Functional analysis of LIPID TRANSFER PROTEIN 6 (LTP6) in pennycress and Arabidopsis reveals divergent roles in oil storage and seed coat development.

In angiosperms, lipid transfer proteins (LTPs) perform multiple roles, including the shuttling of lipids between organelles and to/through the apoplast. In pennycress (Thlaspi arvense L.), the LIPID TRANSFER PROTEIN 6 (TaLTP6) was identified as highly expressed in developing embryos, especially in high-oil accessions. Ectopic expression of pennycress LTP6 (TaLTP6) in Nicotiana benthamiana and Arabidopsis thaliana leaf mesophyll cells induced the proliferation of cytoplasmic lipid droplets (LDs), suggesting a role in neutral lipid accumulation. GFP-tagged TaLTP6 localized predominantly to LDs and endoplasmic reticulum (ER)/LD contact sites, while its Arabidopsis homolog, AtLTP6, localized to the ER and apoplast. Domain-swapping experiments revealed that their N-terminal regions determined these subcellular localizations. Loss-of-function mutants of Arabidopsis ltp6 exhibited major disruptions in LD organization in mature embryos, characterized by large lipid aggregates, and reduced seed oil content. Proteomics analysis revealed mislocalization of LD- and ER-associated proteins in ltp6 mutants, suggesting impaired LD biogenesis. Further, Arabidopsis ltp6 seeds exhibited reduced mucilage extrusion and impaired germination, pointing to a secondary role for AtLTP6 in seed coat function. Complementation of Arabidopsis ltp6 with TaLTP6 restored LD morphology and seed oil levels, but did not rescue mucilage and germination defects, indicating functional divergence between the two homologs. We conclude that LTP6 plays a dual role in seeds: (1) participation in embryo lipid storage, and (2) contribution to seed coat integrity and germination. The embryo-specific expression of TaLTP6 in pennycress suggests that it retained its evolutionary role in lipid storage, but lost functions related to seed coat development and germination.