Functional diversity of sunflower DGAT1 and DGAT2 enzymes underlying seed triacylglycerol biosynthesis

Abstract

Introduction Triacylglycerols (TAGs) are the main storage lipids in oilseeds, providing energy and carbon during seed germination, and serving as valuable feedstocks for food, feed, and industrial applications. In plants, the terminal acylation of diacylglycerol (DAG) to form TAG is catalyzed by diacylglycerol acyltransferases (DGATs) or phospholipid:diacylglycerol acyltransferase (PDAT). Although sunflower (Helianthus annuus) is one of the world´s leading oilseed crops, the contributions of individual DGAT isoforms to seed TAG biosynthesis remain poorly characterized. Methods We identified six DGAT genes in sunflower —three DGAT1, two DGAT2, and one DGAT3—and examined their genomic organization, phylogenetic relationships, predicted structural features, expression profiles, subcellular localization, in planta functional behaviour and TAG profiles in the TAG deficient yeast strain H1246. Results Gene expression analysis revealed that HaDGAT2A and HaDGAT2D transcripts predominated in developing seeds during the main oil accumulation phase, whereas HaDGAT1A and HaDGAT1B showed lower but detectable expression. Confocal microscopy confirmed the endoplasmic reticulum localization of representative HaDGAT1A and HaDGAT2A isoforms, consistent with their potential involvement in ER-associated TAG assembly. Transient expression in Nicotiana benthamiana leaves revealed functional differences between sunflower DGAT isoforms, HaDGAT1 isoforms were associated with TAG profiles enriched in less unsaturated molecular species, whereas HaDGAT2 isoforms, particularly HaDGAT2D, were associated with increased TAG accumulation and a greater representation of polyunsaturated TAG species under the heterologous conditions tested. Functional complementation in yeast further showed that the analyzed sunflower DGAT isoforms restored TAG synthesis in the H1246 mutant and produced distinct TAG molecular profiles, supporting functional diversification among DGAT1 and DGAT2 isoforms. Discussion These results indicate that sunflower DGAT1 and DGAT2 paralogs contribute differently to TAG accumulation and composition in heterologous systems and identify HaDGAT2A and HaDGAT2D as relevant candidate isoforms for future studies on sunflower seed oil biosynthesis. Direct biochemical assays will be required to confirm the intrinsic substrate specificity of each isoform.