Kinase Activities Analysis And Localization Of ZmCCaMK Induced By Abscisic Acid In Leaves Of Maize Plants

The phytohormone abscisic acid (ABA) plays regulatory roles in plant growth and development processes. ABA accumulation and subsequent physiological effects stimulation require a complex interplay of signaling cascades. The Calcium and Calmodulin dependent protein kinases (CCaMK) cascades have been reported to function in various signal transduction pathways in eukaryotes. It is very important to study CCaMK function in abscisic acid signal transduction.In this study the effects of the kinase activities of ZmCCaMK induced by ABA,H2CO2 and NO were investigated.The results as follows:Pretreatment with the 100μM ABA and 10% PEG led to the enhancement in the kinase activities of ZmCCaMK in leaves of maize. Pretreatment with the ABA biosynthetic inhibitor fluridon (FLU) substantially blocked the enhancement in the kinase activities of ZmCCaMK induced by drought stress, suggesting that the increased activities of ZmCCaMK under drought stress is associated with the accumulation of ABA.Immunoprecipitation and in-gel kinase assay further confirm that H2O2 induced ZmCCaMK activities. Pretreatments with H2O2 scavenger or NADPH oxidase inhibitor substantially reduced the ABA-induced increases in the kinase activities of ZmCCaMK. These results suggest that H2O2 are required for ABA induced the kinase activities of the ZmCCaMK in leaves of maize plants.Treatment with Ca2+ channel inhibitor EGTA (5 mM) and LaCl3 (5 mM), CaM inhibitor W7,TFP, CaMK inhibitor KN-93 blocked the enhancement in the kinase activity of ZmCCaMK induced by ABA and H2O2 in leaves of maize. These results suggest that Ca2+ and CaM are required for ABA induced expression of the ZmCCaMK gene in leaves of maize plants.Pretreatment with the 100μM SNP also led to the enhancement in the kinase activities of ZmCCaMK in leaves of maize. Pretreatments with NO scavenger or NOS inhibitor substantially reduced the ABA-induced increases in the kinase activities of ZmCCaMK. These results suggest that NO are required for ABA induced expression of the ZmCCaMK gene in leaves of maize plants.To investigate subcellular locations of the ZmCCaMK, ORFs of the ZmCCaMK were inserted into sites of pXZP008 vector. The constructs were transformed into maize protoplasts. The results showed that ZmCCaMK was mainly localized to the cytoplasm.