Molecular Mechanism Underlying Phosphorylation Status Of CatC Modulating Salinity Tolerance In Rice

As one of the most important crop plants,rice is seriously threatened by soil salinity.Salt stress can cause ion toxicity,osmotic stress and oxidative stress,which seriously threaten the growth and yield of rice.Catalase（CAT）,as an important scavenging enzyme of reactive oxygen species（ROS）,can eliminate H₂O₂ accumulated under stress and maintain H₂O₂ homeostasis,which plays an important role in plant growth and stress response.Previous studies have found that catalase C（CatC）plays an important role in maintaining ROS homeostasis under salt stress in rice,and phosphorylation is also found to play an important role in regulating its function.However,kinases and phosphatases involved in regulating CatC phosphorylation status under salt stress are rarely reported.In this study,the target site of CatC phosphorylated by a protein kinase STRK1（salt tolerance receptor kinase 1）identified previously and its effect on CatC function were further explored.Meanwhile,a phosphatase of catalase 1（PC1）was identified from the CatC complex,and the response of PC1 transgenic rice to salt stress as well as the molecular mechanism underlying PC1regulating CatC phosphorylation status and function was analyzed.The main results are as follows:（1）STRK1 phosphorylates CatC at Tyr210 and activates its activity,and this phosphorylation signal transduction is dependent on the phosphorylation of Tyr240 in STRK1.Firstly,the phosphorylation sites of CatC and STRK1 were identified by mass spectrometry.Phosphorylation analyses in vitro and in vivo revealed that Tyr210 is the target site of CatC phosphorylated by STRK1,demonstrating that STRK1 is the only one tyrosine kinase of CatC reported so far in plants.Subsequently,the phosphorylation sites of STRK1,which play important roles in phosphorylation signal transduction,were identified.The capacitiy for phosphorylating CatC was found to be significantly reduced after the Tyr240 mutation in STRK1,suggesting that Tyr240 plays an important role in STRK1-mediated signal transduction.In addition,the STRK1 knockout mutant strk1 was constructed,and strk1mutants were found to be the same sensitive to salt and oxidative stress as STRK1-RNAi lines,further demonstrating that STRK1 positively regulates rice salt tolerance.（2）The phosphorylation status of Tyr210 regulates CatC enzyme activity and protein stability.The effects of phosphorylation status of Tyr210 on the subcellular localization,protein stability and enzyme activity of CatC were analyzed.It was found that phosphorylation status of Tyr210 did not affect the subcellular localization of CatC in rice protoplasts,which were located in peroxisome.However,the stability and enzyme activity of CatC phosphorylated at Tyr210 were significantly higher than those of CatC not phosphorylated at Tyr210,suggesting that the phosphorylated status of Tyr210 mainly regulates the enzyme activity and protein stability of CatC.In addition,it was found that mutations of phosphorylation sites in both STRK1 and CatC had no obvious effect on STRK1-CatC interaction,indicating that the phosphorylation status of STRK1 and CatC do not affect their interaction.（3）A protein phosphatase PC1,which dephosphorylates catalase,was identified for the first time and was found to negatively regulate the tolerance to salt and oxidative stress in rice.A phosphatase PC1 was identified from the CatC complex by immunoprecipitation（Co-IP）-mass spectrometry（MS）strategy,and the interaction of PC1-CatC was confirmed to take place at peroxisome.It was also found that PC1 co-incubated with phosphorylated CatC could effectively reduce the serine phosphorylation signal of CatC,but not for the phosphorylation signal of threonine and tyrosine,suggesting that PC1 could dephosphorylate the phosphorylated serine residues in CatC.Analysis of salt response found that PC1over-expressi exhibited more sensitive to salt and oxidative stress,while the knockout mutant pc1 showed more tolerant to salt and oxidative stress,indicating that PC1 negatively regulates the tolerance to salt and oxidative stress in rice.Particularly,when treated with salt stress at booting stage,the effective panicles and yields of pc1 were significantly higher than those of wild type,while those of PC1 over-expression lines exhibited the opposite results,suggesting that the loss-of-function mutants or alleles of PC1 have a potential application value in salt-tolerant rice breeding.（4）PC1 regulates CatC polymerization status and enzyme activity by dephosphorylation of CatC at Ser9.By synthesizing a phosphorylated peptide at a specific serine site of CatC and co-incubating with PC1 in vitro,it was found that PC1 could dephosphorylate Ser9 of CatC,while its adjacent Ser10 and other serine residues could not be dephosphorylated,indicating that Ser9 is the target site of CatC dephosphorylated by PC1 and the dephosphorylation process is highly specific.Subsequent studies showed that Ser9 phosphorylation significantly enhanced PC1-CatC interaction and enzyme activity of CatC,but had no effect on CatC subcellular localization and protein stability.Further studies showed that Ser9phosphorylation promoted CatC tetramer formation and increased its enzyme activity.In addition,it was found that salt stress inhibited the dephosphorylation activity of PC1,thereby maintaining Ser9 phosphorylation status and high enzyme activity of CatC,which improved the ROS scavenging ability,and ultimately enhanced the salt tolerance in rice.（5）STRK1 and PC1 antagonistically regulate CatC phosphorylation status and H₂O₂homeostasis,thereby participating in the regulation of salt stress response and growth in rice.Based on the above results,the molecular mechanism underlying CatC phosphorylation status regulating rice salt tolerance and growth was proposed.When rice plants are suffered from salt stress,STRK1 and other unknown kinases are activated immediately,and CatC is recruited and phosphorylated at the plasma membrane.Subsequently,the monomer forms of CatC with low activity are polymerized into the tetramer form with high activity to eliminate the excess H₂O₂ accumulated due to salt stress.Meanwhile,the activities of PC1 and other unknown phosphatase are inhibited,which promotes rice plants to be in the salt stress tolerance state.When the salt stress disappeared,PC1 and other unknown phosphatase are quickly activated and then dephosphorylated CatC at peroxisome,which promotes the tetramer form of CatC with high activity is depolymerized into the monomer forms of CatC with low activity to maintain an opportune H₂O₂ level required as a signal molecule for normal growth and development in rice.Simultaneously,the activities of STRK1 and other unknown kinase are inhibited,which promotes rice plants to alter from salt tolerance status to normal growth and development.In conclusion,these results revealed that the kinase STRK1 and the phosphatase PC1 antagonistically regulate the phosphorylation status of CatC,as well as the regulatory network of the transition from the salt tolerance to growth state in rice.Although STRK1 and PC1 act on CatC at various amino acid residues,the elucidation of this regulatory network lays a good foundation for subsequent systematic revelation of the molecular mechanism underlying ROS participating in the regulation of stress response in plants,and also provides gene resources and theoretical guidance for the salt-tolerant rice breeding.