ZmMPK5 is required for the NADPH oxidase-mediated self-propagation of apoplastic H2O2 in brassinosteroid-induced antioxidant defence in leaves of maize

Abstract

Brassinosteroids (BRs) have been shown to induce hydrogen peroxide (H(2)O(2)) accumulation, and BR-induced H(2)O(2) up-regulates antioxidant defence systems in plants. However, the mechanisms by which BR-induced H(2)O(2) regulates antioxidant defence systems in plants remain to be determined. In the present study, the role of ZmMPK5, a mitogen-activated protein kinase, in BR-induced anitioxidant defence and the relationship between the activation of ZmMPK5 and H(2)O(2) production in BR signalling were investigated in leaves of maize (Zea mays) plants. BR treatment activated ZmMPK5, induced apoplastic and chloroplastic H(2)O(2) accumulation, and enhanced the total activities of antioxidant enzymes. Such enhancements were blocked by pre-treatment with mitogen-activated protein kinase kinase (MAPKK) inhibitors and H(2)O(2) inhibitors or scavengers. Pre-treatment with MAPKK inhibitors substantially arrested the BR-induced apoplastic H(2)O(2) production after 6 h of BR treatment, but did not affect the levels of apoplastic H(2)O(2) within 1 h of BR treatment. BR-induced gene expression of NADPH oxidase was also blocked by pre-treatment with MAPKK inhibitors and an apoplastic H(2)O(2) inhibitor or scavenger after 120 min of BR treatment, but was not affected within 30 min of BR treatment. These results suggest that the BR-induced initial apoplastic H(2)O(2) production activates ZmMPK5, which is involved in self-propagation of apoplastic H(2)O(2) via regulation of NADPH oxidase gene expression in BR-induced antioxidant defence systems.

Fig. 1.

Induction of the activity and gene expression of ZmMPK5 in leaves of maize plants exposed to brassinosteroid (BR) treatment. (A) Time course of BR-induced MBP kinase activation. The detached plants were treated with 10 nM BR for various times as indicated. (B) BR-induced ZmMPK5 activation. The detached plants were treated with 10 nM BR for 30 min. Protein extracts from control (Con)- or BR-treated leaves were immunoprecipitated with ZmMPK5 and then subjected to an in-gel kinase assay. (C) Time course of BR-induced gene expression of ZmMPK5. The detached plants were treated with 10 nM BR for various times as indicated. The plants treated with distilled water under the same conditions during the whole period served as controls. Relative expression levels of ZmMPK5, analysed by qRT-PCR, are normalized to Zmactin transcript levels. (A) and (B) All experiments were repeated at least three times with similar results. (C) 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.

Fig. 2.

Effects of pre-treatment with mitogen-activated protein kinase kinase (MAPKK) inhibitors on the activities of ZmMPK5 and antioxidant enzymes in leaves of maize plants exposed to brassinosteroid (BR) treatment (A) The ZmMPK5 immunoprecipitation kinase activity assay. (B) The total activities of the antioxidant enzymes ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), and catalase (CAT). The detached plants were treated as follows: 1, distilled water (control); 2, 10 nM BR; 3, 100 μM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059)+10 nM BR; 4, 10 μM 1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene (U0126)+10 nM BR. The detached plants were pre-treated with various inhibitors or distilled water for 4 h, and then exposed to 10 nM BR treatment for 30 min (A) or 12 h (B). (A) Experiments were repeated at least three times with similar results. (B) 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.

Fig. 3.

Histochemical detection of brassinosteroid (BR)-induced hydrogen peroxide (H₂O₂) accumulation with 3,3-diaminobenzidine (DAB) staining in leaves of detached maize plants. The detached plants were pre-treated with 100 μM diphenylene iodonium (DPI), 200 U of catalase (CAT), 1 mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or distilled water for 4 h, and then exposed to 10 nM BR treatment for 1 h. The plants treated with distilled water under the same conditions during the whole period served as controls. Experiments were repeated at least five times with similar results. (This figure is available in colour at JXB online.)

Fig. 4.

Cytochemical localization of brassinosteroid (BR)-induced hydrogen peroxide (H₂O₂) accumulation in mesophyll cells of maize leaves with CeCl₃ staining and transmission electron microscopy. (A) H₂O₂ accumulation in apoplasts. (B) H₂O₂ accumulation in chloroplasts. (C) H₂O₂ accumulation in mitochondria and peroxisomes. The detached plants were treated with 10 nM BR for various times as indicated. All experiments were repeated at least three times with similar results. Arrows indicate CeCl₃ precipitates. C, chloroplast; CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole; IS, intercellular space. Bar=1 μm.

Fig. 5.

Effects of pre-treatment with reactive oxygen species (ROS) inhibitors or scavengers on the activity of ZmMPK5 and hydrogen peroxide (H₂O₂) accumulation in leaves of maize plants exposed to brassinosteroid (BR) treatment. (A) The ZmMPK5 immunoprecipitation kinase activity. The detached plants were pre-treated with 100 μM diphenylene iodonium (DPI), 200 U of catalase (CAT), 1 mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), or distilled water for 4 h, and then exposed to 10 nM BR treatment for 30 min. (B, C) Cytochemical localization of BR-induced H₂O₂ accumulation. The detached plants were pre-treated with 100 μM DPI, 200 U of CAT, 1 mM DCMU, or distilled water for 4 h, and then exposed to 10 nM BR treatment for 1 h (B) or 6 h (C). The plants treated with distilled water under the same conditions during the whole period served as controls. Experiments were repeated at least three times with similar results. Arrows indicate CeCl₃ precipitates. C, chloroplast; CW, cell wall; IS, intercellular space. Bar=1 μm.

Fig. 6.

Time-course analysis of the effects of mitogen-activated protein kinase kinase (MAPKK) inhibitors on the apoplastic hydrogen peroxide (H₂O₂) production in leaves of maize plants exposed to brassinosteroid (BR) treatment. The detached plants were pre-treated with 100 μM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 10 μM 1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene (U0126), or distilled water for 4 h, and then exposed to 10 nM BR treatment for various times as indicated. The plants treated with distilled water under the same conditions during the whole period served as controls. H₂O₂ production in leaves was detected by CeCl₃ staining. Experiments were repeated at least three times with similar results. Arrows indicate CeCl₃ precipitates. C, chloroplast; CW, cell wall; M, mitochondrion; N, nucleus; IS, intercellular space. Bar=1 μm.

Fig. 7.

Time-course analysis of the effects of mitogen-activated protein kinase kinase (MAPKK) inhibitors on the chloroplastic hydrogen peroxide (H₂O₂) production in leaves of maize plants exposed to brassinosteroid (BR) treatment. The detached plants were pre-treated with 100 μM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 10 μM 1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene (U0126), or distilled water for 4 h, and then exposed to 10 nM BR treatment for various times as indicated. The plants treated with distilled water under the same conditions during the whole period served as controls. H₂O₂ production in leaves was detected by CeCl₃ staining. Experiments were repeated at least three times with similar results. Arrows indicate CeCl₃ precipitates. C, chloroplast. Bar=1 μm.

Fig. 8.

Time-course analysis of ZmrbohA–ZmrbohD expression in leaves of maize plants exposed to brassinosteroid (BR) treatment. The detached plants were treated with 10 nM BR for various times as indicated. Relative expression levels of ZmrbohA–ZmrbohD genes, analysed by qRT-PCR, are normalized to the Zmactin transcript levels. 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.

Fig. 9.

Effects of pre-treatment with the apoplastic hydrogen peroxide (H₂O₂) inhibitor or scavenger and the mitogen-activated protein kinase kinase (MAPKK) inhibitors on the expression of ZmrbohA–ZmrbohD in leaves of maize plants exposed to brassinosteroid (BR) treatment. (A) Effects of pre-treatment with the apoplastic H₂O₂ inhibitor or scavenger. The detached plants were pre-treated with 100 μM diphenylene iodonium (DPI), 200 U of catalase (CAT), or distilled water for 4 h, and then exposed to 10 nM BR treatment for 30 min and 120 min, respectively. (B) Effects of pre-treatment with MAPKK inhibitors. The detached plants were pre-treated with 100 μM 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 10 μM 1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene (U0126), or distilled water for 4 h, and then exposed to 10 nM BR for 30 min and 120 min, respectively. The plants treated with distilled water under the same conditions during the whole period served as controls. Relative expression levels of ZmrbohA–ZmrbohD genes, analysed by qRT-PCR, are normalized to Zmactin transcript levels. 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.