Identified The Interact Protein With OsDMI3 In ABA-induced Antioxidant Defense Signaling

The classic plant hormone abscisic acid(ABA)mediates lots of physiology process and play an critical role in adaptive response to environment siresse,Ca2+ is a universal second messenger and required for ABA-induced antioxidant defense.Transient Ca2+ elevations are sensed by several Ca2+ sensors to regulate diverse downstreamtargets.Calmodulin(CaM)as important Ca2+sensor do not have activity itself.Ca2+-binding to CaMinduces exposure of hydrophobic clefts that can interactwith CaM-binding proteins(CaMBPs)to result in various physiological responses.CCaMK which belongs to CaMBPs are has been also shown to be involved in the responses of ABA signaling.Resent study showed that OsCCaMK(asDMI3)is required for ABA-induced antioxidant defense and oxidative stress tolerance under water stress.However,the molecular mechanisms by which how ABA induces the activation of CCaMK and the activated CCaMK regulates the antioxidant defense in ABA signaling remain to be determined.Clarify the role of OsDMI3 interact protein in the ABAsignaling is important for mechanism of CCaMK in plant response to stress.In the present study,using the full-length of OsDMI3 as bait by the Y2H interaction screening approach,we identified several protein interact with OsDMI3.There may be some false positives due to Y2H system.To verify the authenticity of the interaction between OsDMI3 and OsUGD,OsBIP130,OsRab5A,BiFC(Bimolecular fluorescence complementation),GST pull-down,Co-IP(Co-immunoprecipitation)assays were tested in this work.It vestigated that OsUGD,OsBIP 130 and OsRab5A did interact with OsDMI3 both in vitro and in vivo.The expression in rice leaves shown that OsUGD,OsBIP130 and OsRab5A were increased under ABA treatment identified by relative quantitative real-time PCR analysis.It showed that the transcripts of OsUGD reached the maximum at 60 min,Treatments with ABA induced a biphasicresponse in the expression of OsBIP130,in which the first peak occurred after 30 minand the second peak appeared within 90 min of treatments.ABA induced a rapid increase in the expression ofOsRab5A in 30 min after treatment.Using protoplast transient system,we demonstrated that OsUGD?OsBIP130 failed to induce the activities of SOD and CAT in the ABA signaling in the protoplasts transiently silencing OsUGD and OsBIP130..It suggested that OsUGD?OsBIP130 may participate in the ABA induced antioxidant defense signaling.However,OsRab5A had very little effect on the ABA-induced increase in the activity of SOD and CAT in the protoplasts transiently silencing OsRab5A.In conclusion,we identified that OsDMI3 interact with OsUGD,OsBIP 130 and OsRab5A.Treatments with ABA,induced the expressionof OsUGD,OsBIP130 and OsRab5A.The analysis the RNA interference(RNAi)silencing protoplasts showed that OsUGD and OsBIP 130 are required for ABA-inducedincreases in the expression and the activities of superoxide dismutase(SOD)and catalase(CAT),and OsRab5A may not be involved.This study lays the foundation for further identification the role of OsDMI3 in ABA induced antioxidant defense signaling.