Volume 5, Issue 6, Pages 1359-1374 (November 2012) OsDMI3 Is a Novel Component of Abscisic Acid Signaling in the Induction of Antioxidant Defense in Leaves of Rice  Ben Shi, Lan Ni, Aying Zhang, Jianmei Cao, Hong Zhang, Tingting Qin, Mingpu Tan, Jianhua Zhang, Mingyi Jiang  Molecular Plant  Volume 5, Issue 6, Pages 1359-1374 (November 2012) DOI: 10.1093/mp/sss068 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 ABA, H2O2, and PEG Induce the Expression of OsDMI3 and the Activity of OsDMI3 in Rice Leaves. (A) Expression analysis of OsDMI3 in leaves of rice plants exposed to ABA, H2O2, and PEG treatments. The rice seedlings were treated with ABA (100μM), H2O2 (10mM), and PEG (10%) for various times as indicated. Relative expression levels of OsDMI3 gene were analyzed by real-time quantitative PCR. Values are means ±SE of three different experiments. Means denoted by the same letter did not significantly differ at P<0.05 according to Duncan’s multiple range test. (B) Induction in the activity of OsDMI3 by ABA, H2O2, and PEG. The plants were treated as described in (A). OsDMI3 was immunoprecipitated from leaves after treatments and the activity of OsDMI3 was measured by immunoprecipitation kinase assay using histone S-III as a substrate. Corresponding Coomassie staining was also shown as indicated. Experiments were repeated at least three times, with similar results. (C) Kinase activity assay of immunocomplexes after immunoprecipitation. Precipitation was performed in the absence or presence of competitor peptides corresponding to the C-terminal portion or the N-terminal peptide of OsDMI3. Kinase activity was measured using an in-gel kinase assay. Corresponding Coomassie staining was also shown as indicated. Experiments were repeated at least three times, with similar results. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 H2O2 Is Required for the ABA-Induced Activation of OsDMI3 under Water Stress. (A, B) Effect of pretreatment with the ABA biosynthetic inhibitor fluridone on the expression of OsDMI3 (A) and the activity of OsDMI3 (B) in rice leaves exposed to PEG treatment. The fluridone-treated and -untreated seedlings were exposed to 10% PEG treatment for 1h. 100μM ABA was added to overcome the effects of fluridone. (C, D) Effects of pretreatments with the ROS manipulators DMTU and DPI on the expression of OsDMI3 (C) and the activity of OsDMI3 (D) in rice leaves exposed to ABA treatment. The rice seedlings were pretreated with 5mM DMTU and 100μM DPI for 4h, and then exposed to 100μM ABA for 30min (C) or 60min (D). Values are means ±SE of three different experiments. Means denoted by the same letter did not significantly differ at P<0.05 according to Duncan’s multiple range test. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Ca2+ and CaM Are Required for the ABA-Induced Activation of OsDMI3 in Rice Leaves. (A) Effects of pretreatments with Ca2+ inhibitors, CaM antagonists, and CaMK inhibitors on the activity of OsDMI3 in leaves of rice seedlings exposed to ABA treatment. The rice plants were pretreated with 5mM EGTA, 5mM LaCl3, 300μM W7, 300μM W5, 10µM KN-92, and 10µM KN-93 for 4h, and then exposed to 100μM ABA treatment for 1h. Plants treated with distilled water under the same conditions served as the control. After treatments, the activity of OsDMI3 was analyzed by immunoprecipitation kinase assay. Values are means ±SE of three different experiments. Means denoted by the same letter did not significantly differ at P<0.05 according to Duncan’s multiple range test. (B) CaCl2-induced changes in the activity of OsDMI3 in rice leaves. The rice seedlings were treated with 10mM CaCl2 for various times as indicated and the activity of OsDMI3 was analyzed by immunoprecipitation kinase assay. Experiments were repeated at least three times, with similar results. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Subcellular Localization of OsDMI3 in Rice Protoplasts. (A, B) Constructs carrying 35S:OsDMI3–YFP, OsDMI3:OsDMI3–YFP, or 35S:YFP were introduced into protoplasts prepared from the leaves and stems of rice by PEG-calcium-mediated transformation. Transfected protoplasts were observed after 16-h incubation by a laser confocal microscope. Nuclei are shown with DAPI staining (blue, (A)). The plasma membrane was labeled with FM4-64 (red, (B)). Experiments were repeated at least five times with similar results. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 OsDMI3 Is Required for the ABA-Induced Up-Regulation in the Expression and the Activities of SOD and CAT in Rice Protoplasts. (A) The transient expression of OsDMI3 in protoplasts. The protoplasts transfected with 35S-OsDMI3–YFP plasmid were observed after 16-h incubation by a laser confocal microscope (bottom). The protoplasts transfected with the empty vector were used as control (top). (B) The transient RNAi silencing of OsDMI3 in protoplasts. Protoplasts were transfected with dsRNA against OsDMI3 (RNAi) or with water (control) and incubated for 24h. Silencing of OsDMI3 was analyzed by RT–PCR. GAPDH was amplified as a control for amount of a template. (C) The activity of OsDMI3 in rice protoplasts. The protoplasts transiently expressing OsDMI3 (top) or transiently silencing OsDMI3 (bottom) were treated with 10µM ABA for 5min, and the activity of OsDMI3 was analyzed by immunoprecipitation kinase assay. Corresponding Coomassie staining was also shown as indicated. (D) The expression of SodCc2 and CatB in the protoplasts transiently silencing OsDMI3. The protoplasts were treated with 10µM ABA for 5min, and the relative expression levels of SodCc2 and CatB were analyzed by real-time quantitative PCR. (E, F) The activities of SOD and CAT in the protoplasts transiently expressing OsDMI3 (E) or transiently silencing OsDMI3 (F). The protoplasts were treated with 10µM ABA for 5min, and the activities of SOD and CAT were measured. In (A–C), experiments were repeated at least three times, with similar results. In (D–F), values are means±SE of three different experiments. Means denoted by the same letter did not differ significantly at P<0.05 according to Duncan’s multiple range test. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Isolation and Characterization of Tos17 Insertion Mutant of OsDMI3. (A) Gene structure of OsDMI3 and the Tos17 insertion site in NF8513. The exons and introns are indicated by boxes and lines, respectively. Insertion site of Tos17 in the mutant line is indicated by flagpole showing the name of the mutant line. (B) Identification of homozygous (–/–) Tos17 insertion mutant by PCR. Distinction of homozygous (–/–), heterozygous (+/–) mutant plants, and wild-type (+/+) plants by PCR using Tos17-flanking, Tos17-, and OsDMI3-specific primers. WT, wild-type Nipponbare. (C) RT–PCR analysis of OsDMI3 transcripts in the wild-type and homozygous NF8513 mutant. RT–PCR was performed using a primer pair designed from exons flanking the insertion site. The allele in NF8513 produced a band of a smaller size due to the deletion of exon 3. (D) OsDMI3 activity in the wild-type and homozygous NF8513 mutant. The activity of OsDMI3 was analyzed by immunocomplex kinase assay using histone S-III as the substrate. Corresponding Coomassie staining was also shown as indicated. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 The OsDMI3 Mutant Shows Sensitivity to Water Stress and Oxidative Stress. (A) Water stress tolerance of OsDMI3 mutant and wild-type plants. The rice seedlings were treated with 20% PEG for 9d. (B, C) The content of MDA (B) and the percent leakage of electrolyte (C) in the mutant and the wild-type. The rice seedlings were treated with 15% PEG and 100mM H2O2 for 24h, and then leaves were sampled for the determination of MDA content (B) and electrolyte leakage (%) (C). (D) The activities of SOD and CAT in the mutant and the wild-type. The rice seedlings were treated with 15% PEG and 100mM H2O2 for 12h, and then leaves were sampled for the determination of the activities of SOD and CAT. In (A), experiments were repeated at least three times, with similar results. In (B–D), values are means±SE of three different experiments. Means denoted by the same letter did not differ significantly at P<0.05 according to Duncan’s multiple range test. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 8 OsDMI3 Mediates the ABA-Induced Production of H2O2. (A) H2O2 fluorescence in the protoplasts transiently silencing OsDMI3. The protoplasts were treated with 10μM ABA (+ABA) or the incubation medium (–ABA) for 5min, and then loaded with H2DCF-DA for 10min. The protoplasts transfected with water were used as controls. H2O2 was visualized by confocal microscopy. (B) Changes in the fluorescence intensity in (A). (C) Time-course analysis of leaf H2O2 production in the mutant of OsDMI3 and the wild-type exposed to ABA treatment. The plants were excised at the base of the stem and the detached plants were treated with 100µM ABA or distilled water for various times as indicated. H2O2 production in leaves was detected by DAB staining. (D) Expression analysis of OsrbohB, OsrbohE, and OsrbohI in the protoplasts transiently silencing OsDMI3. The protoplasts were treated with 10µM ABA for 5min, and the relative expression levels of OsrbohB, OsrbohE, and OsrbohI were analyzed by real-time quantitative PCR. In (A) and (C), experiments were repeated at least three times with similar results. In (B) and (D), values are means±SE of three different experiments. Means denoted by the same letter did not differ significantly at P<0.05 according to Duncan’s multiple range test. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 9 Model Summarizing the Interactions of NADPH Oxidase, H2O2, and CCaMK in ABA-Induced Antioxidant Defense under Water Stress. Water stress induces ABA accumulation, and the accumulation of ABA activates H2O2 production by NADPH oxidase. The production of H2O2 induces the activation of CCaMK, thus resulting in the up-regulation of antioxidant defense enzymes. The activation of CCaMK also enhances H2O2 production by NADPH oxidase, forming a positive amplification loop. The up-regulation in the activities of antioxidant enzymes enhances the ability of cells to scavenge ROS, and reduces the oxidative damage caused by a severe water stress. Molecular Plant 2012 5, 1359-1374DOI: (10.1093/mp/sss068) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions