Functional Identification Of OsTIL1 In Chilling Acclimation And Lipid Metabolism Of Rice Seedlings

Rice is one of the most important food crops.As a temperature-loving plant,rice is susceptible to low temperature stress leading to yield reduction,and improving cold tolerance is the goal that breeders have been pursuing.With the further extension of the application of candidate gene association study(CGAS),many researchers have applied this technology to the screening of plant cold tolerance SNP loci.Temperature-induced lipocalin 1(TIL1)is a family of apolipoproteins in eukaryotic cells,which is highly conserved and plays an important role in plant response to environmental stress.Glycerolipids and glycerophospholipids are the main components of plant chloroplast membranes and cell membranes,respectively.Its role is to maintain the fluidity and functional integrity of the membrane,and the changes in the content and composition of membrane lipids reflect the cold tolerance of plants.In this paper,Os TIL1 was used as a candidate gene to conduct GWAS analysis on the survival rate of 351 rice core germplasm seedlings under chilling injury and low temperature acclimation treatments.On this basis,the laboratory-created Os TIL1-overexpressing and gene-edited homozygous line and wild-type japonica rice variety Zhonghua 11(ZH11)seedlings were used as materials to determine the survival rate and physiological indexes of cold tolerance under chilling injury and low temperature acclimation treatments.Variety.Dynamic changes in the lipidome were identified and the positive regulatory role of Os TIL1 in rice chilling injury and low temperature acclimation was elucidated.The main results are as follows:1.Candidate gene association analysis showed that,under the treatments of chilling injury and low temperature acclimation,japonica rice germplasm was more tolerant to low temperature and had stronger low temperature acclimation ability than indica rice germplasm.The polymorphism in the promoter region of Os TIL1 gene is associated with chilling injury and low temperature acclimation.2.The agronomic characters of the transgenic lines showed that 3 Os TIL1 overexpression lines and 2 gene-edited lines grew and developed normally,and the yield was not significantly different from the wild type Zhonghua 11.3.The measurement of physiological indexes of rice seedlings from chilling injury and low temperature acclimation showed that the survival rate of Os TIL1 gene-edited lines was significantly lower than that of wild-type lines,and the survival rate of Os TIL1 overexpressed lines after 14-day cold injury and low temperature acclimation treatment of soil cultured seedlings.rates were not significantly different from wild-type strains).Under chilling injury and low temperature acclimation,the malondialdehyde(MDA)and hydrogen peroxide(H2O2)contents of Os TIL1 gene-edited lines were significantly higher than those of wild-type lines,while Os TIL1-overexpressing lines had no significant difference with wild-type lines or significantly lower than that of wild-type strains.In terms of proline and soluble sugar content,Os TIL1 gene-edited lines were significantly lower than wild-type lines,while Os TIL1-overexpressing lines were significantly higher than wild-type lines.Therefore,Os TIL1 positively regulates the cold tolerance and low temperature acclimation ability of rice seedlings.4.The lipidomic analysis of rice seedlings under cold stress and low temperature acclimation showed that a total of 9 lipids were detected.Under cold stress,the contents of PA and DAG in the Os TIL1 gene-edited line were significantly higher than those in the wild-type line,and the contents of DGDG,MGDG,PS,PC,PE and PG were significantly lower than those in the wild-type line,and the content of PI was significantly higher than that in the wild-type line.There was no significant difference between the two lines;the content of 9 lipids in the Os TIL1 gene overexpression line was not significantly different from that in the wild-type line.Under low-temperature acclimation conditions,the PA content of Os TIL1 gene-edited lines was significantly higher than that of wild-type lines,and the contents of DGDG,MGDG,PE and PG were significantly lower than those of wild-type lines,and the contents of the other four lipids were similar to those of wild-type lines.There was no significant difference in the lines.Therefore,knockout of Os TIL1 affects the process of membrane lipid remodeling in rice seedlings under cold stress and low temperature acclimation.The lipid double bond index(DBI)and carbon chain length(ACL)measurements showed that both membrane lipid DBI and ACL were significantly positively correlated with their total content.Furthermore,the lipid fractions 36:6,36:5and 34:3 were more correlated with lipid DBI.Among them,lipid 36:6 had the largest correlation coefficient(R = 0.98),indicating that Os TIL1 positively regulates membrane fluidity mainly by affecting lipid molecule 36:6.