| Abscisic acid(ABA)is a classical plant hormone,which plays crucial roles in regulating plant growth and development and plant responses to environmental stresses.Calcium/calmodulin-dependent protein kinase(CCaMK)is an important component in ABA signaling.The rice CCaMK(OsDMI3)has been shown to be involved in ABA-induced antioxidant defense,and enhance plant tolerance to water stress and oxidative stress.However,the molecular mechanism by which how OsDMI3 regulates ABA-induced antioxidant defense remains to be determined.In a previous study,a new putative OsDMI3interacting protein,OsUXS3,was obtained by screening a rice cDNA library using yeast twohybrid(Y2H)assay and OsDMI3 as bait.OsUXS3 is a member of the uridine diphosphate glucuronide decarboxylase family in rice.It catalyzes the synthesis of UDP-xylose,and plays an important role in the synthesis of xylan.In this study,we identified the interaction between OsDMI3 and OsUXS3 in vivo and in vitro.We also investigated whether OsUXS3 can phosphorylate OsDMI3,and if so,what are the site and role of OsDMI3-mediated phosphorylation of OsUXS3.Moreover,we also investigated the role of OsUXS3 in rice tolerance to oxidative stress.The main results of this paper are as follows:Using firefly luciferase complementation imaging(LCI)assay,glutathione S-transferase(GST)pull-down assay,and co-immunoprecipitation(Co-IP)assay,we demonstrated there is an interaction between OsUXS3 and OsDMI3 in vivo and in vitro.Further,Y2H assay and LCI analysis proved that OsDMI3 interacts specifically with OsUXS3.An in vitro kinase assay showed that OsDMI3 phosphorylated OsUXS3.liquid chromatography-tandem mass spectrometry(LC-MS/MS)analyses and in vitro kinase assays revealed that Ser-245 in OsUXS3 is the phosphorylated site by OsDMI3 in vitro.Transient expression analyses of rice protoplasts indicated that OsDMI3-mediated phosphorylation enhances the protein stability of OsUXS3.The mutant of osuxs3 knockout(KO)was generated by CRISPR/Cas9 system,and the role of OsUXS3 in tolerance to oxidative stress was investigated.Compared with the wild type,the osuxs3 KO mutant had reduced survival rate,increased lipid peroxidation,and increased H2O2 content in rice leaves exposed to oxidative stress.These results indicated that the loss of OsUXS3 reduces tolerance to oxidative stress in rice.At the same time,the changes in the activities of the antioxidant enzymes superoxide dismutase(SOD)and catalase(CAT)in the mutant were detected,and it was found that the ABA-induced CAT activity in osuxs3 KO was affected.In order to further verify the role of OsUXS3 in ABA-induced antioxidant protection,the protein changes in ABA-treated osuxs3 KO and wild-type plants were analyzed by proteomics,and it was found that the expression of OsCATB protein induced by ABA was suppressed in osuxs3 KO rice plants.Western blot analyses confirmed that OsUXS3 can regulate OsCATB protein expression in ABA signalings.Using the rice protoplast transient expression system,it was found that OsDMI3-mediated OsUXS3 phosphorylation plays an important role in the ABA-induced increase of OsCATB protein. |
