Molecular Mechanism Of OsEDS1 In Regulating High Temperature Response In Rice

High-temperature stress is one of the main abiotic stresses limiting crop yield.Environmental temperature exceeding the optimumal temperature triggers plants’physiological and biochemical perturbations,such as reactive oxygen species（ROS）burst,resulting in high temperature stress.Heat-triggered ROS accumulation is cleared quickly to prevent oxidation-induced cell damage by ROS scavenging systems.Among the ROS,hydrogen peroxide（H₂O₂）is the main active oxygen involved in cell signal transduction.Enhanced disease susceptibility 1（EDS1）encodes a lipase-like protein.Its positive roles in the plant immune regulatory network have been well elucidated.The function of EDS1 in plant immunity and adaptation to waterlogging stress has been studied,but whether EDS1 is involved in adaptation to other abiotic stresses remains unknown.Therefore,this study aims to uncover the molecular mechanism of OsEDS1 in regulating high-temperature response,which will expand our understanding of EDS1 function.The main results are as follows:（1）oseds1 mutant showed significantly increased heat sensitivity at the seedling stage.OsEDS1 expression was induced by high-temperature.oseds1 mutant showed a high-temperature sensitive phenotype at the seedling stage.The complementary lines E^WT restored high-temperature sensitive phenotype of oseds1 mutant.The N-terminus of OsEDS1 protein is homologous to the lipase of eukaryotes,and the serine residue at position 143 of the OsEDS1protein is embedded in the motif similar to the GXSXG motif（X representing any amino acid）of eukaryotic lipases catalytic triad.E^S143L lines restored the high temperature sensitive phenotype of oseds1 mutant.These results above demonstrated that the full lenth mutation of OsEDS1 was the gene responsible for the thermosensitive phenotype of oseds1 plant,and the conserved S143 residue appeared not required for the OsEDS1-mediated heat tolerance.（2）Heat stress caused decreased catalase activity and H₂O₂ over accumulation in oseds1mutant.Heat stress induced H₂O₂accumulation in oseds1 mutant analyzed by DAB staining and quantification.The H₂O₂ content of E^WT lines and E^S143L lines was similar to that of wild type,but significantly lower than that in oseds1 mutant.Catalase activity assay showed that high temperature decreased catalase activity in oseds1 mutant.The catalase activity of oseds1mutant was higher than that of wild type before high-temperature treatment,but was lower than that of wild type after high-temperature treatment.The catalase activity of E^WT lines and E^S143Llines was similar to that of wild type.These results sμggested the mutation of OsEDS1 reduces the catalase activity and results in excessive accumulation of H₂O₂ in cells,thus decreasing the tolerance of rice to high-temperature.（3）OsEDS1 interacted with catalase B/catalase C（Os Cat B/C）to regulate H₂O₂ levels in cells.Heat stress induced the expression of all three catalase gene Os Cat A,Os Cat B and Os Cat C.The expression patterns of Os Cat A/B/C in wild type ZH11 and oseds1 mutant were similar,sμggesting that OsEDS1 affected catalase activity may not throμgh modulating their expression.The interaction between OsEDS1 and Os Cat B/Os Cat C in vitro and in vivo was verified by yeast two-hybrid assays,pull-down assays,split-luciferase complementation and bimolecular fluorescence complementation assays.oscatc mutant exhibited a heat-sensitive phenotype.Meanwhile,H₂O₂ content was increased and catalase activity was lower in oscatc mutant compared with wild type.Protein degradation assays showed that OsEDS1 stabilized Os Cat C.Enzyme activity assays showed that MBP-OsEDS1 promoted the activity of GST-Os Cat C and catalase activity of wild type ZH11 and oseds1 mutant.These results suggested OsEDS1interacted with and stabilizated Os Cat B/C,thus promoting their catalase activities to regulate H₂O₂ content in cells.（4）Overexpression of OsEDS1 improved the heat tolerance of rice.Compared with wild type,OsEDS1-oe showed a less heat-sensitive phenotype.The content of H₂O₂ in OsEDS1-oe was lower,but the catalase activity was higher.Under natural and artificial high temperature conditions,the wild type and OsEDS1-oe lines remained green,while the oseds1 mutant dried up and died.Compared with wild type,oseds1 mutant showed a defective phenotype in seed setting,grain weight and plant yield,while OsEDS1-oe lines showed some advantages on these agronomic traits.These results indicated that overexpression of OsEDS1 reduced the sensitivity to heat stress in the seedling stage and reproductive stage.These results demonstrated that OsEDS1 positively regulates rice thermotolerance by interacting with and promoting catalase-mediated H₂O₂ scavenging activities to control ROS homeostasis.This research greatly expands the understanding of OsEDS1 gene function and provides genetic resources for breeding heat-tolerant rice varieties.