The role of mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense was investigated in leaves of maize (Zea mays) plants. Treatments with ABA or H 2 O 2 induced the activation of a 46-kD MAPK and enhanced the expression of the antioxidant genes CAT1, cAPX, and GR1 and the total activities of the antioxidant enzymes catalase, ascorbate peroxidase, glutathione reductase, and superoxide dismutase. Such enhancements were blocked by pretreatment with several MAPK kinase inhibitors and reactive oxygen species inhibitors or scavengers. Pretreatment with MAPK kinase inhibitors also substantially arrested the ABA-induced H 2 O 2 production after 2 h of ABA treatment, but did not affect the levels of H 2 O 2 within 1 h of ABA treatment. Pretreatment with several inhibitors of protein tyrosine phosphatase, which is believed to be a negative regulator of MAPK, only slightly prevented the ABA-induced H 2 O 2 production, but did not affect the ABA-induced MAPK activation and ABA-enhanced antioxidant defense systems. These results clearly suggest that MAPK but not protein tyrosine phosphatase is involved in the ABA-induced antioxidant defense, and a cross talk between H 2 O 2 production and MAPK activation plays a pivotal role in the ABA signaling. ABA-induced H 2 O 2 production activates MAPK, which in turn induces the expression and the activities of antioxidant enzymes. The activation of MAPK also enhances the H 2 O 2 production, forming a positive feedback loop.

Summary• The role of nitric oxide (NO) and the relationship between NO, hydrogen peroxide (H 2 O 2 ) and mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize ( Zea mays ) plants were investigated.• Both ABA and H 2 O 2 induced increases in the generation of NO in mesophyll cells of maize leaves, and H 2 O 2 was required for the ABA-induced generation of NO. Pretreatment with NO scavenger and nitric oxide synthase (NOS) inhibitor substantially reduced the ABA-induced production of NO, and partly blocked the activation of a 46 kDa MAPK and the expression and the activities of several antioxidant enzymes induced by ABA. Treatment with the NO donor sodium nitroprusside (SNP) also induced the activation of the MAPK, and enhanced the antioxidant defense systems.• Conversely, SNP treatment did not induce the production of H 2 O 2 , and pretreatments with NO scavenger and NOS inhibitor did not affect ABA-induced H 2 O 2 production.• Our results suggest that ABA-induced H 2 O 2 production mediates NO generation, which, in turn, activates MAPK and results in the upregulation in the expression and the activities of antioxidant enzymes in ABA signaling.

SummaryIn this study it was found that ZFP36 is required for ABA-induced antioxidant defence and for the regulation of the cross-talk between NADPH oxidase, H2O2, and MAPK in ABA signalling.

In maize (Zea mays), abscisic acid (ABA)-induced H2O2 production activates a 46 kDa mitogen-activated protein kinase (p46MAPK), and the activation of p46MAPK also regulates the production of H2O2. However, the mechanism for the regulation of H2O2 production by MAPK in ABA signalling remains to be elucidated. In this study, four reactive oxygen species (ROS)-producing NADPH oxidase (rboh) genes (ZmrbohA–D) were isolated and characterized in maize leaves. ABA treatment induced a biphasic response (phase I and phase II) in the expression of ZmrbohA–D and the activity of NADPH oxidase. Phase II induced by ABA was blocked by pretreatments with two MAPK kinase (MPKKK) inhibitors and two H2O2 scavengers, but phase I was not affected by these inhibitors or scavengers. Treatment with H2O2 alone also only induced phase II, and the induction was arrested by the MAPKK inhibitors. Furthermore, the ABA-activated p46MAPK was partially purified. Using primers corresponding to the sequences of internal tryptic peptides, the p46MAPK gene was cloned. Analysis of the tryptic peptides and the p46MAPK sequence indicate it is the known ZmMPK5. Treatments with ABA and H2O2 led to a significant increase in the activity of ZmMPK5, although ABA treatment only induced a slight increase in the expression of ZmMPK5. The data indicate that H2O2-activated ZmMPK5 is involved in the activation of phase II in ABA signalling, but not in phase I. The results suggest that there is a positive feedback loop involving NADPH oxidase, H2O2, and ZmMPK5 in ABA signalling.