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.

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.

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 antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination, but the mechanism of antagonism during this process is not known. The possible links among ABA, reactive oxygen species (ROS), ascorbic acid (ASC), and GA during rice seed germination were investigated. Unlike in non-seed tissues where ROS production is increased by ABA, ABA reduced ROS production in imbibed rice seeds, especially in the embryo region. Such reduced ROS also led to an inhibition of ASC production. GA accumulation was also suppressed by a reduced ROS and ASC level, which was indicated by the inhibited expression of GA biosynthesis genes, amylase genes, and enzyme activity. Application of exogenous ASC can partially rescue seed germination from ABA treatment. Production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. It can be concluded that ABA regulates seed germination in multiple dimensions. ROS and ASC are involved in its inhibition of GA biosynthesis.

The role of ABA in brassinosteroid (BR)-induced stress tolerance and the relationship between BR, nitric oxide (NO) and ABA under water stress induced by polyethylene glycol (PEG) were investigated in leaves of maize (Zea mays) plants. Water stress led to oxidative damage. Pre-treatment with the BR biosynthetic inhibitor brassinazole (Brz) aggravated the oxidative damage induced by PEG treatment, which was alleviated by the application of BR or ABA. Pre-treatment with the ABA biosynthetic inhibitor fluridone also aggravated the oxidative damage induced by PEG treatment; however, this was barely alleviated by the application of BR. BR treatment increased the content of ABA and up-regulated the expression of the ABA biosynthetic gene vp14 in maize leaves, which was blocked by pre-treatments with the NO scavenger cPTIO (2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and the nitric oxide synthase inhibitor l-NAME (N(G)-nitro-l-arginine methyl ester. Moreover, BR treatment induced increases in the generation of NO in mesophyll cells of maize leaves, and treatment with the NO donor sodium nitroprusside (SNP) up-regulated the content of ABA and the expression of vp14 in maize leaves. Our results suggest that BR-induced NO production and NO-activated ABA biosynthesis are important mechanisms for BR-enhanced water stress tolerance in leaves of maize plants.