Roles of Defense Hormones in the Regulation of Ozone-Induced Changes in Gene Expression and Cell Death  Enjun Xu, Lauri Vaahtera, Mikael Brosché  Molecular Plant  Volume 8, Issue 12, Pages 1776-1794 (December 2015) DOI: 10.1016/j.molp.2015.08.008 Copyright © 2015 The Author Terms and Conditions

Figure 1 Apoplastic ROS Regulated Genes in Col-0 and coi1 ein2 sid2. (A) RNA-seq samples analyzed by principal component analysis (PCA) on the top 10 000 genes with most variable expression. The first (PC1) and second (PC2) principal components contribute to 70% and 14% of the total variation, respectively. (B) Venn diagram comparison of O3-regulated genes in Col-0 and coi1 ein2 sid2. Genes were selected at q < 0.05. (C) Enrichment of selected GO categories in Col-0 and coi1 ein2 sid2. Arrows indicate genes with increased expression (↑) and decreased expression (↓). A full list of enriched GO categories can be found in Supplemental Table 2. (D) Promoter element enrichment in Col-0 and coi1 ein2 sid2. Promoter elements marked with * were identified as ROS responsive (Geisler et al., 2006; Wang et al., 2013). A full list of enriched promoter elements can be found in Supplemental Table 2. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 2 Cluster Analysis of O3 Genotype × Treatment Regulated Genes. Genes with two-fold difference in expression in genotype × treatment comparison between Col-0 and coi1 ein2 sid2 were further analyzed by cluster analysis with array data from the ATH1 chip including treatments with SA, ethylene, ABA, MeJA, and BTH (see Methods for full list of experiments). Log-transformed fold changes were subjected to bootstrapped Bayesian hierarchical clustering. Five distinct clusters were identified (the full gene list for each cluster is provided in Supplemental Table 3). Magenta and green indicate increased and decreased expression compared with untreated or wild-type plants, respectively. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 3 Early Apoplastic ROS Signaling Is Mostly Independent of SA, JA, and Ethylene Signaling. Gene expression was measured with qPCR after a 2-h O3 treatment. Log2-transformed fold change of selected marker genes in sid2 and NahG (SA signaling), coi1-16 (JA signaling), and ein2 (ethylene signaling) single mutants, and corresponding double and triple mutants, from four biological replicates are shown. Statistical analysis was performed with a linear mixed model (P < 0.05). Error bars depict the standard deviation; a, b, c, and d represent comparison of the O3 effect on different genotypes. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 4 The Role of ABA and TFs in Apoplastic ROS Signaling. Gene expression was measured with qPCR after a 2-h O3 treatment. Log2-transformed fold change of selected marker genes in abi1-1 (ABA insensitive), aos (JA deficient), ERF6-4D (gain-of-function of ERF6), ERF6-EAR (dominant negative ERF6), anac017-1 and anac017-3 (loss-of-function alleles), anac017-2 (gain-of-function allele), and tga2 tga5 tga6 (SA- and JA-dependent signaling) mutants from three or four biological replicates are shown; error bars depict the standard deviation; a and b represent comparison of the O3 effect on different genotypes. Statistical analysis was performed with a linear mixed model (P < 0.05). Error bars depict the standard deviation; a, b, c, d, and e represent comparison of the O3 effect on different genotypes. Gain-of-function alleles are shaded gray. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 5 Apoplastic ROS Regulated Genes in Col-0 and tga2 tga5 tga6. (A) RNA-seq samples analyzed by principal component analysis (PCA) on the top 10 000 genes with most variable expression. The first (PC1) and second (PC2) principal components contribute to 75% and 10% of the total variation, respectively. (B) Venn diagram comparison of O3-regulated genes in Col-0 and tga2 tga5 tga6. Genes were selected at q < 0.05. (C) Enrichment of selected GO categories in Col-0 and tga2 tga5 tga6. Arrows indicate genes with increased expression (↑) and decreased expression (↓). A full list of enriched GO categories can be found in Supplemental Table 5. (D) Promoter element enrichment in Col-0 and tga2 tga5 tga6. Promoter elements marked with * were identified as ROS responsive (Geisler et al., 2006; Wang et al., 2013). A full list of enriched promoter elements can be found in Supplemental Table 5. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 6 Similarities and Differences in Gene Expression between the Two Studied Triple Mutants Compared with Wild-type. (A) Overlap of the genes with higher expression in coi1 ein2 sid2 and tga2 tga5 tga6 than in the wild-type with and without O3 treatment. (B) Overlap of the genes with lower expression in coi1 ein2 sid2 and tga2 tga5 tga6 than in the wild-type with and without O3 treatment. Enriched gene ontology (GO) categories characteristic for each gene set are marked. The circled gene set was too small for meaningful GO analysis but contained numerous classical SA marker genes. Gene identities in overlapping gene groups can be found in Supplemental Table 5. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 7 The Redundancy of Different WRKY TFs Might Mask Their Role in Apoplastic ROS Signaling. Gene expression was measured with qPCR after a 2-h O3 treatment. Log2-transformed fold change of selected marker genes in wrky25 wrky33 and wrky18 wrky40 wrky60 from three or four biological replicates are shown; error bars depict the standard deviation; a and b represent comparison of the O3 effect on different genotypes. Statistical analysis was performed with a linear mixed model (P < 0.05). Error bars depict the standard deviation; a and b represent comparison of the O3 effect on different genotypes. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 8 Comparison of Gene Expression in wrky Mutants and Apoplastic ROS Signaling. From the RNA-seq data, O3-regulated genes with at least three W-boxes were identified (323 genes). These genes were analyzed in gene expression datasets from coi1-16 sid2 ein2 and different WRKY TF mutants in response to BTH, and biotic and abiotic stresses. In addition, mutants undergoing spontaneous cell death were included (see Methods for a full list of experiments). Log2-transformed fold changes were subjected to bootstrapped Bayesian hierarchical clustering. Six distinct clusters were identified (the full gene list for each cluster is provided in Supplemental Table 6). Magenta and green indicate increased and decreased expression compared with untreated or wild-type plants, respectively. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 9 Regulation of Apoplastic ROS-Induced Cell Death in coi1-16. The indicated genotypes were exposed to O3 for 6 h (350 nl l−1). Cell death was quantified with ion leakage 22 h after the O3 treatment. Bars represent the means of four biological repeats with the standard deviation (n = 20). A two-way ANOVA with the Tukey test (P < 0.05) was used for statistical analysis of the results; a, b, c, d, e, and f represent comparison of different genotypes after the O3 treatment; A represents comparison of genotypes in fresh air. * and n.s represent comparison between clean air and the O3 treatment in each genotype. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions

Figure 10 RBOHF Regulates Apoplastic ROS-Induced H2O2 accumulation in coi1-16. H2O2 accumulation visualized by DAB staining after a 4-h O3 treatment (350 nl l−1) in the indicated genotypes. The experiment was repeated two times; representative photos are shown. Scale bar = 9 mm. Molecular Plant 2015 8, 1776-1794DOI: (10.1016/j.molp.2015.08.008) Copyright © 2015 The Author Terms and Conditions