Characterization of the role of Arabidopsis long-chain acyl-coenzyme A synthetase 8 in seed oil biosynthesis

Seed storage lipids are major carbon reserves that accumulate in developing embryos, and are stored in the form of triacylglycerols (TAGs) within specialized organelles called oil bodies. Even though several key factors that control oil yield have been identified, many rate-limiting steps remain to be established. One such step may be the export of acyl chains from the plastid where they are synthesized, to the ER where they are used for the production of TAGs. Since acyl chain export and utilization requires activation of acyl chain precursors to acyl-coenzyme A (CoA), I hypothesized that long-chain acyl-CoA synthetase (LACS) enzymes which catalyze acyl-CoA biosynthesis may be rate-limiting activities for the accumulation of TAGs in the seed. Based on the available information, I predicted that of the nine LACS enzymes described in Arabidopsis thaliana, three enzymes, LACS8, LACS9 and LACS1, may be involved in supplying acyl-CoAs for TAG formation. Using a reverse genetic approach, I then examined the contribution of LACS8 to TAG accumulation in developing seeds, and investigated whether LASC9 and LACS1 have overlapping functions with LACS8 in TAG production. Expression analyses using in situ hybridization, β-glucuronidase (GUS) activity assays in transgenic plants expressing LACS8promoter-β-glucuronidase (pLACS8::GUS) construct, and quantitative polymerase chain reaction (PCR) demonstrated that LACS8 is transcribed predominantly in the developing embryo. Yellow fluorescent protein (YFP)-tagged LACS8 was localized to the endoplasmic reticulum (ER) in Arabidopsis by confocal microscopy. Identification and characterization of loss-of-function lacs8 mutants did not reveal any reductions in the amount of TAG produced. Similarly, introduction of additional copies of the LACS8 gene did not result in increased TAG accumulation in transgenic seeds. In contrast, analyses of lacs8lacs9, lacs8lacs1 and lacs1lacs9 double mutants and lacs1lacs8lacs9 triple mutants revealed altered TAG levels. The most pronounced TAG decreases were detected in lacs1lacs9 and lacs1lacs8lacs9 mutants, an indication that LACS1 and LACS9 have redundant functions in supplying adequate acyl-CoA amounts for TAG biosynthesis. I also tried to increase the TAG yield through seed-specific expression of LACS8, LACS9 and FATB genes, but the oil content of all the generated transgenic plants in the T2 generation was in the wild type range.Science, Faculty ofBotany, Department ofGraduat