UNDERSTANDING GENETIC AND ENZYMATIC INFLUENCES ON LIPID METABOLIC PATHWAYS AND TRIACYLGLYCEROL BIOSYNTHESIS: REPLACEMENT OF OIL  BIOSYNTHETIC ENZYMES REQUIRES INTACT CIS-REGULATORY ELEMENTS AND  COUPLED PROTEIN INTERACTIONS

Sean Thomas McGuire

Lipid metabolic processes are essential in all plant tissues to maintain cell viability, with the pollen and seeds of many species requiring the accumulation of storage oil, in the form of triacylglycerol (TAG). The rapid agricultural developments of the 20th century have made seed oils a primary source of calories. In addition to their nutritional value, plant oils from non-food crops are used for various industrial purposes, including biofuels, cosmetics, resins, varnishes and plasticizers; thus, necessitating a thorough understanding of the biochemical mechanisms responsible for seed oil accumulation to maintain product demand. To date, seed metabolic engineering has largely focused on modifying existing pathways by transgene introduction to accrue industrially relevant oils, with the enzymes acyl-CoA:diacylglycerol acyltransferase (DGAT1) and phospholipid:diacylglycerol acyltransferase 1 (PDAT1) that share responsibility for TAG biosynthesis, as primary targets. These enzymes have different substrate selectivities that control the final oil outcome. Many strategies to express selective acyltransferases from different plant systems may cause aberrant phenotypes, since TAG fatty acid (FA) compositions are altered in different tissues when these genes are constitutively expressed. Previous attempts to use exogenous DGAT1 or PDAT1 to control oil compositions has only been done by layering gene expression ‘on-top’ endogenous machinery; consequently, creating metabolic competition between introduced and native pathways. Therefore, the primary investigation of this dissertation was to evaluate if Arabidopsis DGAT1 and PDAT1 could be fully removed and replaced. The first hurdle to overcome was the pollen lethality of dgat1-1/pdat1-2 double mutant plants. We found that to complement the lethality, the AtDGAT1 first intron and full-length promoter are the minimal regulatory elements needed to control AtDGAT1 expression in the pollen and developing embryo. Next, we verified that this cis-regulatory architecture can control expression of heterologous DGAT1s from Camelina sativa and Physaria fendleri as they wholly integrate into the Arabidopsis lipid metabolic network; however, in contrast, the Ricinus communis DGAT1 causes unique perturbations to the transcriptional landscape, oil and FA profiles. Here we also discovered that DGAT1 from diverse plant species have dissimilar protein:protein interactions, adding another layer of incompatibility that must be considered for optimized oilseed engineering. Together, these results establish a new tool for both understanding the function of DGAT1 in specialized tissues and producing designer oilseed FA compositions. Beyond the main oil biosynthetic enzymes, many enzyme families putatively associated with lipid metabolism remain largely unexplored. Thus, the secondary focus of this dissertation was to explore and characterize GDSL-esterase/lipase proteins (GELPs) loss-of-function mutants and their effects on seed lipid accumulation. By lipidomics, quantitative lipid analysis, and confocal microscopy we reveal that seed-enriched GELPs are involved in lipid metabolism in early seed development and localize to the main lipid biosynthetic hub, the endoplasmic reticulum. Collectively, this body of research provides details on previously unknown regulatory mechanisms controlling the expression and function of oil biosynthetic proteins and further suggests that the GELP enzyme family has differential lipid metabolic responses in developing seeds.

UNDERSTANDING GENETIC AND ENZYMATIC INFLUENCES ON LIPID METABOLIC PATHWAYS AND TRIACYLGLYCEROL BIOSYNTHESIS: REPLACEMENT OF OIL BIOSYNTHETIC ENZYMES REQUIRES INTACT CIS-REGULATORY ELEMENTS AND COUPLED PROTEIN INTERACTIONS

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