22°C for 2 weeks and then treated at −4°C for 0.5 h for non-acclimated plants (NA) and at −6°C or −7°C for 1 h for acclimated plants (CA; 3 days at 4°C). At least three plates were treated in each treatment. The representative pictures were taken after recovery. (B and D) Survival rates of BIN2-overexpressing plants (B) and the bin2-3 bil1 bil2 mutant (D). (E and F) The electrolyte leakage assays of BIN2-overexpressing plants (E) and the bin2-3 bil1 bil2 mutant (F) at the indicated freezing temperatures. Plants without freezing treatment were used as control, and represented by “CK”. In (B), (D), (E), and (F), the data are the mean values of three replicates ± SD. ∗P < 0.05 and ∗∗P < 0.01 (t-test) indicate a significant difference between mutants and the WT plants. At least three independent experiments were done with similar results. See also Supplemental Figures 1 and 2. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 2 Freezing Phenotypes of bzr1-1D and bes1-D Mutants. (A and B) Freezing phenotypes of bzr1-1D (A) and bes1-D (B) mutants. The wild-type (WT), bzr1-1D, and bes1-D mutants were grown on 1/2 MS plates at 22°C for 2 weeks and then treated at −4°C for 0.5 h or 1 h for non-acclimated plants (NA) and at −7°C for 0.5 h or 1 h for acclimated plants (CA; 3 days at 4°C). Plants without freezing treatment were used as control, and represented by “CK”. (C and D) Survival rates of bzr1-1D (C) and bes1-D (D) under non-acclimated and acclimated conditions described in (A) and (B). (E and F) The electrolyte leakage assays of bzr1-1D (E) and bes1-D (F) mutant at the indicated freezing temperatures. In (C), (D), (E), and (F), the data are the mean values of three replicates ± SD. ∗P < 0.05 and ∗∗P < 0.01 (t-test) indicate a significant difference between mutants and WT. At least three independent experiments were done with similar results. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 3 Cold Treatment Induces the Accumulation of Dephosphorylated BZR1. (A and B) Expression of BIN2 (A) and BZR1 (B) gene in the wild-type (WT) plants treated at 4°C by real-time qPCR. The 2-week-old plants grown on 1/2 MS plates at 22°C were treated at 4°C for the indicated time. Data are mean ± SD of three technical replicates. (C) Protein level of BIN2 in the BIN2-overexpressing plants under cold stress. The 2-week-old plants grown on 1/2 MS plates at 22°C were treated at 4°C for the indicated time, and total proteins were extracted and subjected to immunoblot analysis with anti-Myc antibody. (D) Protein level of BZR1 in the WT plants under cold stress. The 2-week-old plants grown on 1/2 MS plates at 22°C were treated at 4°C for the indicated time, and total proteins were extracted and subjected to immunoblot analysis with anti-BZR1 antibody. (E) Protein level of BZR1 in the WT, bin2-3 bil1 bil2, and BIN2-overexpressing plants under cold stress. The 2-week-old plants grown on 1/2 MS plates at 22°C were treated at 4°C for 0, 1, 3, and 6 h, and total proteins were extracted and subjected to immunoblot analysis with anti-BZR1 antibody. “−/+P” represents dephosphorylated BZR1 versus phosphorylated BZR1. At least three independent experiments were done with similar results. See also Supplemental Figure 3. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 4 Analysis of BZR1 Target Genes. (A) Functional categorization analysis of BZR1 candidate target genes. (B) Venn diagram showing overlapping target genes of BZR1 and BES1 in plant response to cold stress. The shared targets are listed in Supplemental Table 1. See also Supplemental Figure 4 and Supplemental Table 1. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 5 BZR1 Binds to the Promoters of CBF1 and CBF2. (A) Schematics showing the promoter structures of CBF1 and CBF2 genes. The upstream regions and part of coding regions are shown with a white box and black box, respectively. The solid arrowheads indicate the sites containing E box and the hollow arrowheads indicate the sites containing BRRE in the CBF1 and CBF2 promoters. Hatched boxes represent the fragments amplified in the ChIP assay. (B) ChIP assay of BZR1 binding to the promoters of CBF1 and CBF2 genes. Chromatin from Col and 35S:BZR1-Myc seedlings were immunoprecipitated with anti-Myc antibody or without antibody. The amounts of the indicated DNA in the immune complex were tested by real-time qPCR, and a Tubulin2 fragment was amplified as control. The Relative Enrichment was calculated as Input% (indicated DNA)/Input% (control). The data are the mean values of three technical replicates ± SD from one experiment. At least three independent experiments were performed with similar results. (C) EMSA assay of BZR1 binding to the promoters of CBF1 and CBF2 genes. Biotin-labeled fragments representing parts of CBF1 and CBF2 promoters that contain an E box were incubated with MBP or MBP-BZR1 protein. Competitor or mutated competitor fragments were added in 100× or 300× excess to analyze the specificity of binding. (D) GUS transactivation assays in Arabidopsis protoplasts. The promoters of CBF1 (left) or CBF2 (right) fused to the GUS gene as reporters that were transiently expressed in protoplasts either with effector BZR1 or with vector as control at 22°C for 16 h and then were treated at 22°C or 4°C for another 3 h. See also Supplemental Figure 5. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 6 Genetic Interaction of BZR1 and CBF Genes. (A and B) Expression of CBF genes (A) and CBF target genes (B) in the bzr1-1D mutant under cold stress. The 2-week-old plants grown on 1/2 MS plates at 22°C were treated at 4°C for the indicated time. Total RNAs were extracted and subjected to real-time qPCR analysis. The expression levels of the genes in the wild-type (WT) at 22°C were set as 1.0. Data are mean of three replicates ± SD. ∗P < 0.05, ∗∗P < 0.01 (t-test) indicate a significant difference between mutants and the WT plants. (C–E) Freezing phenotypes (C), survival rates (D), and electrolyte leakage (E) of bzr1-1D cbfs-1 quadruple mutant under non-acclimated and acclimated conditions. The WT and bzr1-1D cbfs-1 quadruple mutants were grown on 1/2 MS plates at 22°C for 2 weeks and then treated at gradient temperatures for non-acclimated (NA) and acclimated plants (CA; 3 days at 4°C). Plants without freezing treatment were used as control, and represented by “CK”. At least three plates were treated in each treatment. The representative pictures were taken after recovery (C). In (D) and (E), data are mean of three replicates ± SD. Similar results were obtained from three independent experiments. See also Supplemental Figure 6. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 7 Expression of CBF Genes and CBF Target Genes in the bin2-3 bil1 bil2 Mutant. Expression of CBF genes (A) and CBF target genes (B) in the bin2-3 bil1 bil2 mutant under cold stress. The 2-week-old plants grown on 1/2 MS plates at 22°C were treated at 4°C for the indicated time. Total RNAs were extracted and subjected to real-time qPCR analysis. Data are mean of three replicates ± SD. ∗P < 0.05, ∗∗P < 0.01 (t-test) indicate a significant difference between mutants with the wild-type (WT) plants. Similar results were obtained from three independent experiments. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 8 Transcriptome Profiling Analyses Identify Cold-Regulated BZR1-Dependent Genes. (A) Venn diagrams indicating the number and overlap of genes with higher level in bzr1-1D than Col-0 with or without cold treatment for 3 h (represented by bzr1-1Dc/WTc Induced Genes or bzr1-1Dw/WTw Induced Genes, respectively), genes induced by cold treatment for 3 h in Col-0 (WTc/WTw Induced Genes), and genes regulated by CBFs (CBF Regulated Genes) (I); the number and overlap of genes with lower level in bzr1-1D than Col-0 with or without cold treatment for 3 h (represented by bzr1-1Dc/WTc Repressed Genes or bzr1-1Dw/WTw Repressed Genes, respectively) and genes induced by cold treatment for 3 h in Col-0 and genes regulated by CBFs (II); the number and overlap of genes with lower level in bzr1-1D than Col-0 with or without cold treatment for 3 h, genes repressed by cold treatment for 3 h in Col-0 (WTc/WTw Repressed Genes), and genes regulated by CBFs (III); the number of genes and overlap with higher level in bzr1-1D than Col-0 with or without cold treatment for 3 h and genes repressed by cold treatment for 3 h in Col-0 and genes regulated by CBFs (IV). (B) Heatmap representing fold change (log2) in transcription level for genes belonging to indicated groups branded with red bold numbers in (A). (C) Expression of PYL6, WRKY6, SAG21, SOC1, JMT, and ESM1 genes in the wild-type (WT) plants under cold stress. The 2-week-old WT plants grown on 1/2 MS plates at 22°C were treated at 4°C for 0, 3, 6, and 12 h. Total RNAs were extracted and subjected to real-time qPCR analysis. The expression levels of the genes in the WT at 22°C were set as 1.0. Data are mean ± SD of three technical replicates. ∗P < 0.05 and ∗∗P < 0.01 (t-test) indicate a significant difference between mutants and the WT plants. Similar results were obtained from three biological replicates. See also Supplemental Figures 7–9 and Supplemental Tables 3–16. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 9 WRKY6 Positively Regulates Plant Freezing Tolerance. (A) Schematics showing the promoter structures of WRKY6 genes. The upstream regions and part of coding regions are shown with a white box and black box, respectively. The solid arrowheads indicate the sites containing E box in the WRKY6 promoters. Hatched boxes represent the fragments amplified in the ChIP assay. (B) ChIP assay of BZR1 binding to the promoters of WRKY6 genes. Chromatin from 35S:BZR1-Myc seedlings were immunoprecipitated with anti-Myc antibody or without antibody. The amounts of the indicated DNA in the immune complex were tested by real-time qPCR, and a Tubulin2 fragment was amplified as control. The Relative Enrichment was calculated as Input% (indicated DNA)/Input% (control). The data are the mean values of three technical replicates ± SD from one experiment. *P < 0.05 and **P < 0.01 (t-test) indicate a significant difference. At least three independent experiments were performed with similar results. (C) EMSA assay of BZR1 binding to the promoters of WRKY6 genes. Biotin-labeled fragments representing parts of WRKY6 that contain an E box were incubated with MBP or MBP-BZR1 protein. Competitor or mutated competitor fragments were added in 100× or 300× excess to analyze the specificity of binding. (D–F) Freezing phenotypes (D), survival rates (E), and electrolyte leakage (F) of wrky6 mutants. The wild-type (WT), wrky6-1, and wrky6-2 mutants were grown on 1/2 MS plates at 22°C for 2 weeks and then treated at −4°C for 0.5 h for non-acclimated plants (NA) and at −6°C for 1 h for acclimated plants (CA; 3 days at 4°C). Plants without freezing treatment were used as control, and represented by “CK”. In (E) and (F), the data are the mean ± SD of three replicates. *P < 0.05 and **P < 0.01 (t-test) indicate a significant difference between mutants and WT. At least three independent experiments were done with similar results. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions

Figure 10 The Proposed Model for BZR1 Modulating Freezing Tolerance in Arabidopsis. Cold induces the accumulation of dephosphorylated BZR1, and activation of BZR1 induces the expression of CBF1/2 and CBF-independent genes by binding to the conserved E-box/BRRE motifs of their promoters, while BIN2 negatively regulates plant response to cold stress by inhibiting cold-induced dephosphorylated BZR1 protein. BZR1 also directly regulates some COR genes, including WRKY6, SAG21, and SOC1, which are independent of CBFs, to modulate plant freezing tolerance. Molecular Plant 2017 10, 545-559DOI: (10.1016/j.molp.2017.01.004) Copyright © 2017 The Author Terms and Conditions