Functional Identification Of OsTIL2 In Heat Tolerance And Lipid Metabolism In Rice

Under the climate warming circumstance,the global temperature is generally rising,and the extreme temperature has seriously affected the growth and development of plants in the whole growth period.To cope with high temperature stress,rice has evolved multiple tolerance mechanisms to reduce intracellular reactive oxygen species（ROS）levels to maintain the stability of the cell membrane system and improve tolerance to high temperature.Therefore,it is of great theoretical and practical significance to study the mechanism of rice response to high temperature stress.In plants,temperature-induced apolipoproteins（TILs,temperature-induced lipocalins）are a class of highly conserved and lipid-binding and transporting proteins in eukaryotic cells,which play an important role in plant responses to different biotic and abiotic stresses.In this paper,by using OsTIL2 overexpression and gene editing homozygous lines as materials,the agronomic traits of the transgenic lines were investigated,and the heat tolerance of rice throughout the growth period（germination,seedling and flowering stages）was identified.Antioxidant enzyme activity and its lipidomic changes under high temperature stress were detected,the main results are as follows:1.OsTIL2（LOC＿Os08g34150）encodes a protein containing 179 amino acid residues.This protein is localized in the cell membrane and cytoplasm in rice protoplasts.Moreover,OsTIL2 was up-regulated by high temperature in rice seedlings.2.OsTIL2 did not affect rice growth and yield under normal condition.In the 2021planting season,three homozygous overexpression lines（OE-1,OE-3 and OE-9）and three gene editing homozygous lines（C-2,C-3 and C-10）and wild type had no significant difference in plant height,ear length,effective tiller number,grain number per ear,seed setting rate and 1000-grain weight.3.OsTIL2 negatively regulated heat tolerance in rice throughout the growth period.At germination stage,under the control treatment（30℃for 7 d）,the root length and shoot length of each line were not significantly different.After treatment at 40°C for 2days,the root and shoot lengths of OsTIL2 overexpressed and gene-edited lines were significantly different from those of the wild type.At the seedling stage,the 14-day hydroponic seedlings were treated at 42°C for 2 days and then recovered at 28°C for 7days.Compared with the wild type,the OsTIL2 overexpressing line showed a reduced survival rate,while the gene-edited line had a higher survival rate.After high temperature37°C/30°C（day/night）treatment for 10 days,heat tolerance index of OsTIL2 overexpression lines was lower than that of wild type wehereas that of OsTIL2gene-edited lines was higher than that of wild type.4.Physiological index measurement results showed that,under normal conditions,physiological indexes of OsTIL2 overexpressed,gene edited lines and wild type were not significantly different at seedling and flowering stages.Under high temperature stress,the enzyme activities of SOD,POD,APX and CAT in OsTIL2 gene edited lines were higher than those in wild type and overexpression lines;The soluble sugar content of the lines was higher;the MDA and H₂O₂contents of the OsTIL2 gene edited lines were lower than those of the wild type and overexpressing lines,and these results were consistent with the results at the seedling and flowering stages,indicating that OsTIL2 negatively regulates the heat tolerance in rice.5.Lipidomic changes of OsTIL2 transgenic lines and wild type at seedling stage were determined by UPLC-MS/MS.Under normal condition,there was no significant difference in lipid species and contents between the transgenic lines and the wild type.Under high temperature stress,the total lipid content of each lines decreased,but the degree of decrease was different.OsTIL2 overexpressing lines had higher DBI and shorter ACL compared with wild-type and gene edited lines,indicating a stronger membrane lipid fluidity and less resistance to heat stress.Correlation analysis showed that,DAG36:6,PA36:6,MGDG36:6,DGDG36:6 components were significantly positively correlated with lipid DBI,and 36:2 glycerophospholipid was significantly positively correlated with membrane lipid DBI.These results showed that,OsTIL2 mainly affected the contents of36:6 glyceroglycolipid and 36:2 glycerophospholipid at high temperature,thereby negatively regulating the heat tolerance in rice.