The Roles Of Ca²⁺-dependent And Calmodulin Stimulated Protein Kinase In Abscisic Acid-Induced Antioxidant Defense In Leaves Of Maize Plants

The phytohormone abscisic acid (ABA) controls many important aspects of plant growth and development, but its main function is to regulate plant adaptive responses to various adverse environmental conditions. ABA can cause the generation of reactive oxygen species (ROS) in various plant cells or tissues, induce the expression of antioxidant genes, and enhance the capacity of antioxidant defense systems. Both ABA and H2O2 induce the activities of total protein kinases and Calcium-dependent protein kinases enhancements significantly, CaMKs(calmodulin dependent protein kinases) contributing to these processes evidently. Thus CaMK may adjust physiological processes of plant in response to adverse stress. ROS play important roles in the ABA-induced antioxidant defense. However, the detailed mechanism about how ABA induces antioxidant defense in plant cells remains to be elucidated.In the present study, the role of CaMK as well as its relationships between ABA and H2O2 in the ABA-induced antioxidant defense were investigated maize plants(Zea mays L.). Applied in vitro kinase assays, the results show that treatments with ABA or H2O2 induced the activities of total protein kinases and Calcium-dependent protein kinases enhancements significantly.However these effects were compromised by pretreatment with several reactive oxygen species inhibitors or scavengers. Protein kinases inhibitors prevented not only the ABA or H2O2-induced Kinase activity increases but also ABA or H2O2 induced antioxidant enzyme activity increases. Our results indicated that Protein phosphorylations caused by ABA or H2O2 precede ABA or H2O2 induced antioxidant defense obviously.Using Histoneâ…¢-S as a substrate, in-gel kinase assays were performed on protein extracts from the leaves of maize plants. Treatment with ABA or H2O2 induced the activities of a set of protein kinases with molecular weight 66 kD,52 kD,49 kD and 35 kD enhancement significant respectively, but the 52 kD protein kinase for more evidently. PEG(polyacrylamide gel electrophoresis) treatment only cause 52 kD protein kinase activation. These protein kinases activation presented time course and dose dependent manners. Furthermore, our results indicate that the 52 kD protein kinase has the characterization of calmodulin stimulated activity and being sensitivity to CaMK II inhibitor KN93 or calmodulin antagonist W-7. Treatments with ABA or H2O2 not only induced the activation of the 52 kD protein kinase, but also enhanced the total activities of the antioxidant enzymes catalase, ascorbate peroxidase, glutathione reductase, and superoxide dismutase. Such enhancements were blocked by pretreatment with a CaMK inhibitor and reactive oxygen species inhibitors or scavengers.In conjunction with using 3,3-diaminobenzidine (DAB) and CeCl3 staining respectively, data show that pretreatment with the CaMK inhibitor also substantially arrested the ABA-induced H2O2 production.ABA induced ROS production is required for 52 kD protein kinase activation. Additionally, our results show that repressing 52 kD protein kinase activation prevents ABA induced ROS production and antioxidant enzymes activity increases. So We propose that 52 kD CaMK is involved in ABA-induced antioxidant defense and that cross-talk between CaMK and H2O2 plays a pivotal role in ABA signaling. CaMK might act both upstream and downstream of H2O2, but mainly acts between ABA and H2O2 in ABA-induced antioxidant defensive signaling.Water stress can enhance the total protein kinase activities and induce antioxidant defense, but water stress induced endogenous accumulation of ABA and H2O2 are required in these courses. Time course analyses indicate that kinases activity enhancements also precede antioxidant enzyme activity increases, especially for calcium dependent protein kinases in water stressed leaves. Pretreatment with kinase inhibitors decrease water stress induced antioxidant enzyme activity increases and ROS production. These data suggest that Protein Phosphorytions involve in water stress-induced antioxidant defense in plant cells ubiquitiously, and that other different protein kinases may contribute to water stress-induced antioxidant defense. We infer that 52kD protein kinase is a concrete CaMK involved in antioxidant defense.