Functional Analysis Of ZmCCaMK Autophosphorylation And ZmBSK1 Phosphorylating ZmCCaMK In BR-induced Antioxidant Defense

Plant steroid hormones,brassinosteroids(BRs),were considered to regulate plant growth and development and were known to alleviate various biotic and abiotic stresses on plants.Under long-term abiotic stress conditions,such as drought stress,excess reactive oxygen species(ROS)were accumulated in plants,which further caused oxidative stress and cell death.BR enhanced the activities of antioxidant defense enzymes superoxide dismutase(SOD),ascorbate peroxidase(APX),catalase(CAT)and glutathione reductase(GR)to scavenge excess ROS which caused by drought stress and protect plants from oxidative damage.Previous studies suggested that calcium/calmodulin-dependent protein kinase(CCaMK)was involved in BR-induced antioxidant defense and increased tolerance of rice plants to drought stress.However,the detailed mechanisms by which ZmCCaMK functioned in this process were still unknown.In this study,we investigated the mechanisms by which ZmCCaMK autophosphorylation and phosphorylation modifications regulated BR-induced antioxidant defense and drought tolerance of plants.Results were as follows:To study whether the autophosphorylation of ZmCCaMK is involved in modulating BR-induced antioxidant defense in maize,seven autophosphorylation residues in ZmCCaMK were identified firstly using an in vitro in-gel kinase assay and liquid chromatography-tandem mass spectrometry(LC-MS/MS).All of them were mutanted to alanine,then they were transiently expressed in maize protoplasts to detect the activities of antioxidant defense enzymes SOD and APX,respectively.The results showed that transient overexpression of T420A and S454A in maize protoplasts obviously blocked the increases in activities of SOD and APX when compared with ZmCCaMK,treatment with exogenous BR could not increase activities of SOD and APX.Simultaneously,we also analyzed the hydrogen peroxide(H2O2)content and the gene expression level of maize respiratory burst oxidase homologues(ZmRbohs)in maize protoplasts.We found that transient overexpression of T420A and S454A in maize protoplasts strongly decreased H2O2 accumulation and the gene expression level of ZmRbohs,exogenous BR could not restore them to ZnCCaMK.To further investigate the mechanisms by which Thr420 and Ser454 affected BR-induced antioxidant defense in maize,we performed an in vitro kinase assay to study the effects of phosphorylation status of Thr420 and Ser454 on ZmCCaMK kinase activity.Our results showed that T420A and S454A significantly reduced the kinase activities of ZmCCaMK,including the autophosphorylation and substrate phosphorylation activities,indicating that Thr420 and Ser454 were involved in regulating the kinase activities of ZmCCaMK.To further study whether the phosphorylation of Thr420 and Ser454 function in plants in response to drought stress,we constructed various transgenic tobacco plants and analyzed the drought tolerance of plants.Results showed that T420D and S454D overexpression transgenic plants had higher survival rate and lower oxidative damage than ZmCCaMK plants when exposed to drought stress.Furthermore,overexpressing T420D and S454D in tobacco plants increased drought stress-induced activities of SOD and APX,thus enhancing antioxidant defense.Taken together,Thr420 and Ser454 of ZmCCaMK were crucial for BR-induced antioxidant defense and positively regulated drought tolerance of plants.To investigate the mechanisms by which ZmCCaMK positively regulated BR-induced antioxidant defense and drought tolerance of plants,maize BR-signaling kinase 1(ZmBSK1)was screened to interact with ZmCCaMK in previous studies.In our study,GST-pull down,Co-Immunoprecipitation(Co-IP)and firefly luciferase complementation imaging(LCI)assay were used to confirm the interaction between ZmCCaMK and ZmBSK1,results showed that ZmCCaMK directly interacted with ZmBSK1 in vivo and in vitro,exogenous BR treatment enhanced the interaction between them.Subcellular localization showed that ZmCCaMK interacted with ZmBSK1 in cell membrane.To study whether ZmBSKl is involved in BR-induced antioxidant defense in maize,we firstly detected the kinase activity of ZmBSK1 in maize leaves.Immunoprecipitated in-gel kinase assay indicated that the activity of ZmBSK1 could be upregulated by exogenous BR.Transiently overexpressed or silenced ZmBSK1 in maize protoplasts to analyze the activities of antioxidant defense enzymes SOD and APX,results showed that transient overexpression of ZmBSK1 significantly increased the activities of SOD and APX when compared with control,treatment with exogenous BR could further increase that activities.On the contrary,transient silencing of ZmBSK1 resulted decreased activities of SOD and APX,treatment with exogenous BR could not increase that activities.OE-ZmBSK1(overexpression)and RNAi-ZmBSK1(RNA interference)transgenic maize plants were constructed to further study the effects of ZmBSK1 on activities of SOD and APX.Under normal conditions,there had no differences among transgenic and wild-type(WT)plants.When treated with exogenous BR,the activities of SOD and APX in OE-ZmBSK1 plants were higher than in WT.These results showed that ZmBSK1 was involved in BR-induced antioxidant defense in maize.Additionally,we analyzed the roles of ZmBSK1 in drought tolerance of maize plants.Immunoprecipitated in-gel kinase assay indicated that the activity of ZmBSK1 could be induced by polyethylene glycol(PEG)and drought stress.ZmBSK1 overexpression transgenic plants had higher survival rate and activities of SOD and APX than WT plants when exposed to drought stress.These results showed that ZmBSK1 positively regulated drought tolerance of maize plants.To clarify how the interaction between ZmCCaMK and ZmBSK1 affected BR-induced antioxidant defense in maize,we firstly studied the phosphorylation between them.An in vitro in-gel kinase assay and LC-MS/MS showed that ZmBSK1 phosphorylated ZmCCaMK mainly at Ser67,but ZmCCaMK could not phosphorylate ZmBSK1.And then,the phosphorylation of ZmCCaMK in ZmBSK1 transgenic maize plants were detected by immunoblotting with anti-phosphoserine67 antibody,the kinase activity of ZmCCaMK in maize were detected by immunoprecipitated in-gel kinase assay using anti-ZmCCaMK antibody.Results showed that BR induced the increases of Ser67 phosphorylation and ZmCCaMK activity were depended on ZmBSK1.To study the relationship between Ser67 phosphorylation and ZmCCaMK activity,electrophoresis migration shift assay(EMSA),45Ca2+overlay assay,Calmodulin(CaM)-binding assay and Co-IP assay were performed to detect the effects of Ser67 phosphorylation on binding of ZmCCaMK to Ca2+ and Ca2+/CaM.Results showed that Ser67 phosphorylation enhanced the binding capacity of ZmCCaMK to Ca2+ and Ca2+/CaM,thus promoting its autophosphorylation and substrate phosphorylation activities.Subsequently,we analyzed the activities of SOD and APX in ZmCCaMK transgenic plants,we found that Ser67 phosphorylation positively regulated BR-induced antioxidant defense in maize.For ZmCCaMK transgenic plants,when exposed to drought stress,S67D plants had higher survival rate,lower oxidative damage and higher activities of antioxidant defense enzymes than ZmCCaMK and WT plants.In conclusion,ZmCCaMK-Ser67 phosphorylation by ZmBSK 1 affected ZmCCaMK kinase activity,thereby positively regulating BR-induced antioxidant defense and tolerance of maize plants to drought stress.The above conclusions provided important guidance for constructing transgenic plants using molecular biology methods,thereby improving the activities of antioxidant defense enzymes,reducing oxidative damage of crops under abiotic stresses such as drought,and enhancing stress tolerance of crops.