Functional Analysis Of Lipid Metabolism-Related Genes GmPLD? And AtGPATs In Oil Synthesis And Development Of Seeds In Arabidopsis

Plant oil is the resource of energy and substrates for maintaining plant growth and development.It can be used for food,production of chemical products and fuel substitutes,which are essential for human life.Although biochemical pathways of lipid biosynthesis are relatively clear,there are few successful examples of improving seed oil content and quality through molecular biotechnology.The purpose of this study is to find the genes that can be used to improve the oil content and quality of crop seeds after dissecting their functions and mechanisms.Plant phospholipase D(PLD)hydrolyzes glycerophospholipids to produce phosphatidylic acid(PA),functioning in glycerolipid metabolism and signaling.Glycerol-3-phosphate acyltransferase(GPAT)catalyzes the esterifcation of a fatty acyl moiety from acyl-CoA or acyl-ACP to the sn-1 or sn-2 position of glycerol-3-phosphate(G3P),which is important for the biosynthesis of glycerolipids and extracellular lipids.Through a series of screening,we found that the soybean phospholipase Dy(GmPLD?)and Arabidopsis glycerol-3-phosphate acyltransferase(AtGPAT1 and AtGPAT2)played important roles in seed oil biosynthesis,and had potential application value.Besides,we also studied the other physiological and biochemical functions of these genes.The main results are as follows:Overexpression of soybean GmPLD? gene in Arabidopsis resulted in significant increase of seed w eight and oil content but decrease of oil body size compared with that of the control plants,Arabidopsis wild type(WT)and empty vector transgenic(EV)plants.Analysis of seed fatty acid composition showed that overexpression of GmPLD? altered the seed fatty acid composition.The content of palmitic(16:0)and palmitoleic(16:1?9)acids decreased,while linolenic(18:3?9,12,15),arachidic(20:0),eicosenoic(20:1?11),eicosadienoic(20:2?11,14),docosanoic(22:0)and erucic(22:1?13)acids increased in the seeds of GmPLD?-OE plants as compared with those of WT and EV plants.In addition,overexpression of GmPLD),decreased the membrane lipid content of mature leaves,but had no significant effect on the membrane lipid content of seeds.Further analysis of the effect of overexpression of GmPLD? on the expression of genes related to glycerolipid biosynthesis in Arabidopsis development pods showed that overexpression of GmPLD? led to the increase of expression of some genes in the glycerolipid biosynthesis pathway,which preliminarily explained how GmPLD? overexpression increased seed oil content and changed seed fatty acid composition.To further understand the mechanism of GmPLD? in regulating oil biosynthesis,we determined the biochemical and molecular characteristics of GmPLD?.Our results might suggest that GmPLD? was localized in mitochondria.GmPLD? exhibited typical PLD activity,and its activity was dependent on oleate and affected by Ca2+,PIP2.The C2 and HKD domains were critical for GmPLD activity.Besides,the residues Lys-600 and Ser-407 were also important for its PLD activity.GUS staining anaysis indicated that AtGPAT1 and AtGPA T2 genes were expressed in embryo,seedling,rosette leaf,inflorescence,silique and geminated seed respectively.Both proteins were sub-located in mitochondrion.Mutation of AtGPAT1 decreased the seed oil content significantly,and the content of palmitic(16:0),stearic(18:0),and docosanoic(22:0)acids also reduced markedly while oleic acid(18:1?9)increased significantly.Knockout of AtGPAT2 had no markable effect on seed oil content but changed the fatty acid composition significantly with decreased content of oleic acid(18:1?9)and increased content of palmitic(16:0)and linolenic(18:3?9,12,15)acids.Knockout of both AtGPATl and AtGPAT2 resulted in more severe reduction of seed oil content and change of fatty acid composition than single mutation of each gene.The content of stearic(18:0),linolenic(18:3?9,12,15),eicosenoic(20:1?11),eicosadienoic(20:2?11,14),and erucic(22:1?13)acids decreased significantly while palmitic(16:0),palmitic(16:1?9),oleic(18:1?9),and linoleic(18:2?9,12)acids increased markedly in the seeds of the double mutant.These results indicate that both AtGPAT1 and AtGPAT2 play important roles in seed oil biosynthesis,and the effect of AtGPAT1 on seed oil content and fatty acid composition is greater than that of AtGPAT2.In addition,AtGPAT1 and AtGPAT2 are involved in the late embryo development,and knockout of both genes caused the abnormal development of the late embryo,resulting in the morphological deformities of seeds,cotyledons and some true leaves.Knockout of both genes also caused the defects in the formation of leaf cuticles.In conclusion,we found that GmPLD?,AtGPAT1 and AtGPAT2 played important roles in seed oil biosynthesis,but their roles in regulating fatty acid composition were different.AtGPATl and AtGPAT2 also functioned redundantly in late embryo development and the formation of leaf cuticles.These results may be useful for production of oil crops with high oil content and quality using genetic engineering approaches.