Phylogenomics and functional analysis of Glycerol‐3‐Phosphate Acyltransferase (<scp>GPAT</scp>) Genes: A critical role in lipid biosynthesis

The tung tree (Vernicia fordii Hemsl.), an economically important woody plant, is widely planted for the production of high-quality tung oil. Glycerol-3-phosphate acyltransferases (GPATs), the rate-limiting enzymes in triacylglycerol synthesis, play an important role in seed oil biosynthesis. In this study, we performed a genome-wide analysis of VfGPATs. A total of 9 VfGPATs were identified from the whole tung genome, and phylogenetic analysis divided the VfGPATs into three major clades: clade II (VfGPAT9), clade III (VfATS1) and clade IV (VfGPAT1 ~ 8). Subcellular localization analysis revealed that five VfGPATs (1, 5, 6, 8, and 9) are localized in the endoplasmic reticulum, and four VfGPATs (3-1, 3-2, 3-3, and ATS1) are localized in the chloroplast. Overexpression of VfGPATs in Arabidopsis thaliana revealed that the oil content in VfGPAT8- and VfGPAT9-transgenic plants were significantly increased by 26.60 and 55.94% compared to the wild-type. Transient expression of VfGPAT8 + VfFADX and VfGPAT9 + VfFADX could promote the synthesis of α-eleostearic acid and enhance the accumulation of lipid droplets in tobacco (Nicotiana benthamiana) leaves. We further tested the enzymatic activities of VfGPAT8 and VfGPAT9 with the yeast double mutant strain ZAFU1. The results showed that VfGPAT8 complemented the phosphatidate biosynthetic defect in the double mutant, while VfGPAT9 could not, suggesting that VfGPAT8 has a high acetyltransferase activity. However, altering serine (S) residue at position 113 of VfGPAT9 to threonine (T) could restore its enzymatic activity. This study provided important insights into the evolutionary history of VfGPATs and will promote the genetic improvement of tung trees and related species.