Label-Free Proteomics of Starch Granule-Associated Proteins Reveals Starch Molecular Modification during Rice Grain Filling

Molecular weight (MW) of rice starch molecules initially increased and then decreased during grain filling, while concurrently the molecular size of amylopectin (AP) decreased (from 173.5 to ∼110 nm) and its MW increased. These dynamic structural changes may be linked to changes in starch granule-associated proteins (SGAPs) during starch synthesis. Label-free quantitative proteomics of SGAPs identified 286 differentially expressed proteins (DEPs) between early/midfilling but only seven DEPs between mid/late-filling stages. Key findings include: (1) Direct synthesis/Modification: Co-increased abundances of debranching and branching enzymes, coupled with reduced granule-bound starch synthase, correlated with a more uniform AP structure, smaller size, and higher MW. (2) Indirect effects: Reduced SGAPs for glycolysis versus highly expressed SGAPs for lipid metabolism, tubulin, and ribosomal proteins during early filling suggest an enhanced starch synthesis rate, intracellular transport, and protein translation function in the amyloplast. This study provides detailed SGAPs proteomics data identifying specific enzymes modulating starch granule structure during development.