Functional Analysis Of AtABCG5 Transporter Regulating Fatty Acids Levels In Arabidopsis Thaliana

In plant cells or tissues,fatty acids are the important precursors of various lipid compounds,such as neutral lipids,waxes,phospholipids,glycolipids,and cholesterol esters,as well as seed oil,which provide abundant nutrients and energy for humans.The fatty acids are synthesized in the plastid and then transported to the endoplasmic reticulum for lipids assembly to form oil bodies that are stored in seeds or that synthesize cutin and wax precursors.Therefore,the lipid synthesis process relies on transporters to affect the transport and delivering of fatty acids between organelles.At present,it is found that Arabidopsis thaliana FAX1 localized at the inner membrane of the plastids was involved in the efflux of fatty acids in the plastids,leading to the reduction of lipids in the plastids,the increase of lipid content in the endoplasmic reticulum,and the increase of triacylglycerol content in flowers and leaves.Arabidopsis thaliana AtABCA9 localized at the endoplasmic reticulum membrane was involved in Triacylglycerol(TAG)biosynthesis at the ER during the seed-filling stage for promoting the synthesis of seed oil.Trigalactosyldiacylglycerol(TGD)protein TGD1-5 transporter complex promoted lipids transport from the outer membrane to the inner membrane.Most of the ABCG transporters that localized at the plasma membrane transport cutin or waxy precursors to synthesize the wax of the plant stem and leaf epidermis.However,little research has been reported on the function of ABCG subfamily proteins in intracellular lipid synthesis pathways.In this study,we concerned the lipid synthesis pathway as the core regulation,based on the phenotypic observation and the screening theory of atabca9 mutant,in which the early seedling growth of oil seed plants depends on the lipid stored in seeds on the 1/2 MS medium without sucrose.We screened the similar sensitive phenotypes of atabcg5 that seems like atabca9 mutants,then cloned AtABCG5 and its promoter from Arabidopsis thaliana,and found the molecular mechanism of AtABCG5 in regulating plant seed size and fatty acid content in Arabidopsis thaliana.The main research results were as follows:1.We showed that the growth of atabcg5 roots were inhibited and developmental retardation under the control of 1/2MS medium without sucrose.In nutrient soil,the mutant grew slowly and flowering was delayed.During the growth and development of silique,it appeared seed development defects in late stages.Most of the mature mutant seeds are brown in color,smaller in size,and defective in cotyledons.The thousand-grain weight of dry seeds was 19% lower in mutant seeds compared with WT seeds.These results indicated that AtABCG5 played an important role in normal morphological size of seed and normal growth of early seedlings.2.The total lipid was extracted by the soxhlet method.It was found that the total lipid of per dry seed was 15.6% lower in mutant compared with WT.We determined the fatty acid contents in Arabidopsis seeds and found C18:1 and C18:3 significantly decreased by 39.8% and 14.8%,respectively.We analyzed some key genes in the lipid synthesis pathway in mutant and wild-type siliques via quantitative real-time RT-PCR,finding that the expression of lipid synthase gene DGAT1,FAD3,FATA,LACS2,GPAT4 and FAE1 was down-regulated,and the expression of suberin monomer transporter gene AtABCG20 was down-regulated.These results indicated that AtABCG5 was involved in fatty acid transport and lipid accumulation during seed formation.3.AtABCG5 and its promoter were cloned and analyzed.It was found that AtABCG5 contains no intron and encodes a half-molecule ABC transporter protein.It has typical structural characteristics of "NBD-TMD" half-molecular transporter.Phylogenetic tree analysis showed that the protein has a close evolutionary relationship with Capsella rubella and Brassica napus ABCG proteins.ProAtABCG5 has multiple MYB binding sites and ABA response CIS elements.4.Expression profiling via quantitative real-time RT-PCR analysis showed that AtABCG5 was expressed in all tissues of Arabidopsis thaliana,with the highest expression in leaves,siliques and flowers,and the lowest expression in roots.Moreover,the promoter fragment of AtABCG5 was fused with a GUS reporter gene and transformed into Arabidopsis thaliana,and GUS staining indicated that AtABCG5 was highly expressed in the later stage of siliques development(S3 phase),leaf stomata,anther and phloem of stem.These results showed that AtABCG5 may be involved in seed formation and other important parts processes.5.In order to determine the subcellular localization of AtABCG5 transporter,the plant expression vector Pro35S:AtABCG5-EGFP was constructed to express in tobacco and Arabidopsis thaliana protoplasts transiently.Confocal laser scanning microscopy revealed that AtABCG5 coincided with chlorophyll autofluorescence and AtABCG5 was located at the chloroplast.The results suggested that AtABCG5 functions as a transporter at the chloroplast.6.To verify the biological function of AtABCG5,plant overexpression vector(named pCXSN-AtABCG5)was constructed and transfected into Arabidopsis thaliana for stable expression.It was found that overexpression of AtABCG5 in wild type accelerated the growth of Arabidopsis thaliana,earlier flowering and increased its biomass,indicating that overexpression of AtABCG5 promoted plant growth and biomass accumulation.7.Further phenotypic observation of AtABCG5 transgenic and wild type showed that the AtABCG5-overexpressing plants produced enlarged seeds,and the thousand-seed weight increased by 20-40% than that of wild type after seed maturation.The triacylglycerol TAG in the seeds of overexpressing four lines increased,among which C18:1,C18:3 and C20:1 increased by up to 37.0%,52.4% and 34% respectively.The results showed that overexpression of AtABCG5 increased seed size and significantly increased the content of triacylglycerol TAG and unsaturated fatty acids in seeds.To sum up,on the one hand,the deficiency of Arabidopsis thaliana ABCG5 results in smaller and darkening seeds,the decrease of lipid content,the blockage of lipid synthesis pathway,and the decrease of expression of key enzymes in negative feedback regulation pathway.On the other hand,AtABCG5 gene is highly expressed in the late stage of seed formation,and AtABCG5 transporter is located at the chloroplast.Overexpression of AtABCG5 promoted the accumulation of lipids and unsaturated fatty acids,proving that AtABCG5 participates in lipid synthesis in plant seeds.The results of this study enriched the molecular mechanism of plastid fatty acid output in lipid synthesis pathway,and provided a new theoretical basis and ideas for cultivating high-oil contents and high-quality oil crops.