The Phosphatase PHO Affects Rice Abiotic Salt Tolerance By Regulating The Phosphorylation Status Of CatC

Rice is one of the most important food crops.Rice feeds most people in China and even half of the world's people.However,land salinization,insufficient fresh water resources,soil erosion and other problems occur frequently recent years,so it is important to improve stresses resistance of rice.We identified a phosphatase PHO that interacts with CatC,a key enzyme in ROS scavenging,and speculated that it might affect the stresses tolerance of rice via regulating the phosphorylation status of CatC.In order to further study the molecular mechanism that PHO involve in rice stress response,we carried out a series of physiological and biochemical experiments,the results are as follows:?1?In order to analysis the expression pattern of PHO,the GFP fused PHO was transferred into rice protoplasts.The fluorescence distribution showed that PHO was mainly expressed in the cytoplasm,but its accurate subcellular localization need to analysis by cell components separation.At the same time,the expression of PHO gene under salt,drought and hydrogen peroxide stress was analyzed by fluorescence quantitative PCR.It was found that the expression level of PHO gene was up-regulated under abiotic stresses.The expression of PHO is induced by abiotic stress,suggesting that it may function in rice stress response.?2?The yeast two-hybrid analysis indicated that PHO interact with rice CATs,and had the strongest interaction with CatB.In addition,PHO was also found to interact with catalase domain of CatC,and the interaction with the phosphorylated CatC was significantly higher than that of CatC under normal conditions,indicating that PHO specifically recognizes the phosphorylated CatC,which is an important part of the CatC dephosphorylation regulation.?3?To verify whether PHO can dephosphorylate CatC,in vitro dephosphorolation assay found that PHO is a Ca²⁺-dependent phosphatase and can dephosphorylate serine sites of CatC.Further catalase activity analysis of CatC before and after dephosphorylation revealed that the enzyme activity decreased after dephosphorylation,indicating that PHO can regulate the phosphorylation status of CatC to affect its catalase activity.?4?In order to verify the function of PHO in stress response,we constructed a binary expression vector of PHO and a CRISPR/Cas9 mutation vector,which were transferr into rice by Agrobacterium-infected transformation and successfully obtained PHO transgenic lines.Salt stress treatment of the overexpressed lines found that salt tolerance of the PHO overexpression rice markedly decreased,which was consistent with the previous prediction.It was demonstrated that PHO participates in salt stress response of rice by regulating the activity of CatC.In summary,we found a phosphatase PHO interacts with CatC and can dephosphorylate serine sites of CatC to reduce its catalase activity.It was the first and the only phosphatase that can dephosphorylate CatC so far.These findings help us understanding the influence of phosphorylation modificationon CAT's function.So study on PHO is significant for both science and application field.Further phenotype analysis under salt stress found that over-expression of PHO decreased rice salt stress tolerance.The results indicated PHO negative regulate rice salt stress tolerance and can be used to breed high stress tolerance rice varieties.