Molecular Mechanism Of Phosphorylation Of NADPH Oxidase By OsDMI3 To Regulate The Production Of ROS In ABA Signaling In Rice

Rapid accumulation of the plant hormone abscisic acid（ABA）is a typical marker of plant cell response to water stress such as drought and salinity.As a stress signal,ABA plays a key role in regulating water balance and inducing stress tolerance in plants.In ABA signaling,ABA binds to its receptor to release the kinase activity of SNF1-associated protein kinase 2（SnRK2s）inhibited by type 2C protein phosphatase（PP2Cs）,which is activated by autophosphorylation and then phosphorylates its downstream target molecules,such as NADPH oxidase（also known as the respiratory burst oxidase homologue,Rboh）.These induce a series of transcriptional or kinase regulation in response to ABA.Rboh is the main source of reactive oxygen species（ROS）,which plays an important role in ABA signaling pathway in plants.Ca²⁺/CaM-dependent protein kinase（CCaMK）is an important component of ABA signal transduction.CCaMK in rice（OsDMI3）has been reported to be involved in ABA-induced antioxidant protection and enhance rice tolerance to water and oxidative stress.Previous studies in our laboratory have shown that OsDMI3 is required for the accumulation of H₂O₂at a higher level in ABA signaling,but not for the initial production of H₂O₂,indicating that ABA-induced activation of OsDMI3 can induce ROS production,thus amplifying ROS signal.However,the molecular mechanism of how OsDMI3 regulates ROS in ABA signaling remains to be clarified.Thus,in this study,the interactions between OsDMI3 and OsRbohB/OsRbohE in rice were identified,and OsRbohB/OsRbohE were clarified as the substrate of OsDMI3 phosphorylation in vivo and in vitro,then its phosphorylation site was clarified.Finally,we analyzed the role of OsDMI3-mediated phosphorylation of OsRbohB/OsRbohE protein in ABA regulated physiological response and water stress.The main results are as follows:It was found that ABA could induce the increase of NADPH oxidase activity and H₂O₂content in WT rice leaves,and there were two rises over time,which increased significantly and reached the maximum at 15 min,then increased again after 90 min of ABA treatment and decreased with time.When OsDMI3 knockout,NADPH oxidase activity and H₂O₂content were not up-regulated at 90 min of ABA induction,indicating that OsDMI3 regulated the increase of NADPH oxidase activity and H₂O₂content at 90min of ABA induction.It was found that OsRbohB and OsRbohE jointly responded to the ABA-induced increase in NADPH activity and H₂O₂content by using osrbohb,osrbohe single mutants and osrbohb/e double mutants,and OsRbohB was dominant among them.The interaction between OsDMI3 and OsRbohB N-terminal/OsRbohE N-terminal in vivo and in vitro were determined by Yeast two-hybrid（Y2H）,GST-pull Down,Luciferase complementary Imaging（LCI）and Co-immunoprecipitation（Co-IP）analysis.OsRbohB and OsRbohE full-length proteins were constructed and found to interact with OsDMI3.LCI and Y2H analysis showed that the N-terminus of OsRbohB（229-355 aa）interacted with OsDMI3 EF Hands（337-516 aa）,and the N-terminus of OsRbohE（169-290 aa）interacts with OsDMI3（301-336 aa）.Moreover,Exogenous ABA could enhance the interaction between OsDMI3 and OsRbohB/OsRbohE.OsDMI3 only interacted with OsRbohB N-terminal and OsRbohE N-terminal,but did not interacted with other OsRbohs N-terminal,indicating the specificity of OsDMI3’s interaction with OsRbohB and OsRbohE.OsDMI3 directly phosphorylated OsRbohB_N/OsRbohE_Nin vitro by gel kinase activity analysis.LC-MS/MS and phosphorylation analysis in vitro results showed that OsDMI3phosphorylated OsRbohB_Nat Ser191 and OsRbohE_Nat Ser130.Then,OsRbohB and OsRbohE knockout and overexpression materials as well as OsRBOHB^S191and OsRBOHE^S130point mutation overexpression materials were constructed.Phosphorylation of OsRbohB S191 positively regulated the increase of NADPH oxidase activity and H₂O₂content in ABA signaling,while phosphorylation of OsRbohE S130 had no significant effect on NADPH oxidase activity and H₂O₂content.ABA induced phosphorylation of OsRbohB S191 by using the phosphorylation antibody OsRbohB^S191（P）and the phosphorylation of S191 was regulated by OsDMI3.Further studies showed that the increases in NADPH enzyme activity and H₂O₂content induced by phosphorylation of OsRbohB S191 were also regulated by OsDMI3 in ABA signaling.Water stress（PEG treatment）also induced phosphorylation of OsRbohB S191,which was regulated by endogenous ABA.Further genetic evidence revealed the role of OsRbohB/OsRbohE phosphorylation by OsDMI3 in ABA signaling and drought and oxidation tolerance.Phosphorylation of OsRbohB S191 positively regulated the ABA sensitivity of rice seed germination and root growth,while phosphorylation of OsRbohE S130 did not affect the ABA sensitivity of seed germination and root growth.Phenotypic analysis and physiological analysis showed that the phosphorylation of OsRbohB S191 positively regulated the tolerance of rice seedlings to drought and oxidative stress,while the phosphorylation of OsRbohE S130 had no significant effect on the tolerance of rice seedlings to drought and oxidative stress.OsRBOHB overexpression and OsRBOHB^S191point mutation overexpression materials were constructed using os DMI3 mutants as background,and osrbohb knockout material was constructed based on OsDMI3 overexpression material.The role of phosphorylation of OsRbohB by OsDMI3 in ABA signaling and drought and oxidation tolerance was investigated by using the above materials.Phenotypic analysis and physiological analysis showed that the OsDMI3-OsRbohB phosphorylation pathway positively regulated the sensitivity of seed germination and root growth to ABA,and the tolerance of rice seedlings to drought and oxidative stress.Finally,whether SnRK2s（SAPK8/9/10 in rice）,a major component of the ABA signaling pathway,regulates the OSDMI3-OsRbohB pathway.The results showed that SAPK8/9/10 could regulate the OsDMI3 kinase activity and the phosphorylation of OsRbohB S191 induced by ABA treatment for 90 min,thus further affecting the NADPH oxidase activity and H₂O₂content in rice leaves.The results also show that the NADPH oxidase activity and H₂O₂content are regulated by SAPK8/9/10 when treated with ABA for15 min.The conclusion is that OsDMI3 induces H₂O₂production by phosphorylation of OsRbohB S191 in ABA signaling,further amplifying ROS signal.This process is regulated by SAPK8/9/10.And this signal transduction pathway plays an important role in the physiological response of ABA regulation and drought tolerance and oxidative stress tolerance of rice.