Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy Durrett / In plants including the members of Brassicaceae family, such as Arabidopsis thaliana and Brassica juncea, seed storage reserves, which include lipids and proteins, accumulate in seeds during development. Triacylglycerols (TAG) are the major storage lipids found in the developing seeds, petals, pollen grains, and fruits of plants. In Arabidopsis seeds, acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) is the major enzyme contributing to TAG biosynthesis. In Arabidopsis, dgat1-1 mutants retain 60-80% seed TAG content due to the involvement of phospholipid: diacylglycerol acyltransferase (PDAT1) in acyl-CoA independent TAG biosynthesis. My study focuses on the elucidation and functional characterization of novel genes involved in the regulation of the TAG biosynthesis pathway. In developing seeds of the dgat1-1 mutant, altered fatty acid composition was observed with reduced TAG content and increased polar lipid content as compared to wild type. RNA-Seq of developing Arabidopsis seeds was employed to detect differentially expressed genes in dgat1-1. An empirical analysis for differential gene expression revealed a significant number of differentially expressed genes among all developmental stages in dgat1-1. Significant changes in gene expression profile were detected in lipid-related genes such as lipases and desaturases. RT-PCR was used to confirm the differential expression of major lipid-related genes including DGAT1, PDAT, and FAD2. Lipid profiling of T-DNA insertion mutants for differentially expressed genes revealed significant changes in lipid content and composition. Mutations in a member of the α, β-hydrolase family, encoded by gene named PLIP1, resulted in smaller seed and an altered seed oil phenotype. Also, combining the dgat1-1 and plip1-2 mutations resulted in a lethal phenotype, demonstrating the important role of this enzyme in embryo development and TAG biosynthesis. To identify key components in the regulation of storage lipid biosynthesis, correlation analysis using differential transcript abundance and lipid profile during different stages of seed development from dgat1-1 and wild type lines of Arabidopsis was performed. Using clustering analysis with Pearson correlation coefficient and single linkage identified one cluster of genes which included PLIP1, FAD2, FAD3, and PDCT . Similar analysis using combined data from the neutral and polar fractions resulted in clustering of lipids containing polyunsaturated fatty acids. To investigate the reduced seed germination phenotype for mature seeds of dgat1-1 and non-germinating green seed phenotype of dgat1-1 plip1-2 lines, differential expression (DE) analysis for genes involved in hormone metabolism was performed. Upregulation of expression was observed for genes involved in promoting abscisic acid (ABA) response, which led us to specuate the role of altered hormone metabolism in delayed germination of dgat1-1 seeds. Development of allopolyploid Brassica species from its diploid progenitors involves duplication, loss, and reshuffling of genes leading to massive genetic redundancy. It leads to selective expression or newly acquired role for duplicated homeologs. Differential expression (DE) analysis for homoeologous genes from A and B subgenomes of allopolyploid B. juncea implicated in FA synthesis, acyl editing, and TAG biosynthesis and metabolism was performed. Differential expression (DE) analysis identified the transcriptional dominance of A subgenome homoeologs. Identification of these homoeologs will enable their use in breeding programs directed towards improvement of lipid content and composition in seeds.

L’hydrolyse des triglycérides (TG) du tissu adipeux est initialisée par l’action de l’adipocyte TG lipase (ATGL), activée par son cofacteur la protéine CGI-58. Des mutations du gène codant les protéines CGI-58 ou ATGL sont respectivement à l’origine du syndrome de Chanarin-Dorfmann et de maladies de stockagede lipides neutres. La protéine CGI-58 appartient à la famille des a/ß-hydrolases, elle en possède la triade catalytique Ser-Asp/Glu-His caractéristique des carboxylester hydrolases, ainsi que le motif HX4D, caractéristique d’une activité acyltransférase. A ce jour, le rôle de la protéine CGI-58, chez les mammifères ou chez les plantes, n’est pas totalement éclairci, de même que son activité enzymatique. Dans le but de mieux comprendre le rôle de cette protéine CGI-58 dans le métabolisme lipidique, nous avons développé une nouvelle stratégie, en générant notamment de nouveaux plasmides qui nous ont permis d’exprimer et de purifier la protéine CGI-58 de souris et de plantes, ainsi que l’ATGL murine, dans différents souches d’E. coli, pour tester l’activité in vitro de ces protéines. De plus, nous avons mis en place, en utilisant ces plasmides générés et différentes souches E. coli, un système qui nous a permis de tester in vivo, dans E. coli, l’activité acyltransférase (LPAAT et/ou LPGAT) de la protéine CGI-58. En utilisant ces différentes techniques, nous avons pu montrer, aussi bien in vivo qu’in vitro, que la protéine CGI-58 de plante, ainsique celle de mammifère, ne possède ni activité LPAAT ni activité LPGAT, et que la protéine CGI-58 de plante est dépourvue d’activité TG lipase ou phospholipase. Cependant, nous avons montré, en analysant, par chromatographie sur couche mince et par spectrométrie de masse, des extraits lipidiques des différentes souches d’E. coli exprimant la protéine CGI-58, que l’expression de la protéine de plante, et non celle de mammifère, aboutit à une diminution du taux de phosphatidylglycérol (PG) dans les différentes souchestestées, et a contrario nous avons montré que la mutation de la sérine, ainsi que la mutation de l’histidine de la triade catalytique potentielle, restaure le phénotype sauvage. Ces résultats nous ont permis de proposer que la protéine CGI-58 de plante est impliquée dans le métabolisme du PG / Triglycerides (TG) hydrolysis in adipose tissue is initialized by the action of the adipose TG lipase (ATGL) and its cofactor, the CGI-58 protein. Mutations in the gene coding the CGI-58 or ATGL proteins are the cause of the syndrome of Chanarin-Dorfman and of neutral lipids storage disease, respectively. CGI-58 protein belongs to the a/ß-hydrolase family, harboring the catalytic triad Ser-Asp/Glu-His, characteristic of carboxylester hydrolases, as well as the HX4D motif, characteristic of acyltransferase activity. Nowadays, the role and the enzymatic activity of the CGI-58 protein, in mammalians and plants, are not quite clear. In order to better understand the function of CGI-58 protein in lipid metabolism, we developed a new strategy using a new set of plasmids that enabled us, with the use of pET28b(+) plasmid, to express and purify the CGI-58 protein of mice and plants as well as the murine ATGL. These proteins were expressed in different E. coli strains, to test in vitro their activity. In addition, we have set up, using the generated plasmids and different E. coli strains, a system that allowed us to test the in vivo acyltransferase activity (LPAAT and/or LPGAT) of the CGI-58 proteins expressed in E. coli. Using these techniques, we demonstrated in vivo and in vitro that both mice and plant CGI-58 proteins, are neither able to catalyze a LPAAT or a LPGAT reaction, and that the plant CGI-58 protein is devoid of TG lipase or phospholipase activity. However we have shown, by analyzing lipid extracts, by thin layer chromatography and by mass spectrometry of different E. coli strains expressing the CGI-58 protein, that the expression of the plant CGI-58 protein, but not the mice one, results in a decrease of phosphatidylglycerol (PG) content in the different strains tested. However the mutation of the serine or histidine residue of the putative catalytic triad restores the wild-typephenotype. These results allowed us to propose that plant CGI-58 protein is involved in the metabolism of PG

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