sn-Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-1 position of sn-glycerol-3-phosphate to produce lysophosphatidic acid and Coenzyme A. GPATs are involved in several lipid synthetic pathways and play important physiological roles in plant development. The present doctoral thesis includes three related studies, which aim to molecularly and functionally characterize several plant GPAT genes and the encoded enzymes. The first study characterized three endoplasmic reticulum-bound GPAT4s encoded by three homologous GPAT4 genes of Brassica napus (oilseed rape), focusing primarily on their functional divergence and physiological roles in plant development and lipid biosynthesis. The three homologous GPAT4 genes exhibited different expression patterns and altered epigenetic features. Phenotypic rescue of a gpat4 gpat8 Arabidopsis double mutant and analysis of the gpat4 RNAi B. napus lines suggested physiological roles for the GPAT4s in cuticle formation of the rosette leaves, early flower development, pollen development and storage lipid biosynthesis. The second study investigated stable internal reference genes for gene expression studies in B. napus. This project identified four reliable reference genes to be used in gene expression analysis of BnGPAT4 homologues in both vegetative tissues and developing seeds. The third study focused on molecular cloning and biochemical characterization of a soluble plastidial GPAT isolated from a chilling-tolerant plant, western wallflower (Erysimum asperum). A truncated form of recombinant EaGPAT, with the putative transit peptide deleted, was functionally expressed in yeast. A series of enzymatic assays were performed in order to determine the optimum in vitro reaction conditions for the recombinant EaGPAT. The recombinant EaGPAT was further assayed with different acyl-CoAs and exhibited a substrate preference for 18 carbon unsaturated acyl-CoAs. With this substrate preference, the EaGPAT could potentially be used as a biotechnological tool for improving plant chilling-tolerance or increasing unsaturated fatty acid content of seed oil. Overall, the present doctoral studies revealed the functional divergence and important physiological roles of the GPAT4s in B. napus, and biochemically characterized a plastidial GPAT from E. asperum. The knowledge obtained from these studies provides new insights into the role of GPAT in plants and will be useful for further development of biotechnological approaches to modify seed oil biosynthesis in oleaginous crops. / Plant Science

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Approximately two-thirds of US adults are overweight or obese, and obesity is also becoming more prevalent in children and adolescents. Similar to adults, obese children are at a higher risk of developing health problems due in part to dysfunctional immune surveillance. Obesity has been shown reduce the generation of new T-cells by accelerating thymic aging in an adult mouse. This study therefore aimed at determining whether similar diet induced obesity (DIO) changes can be induced in a young mouse. Comparisons made between lean and DIO C57Bl/6 mice showed a significant increase in thymic weight, decrease in thymic cellularity and thymic output, and impaired T-cell development at the double negative stage. We associate these alterations with changes in thymic architecture and accumulation of lipid droplets within the thymic cortex and medulla of the obese mice. The above observations indicate that DIO can induce fat accumulation and reduce thymic function at a young age. Resveratrol, a natural polyphenolic compound, was then used to regulate fat metabolism in an attempt to reduce these DIO changes we observed. Resveratrol induces fat oxidation via 5' adenosine monophosphate-activated protein kinase (AMPK), and its reciprocal regulation of glycerol-3-phosphate acyltransferase-1 (GPAT-1) and carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzymes required for glycerophospholipid biosynthesis and oxidation, respectively. Through resveratrol feeding, we were able to prevent the effects of DIO on thymic architecture and thymic T-cell proliferation. This was achieved by manipulating AMPK into inhibiting GPAT-1 and enhancing CPT-1 activity. Since the expression of GPAT-1 was upregulated in the obese mice, we investigated whether deleting GPAT-1 altogether might prevent the thymic involution, by inhibiting synthesis of glycerophospholipids and triacylglycerol. Instead, we found that GPAT-1 deletion slowed thymic growth and reduced cellularity in young mice, which we associated with impaired thymic T-cell function and development, suggesting that the deleterious effects of GPAT-1 deficiency may be due to perturbations in thymic T-cell activation and signaling. These data provide a novel link between lipid metabolism and T-cell development, and identify the use of the naturally-occurring resveratrol to reduce lipid accumulation within the involution-prone thymus, thus providing a useful approach to preventing a decline in thymic function in childhood. / text