Location: Commodity Utilization Research
Project Number: 6054-41000-113-008-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Mar 1, 2023
End Date: Feb 28, 2026
1. Evaluate the effect of different PC synthesis/turnover mutants and seed-specific knockdown combinations on Arabidopsis seed oil yield and FA composition. 2. In vivo isotopic tracing and mathematical modeling of acyl flux through multiple possible substrate pools into TAG molecular species with different fatty acid compositions. 3. Assess lipid metabolic enzymes for localization to distinct, spatially-separated lipid biosynthetic metabolons within the endoplasmic reticulum (ER) membrane network.
Our long-term goals are to understand the control of acyl fluxes through the lipid biosynthetic network into TAG, and use this information to develop quantitative models of lipid biosynthesis for predictive bioengineering, and to ultimately optimize plant oil production for food, fuels, or chemical feedstocks. In this proposal we will investigate two distinct hypotheses for the control of TAG biosynthesis from multiple different diacylglycerol (DAG) pools, each of which can produce TAG with different fatty acid compositions. Hypothesis 1: PC production out-competes TAG biosynthesis for de novo DAG, promoting TAG synthesis from PC-derived DAG. Hypothesis 2: ER lipid metabolism is organized into separate distinct metabolons with PC as a “DAG carrier” between separate sites of: (1) de novo DAG and PC synthesis; and (2) PC turnover to produce PC-derived DAG for TAG biosynthesis. Here we propose a multi-faceted approach that combines the power of: plant genetics and molecular biology (mutant and tissue specific gene knockdowns); lipid flux analysis (in vivo isotopic labeling and computational modeling); cell biology (in vivo localization and protein-protein interaction); and proteomic analysis of isolated TAG-producing metabolons, to reveal the function of specific genes within the 3-dimensional context of the spatially organized lipid metabolic network. We will generate rod1/aapt1 and rod1/aapt2 double mutants to reduce the conversion rate of de novo DAG to PC, proportionate to the relative AAPT isoform expression levels in developing seeds. More recently developed CRISPR AAPT2 constructs are also being tested, using our published methods. We will also utilize AAPT1/AAPT2 RNAi partial gene silencing in rod1 as a complementary approach, using a seed-specific knockdown approach that we have successfully used to characterize the role of GPAT9 in seed oil production. We have already transformed rod1 with seed-specific RNAi knockdown constructs for AAPT1 or AAPT2 individually and are screening T1 lines; the double AAPT RNAi knockdown construct is in production. Knockouts and knockdowns of homozygous lines will be confirmed by qPCR, and seed oil levels and FA composition will be determined by gas chromatography.