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In plants, Ca 2+ /calmodulin-dependent protein kinase (CCaMK) is a positive regulator of abscisic acid (ABA) responses, including root growth, antioxidant defense, and tolerance of both water stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, we show a direct interaction between DMI3 (Doesn't Make Infections 3), a rice ( Oryza sativa ) CCaMK and PP45, a type 2C protein phosphatase in rice (PP2C). This interaction involves the CaM binding domain of DMI3 and the PP2C domain of PP45. In the absence of ABA, PP45 directly inactivates DMI3 by dephosphorylating Thr-263 in DMI3. However, in the presence of ABA, ABA-induced H 2 O 2 production by the NADPH oxidases RbohB/E inhibits the activity of PP45 not only by inhibiting the expression of PP45 but also by oxidizing Cys-350 and Cys-428 residues to form PP45 intermolecular dimers. ABA-induced oxidation of Cys-350 and Cys-428 in PP45 blocked the interaction between PP45 and DMI3 and substantially prevented PP45-mediated inhibition in DMI3 activity. Genetic analysis indicated that PP45 is an important negative regulator of ABA signaling. These results reveal important pathways for the inhibition of DMI3 under the basal state and for its ABA-induced activation in rice., (© 2019 American Society of Plant Biologists. All rights reserved.)

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.

Nitric oxide (NO) is a bioactive molecule involved in many biological events, and has been reported as pro-oxidant as well as anti-oxidant in plants. In the present study, the sources of NO production under water stress, the role of NO in water stress-induced hydrogen peroxide (H2O2) accumulation and subcellular activities of anti-oxidant enzymes in leaves of maize (Zea mays L.) plants were investigated. Water stress induced defense increases in the generation of NO in maize mesphyll cells and the activity of nitric oxide synthase (NOS) in the cytosolic and microsomal fractions of maize leaves. Water stress-induced defense increases in the production of NO were blocked by pretreatments with inhibitors of NOS and nitrate reductase (NR), suggesting that NO is produced from NOS and NR in leaves of maize plants exposed to water stress. Water stress also induced increases in the activities of the chloroplastic and cytosolic anti-oxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), and the increases in the activities of anti-oxidant enzymes were reduced by pretreatments with inhibitors of NOS and NR. Exogenous NO increases the activities of water stress-induced subcellular anti-oxidant enzymes, which decreases accumulation of H2O2. Our results suggest that NOS and NR are involved in water stress-induced NO production and NOS is the major source of NO. The potential ability of NO to scavenge H2O2 is, at least in part, due to the induction of a subcellular anti-oxidant defense.

* 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.

* 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 water stress-induced H(2)O(2) production in the leaves of ABA-deficient vp5 mutant and wild-type maize (Zea mays L.) plants were examined, using 3,3-diaminobenzidine and CeCl(3) staining, respectively, and the roles of endogenous ABA in the production of H(2)O(2) induced by water stress were assessed. Water stress induced by polyethylene glycol resulted in the accumulation of H(2)O(2) in mesophyll cells, bundle-sheath cells and vascular bundles of wild-type maize leaves, and the accumulation was substantially blocked in the mutant maize leaves exposed to water stress. Pre-treatments with several apoplastic H(2)O(2) manipulators abolished the majority of H(2)O(2) accumulation induced by water stress in the wild-type leaves. The subcellular localization of H(2)O(2) production was demonstrated in the cell walls, xylem vessels, chloroplasts, mitochondria and peroxisomes in the leaves of wild-type maize plants exposed to water stress, and the accumulation of H(2)O(2) induced by water stress in the cell walls and xylem vessels, but not in the chloroplasts, mitochondria and peroxisomes, was arrested in the leaves of the ABA mutant or the ABA biosynthesis inhibitor (tungstate)-pre-treated maize plants. Pre-treatments with the apoplastic H(2)O(2) manipulators also blocked the apoplastic but not the intracellular H(2)O(2) accumulation induced by water stress in the leaves of wild-type plants. These data indicate that under water stress, the apoplast is the major source of H(2)O(2) production and ABA is a key inducer of apoplastic H(2)O(2) production. These data also suggest that H(2)O(2) generated in the apoplast could not diffuse freely into subcellular compartments.

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.

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense and enhance the tolerance of plants to drought stress. However, its downstream molecular events are poorly understood. Here, we identify a NAC transcription factor, ZmNAC84, in maize (Zea mays), which physically interacts with ZmCCaMK in vitro and in vivo. ZmNAC84 displays a partially overlapping expression pattern with ZmCCaMK after ABA treatment, and H2O2 is required for ABA-induced ZmNAC84 expression. Functional analysis reveals that ZmNAC84 is essential for ABA-induced antioxidant defense in a ZmCCaMK-dependent manner. Furthermore, ZmCCaMK directly phosphorylates Ser-113 of ZmNAC84 in vitro, and Ser-113 is essential for the ABA-induced stimulation of antioxidant defense by ZmCCaMK. Moreover, overexpression of ZmNAC84 in tobacco (Nicotiana tabacum) can improve drought tolerance and alleviate drought-induced oxidative damage of transgenic plants. These results define a mechanism for ZmCCaMK function in ABA-induced antioxidant defense, where ABA-produced H2O2 first induces expression of ZmCCaMK and ZmNAC84 and activates ZmCCaMK. Subsequently, the activated ZmCCaMK phosphorylates ZmNAC84 at Ser-113, thereby inducing antioxidant defense by activating downstream genes.

Nitric oxide (NO), hydrogen peroxide (H2O2), and calcium (Ca2+)/calmodulin (CaM) are all required for abscisic acid (ABA)-induced antioxidant defence. Ca2+/CaM-dependent protein kinase (CCaMK) is a strong candidate for the decoder of Ca2+ signals. However, whether CCaMK is involved in ABA-induced antioxidant defence is unknown. The results of the present study show that exogenous and endogenous ABA induced increases in the activity of ZmCCaMK and the expression of ZmCCaMK in leaves of maize. Subcellular localization analysis showed that ZmCCaMK is located in the nucleus, the cytoplasm, and the plasma membrane. The transient expression of ZmCCaMK and the RNA interference (RNAi) silencing of ZmCCaMK analysis in maize protoplasts revealed that ZmCCaMK is required for ABA-induced antioxidant defence. Moreover, treatment with the NO donor sodium nitroprusside (SNP) induced the activation of ZmCCaMK and the expression of ZmCCaMK. Pre-treatments with an NO scavenger and inhibitor blocked the ABA-induced increases in the activity and the transcript level of ZmCCaMK. Conversely, RNAi silencing of ZmCCaMK in maize protoplasts did not affect the ABA-induced NO production, which was further confirmed using a mutant of OsCCaMK, the homologous gene of ZmCCaMK in rice. Moreover, H2O2 was also required for the ABA activation of ZmCCaMK, and pre-treatments with an NO scavenger and inhibitor inhibited the H2O2-induced increase in the activity of ZmCCaMK. Taken together, the data clearly suggest that ZmCCaMK is required for ABA-induced antioxidant defence, and H2O2-dependent NO production plays an important role in the ABA-induced activation of ZmCCaMK. [ABSTRACT FROM PUBLISHER]

Using pharmacological and biochemical approaches, the role of protein phosphorylation and the interrelationship between water stress-enhanced kinase activity, antioxidant enzyme activity, hydrogen peroxide (H2O2) accumulation and endogenous abscisic acid in maize ( Zea mays L.) leaves were investigated. Water-stress upregulated the activities of total protein phosphorylation and Ca2+-dependent protein kinase, and the upregulation was blocked in abscisic acid-deficient vp5 mutant. Furthermore, pretreatments with a nicotinamide adenine dinucleotide phosphate oxidase inhibitor and a scavenger of H2O2 significantly reduced the increased activities of total protein kinase and Ca2+-dependent protein kinase in maize leaves exposed to water stress. Pretreatments with different protein kinase inhibitors also reduced the water stress-induced H2O2 production and the water stress-enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. The data suggest that protein phosphorylation and H2O2 generation are required for water stress-induced antioxidant defense in maize leaves and that crosstalk between protein phosphorylation and H2O2 generation may occur. [ABSTRACT FROM AUTHOR]

Using pharmacological and biochemical approaches, the role of maize polyamine oxidase (MPAO) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize ( Zea mays L.) plants was investigated. Exogenous ABA treatment enhanced the expression of the MPAO gene and the activities of apoplastic MPAO. Pretreatment with two different inhibitors for apoplastic MPAO partly reduced hydrogen peroxide (H2O2) accumulation induced by ABA and blocked the ABA-induced expression of the antioxidant genes superoxide dismutase 4 and cytosolic ascorbate peroxidase and the activities of the cytosolic antioxidant enzymes. Treatment with spermidine, the optimum substrate of MPAO, also induced the expression and the activities of the antioxidant enzymes, and the upregulation of the antioxidant enzymes was prevented by two inhibitors of MPAO and two scavengers of H2O2. These results suggest that MPAO contributes to ABA-induced cytosolic antioxidant defense through H2O2, a Spd catabolic product. [ABSTRACT FROM AUTHOR]