In oilseeds, energy-rich carbon is stored as triacylglycerols in organelles called lipid droplets (LDs). While several of the major biogenetic proteins involved in LD formation have been identified, the full repertoire of LD proteins and their functional roles remains incomplete. Here, we show that the low-abundance, seed-specific LD protein LIPID DROPLET PROTEIN OF SEEDS (LDPS) contains an amphipathic α-helix and proline hairpin motif that serves as an LD-targeting signal and a separate region that binds to the LD protein OLEOSIN 1 (OLEO1). Loss of LDPS function results in smaller LDs and less seed oil in comparison with wild type, while overexpression of LDPS results in an increase in LD size and seed oil content. Loss of LDPS function also results in an inability of LDs to undergo fusion during postgerminative seedling growth. Analysis of oleo1 and ldps single- and double-mutant seeds and freeze–thaw treatment of seeds revealed that OLEO1 suppresses the ability of LDPS to promote larger LDs. Collectively, our results identify LDPS as an important player in LD biology that functions together with OLEO1 to determine LD size in Arabidopsis (Arabidopsis thaliana) seeds and seedlings through a process that involves LD–LD fusion.

Jojoba (Simmondsia chinensis) is a desert shrub with an unusual capacity to store liquid wax esters (WEs) in its seeds instead of triacylglycerols (TAGs) like most oilseed crops. To examine the factors that are important for WE compartmentalization in jojoba, we reconstituted WE biosynthesis and packaging in the leaves of Nicotiana benthamiana. Using this system, we screened jojoba proteins for their ability to support lipid droplet (LD) formation. A specific LIPID DROPLET-ASSOCIATED PROTEIN (LDAP) isoform, ScLDAP1, was identified as a key factor in the efficient compartmentalization of WEs in plant cells. LDAP1 isoforms from other plants (e.g. Arabidopsis thaliana [AtLDAP1]) did not support WE partitioning from the endoplasmic reticulum into LDs, although both AtLDAP1 and ScLDAP1 were targeted specifically to LD monolayer surfaces. ScLDAP1-mediated selective, efficient WE partitioning was facilitated by an amphipathic α-helix near its C-terminus, and mutational analysis identified 1 amino acid residue within this helix that was both necessary and sufficient for proper WE packaging into cytoplasmic LDs. Taken together, our results provide a mechanistic link between the biosynthesis and storage of WEs in plant cells, and will inform future biotechnology strategies for the efficient packaging of various neutral lipid types as demonstrated here for WEs in transgenic seeds.

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 Arabidopsis thaliana. To expand our knowledge of the plant LD proteome, the LD-associated protein (LDAP) family from the tung tree (Vernicia fordii), whose seeds are rich in a commercially valuable TAG containing the conjugated fatty acid α-eleostearic acid (C18:3Δ^{9cis,11trans,13trans} [α-ESA]), was identified and characterized. Based on the tung tree transcriptome, three LDAP isoforms (VfLDAP1-3) were elucidated and the encoded proteins distinctly clustered into three clades along with their respective isoforms from other angiosperm species. Ectopic expression of the VfLDAPs in Nicotiana benthamiana 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 VfLDAP3 and, to a lesser degree, VfLDAP2, 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.