Identification and Characterization of Lipid Droplet-Associated Protein (LDAP) Isoforms from Tung Tree (<i>Vernicia fordii</i>)

Lipid droplets (LDs) are cytoplasmic organelles responsible primarily for the storage of neutral lipids, such as triacyclglycerols (TAGs). Derived from the endoplasmic reticulum bilayer, LDs are composed of a hydrophobic lipid core encased by a phospholipid monolayer and surface-associated proteins. To date, only a relatively few LD ‘coat’ proteins in plants have been identified and characterized, most of which come from studies of the model plant <i>Arabidopsis thaliana</i>. To expand our knowledge of the plant LD proteome, the LD-associated protein (LDAP) family from the tung tree (<i>Vernicia fordii</i>), whose seeds are rich in a commercially valuable TAG containing the conjugated fatty acid α-eleostearic acid (C18:3Δ^{9<i>cis</i>,11<i>trans</i>,13<i>trans</i>} [α-ESA]), was identified and characterized. Based on the tung tree transcriptome, three <i>LDAP</i> isoforms (<i>VfLDAP1-3</i>) were elucidated and the encoded proteins distinctly clustered into three clades along with their respective isoforms from other angiosperm species. Ectopic expression of the <i>VfLDAPs</i> in <i>Nicotiana benthamiana</i> leaves revealed that they localized specifically to LDs and influenced LD numbers and sizes, as well as increasing TAG content and altering TAG fatty acid composition. Interestingly, in a partially reconstructed TAG-ESA biosynthetic pathway, the co-expression of <i>VfLDAP3</i> and, to a lesser degree, <i>VfLDAP2</i>, significantly increased the content of α-ESA stored within the LDs. These results suggest that the VfLDAPs can influence the steady-state content and composition of TAG in plant cells and that certain LDAP isoforms may have evolved to more efficiently package TAGs into LDs containing unusual fatty acids, such as α-ESA.