The Mechanisms Of Ca²⁺/CaM And ZmCCaMK In BR-Induced Antioxidant Denfense

Brassinosteroids (BRs) are a family of naturally occurring steroidal plant hormones response to environmental stresses, such as drought, salinity and copper stress. Several studies have shown exogenously BR enhanced the tolerance to the environmental stress is associated with the induction of antioxidant defence systems. However, the mechanism of the BR-induced antioxidant defense in plants has seldomly been reported. In this report, the role of Calcium and Calmodulin dependent kinase (CCaMK), and Ca2+/CaM in the BR-induced antioxidant defense was studied using maize(Zea maize, L) Nongda 108 as material.In order to investigate the roles of ZmCCaMK in brassinosteroid-induced antioxidant defense in leaves of maize(Zea mays L.) plants. Firstly, we investigate the effect of BR or PEG on the expression of ZmCCaMK in leaves of maize plants, relative quantitative real-time PCR analysis further confirm that 10 nM BR,10%PEG, are able to enhance the expression of ZmCCaMK, moreover, the expression of ZmCCaMK was even rapidly upregulated by the treatment of 10 nM BR in protoplasts. Transient expression of ZmCCaMK in protoplasts resulted in significant increases in the activities of the antioxidant enzymes SOD and APX, and treatment with 10 nM BR induced significant increases in d the activities of SOD and APX compared to the control protoplasts. However, RNAi-mediated silencing of ZmCCaMK decreased the activities of the antioxidant enzymes SOD and APX, and BR treatment had little effect on the activities of SOD and APX. These results indicated unequivocally that ZmCCaMK is required for BR-induced antioxidant defence in maize.In order to investigate the roles of Ca2+/CaM in brassinosteroid-induced antioxidant defense in maize(Zea mays L.) plants. Treatment with BR led to significant increases in the levels of cytosolic Ca2+([Ca2+]i) and the content of CaM in protoplasts of maize plants. Moreover, pre-treatment with Ca2+ and CaM inhibitors and antagonists nearly fully inhibited the BR-induced antioxidant defense in protoplasts of maize plants. These results clearly suggest that Ca/CaM is involved in the BR-induced antioxidant defense in protoplasts of maize plants.In order to investigate the relationship between Ca2+/CaM and ZmCCaMK in brassinosteroid-induced antioxidant defense in maize(Zea mays L.) plants. Pre-treatment with Ca2+ channel inhibitor EGTA and LaCl3, CaM inhibitor W7?TFP blocked the enhancement in the expression of ZmCCaMK induced by BR in leaves of maize. These results suggest that Ca2+/CaMare required for BR induced expression of the ZmCCaMK in leaves of maize plants. Transient expression of ZmCCaMK in protoplasts resulted in a significant increase in the concentration of cytosolic calcium and the content of CaM, which were further enhanced by BR treatment. Conversely, RNAi-mediated silencing of ZmCCaMK caused an obvious decrease in the concentration of cytosolic calcium and the content of CaM, and BR treatment failed to induce the cytosolic Ca2+ concentration elevation. From the results above, there is a cross-talk between ZmCCaMK and Ca2+/CaM in BR signalingThese results indicate that ZmCCaMK is involved in BR-induced antioxidant defence, and Ca2+/CaM play a important role in BR signaling, and there is a cross-talk between ZmCCaMK and Ca2+/CaM in BR signaling in maize.