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Drought stress is a crucial environmental factor that limits plant growth, development, and productivity. Autophagy of misfolded proteins can help alleviate the damage caused in plants experiencing drought. However, the mechanism of autophagy‐mediated drought tolerance in plants remains largely unknown. Here, we cloned the gene for a maize (Zea mays) selective autophagy receptor, NEXT TO BRCA1 GENE 1 (ZmNBR1), and identified its role in the response to drought stress. We observed that drought stress increased the accumulation of autophagosomes. RNA sequencing and reverse transcription‐quantitative polymerase chain reaction showed that ZmNBR1 is markedly induced by drought stress. ZmNBR1 overexpression enhanced drought tolerance, while its knockdown reduced drought tolerance in maize. Our results established that ZmNBR1 mediates the increase in autophagosomes and autophagic activity under drought stress. ZmNBR1 also affects the expression of genes related to autophagy under drought stress. Moreover, we determined that BRASSINOSTEROID INSENSITIVE 1A (ZmBRI1a), a brassinosteroid receptor of the BRI1‐like family, interacts with ZmNBR1. Phenotype analysis showed that ZmBRI1a negatively regulates drought tolerance in maize, and genetic analysis indicated that ZmNBR1 acts upstream of ZmBRI1a in regulating drought tolerance. Furthermore, ZmNBR1 facilitates the autophagic degradation of ZmBRI1a under drought stress. Taken together, our results reveal that ZmNBR1 regulates the expression of autophagy‐related genes, thereby increasing autophagic activity and promoting the autophagic degradation of ZmBRI1a under drought stress, thus enhancing drought tolerance in maize. These findings provide new insights into the autophagy degradation of brassinosteroid signaling components by the autophagy receptor NBR1 under drought stress. [ABSTRACT FROM AUTHOR]

Summary: Mitogen‐activated protein kinase (MPK) is a critical regulator of the antioxidant defence system in response to various stimuli. However, how MPK directly and exactly regulates antioxidant enzyme activities is still unclear. Here, we demonstrated that a NAC transcription factor ZmNAC49 mediated the regulation of antioxidant enzyme activities by ZmMPK5.ZmNAC49 expression is induced by oxidative stress. ZmNAC49 enhances oxidative stress tolerance in maize, and it also reduces superoxide anion generation and increases superoxide dismutase (SOD) activity. A detailed study showed that ZmMPK5 directly interacts with and phosphorylates ZmNAC49 in vitro and in vivo. ZmMPK5 directly phosphorylates Thr‐26 in NAC subdomain A of ZmNAC49. Mutation at Thr‐26 of ZmNAC49 does not affect the interaction with ZmMPK5 and its subcellular localisation.Further analysis found that ZmNAC49 activates the ZmSOD3 expression by directly binding to its promoter. ZmMPK5‐mediated ZmNAC49 phosphorylation improves its ability to bind to the ZmSOD3 promoter. Thr‐26 of ZmNAC49 is essential for its transcriptional activity. In addition, ZmSOD3 enhances oxidative stress tolerance in maize.Our results show that phosphorylation of Thr‐26 in ZmNAC49 by ZmMPK5 increased its DNA‐binding activity to the ZmSOD3 promoter, enhanced SOD activity and thereby improved oxidative stress tolerance in maize. [ABSTRACT FROM AUTHOR]

Summary: Drought stress seriously limits crop productivity. Although studies have been carried out, it is still largely unknown how plants respond to drought stress. Here we find that drought treatment can enhance the phosphorylation activity of brassinosteroid‐signaling kinase 1 (ZmBSK1) in maize (Zea mays).Our genetic studies reveal that ZmBSK1 positively affects drought tolerance in maize plants. ZmBSK1 localizes in plasma membrane, interacts with calcium/calmodulin (Ca2+/CaM)‐dependent protein kinase (ZmCCaMK), and phosphorylates ZmCCaMK. Ser‐67 is a crucial phosphorylation site of ZmCCaMK by ZmBSK1.Drought stress enhances not only the interaction between ZmBSK1 and ZmCCaMK but also the phosphorylation of Ser‐67 in ZmCCaMK by ZmBSK1. Furthermore, Ser‐67 phosphorylation in ZmCCaMK regulates its Ca2+/CaM binding, autophosphorylation and transphosphorylation activity, and positively affects its function in drought tolerance in maize.Our results reveal an important role for ZmBSK1 in drought tolerance and suggest a direct regulatory mode of ZmBSK1 phosphorylating ZmCCaMK. [ABSTRACT FROM AUTHOR]

Drought stress severely limits the growth, development, and productivity of crops, and therefore understanding the mechanisms by which plants respond to drought is crucial. In this study, we cloned a maize NAC transcription factor, ZmNAC49 , and identified its function in response to drought stress. We found that ZmNAC49 is localized in the nucleus and has transcriptional activation activity. ZmNAC49 expression is rapidly and strongly induced by drought stress, and overexpression enhances stress tolerance in maize. Overexpression also significant decreases the transpiration rate, stomatal conductance, and stomatal density in maize. Detailed study showed that ZmNAC49 overexpression affects the expression of genes related to stomatal development, namely ZmTMM , ZmSDD1 , ZmMUTE , and ZmFAMA. In addition, we found that ZmNAC49 can directly bind to the promoter of ZmMUTE and suppress its expression. Taken together, our results show that the transcription factor ZmNAC49 represses ZmMUTE expression, reduces stomatal density, and thereby enhances drought tolerance in maize. [ABSTRACT FROM AUTHOR]

Brassinosteroid (BR)-induced antioxidant defence has been shown to enhance stress tolerance. In this study, the role of the maize 65kDa microtubule-associated protein (MAP65), ZmMAP65-1a, in BR-induced antioxidant defence was investigated. Treatment with BR increased the expression of ZmMAP65-1a in maize (Zea mays) leaves and mesophyll protoplasts. Transient expression and RNA interference silencing of ZmMAP65-1a in mesophyll protoplasts further revealed that ZmMAP65-1a is required for the BR-induced increase in expression and activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Both exogenous and BR-induced endogenous H2O2 increased the expression of ZmMAP65-1a. Conversely, transient expression of ZmMAP65-1a in maize mesophyll protoplasts enhanced BR-induced H2O2 accumulation, while transient silencing of ZmMAP65-1a blocked the BR-induced expression of NADPH oxidase genes and inhibited BR-induced H2O2 accumulation. Inhibiting the activity and gene expression of ZmMPK5 significantly prevented the BR-induced expression of ZmMAP65-1a. Likewise, transient expression of ZmMPK5 enhanced BR-induced activities of the antioxidant defence enzymes SOD and APX in a ZmMAP65- 1a-dependent manner. ZmMPK5 directly interacted with ZmMAP65-1a in vivo and phosphorylated ZmMAP65-1a in vitro. These results suggest that BR-induced antioxidant defence in maize operates through the interaction of ZmMPK5 with ZmMAP65-1a. Furthermore, ZmMAP65-1a functions in H2O2 self-propagation via regulation of the expression of NADPH oxidase genes in BR signalling. [ABSTRACT FROM AUTHOR]

Calcium-dependent protein kinases (CDPKs) have been shown to be involved in abscisic acid (ABA)-mediated physiological processes, including seed germination, post-germination growth, stomatal movement, and plant stress tolerance. However, it is not clear whether CDPKs are involved in ABA-induced antioxidant defence. In the present study, the role of the maize CDPK ZmCPK11 in ABA-induced antioxidant defence and the relationship between ZmCPK11 and ZmMPK5, a maize ABA-activated mitogen-activated protein kinase (MAPK), in ABA signalling were investigated. Treatments with ABA and H2O2 induced the expression of ZmCPK11 and increased the activity of ZmCPK11, while H2O2 was required for the ABA-induced increases in the expression and the activity of ZmCPK11. The transient gene expression analysis and the transient RNA interference (RNAi) test in protoplasts showed that ZmCPK11 is involved in ABA-induced up-regulation of the expression and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX), and in the production of H2O2. Further, ZmCPK11 was shown to be required for the up-regulation of the expression and the activity of ZmMPK5 in ABA signalling, but ZmMPK5 had very little effect on the ABA-induced up-regulation of the expression and the activity of ZmCPK11. Moreover, the transient gene expression analysis in combination with the transient RNAi test in protoplasts showed that ZmCPK11 acts upstream of ZmMPK5 to regulate the activities of antioxidant enzymes. These results indicate that ZmCPK11 is involved in ABA-induced antioxidant defence and functions upstream of ZmMPK5 in ABA signalling in maize. [ABSTRACT FROM AUTHOR]

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]

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 (NG-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. [ABSTRACT FROM AUTHOR]

In search for components of mitogen-activated protein kinase (MAPK) cascades in maize ( Zea mays) involved in response to abscisic acid (ABA) stimulus, a novel MAPK gene, ZmMPK3, from ABA-treated maize leaves cDNA was isolated and characterized. The full length of the ZmMPK3 gene is 1 520 bp and encodes a 376 amino acid protein with a predicted molecular mass of 43.5 kD and a pI of 5.83. ZmMPK3 contains all 11 MAPK conserved subdomains and the phosphorylation motif TEY. Amino acid sequence alignment revealed that ZmMPK3 shared high identity with group-A MAPK in plants. A time course (30–360 min) experiment using a variety of signal molecules and stresses revealed that the transcripts level of ZmMPK3 accumulated markedly and rapidly when maize seedlings were subjected to exogenous signaling molecules: ABA, H2O2, jasmonic acid and salicylic acid, various abiotic stimuli such as cold, drought, ultraviolet light, salinity, heavy metal and mechanical wounding. Its transcription was also found to be tissue-specific regulated. Here, we show that ABA and H2O2 induced a significant increase in the ZmMPK3 activity using immunoprecipitation and in-gel kinase assay. Furthermore, the results showed that the ZmMPK3 protein is localized mainly to the nucleus. These results suggest that the ZmMPK3 may play an important role in response to environmental stresses. [ABSTRACT FROM AUTHOR]

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]

Simple Summary: Current knowledge about the downstream substrate proteins of MAPKs is still limited. Our study screened a new WRKY IIa transcription factor as the substrate protein of ZmMPK6, and its phosphorylation at Thr-59 is critical to the role of ZmWRKY104 in ABA-induced antioxidant defense. Moreover, overexpression ZmWRKY104 in maize enhances the drought tolerance of transgenic plants. These findings define a mechanism for the function of ZmWRKY104 phosphorylated by ZmMPK6 in ABA-induced antioxidant defense and drought tolerance. Mitogen-activated protein kinase (MAPK) cascades are primary signaling pathways involved in various signaling pathways triggered by abiotic and biotic stresses in plants. The downstream substrate proteins of MAPKs in maize, however, are still limited. Here, we screened a WRKY IIa transcription factor (TF) in maize (Zeamays L.), ZmWRKY104, and found that it is a substrate of ZmMPK6. ZmWRKY104 physically interacts with ZmMPK6 in vitro and in vivo. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis results showed that threonine-59 (Thr-59, T59) was the major phosphorylation site of ZmWRKY104 by ZmMPK6. Subcellular localization analysis suggested that ZmWRKY104 acts in the nucleus and that ZmMPK6 acts in the nucleus and cytoplasmic membrane in the cytosol. Functional analysis revealed that the role of ZmWRKY104 in ABA-induced antioxidant defense depends on ZmMPK6. Moreover, overexpression of ZmWRKY104 in maize can enhance drought tolerance and relieve drought-induced oxidative damage in transgenic lines. The above results help define the mechanism of the function of ZmWRKY104 phosphorylated by ZmMPK6 in ABA-induced antioxidant defense and drought tolerance in maize. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

MYB transcription factors play a vital role in response to stress in plant. MYB-CC transcription factors belong to MYB transcription factors, which contain a conserved MYB DNA-binding domain and a coiled-coil (CC) domain. MYB-CC transcription factors participate in the process of plant drought tolerance. However, the underlying molecular mechanisms of ZmMYB-CC in regulating drought tolerance are still largely unknown. Here, we found that ZmMYB-CC10 enhanced drought tolerance by reducing oxidative damage in maize. Further, ZmMYB-CC10 improves the activity of APX and decreases the content of H 2 O 2. Overexpression of ZmMYB-CC10 increases the expression of ZmAPX4 under drought stress. Luciferase assays and Yeast one-hybrid assays (Y1H) showed that ZmMYB-CC10 activates the expression of ZmAPX4 by directly binding to its promoter. Taken together, our results demonstrate that ZmMYB-CC10 enhances tolerance to drought stress by directly activating ZmAPX4 expression, thereby reducing H 2 O 2 content. • ZmMYB-CC10 enhances drought tolerance by reducing oxidative damage in maize. • ZmMYB-CC10 decreases the content of H 2 O 2 by increasing the activity of APX under drought stress. • ZmMYB-CC10 directly binds to ZmAPX4 promotor and activates its expression. [ABSTRACT FROM AUTHOR]

Salinity has become a crucial environmental factor that restricts plant growth, development, and productivity. Nevertheless, the mechanisms by which plants react to salt stress remain inadequately comprehended. In this study, we identified maize brassinosteroid-signaling kinase gene ZmBSK7 which is homologous to AtBSK1. Our results showed that ZmBSK7 is induced by salt stress and ZmBSK7 localizes in the plasma membrane. ZmBSK7 overexpression increases salt tolerance, while its knockdown decreases salt tolerance in maize. ZmBSK7 reduces the malondialdehyde (MDA) content and the percentage of electrolyte leakage, and also elevates the activities of antioxidant enzymes. Furthermore, ZmBSK7 promotes K+ content accumulation and reduces Na+/K+ ratio. Further found that ZmBSK7 physically interacts with K+ efflux antiporter 2 (ZmKEA2) in vivo and in vitro. Salt stress also increased the expression of ZmKEA2. Thus, ZmBSK7 improves salt tolerance in maize by affecting ZmKEA2 expression to promote K+ content accumulation and reduce Na+/K+ ratio. This study enhances the comprehension of BSK proteins and establishes a theoretical foundation for investigating salt stress tolerance in plants. • ZmBSK7 positively enhance the tolerance of salt stress in maize. • Overexpression of ZmBSK7 improves the activities of antioxidant enzymes and maintaining a balance of Na+ and K+. • ZmBSK7 physically interacts with ZmKEA2 in vivo and in vitro. [ABSTRACT FROM AUTHOR]

NAC (NAM, ATAF1/2, and CUC2) transcription factors play vital roles in response to multiple abiotic stresses. Our previous study has demonstrated that ZmNAC84, a maize NAC transcription factor, enhanced the drought tolerance by increasing abscisic acid (ABA)-induced antioxidant enzyme activities of APX and SOD, and Ser-113, a key phosphorylation site, of ZmNAC84 played an important role in this process. However, the target gene of ZmNAC84 in this process is still unknown. Here, we found that ZmNAC84 only regulated the luciferase activity driven by ZmSOD2 promoter in tobacco. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay showed that ZmNAC84 directly bound to the CACGTG motif of ZmSOD2 promoter. Furthermore, phosphorylation of ZmNAC84 at Ser-113 up-regulated the ZmSOD2 expression by enhancing the DNA binding ability of ZmNAC84 to ZmSOD2 promoter and improved the drought tolerance. Taken together, our results demonstrate that ZmNAC84 directly regulates ZmSOD2 expression to enhance drought tolerance and Ser-113 of ZmNAC84 is crucial in this process. • ZmNAC84 directly binds to ZmSOD2 promoter in vivo and in vitro. • Phosphorylation of ZmNAC84 at Ser-113 up-regulates the ZmSOD2 expression. • Phosphorylation of ZmNAC84 at Ser-113 enhances the DNA binding ability of ZmNAC84. [ABSTRACT FROM AUTHOR]

Calcium/calmodulin-dependent protein kinase (CCaMK) has been shown to play important roles in brassinosteroid (BR)-induced antioxidant defense and enhancing the tolerance of plants to drought stress. The autophosphorylation of CCaMK is a key step for the activation of CCaMK, thus promoting substrate phosphorylation. However, how CCaMK autophosphorylation function in BR-induced antioxidant defense is not known yet. Here, seven potential autophosphorylation sites of ZmCCaMK were identified using mass spectroscopy (liquid chromatography-tandem mass spectrometry [LC-MS/MS]) analysis. The transient gene expression analysis in maize protoplasts showed that Thr420 and Ser454 of ZmCCaMK were important for BR-induced antioxidant defense. Furthermore, Thr420 and Ser454 of ZmCCaMK were crucial for improving drought tolerance and alleviating drought induced oxidative damage of plants via overexpressing various mutant versions of ZmCCaMK in tobacco (Nicotiana tabacum). Mutations of Thr420 and Ser454 in ZmCCaMK substantially blocked the autophosphorylation and substrate phosphorylation of ZmCCaMK in vitro. Taken together, our results demonstrate that Thr420 and Ser454 of ZmCCaMK are crucial for BR-induced antioxidant defense and drought tolerance through modulating the autophosphorylation and substrate phosphorylation activities of ZmCCaMK. • Thr420 and Ser454 of ZmCCaMK are crucial for BR-induced antioxidant defense in maize. • Phosphorylation of Thr420 and Ser454 of ZmCCaMK enhance drought tolerance in plants. • T420A and S454A of ZmCCaMK affect the kinase activity of ZmCCaMK in vitro. [ABSTRACT FROM AUTHOR]