Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in Triacylglycerol Biosynthesis

Authors: Shockey, Jay; REGMI, ANUSHOBHA - University Of Southern Mississippi; COTTON, KIMBERLY - Washington State University; ADHIKARI, NEIL - Washington State University; BROWSE, JOHN - Washington State University; BATES, PHILIP - University Of Southern Mississippi

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/19/2015
Publication Date: 11/19/2015
Citation: Shockey, J., Regmi, A., Cotton, K., Adhikari, N., Browse, J., Bates, P.D. 2015. Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in Triacylglycerol Biosynthesis. Plant Physiology. 170:163-179.

Interpretive Summary: The genetics and biochemistry that underpin the pathways for vegetable oil production in plant seeds have become clearer over the past several years. Many genes that produce the enzymes for the middle and late stage reactions have been cloned and characterized in detail. In some cases, specific enzymes have been found that clearly impact the final composition of the oil in some plant species. Despite such progress, the initial reaction of oil synthesis, carried out by an enzyme called glycerol-3-phosphate acyltransferase, or GPAT, has remained mysterious. Many GPAT genes have been cloned, but none match the characteristics of the oil pathway enzyme. In this study, we identify a gene known as GPAT9 that likely fulfills this role in oilseeds and other plants. GPAT9 is essential to plant survival; loss of this gene results in poor fertility and death of reproductive tissues in the flowers of mutant mouse ear cress plants. Reduction of GPAT9 protein levels in these plants directly reduces oil synthesis, and the GPAT9 protein directly interacts with other known proteins of the oil synthesis pathways. All told, the evidence shown here reveals GPAT9 to be the elusive ‘Kennedy pathway GPAT’ and provides one more key tool for studying the biochemistry, regulation, and engineering strategies for oil production in plants.

Technical Abstract: The first step in the biosynthesis of nearly all plant membrane phospholipids and storage triacylglycerols is catalyzed by a glycerol-3-phosphate acyltransferase (GPAT). The requirement for an endoplasmic reticulum (ER) localized GPAT for both of these critical metabolic pathways was recognized more than 60 years ago. However, identification of the gene(s) encoding this GPAT activity has remained elusive. Here we present the results of a series of in vivo, in vitro, and in silico experiments designed to assign this essential function to AtGPAT9. This gene has been highly conserved throughout evolution, and is largely present as a single copy in most plants, features consistent with essential housekeeping functions. A knockout mutant of AtGPAT9 demonstrates both male and female gametophytic lethality phenotypes, consistent with the role in essential membrane lipid synthesis. Significant expression of developing seed AtGPAT9 is required for wild-type levels of triacylglycerol accumulation, and transcript level is directly correlated to the level of microsomal GPAT enzymatic activity in seeds. Finally, the AtGPAT9 protein interacts with other enzymes involved in ER glycerolipid biosynthesis, suggesting the possibility of ER-localized lipid biosynthetic complexes. Together these results suggest that GPAT9 is the ER-localized GPAT enzyme responsible for plant membrane lipid and oil biosynthesis.