Functionanalysis Of ZmCPK11 In The ABA-InducedAntioxidant Defense In MaizeLeaves

ABA (Abscisic acid) is an important plant hormone, plays an important role in plant growth and development as well as the plant to cope with various environmental stress reactions. CDPKs (calcium-dependent protein kinases)were first founded in plants.lt can be directly activated by calcium signals in plant growth and developmentAnd play an important regulatory role adapt to adversity process. CDPKs have an important role in plant calcium signal transduction.CDPKsparticipates the plant carbon and nitrogen metabolism, ion and water transport across membranes, cytoskeletal regulation, regulation of stomatal movement, growth and development regulation and biotic or abiotic stress response reaction.In this study, Calcium-dependent protein kinase ZmCPK11 was identified from maize (Zea mays.) by bioinformatics method and RT-PCR approach. Sequence analysis showed that ZmCPK11 encodes a56.21kD polypeptide with 511 amino acids. By subcellular localization, ZmCPK11 is located in the nucleus and cytoplasm. Real-time quantitative RT-PCR analysis showed that: ABA, H202can increase ZmCPKll gene expression.ZmCPK11, expression increased underlO mM H2O2,100 μM ABA、10mM CaCl2 and PEG-imitating natural water stress (10%) treatment, identified by semi-quantitative and quantitative RT-PCR expression analysis. Western-blot experiments show that 10 mM H2O2,100 μM ABA,10 mM CaCl2 were induced ZmCPKll protein level raised. And ABA-synthesis-inhibiting fluridone arrested ZmCPK11 activation and ZmCPKll gene expression induced by PEG; likewise DMTU, CAT (H2O2 scavenger) and DPI (NADPH oxidase inhibitor) blocked ZmCPK11 activation induced by ABA. This result is similar with the research before of our Lab, we believe the water stress signal goes from PEG to ABA and H2O2.To further investigate the function of ZmCPKll, we constructed a ZmCPK11 gene over-expression vector. To use of the protoplast transient expression system, we found expression ZmCPK11 gene can significantly improve the activity of the enzyme SOD andAPX. Interference with ZmCPK11 gene significantly decreased the activity of SOD and APX. The activity of SOD and APX increased slightly after ABA treatment. The experimental results show that ZmCPK11 involved in ABA-induced antioxidant protection, play an important role in which plants enhanced antioxidant protection and improve the ability to resist abiotic stress.In this study, we attempt to explore the relationship of ZmCPK11 and ZmMPK5 in the ABA signaling pathway. The inhibitor tests showed: treated with CDPK inhibitor TFP, calcium chelator EGTA, ZmMPK5 gene expression and ZmMPK5 activity were significantly reduced; treated with MAPKK inhibitor PD98059 and U0126, the ZmCPK11 gene expression and ZmCPK11 activity did not change significantly. RNAi experiments in theprotoplast showed that: the ZmMPK5 gene expression and ZmMPK5 activitydecreased significantlywhen interference ZmCPK11; ABA-inducedZmCPK11 gene expression and ZmCPK11 activityhad no significant effect when ZmMPK5 interference. Overexpression ZmCPK11 and interferenceZmMPK5 can’tbe increase the antioxidant defense enzyme activities. Interference ZmMPK5 prevent the downstream antioxidant defense reaction to express ZmCPK11 caused. Those resultshowed ZmCPK11 stands upstream of ZmMPK5.