OsVPS34-generated PI3P recruits GPA5/Rab5a to regulate post-Golgi glutelin trafficking in rice endosperm

Seed storage proteins (SSPs) are stored in protein storage vacuoles (PSVs) within plant endosperm cells. In rice, glutelins undergo post-Golgi trafficking via dense vesicles (DVs) to protein body II (PBII). Phosphatidylinositol 3-phosphate (PI3P) regulates endosomal, autophagic, and vacuolar trafficking, yet its role in glutelin transport remains unclear. Here, we characterized the glutelin precursor accumulation14 (gpa14) mutant, which exhibits over-accumulation of 57-kDa glutelin precursors and floury, shrunken endosperm. Map-based cloning identified a single adenine insertion in Vacuolar Protein Sorting 34 (OsVPS34), resulting in a putative truncated protein lacking the PI3Ka and PI3_PI4_kinase domains. OsVPS34 encodes phosphatidylinositol 3-kinase (PI3K), which interacts with other subunits of the PI3K complex to regulate the production of PI3P. PI3P was enriched in the trans-Golgi network (TGN) and pre-vacuolar compartment (PVC), co-localized with Rab5a and GPA5, and was detected in DVs and PBIIs. In gpa14, PI3P levels were reduced, leading to mis-localization and decreased membrane association of Rab5a and GPA5, key regulators of glutelin trafficking. Our findings demonstrate that OsVPS34 is essential for synthesis of PI3P, which plays a crucial role in recruiting GPA5 and Rab5a to DVs for glutelin post-Golgi trafficking in rice endosperm.