The Function Of ZmMAP65-1a And ZmNAC In The Abiotic Stress Tolerance In Maize

Recently, It is proved that microtubules as an important component of cytoskeleton responses to abiotic stress by regulating the dynamic instability. MT dynamic instability is precisely regulated by microtubule associated proteins (MAPs). MAP65 is one of the most abundant plant MAPs.In this study, one maize 65 kDa microtubule-associated protein (MAP65), ZmMAP65-1a was identified from maize (Zea mays. L) by RT-PCR approach. Phylogenetic tree shows that ZmMAP65-1a is most homology with OsMAP65-1(90% homology) and ZmMAP65-la encodes a 65 kDa polypeptide with 584 amino acids and PI value is 4.845. As typical MAP65, ZmMAP65-la protein contains two C-terminal microtubule binding domain:MTB1 (microtubule binding region 1) and MTB2 (microtubule binding region2). MTB2 has sites of phosphorylation by MAPK. It was found by subcellular localization, ZmMAP65-1a is located in the cell cytoskeleton.Treatment with BR increased the expression of ZmMAP65-1a in maize leaves and mesophyll protoplasts. Transient expression and RNA interference silencing of ZmMAP65-la 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. However, transient expression of ZmMAP65-1a in protoplasts had little if any effect on the expression of ZmrbohA-D in either BR-treated or untreated protoplasts. 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.There is already evidence that BR is upstream in ABA signaling. Transient expression and RNA interference silencing of ZmMAP65-1a in mesophyll protoplasts further revealed that ZmMAP65-1a is required for the ABA-induced increase in activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Inhibiting the ABA biosynthesis significantly prevented the BR-induced expression of ZmMAP65-1a, ZmrbohA-D and ZmMPK5 kinase activity. These results indicate that BR-induced antioxidant defence in maize operates through the interaction of ZmMPK5 with ZmMAP65-1a by BR-induced ABA biosynthesis.The plant-specific NAC (NAM, ATAF1/2 and CUC2) proteins constitute a major transcription factor family renowned for their roles in several developmental programs. In this study, a NAC transcription factor obtained through the yeast two-hybrid screen was identified to have some relationship with ZmCCaMK and the function of the potential protein in abiotic stress was analysised.In this study, one NAC transcription factor was identified from maize (Zea mays L.) by bioinformatics method. ZmNAC encodes a 47.94 kDa polypeptide with 431 amino acids and PI value is7.74. As a typical NAC family protein, ZmNAC has a conservative N-terminal domain, NAM structure which in the 43aa-204aa. Subcellular localization analysis in maize protoplasts indicated that ZmNAC was a nuclear protein. Transactivation assay in yeast demonstrated that ZmNAC functioned as a transcriptional activator and C terminus was necessary for the transactivation activity. The electrophoretic mobility shift assay (EMSA) suggested that ZmNAC could bind to promoter region (containing CATGTG sites) of ZmERD. The tissue-specific expression analysis suggested that ZmNAC was constitutively expressed in maize different tissue and much higher in female flower than that in other organs. The results of real-time PCR analysis indicated that ZmNAC was up-regulated by several abiotic stresses and plant hormones.This analysis discusses emphatically ZmNAC response to abiotic stress by ABA. It is showed that ZmNAC is involved in ABA induced antioxidant defense by transient expression, RNA interference silencing and CREST (Chimeric Repressor Silencing Technology) in mesophyll protoplasts. It found that H2O2 as signal molecules is most important in this pathway. The data suggested that ZmNAC is involved in the ABA induced H2O2 production by regulation of the gene expression of NADPH oxidase in ABA signalling. Inhibiting the ABA biosynthesis totally prevented the PEG-induced expression of ZmNAC, and partly prevented the NaCl-induced expression.The results suggest that ZmNAC response to abiotic stress depend on totally or partly ABA.Inhibiting the activity and gene expression of ZmCCaMK significantly prevented the ABA-induced expression of ZmNAC. Compared with single transient expression or silence ZmCCaMK/ZmNAC, transient expression or silence ZmCCaMK/ZmNAC simultaneously is more influence on the activity of ABA induced antioxidant defence enzymes SOD and APX. Likewise, transient expression of ZmNAC enhanced ABA-induced activities of the antioxidant defence enzymes SOD and APX in a ZmCCaMK-dependent manner.It is also proved that ZmCCaMK interacted with ZmNAC affects H2O2 production in protoplast. ZmCCaMK also directly phosphorylated ZmNAC in vitro. ZmNAC has eight sites of phosphorylation by ZmCCaMK.we designed a serious of ZmNAC mutant to test their function on the ABA induced antioxidant defence by point mutation.To identify the function of the ZmNAC under stress conditions, a binary vector containing ZmNAC driven by CaMV35S promoter was constructed and the transgenic tobacco plants over-expression ZmNAC were produced by Arobacterium-mediated transformation. It is proved that the transgenic tobacco displayed obvious water-stress and salt tolerance than wt.