Identification Of The Interaction Of OsDMI3 And Osprx20 Functional Analysis And Osprx20 In Abscisic Acid-induced Production Of H₂O₂ In Rice

As a major phytohormone in plants,abscisic acid mediates lots of physiology process,especially important for adaptation to extream environmental conditions.ABA can accumulate in plant cell when under water stress,leads to stomatal closure in guard cells to let plant avoid adversity.Studies had been proved that in rice OsCCAMK(OsDMI3)is required for ABA-induced antioxidant defense that ABA-induced and oxidative stress tolerance under water stress.But the molecular mechanisms of ABA induces the activation of the protein OsDMI3 and how the activated CCaMK let the antioxidant defense in this signal still need further study.It needs us to find the character of OsDMI3 interact proteins and make sure if they join in the ABA signaling.In order to better study the role of OsDMI3 in the ABA induced antioxidant defense,the yeast two hybrid system was used to screen the OsDMI3 of rice leaf cDNA library to find it's target protein in ABA signaling.One of the screened positive clones were compared to be Osprx20.The analysis found the protein belongs to the plants class III peroxidase,and is one of the rice Osprx family members.Present research shown that the family's member function have something to do with the content of H2O2 in the issue of maintaining the stability.Due to the false positives of Y2H system,and to verify the authenticity of the interaction between OsDMI3 and Osprx20,BiFC(Bi-molecular fluorescence complemention),GST-pull down,Co-IP(Co-immunoprecipitation)assays were tested in this work.The result confirmed that there do have interaction between OsDM13 and Osprx20 both in vitro and in vivo.Based on these results the above results,we can prove that Osprx20 can interact with OsDMI3.In the view of the above mentioned result,to investigate weather the Osprx20 join the ABA-induced signaling and the function of Osprx20 in this signaling.the real-time fluorescent quantitative PCR technology and transient gene expression analysis in rice protoplasts were used.Our results indicated that the expression of Osprx20 in rice leaves were increased under ABA/H2O2 treatment.In ABA treatment the first peak occurred after 90 min and in the H2O2 treatment the first peak occurred after 45 min and the second peak appeared within 90 min.At the same time,using in vitro gel kinase reaction system analysis to confirm that OsDMI3 can phosphorylate Osprx20.These results show that Osprx20 can be induced by ABA,and OsDMI3 acts on the upstream of Osprx20,also phosphorylate Osprx20.Osprx20 belongs to the family of catalase.Amplex(?)Red Hydrogen Peroxide/Peroxidase Assay Kit was used and found that the protein of Osprx20 can directly scavenge H2O2.Peroxidase activity assay to confirmed that Osprx20 catalyzes the reduction of H2O2.Transient expression of Osprx20 in rice protoplasts to investigate the ABA-induced production of H2O2.Connect the CDS region of Osprx20 to mcherry vector.Transform Osprx20-mcherry into rice protoplasts.With treatment of ABA,transient expression of Osprx20 decreases ABA-induced production of H2O2.At the same time,through the analysis of Osprx family phylogenetic tree,found homologous of Osprx20,Osprx22.Clone and using yeast two hybrid system confirm that the family proteins Osprx22 and OsDMI3 are not interaction in yeast.