Characterizationof Phosphatidylcholine:DiacylglycerolCholinephosphotransferases from Soybean (Glycine max)

Plant oils in the form of triacylglycerols (TAGs) have important food and industrial applications. The fatty acid composition of TAGs, especially their degree of unsaturation, affects the oil value and applications. Phosphatidylcholine:Diacylglycerol Cholinephosphotransferase (PDCT) facilitates the exchange of fatty acids between phosphatidylcholine and diacylglycerol, influencing the degree of fatty acid unsaturation. In this study, we identified and characterized two <i>PDCT</i> isoforms from soybean (Glycine max). Phylogenetic and structural analyses revealed that PDCTs are widely conserved across Embryophyta and share key sequence and structural features among species. Subcellular localization assays using transient expression of fluorescent protein-tagged GmPDCTs in Nicotiana benthamiana leaves confirmed their localization to the endoplasmic reticulum. Expression of <i>GmPDCT</i>s in yeast altered lipid unsaturation, while <i>in vitro</i> enzyme assays using yeast microsomal fractions confirmed that both GmPDCTs are catalytically active, preferring unsaturated substrates. Further structural analysis and mutagenesis revealed that the N-terminus and several amino acids within/near the predicted catalytic domains are critical to the PDCT function. Lastly, stable overexpression of <i>GmPDCT</i>s in Arabidopsis thaliana <i>rod1</i> (<i>pdct</i>) mutant plants successfully restored a wildtype lipid phenotype, providing evidence that these genes encode functional PDCTs. Together, these findings provide new insights into PDCT structure–function relationships, offering potential targets for bioengineering strategies aimed at optimizing oil composition.