GhGFS9 promotes proanthocyanidin and anthocyanin accumulation in brown cotton fiber via vesicle trafficking and biosynthetic regulation

Naturally colored cotton (NCC) serves as a green alternative to conventional dyeing processes in the textile industry; however, its limited color spectrum restricts broader application. Proanthocyanidins (PAs) and their derivatives are key pigments in brown cotton fiber, but the precise molecular pathways directing PAs from the endoplasmic reticulum to the central vacuole remain incompletely understood. Green Fluorescent Seed 9 (GFS9), a vesicle trafficking protein, plays an important role in PAs transport. In this work, four GhGFS9 homologs were identified in Gossypium hirsutum , among which GhGFS9-A13 ( GhGFS9 here) exhibited high expression levels in fibers that positively correlated with PAs and anthocyanin contents. Functional characterization demonstrated that GhGFS9 rescued the PAs-deficient phenotype displayed by the Arabidopsis gfs9 mutant. Silencing GhGFS9 via VIGS and RNAi significantly reduced PAs and anthocyanin accumulation in cotton calli, leaves, and fibers, while transient overexpression enhanced the deposition of these pigments. Furthermore, GhGFS9 positively regulated the expression of key biosynthesis and oxidative polymerization genes, including GhF3'H , GhDFR , GhANS , GhLAR , GhANR , and GhTT10 , potentially through a feedback mechanism based on substrate concentration. Collectively, our findings point to a dual function for GhGFS9 in the pigmentation of brown cotton fibers: it facilitates the vesicle-mediated transport of PAs and anthocyanins and concurrently upregulates their biosynthesis, thereby promoting pigment deposition.