Characterization of Fatty Acid Elongation1 (FAE1) in Thlaspi arvense

FAE1 encodes the condensing enzyme 3-ketoacyl-CoA synthase 18 (KCS18), which catalyzes the first and rate-limiting step in the four-reaction cycle of very long chain fatty acid (VLCFA) biosynthesis in plant embryos. In Thlaspi arvense, colloquially known as pennycress, VLCFAs make up a substantial proportion of the total fatty acid (FA) content and play essential roles as components in cellular membranes, storage lipids, and sphingolipids, contributing to both structural integrity and metabolic function. While the role of FAE1 in VLCFA synthesis has been characterized in Arabidopsis thaliana and other Brassicaceae species, the broader metabolic and developmental consequences of its disruption, particularly in oilseed embryos, remain largely unexamined. To address this knowledge gap, this study explores the impact of FAE1 knockout in pennycress embryos through an integrated approach combining physiological, biochemical, and multi-omics techniques. Here, I demonstrate that fae1 pennycress embryos experience a substantial shift in fatty acid (FA) composition, marked by the loss of very long chain fatty acids (VLCFAs) and an increase in long-chain unsaturated FAs. This alteration in lipid composition led to changes in membrane fluidity, enhancing cold-germination efficiency under low-temperature conditions. The disruption of VLCFAs also reduced lipid storage content in pennycress seeds. These changes were associated with extensive metabolic reprogramming, including the accumulation of glucose and ADP-glucose, which, along with the upregulation of genes involved in cellular energy homeostasis, aligned with an increase in starch biosynthesis. Additionally, fae1 embryos exhibited significantly elevated expression of heat shock proteins, emphasizing the impact of eliminating VLCFAs on stress response pathways. Further investigation into metabolism showed wildtype pennycress embryos exhibited a uniquely high carbon conversion efficiency (CCE). Metabolic flux study using 13C-labeling and isotopomer network compartmental analysis (INCA) modeling revealed that elevated ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity plays a central role in driving the high CCE. Additionally, pyruvate kinase emerged as a key enzyme providing carbon substrate for de novo fatty acid synthesis (FAS). Results also showed that the oxidative portion of the pentose phosphate pathway (PPP) supplies reductant, and an alternative mode of the tricarboxylic acid cycle (TCA) cycle contributes carbon for FA elongation. Disruption of FAE1 altered these metabolic fluxes, reducing carbon flow toward FAS, decreasing PPP activity, and limiting CO2 refixation by RuBisCO, lowering the CCE. Moreover, flux analysis of fae1-3 embryos showed that the TCA cycle reverted to its conventional function of generating reductant for ATP production via oxidative phosphorylation rather than primarily serving as a carbon source for elongation. Altogether, these results shed light on the impact of disrupting VLCFA biosynthesis in pennycress embryos. By identifying the metabolic shifts that occur in the absence of FAE1, this study provides a basis for further exploration into optimizing pennycress seed oil for biofuel applications.