Functional Analysis Of ZmSK1 In BR-induced Antioxidant Defense In Maize

Abiotic stresses,including drought,heat and salt stresses,can cause excess reactive oxygen species(ROS)in plants,leading to severe oxidative damages to cells.Plants have evolved enzymatic antioxidant mechanisms by production of different antioxidant enzymes to scavenge excess ROS in cells to maintain normal cellular homeostasis.Brassinosteroid(BR)is a phytohormone and exogenous BR is known to improve the tolerance to abiotic stresses by enhancing antioxidant defense system.Plant GSK3(shaggy/glycogen synthase kinase 3)proteins have been reported as the key factors in the BR-induced signaling pathway and function in a wide range of growth and development processes.However,their detailed functions in plant antioxidant defense are still unknown.In this study,a member of the GSK3 proteins was identified and named as maize ZmSK1.The role of ZmSK1 in maize antioxidant defense induced by BR was investigated.Results obtained in this study are as follows:To investigate the roles of GSK3-like proteins in BR-induced antioxidant defense,we initially identified 11 GSK3-like proteins from MaizeGDB database and were named as ZmSK1,ZmSK2,ZmSK3,ZmSK4 and ZmSK5,respectively.Relative gene expression analysis indicated that the application of exogenous BR could suppress the expressions of ZmSK1-ZmSK5.To explore the functions of ZmSK1-ZmSK5 during BR-induced antioxidant defense,these five proteins were individually transient overexpressing in maize protoplasts and then analyzed the activities of ascorbate peroxidase(APX)and superoxide dismutase(SOD).Transient overexpression of ZmSK1 or ZmSK2 in maize protoplasts strongly suppressed the activities of APX and SOD compared with control.The suppressed APX and SOD activities were partially restored by treatment of exogenous BR.There was no significant effect on the activities of APX and SOD by transiently overexpressed ZmSK3 while there was variance on the activity of SOD by exogenous BR treatment.The results showed that ZmSK4 and ZmSK5 didn’t involved in the process of BR-induced antioxidant defense.Moreover,the results also showed that the effect of overexpression of ZmSK1 on BR-induced antioxidant defense was the most significant.Based on the above results,the later studies were focused on the analysis of ZmSK1 function.Results of pharmacological studies showed that the activities of APX and SOD in maize leaves could be significantly induced under treatment of the GSK3 inhibitor known as bikinin(BK).The activities of APX and SOD in maize leaves could also be induced after silencing ZmSK1 expression through virus induced gene silencing(VIGS).Similarly,the activities of APX and SOD were increased after knocking down ZmSK1 in maize protoplasts which were further enhanced by BR treatment.Thus,we concluded that ZmSK1 is a key factor involved in the BR-induced antioxidant defense,and the activities of the two antioxidant enzymes could be inhibited by overexpressing ZmSK1.Further,in vitro phosphorylation studies using myelin basic protein(MBP)as substrate confirmed that ZmSK1 acted as a protein kinase,and its activity in maize leaves could be inhibited by exogenous BR treatment.In addition,application of exogenous abscisic acid(ABA),hydrogen peroxide(H2O2),sodium chloride(NaCl)or polyethylene glycol(PEG)could up-regulate the transcriptions and activities of ZmSK1.These findings suggested that ZmSK1 participates in maize responses to diverse signaling pathways.In conclusion,the gene expression and activity of ZmSK1 can be inhibited by exogenous BR treatment,and ZmSK1 is a key factor in the BR-induced antioxidant defense pathway and acts as a negative regulator in this process.To clarify the mechanism of ZmSK1 in BR-induced antioxidant defense,previous study identified a maize transcription factor,ZmCPP2,interacting with ZmSK1.In this study,through yeast two-hybrid(Y2H),GST-pull down,co-immunoprecipitation(CoIP)and firefly luciferase complementation imaging assay(LCI),the physical interactions between ZmSK1 and ZmCPP2 was further confirmed.ZmCPP2 encodes 394 aa and contains two conserved CXC domains(aa 84-125 and aa 160-201).Phylogenic analysis identified 13 ZmCPP members in maize,and ZmCPP2 is belonged to Group I.The expression level of ZmCPP2 was up-regulated by exogenous BR treatment in maize leaves.To investigate the potential function(s)of ZmCPP2 in maize antioxidant defense induced by BR,ZmCPP2 was transiently overexpressed or silenced in maize protoplasts followed by analysis of APX and SOD activities.Results showed that transient overexpression of ZmCPP2 resulted in a significant increase of APX and SOD activities compared with controls.The increase of enzyme activities were further enhanced by exogenous BR treatment.In contrast,RNAi-mediated transient silencing of ZmCPP2 in protoplasts suppressed the activities of APX and SOD and the suppression was restored after the BR treatment.These findings indicated that ZmCPP2 is involved in the BR-induced antioxidant defense.In vitro phosphorylation analysis showed that ZmCPP2 could be phosphorylated by ZmSK1.Further analysis using liquid chromatography-mass spectrometry(LC-MS)confirmed that Ser250(S250)in ZmCPP2 was the phosphorylation site.To further dissect the function of ZmSK1 and ZmCPP2 interaction in the BR-induced antioxidant defense,both ZmSKl and ZmCPP2 were transiently overexpressed in maize protoplasts.The results showed that the activities of APX and SOD were much higher with overexpressing both ZmSKl and ZmCPP2 than that overexpressing only ZmSK1,but lower than overexpressing ZmCPP2 alone.However,in the protoplasts with transiently silenced ZmSK1,the transient overexpression of ZmCPP2 could resulted in higher activities of APX and SOD than overexpressing ZmCPP2 alone.These results suggested that ZmCPP2 is involved in the BR-induced antioxidant defense and is accompanied with ZmSK1.To determine the role of the ZmCPP2 phosphorylation site S250 in the BR-induced antioxidant defense,two mutant constructs expressing ZmCPP2S250A or ZmCPP2S250D were made and overexpressed individually in maize protoplasts.Results showed that overexpressing ZmCPP2S250A in protoplasts increased the ability of ZmCPP2 to up-regulate the activities of APX and SOD,while overexpressing ZmCPP2S250D in protoplasts decreased the ability of ZmCPP2 to up-regulate the activities of APX and SOD.These results indicated that phosphorylation at the S250 site by ZmSK1 has a critical role in regulating the BR-induced antioxidant defense.To explore the mechanism of ZmCPP2 in BR-induced antioxidant defense,the genes of antioxidant enzymes which might be modulated were analyzed.Results showed that overexpression of ZmCPP2 in maize protoplasts up-regulated the accumulations of ZmSOD1 ZmSOD2,ZmSOD4 and ZmSOD4A.When ZmCPP2 was co-expressed with an expression vector harboring the promoters of ZmSOD1,ZmSOD2,ZmSOD4 or ZmSOD4A and a firefly luciferase(Luc)gene in tobacco,the luciferase activity was significantly enhanced.In summary,results from this study showed that ZmCPP2 transcription factor is a positive factor in the BR-induced antioxidant defense.Also,ZmSK1 is a negative regulator to modulate the phosphorylation of ZmCPP2 at the S250 site during the BR-induced antioxidant defense.ZmNAGK is another protein that interact with ZmSK1.In this study,the interactions between ZmSK1 and ZmCPP2 was confirmed through Y2H and GST-pull down assays.It was determined that ZmNAGK expressed to high levels in maize leaves but to lower levels in root,stem,female flower and pollen.It was also determined that the relative gene expression of ZmNAGK was significantly up-regulated upon exogenous BR,PEG,NaCl,ABA or H2O2 treatment.Transient overexpression of ZmNAGK in maize protoplasts resulted in significant increase of APX and SOD activities compared with controls which was further enhanced by exogenous BR treatment.In contrast,RNAi-mediated transient silencing of ZmNAGK suppressed the activities of APX and SOD and the suppression was restored by the BR treatment.These results indicated that ZmNAGK is responsive to exogenous BR treatment and is involved in the BR-induced antioxidant defense.Overexpressing ZmNAGK in tobacco plants resulted in a high tolerance to drought stress compared with control group.Further analysis revealed that overexpression of ZmNAGK in tobacco plants could also increase the activities of antioxidant enzymes,and decrease the content of malondialdehyde(MDA)as well as the leakage of electrolyte during drought stress.Moreover,ZmNAGK transgenic tobacco plants accumulated more arginine(Arg)and nitric oxide(NO)than the vector control plants during drought stress.These findings indicated that ZmNAGK is involved in antioxidant defense and plays a crucial role in enhancing plant drought tolerance by regulating the accumulation of NO.