Molecular Docking and Transgenic Expression Unveil the Role of Rice Seed-Specific Oleosin OsOle1 in Lipid Accumulation and Lipid Droplet Enlargement

Among the six oleosin genes present in the rice (Oryza sativa L.) genome, one encodes a seed-specific low molecular weight (16 kDa) protein and another one encodes a seed-specific high molecular weight (18 kDa) protein, which are known to be associated with oil bodies or lipid droplets (LDs). To understand the structure-function relationship of these two oleosins, designated as OsOle1 and OsOle2, we first carried out bioinformatic analyses of both proteins followed by transgenic expression of only OsOle1, which has potential application in lipid biotechnology. Although both oleosins have the conserved proline knot motif (PX₅SPX₃P) in the central hydrophobic domain that facilitates LD anchoring, only OsOle1 possesses the acyltransferase motif (HX₄D) within the C-terminal domain. Molecular docking of a few LD-associated molecules with the predicted structures of two oleosins revealed that OsOle1 has slightly higher binding affinities towards fatty acyl glycerol esters, suggesting its role in triacylglycerol accumulation. In fact, OsOle1 expression with the galactose-inducible promoter in Saccharomyces cerevisiae INVSc1 cells augmented lipid content and promoted early LD formation. Moreover, it successfully restored LD biogenesis in a mutant S. cerevisiae strain impaired in LD formation, producing larger LDs with fewer per cell. Ectopic expression of the OsOle1 gene in rice, under a bran-specific promoter, led to reduced seed dimensions (length, 38–39.5%; breadth, 30–32%; weight, 40%) and delayed inflorescence arrival, indicating a slower developmental pace compared to the untransformed control. However, the transgenic rice lines demonstrated a noteworthy increase in seed lipid content by an average of 57.5% in comparison to the untransformed control and accompanied by a conspicuous enlargement in LD dimensions. Thus, OsOle1 emerges as a positive regulator in the context of lipid accumulation, LD formation, and LD enlargement. These findings suggest OsOle1 as a potential target for further exploration aimed at enhancing the oil content of rice seeds.