;Leaves of maize (Zea mays L.) seedlings were supplied with different concentrations of abscisic acid (ABA). Its effects on the levels of superoxide radical (O 2 -), hydrogen peroxide (H 2 O 2 ) and the content of catalytic Fe, the activities of several antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR), the contents of several non-enzymatic antioxidants such as ascorbate (ASC), reduced glutathione (GSH), a a a a-tocopherol (a a a a-TOC) and carotenoid (CAR), and the degrees of the oxidative damage to the membrane lipids and proteins were examined. Treatment with 10 and 100 m m m mM ABA significantly increased the levels of O 2 -and H 2 O 2 , followed by an increase in activities of SOD, CAT, APX and GR, and the contents of ASC, GSH, a a a a-TOC and CAR in a dose-and time-dependent pattern in leaves of maize seedlings. An oxidative damage expressed as lipid peroxidation, protein oxidation, and plasma membrane leakage did not occur except for a slight increase with 100 m m m mM ABA treatment for 24 h. Treatment with 1,000 m m m mM ABA led to a more abundant generation of O 2 -and H 2 O 2 and a significant increase in the content of catalytic Fe, which is critical for H 2 O 2 -dependent hydroxyl radical production. The activities of these antioxidative enzymes and the contents of a a a a-TOC and CAR were still maintained at a higher level, but no longer further enhanced when compared with the treatment of 100 m m m mM ABA. The contents of ASC and GSH had no changes in leaves treated with 1,000 m m m mM ABA. These results indicate that treatment with low concentrations of ABA (10 to 100 m m m mM) induced an antioxidative defence response against oxidative damage, but a high concentration of ABA (1,000 m m m mM) induced an excessive generation of AOS and led to an oxidative damage in plant cells.

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.

The roles of the plasma-membrane (PM) NADPH oxidase in abscisic acid (ABA)- and water stress-induced antioxidant defense were investigated in leaves of maize ( Zea mays L.) seedlings. Treatment by exogenous ABA (100 micro M ABA) or osmotic stress (-0.7 MPa induced by polyethylene glycol) significantly increased the activity of the PM NADPH oxidase, the production of leaf O(2)(-), the activities of several antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), and the contents of antioxidant metabolites (ascorbate and reduced glutathione). Pretreatment with three different inhibitors of NADPH oxidase (diphenylene iodonium, imidazole and pyridine) or an inhibitor of ABA biosynthesis (tungstate) reduced the increase in the activity of the PM NADPH oxidase and the production of leaf O(2)(-), and the capacity of antioxidant defense systems mediated by ABA. The inhibitory effects above caused by tungstate were reversed by exogenous ABA. These data indicate that NADPH oxidase is involved in the ABA-induced production of active oxygen species (AOS), and our results depict a minimal chain of events initiated by water stress-induced ABA accumulation, which then triggers the production of AOS by membrane-bound NADPH oxidase, resulting in the induction of antioxidant defense systems against oxidative damage in plants.

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.

Summary• Using pharmacological and biochemical approaches, the role of calmodulin (CaM) and the relationship between CaM and hydrogen peroxide (H 2 O 2 ) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize ( Zea mays ) plants were investigated.• Treatment with ABA or H 2 O 2 led to significant increases in the concentration of cytosolic Ca 2 + in the protoplasts of mesophyll cells and in the expression of the calmodulin 1 ( CaM1 ) gene and the content of CaM in leaves of maize plants, and enhanced the expression of the antioxidant genes superoxide dismutase 4 ( SOD4 ), cytosolic ascorbate peroxidase ( cAPX ), and glutathione reductase 1 ( GR1 ) and the activities of the chloroplastic and cytosolic antioxidant enzymes. The up-regulation of the antioxidant enzymes was almost completely blocked by pretreatments with two CaM antagonists.• Pretreatments with CaM antagonists almost completely inhibited ABA-induced H 2 O 2 production throughout ABA treatment, but pretreatment with an inhibitor or scavenger of reactive oxygen species (ROS) did not affect the initial increase in the contents of CaM induced by ABA.• Our results suggest that Ca 2 + -CaM is involved in ABA-induced antioxidant defense, and that cross-talk between Ca 2 + -CaM and H 2 O 2 plays a pivotal role in ABA signaling.

The histochemical and cytochemical localization of abscisic acid (ABA)-induced H(2)O(2) production in leaves of maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine (DAB) and CeCl(3) staining, respectively, and the relationship between ABA-induced H(2)O(2) production and ABA-induced subcellular activities of antioxidant enzymes was studied. H(2)O(2) generated in response to ABA treatment was detected within 0.5 h in major veins of the leaves and maximized at about 2-4 h. In mesophyll and bundle sheath cells, ABA-induced H(2)O(2) accumulation was observed only in apoplast, and the greatest accumulation occurred in the walls of mesophyll cells facing large intercellular spaces. Meanwhile, ABA treatment led to a significant increase in the activities of the leaf chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and pretreatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), the O (2) (-) scavenger Tiron and the H(2)O(2) scavenger dimethylthiourea (DMTU) almost completely arrested the increase in the activities of these antioxidant enzymes. Our results indicate that the accumulation of apoplastic H(2)O(2) is involved in the induction of the chloroplastic and cytosolic antioxidant enzymes. Moreover, an oxidative stress induced by paraquat (PQ), which generates O (2) (-) and then H(2)O(2) in chloroplasts, also up-regulated the activities of the chloroplastic and cytosolic antioxidant enzymes, and the up-regulation was blocked by the pretreatment with Tiron and DMTU. These data suggest that H(2)O(2) produced at a specific cellular site could coordinate the activities of antioxidant enzymes in different subcellular compartments.