Volume 6, Issue 5, Pages 1487-1502 (September 2013) Arabidopsis Di19 Functions as a Transcription Factor and Modulates PR1, PR2, and PR5 Expression in Response to Drought Stress  Wen-Xin Liu, Fei-Cui Zhang, Wen-Zheng Zhang, Lian-Fen Song, Wei-Hua Wu, Yi-Fang Chen  Molecular Plant  Volume 6, Issue 5, Pages 1487-1502 (September 2013) DOI: 10.1093/mp/sst031 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Phenotype Tests for Arabidopsis Di19 Variants under Drought Stress. (A) T-DNA insertion site in di19 mutant. The T-DNA was inserted in the second exon of Di19 genomic DNA. Exons and introns are indicated by black boxes and solid lines, respectively. (B) qRT–PCR analysis of Di19 expression in the wild-type plant (WT), di19 mutant (di19), di19 mutant complementation line (di19/ProDi19:Di19), and Di19-overexpressing line (Super:Di19-7 and Super:Di19-8). The mature leaves of 3-week-old plants were used for RNA extraction. The data represent the mean values of three replicates ± SD. (C) Phenotype tests of Arabidopsis plants under drought stress. Three-week-old plants were grown for 18 d with (Control, upper panel) or without (Drought, lower panel) irrigation. The pictures were taken on day 18. The experiments were repeated three times, with similar results. (D) Water loss measurements for Di19 variants. Weight loss in detached leaves was measured at the time points indicated. Water loss was expressed as a percentage of the initial fresh weight. The data presented are means ± SD of four replicates for one experiment (*P < 0.05 from the fourth time point). (E) qRT–PCR analysis of Di19 expression induced by drought stress. Total RNA was extracted at the times indicated from 3-week-old wild-type mature leaves. The data represent the mean values of three replicates ± SD. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Subcellular Localization and Transcriptional Activation Analysis of Di19. (A) Subcellular localization of Di19. Confocal images of Arabidopsis mesophyll protoplasts transiently expressed with Di9-1:EGFP or pUC:EGFP fusion constructs. The protoplast expressed with pUC:EGFP was used as control. Bars = 10 μm. (B) Transcriptional activation assay of Di19 in yeast. Schematic diagrams of the constructs are shown to the left. The pBD–GAL4 and pGBKT7 vectors were used as positive and negative controls, respectively. NLS, Nuclear Localization Signals sequence; GAL4 AD, GAL4 Activation Domain; DNA-BD, GAL4 DNA-Binding Domain; MCS, Multiple Cloning Site. The black boxes indicate zinc-finger motifs and the gray box indicates NLS sequence. β-Gal, β-galactosidase assay. (C) Transcriptional activation assay of Di19 in Arabidopsis protoplasts. GALDBD is a negative control and VP16 is a positive control for transactivation activity. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 DNA-Binding Assay of Di19. (A) Alignment of representative sequences identified in bacterial one-hybrid experiment. Conserved nucleotides are colored. The sequence logo was generated using Weblogo (see the Methods section for details). (B) EMSA assay for Di19 binding to the DiBS sequence. The biotin-labeled DNA fragment was incubated with the His–Di19 or His protein. Competition for the labeled probe was performed by adding an excess of unlabeled probe. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Arabidopsis Di19 Binds to the Promoters of PR Genes and Promotes Their Expression. (A) Schematic representation of the PR gene locus. PR promoter and gene sequences are represented by horizontal lines and open boxes. Black rectangles mark the relative positions of the DiBS (Di19-binding sequence). The positions and relative sizes of the different ChIP–qPCR fragments covering the locus are marked by horizontal lines below the DiBS. (B) ChIP–qPCR assay for Di19 binding to the DiBS of the PRs promoters in wild-type (WT) and di19 mutant. The 3-week-old plants were irrigated (water) or drought-treated (drought, without irrigation) for 10 d, and then the mature leaves were harvested for ChIP–qPCR. The ChIP signals with (+AB) and without (–AB) anti-Di19 antibody serum are indicated. Three independent experiments were performed, with similar results. Data are the mean values of three replicates ± SD from one experiment. (C) Transient expression of the ProPRs:GUS fusion together with Super:Di19 in Nicotiana benthamiana leaves. ProPRs:GUS fusion together with Super1300 vector was the control. Super:LUC was co-injected as an internal standard in each injection. The data represent the mean values of four replicates ± SD. (D) qRT–PCR analysis of PR expressions in the Di19-overexpressing line (Super:Di19-7) and wild-type plants. Wild-type and Di19-overexpressing plants were grown in soil for 3 weeks, and then the mature leaves were harvested for qRT–PCR. The data represent the mean values of three replicates ± SD. (E) qRT–PCR analysis of PR1, PR2, and PR5 expressions in the di19 mutant and wild-type plants. Three-week-old plants were irrigated (water) or drought-treated (drought, without irrigation) for 14 d, and then the mature leaves were harvested for qRT–PCR. The data represent the mean values of three replicates ± SD. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Phenotype Tests for the PR1-, PR2-, and PR5-Overexpressing Lines. (A–C) qRT–PCR analysis of PR1 (A), PR2 (B), and PR5 (C) expression in the PR-overexpressing lines. The mature leaves of 3-week-old plants were used for RNA extraction. The data represent the mean values of three replicates ± SD. (D) Phenotype tests of Arabidopsis plants under drought stress. Three-week-old plants were grown for 18 d with (Control, upper panel) or without (Drought, lower panel) irrigation. The pictures were taken on day 18. The experiments were repeated for three times, with similar results. (E) Water loss measurements for Arabidopsis different genotypes. Weight loss in detached leaves was measured at the time points indicated. Water loss was expressed as a percentage of initial fresh weight. The data presented are means ± SD of four replicates for one experiment (* P < 0.05 from the fourth time point). The experiments were repeated three times, with similar results. (F) GUS staining in stomata in ProPRs:GUS transgenic lines. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Plants Pre-Treated with INA Display Enhanced Drought Tolerance Phenotype. (A) Drought tolerance tests of di19 mutant and wild-type plants pre-treated with INA. Three-week-old di19 mutant and wild-type plants were treated with (+INA, adding 300 μM INA) or without (–INA, adding water) INA for 3 h, and then the plants were grown with (water) or without (drought) irrigation for 21 d. The pictures were taken on day 21. The experiments were repeated three times, with similar results. (B–D) qRT–PCR analysis of PR1, PR2, and PR5 expressions in di19 mutant and wild-type plants with INA treatment. Three-week-old plants were treated with (+INA) or without (–INA) IAN for 3 h, and then plants continued to grow for 3 d before their mature leaves were harvested for qRT–PCR. The data represent the mean values of three replicates ± SD. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Phenotype Tests of SA Mutant under Drought Stress. (A) Phenotype test of sid2, eds5, and npr1 mutants under drought stress. Three-week-old plants were grown for 18 d with (Control, upper panel) or without (Drought, lower panel) irrigation. The pictures were taken on day 18. The experiments were repeated three times, with similar results. (B, C) qRT–PCR analysis of PR1, PR2, and PR5 expressions in sid2, eds5, and npr1 mutants under drought stress. Total RNA was extracted at the times indicated from 3-week-old plants’ mature leaves. The data represent the mean values of three replicates ± SD. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 CPK11 Enhanced Di19 Transcriptional Activation. (A) BiFC assay of Di19 interaction with CPK11 in vivo. The expressions of Di19 alone (Di19–YN/pUC–SPYCE) and CPK11 alone (CPK11–YN/pUC–SPYCE) were used as controls. (B) Transient expression of the ProPRs:GUS fusion together with Super:Di19, or with Super:Di19 and Super:CPK11 in Nicotiana benthamiana leaves. ProPRs:GUS fusion together with Super1300 vector was the control. Super:LUC was co-injected as an internal standard in each injection. The data presented are means ± SD (n = 4). (C, D) qRT–PCR analysis of CPK11 (C) and PRs (D) expressions in CPK11-overexpressing lines and wild-type plants. The mature leaves of 3-week-old plants were used for RNA extraction. The data represent the mean values of three replicates ± SD. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 9 Working Model of CPK11/Di19/PRs-Regulatory Pathway in Plant Responses to Drought Stress. Arrows ending solid lines denote positive regulation and arrows ending broken lines indicate hypothetical regulation. Details of this schematic model are discussed in the text. Molecular Plant 2013 6, 1487-1502DOI: (10.1093/mp/sst031) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions