The Regulatory Mechanisms Of OsBiPP2C1 And OsDMI3 In ABA-induced Antioxidant Denfense

The plant hormone abscisic acid (ABA) mediates lots of physiology process and play an critical role in adaptive response to environment stresses. ABA induces ROS to regulate various physiological process in plant. Previous studies showed that Ca2+/CaM is required for ABA-induced antioxidant defense. However, it not clear that Ca2+/CaM-dependent protein kinase is involved in the ABA signaling leading to the up-regulation of antioxidant defense system in plants. We had found a gene of rice CCaMK, OsDMI3. Treatment with ABA, H2O2, PEG induced the expression of OsDMI3 and the activity of OsDMI3 in rice leaves. We investigated the role and mechanism of OsDMI3 in the ABA signaling of antioxidant defense systems in plants.We contruscted the OsDMI3 over-expression vector, synthesized dsRNA of OsDMI3. Using the protoplast transient expression assays, we found that OsDMI3 is required for ABA-induced increasing in the expression and the activities of superoxide dismutase(SOD) and catalase(CAT). Moreover, the analysis of the RNAi silencing of OsDMI3 in rice protoplasts showed that higher levels of H2O2 accumlation required OsDMI3 activation in ABA signaling. Our data revealed that OsDMI3 was an important component in ABA-induced antioxidant defense in rice.In order to clear the role of OsDMI3 in ABA-induced antioxidant defense, we used full-length OsDMI3 as a bait to screen the rice leaves cDNA library by Y2H system. Several independent positive colonies were revealed. After plasmids rescue from the clones, the cDNA inserted into pGADT vector was analyzed by sequencing. Three of these clones containing identical nucleotide sequence coding the C-terminal region of a gene. A search for this sequence in rice databases identified this was the PP2C domain of protein phosphatase 2C gene, OsBiPP2Cl. OsBiPP2Cl was predicted to encode a 569 amino acid protein that contained a phosphatase domain at its C-terminal and a relatively long N-terminal extension. However, the homology analysis of the long extension at N-terminal, showed lower similarity of the other plant PP2Cs,and the functions of this domain remained unkown. OsBiPP2Cl was reported that it could be in response to pathogen infection induced by BTH treatment.There may be some false positives due to the Y2H system. To verify the authenticity of the interaction between OsDMI3 and OsBiPP2Cl, BiFC (bimolrcular fluorescence complementation, GST pull-down, Co-IP (Co-immunoprecipitation) assays were tested. It investigated that OsDMI3 did interact with OsBiPP2Cl both in vitro and in vivo. Subcellular localization showed that OsBiPP2Cl has the same subcellular localization with OsDMI32, including nucleus, cytoplasm and plasma membrane.OsBiPP2Cl was the protein phosphatase with recersible phosphorylation. We found that OsBiPP2Cl could dephosphorylate OsDMI3. ABA could infected the interaction between OsDMI3 and OsBiPP2Cl by the analyse of Kinase activity detection, Y2H system, BiFC and Co-IP assays. The majority of protein phosphatase 2CS in Arabidopsis thaliana had been implicated to act as a negative modulators of protein kinase pathways involved in ABA signaling. The expression and activity of OsBiPP2Cl in rice leaf showed that OsBIPP2Cl also be a negative component of ABA signaling. ABA could inhibited the activity of OsBiPP2Cl in brief period of treatment. OsBiPP2Cl could actived OsDMI3 by ABA.The expression and activity of OsBiPP2Cl in rice leaf showed that OsBIPP2Cl as a negative component of ABA signaling. It certified that OsBiPP2Cl was stand upsteam of OsDMI3 in ABA-induced antioxidant defense signaling.In the present study, H2O2 treatment induced the expression of OsDMI3 and the activity of OsDMI3 in ABA signaling. To investigate the effects of H2O2 on the induction of OsBiPP2Cl in leaf, relative quantitative real-time PCR analysis was performed and the Serine/Threonine Phosphatase Assay System was carried. It revealed that H2O2 inhibit the activity of OsBiPP2Cl. Analysis of the Co-IP assay in the rice leaves, we found that the interaction between OsDMI3 and OsBiPP2Cl could be regulated by ABA-induced H2O2. H2O2 weaken the activity of OsBiPP2C to releas OsDMI3.OsDMI3 could up-regulate antioxidant defense enzyme.Using protoplasts transient system, we demonstrated that OsBiPP2Cl was the negative factor of ABA-induced antioxidant defense. ABA treatment failed to induce the increases in the expression and the activities of SOD and CAT in the protoplasts expressing transiently OsBiPP2Cl. It suggested that OsBiPP2Cl interact with OsDMI3 and dephosphatase OsDMI3 under normal condition. When the stress stimulated the plant cell to accumulated ABA-induced H2O2. it inhibited the OsBiPP2Cl and actived OsDMI3. OsDMI3 up-regulated the activities of antioxidant enzymes and accumulated H2O2 to enhance the ability of scavenging ROS in plant cells. The activities of OsBiPP2Cl would be recoverd when the cell removed excess ROS. OsBiPP2Cl interactd with OsDMI3 again. OsBiPP2Cl was the key of ABA regulating OsDMI3 to active antixiodant denfense.