An R2R3 MYB transcription factor GhMYB5: regulator of CHS expression and proanthocyanin synthesis in brown cotton (Gossypium hirsutum L.).

Background: Cotton (Gossypium spp.) serves as a vital global crop for textile production, with naturally pigmented brown cotton has gained industrial interest due to its eco-friendly coloration. However, the molecular mechanisms underlying variation in color intensity in brown cotton fibers remain poorly characterized. In this study, we investigated the genetic and biochemical basis of fiber color differentiation in hybrid-derived cotton lines spanning white, light-brown, middle-brown, and deep-brown phenotypes.

Results: Biochemical quantification revealed significantly elevated proanthocyanidin levels in brown cotton fibers compared to those in white counterparts, with their content positively correlating with color intensity. Transcriptomic profiling identified significant activation of flavonoid biosynthesis pathway genes in pigmented fibers. Among differentially expressed transcription factors, GhMYB5 (Ghir_D07G002110), an R2R3-type MYB regulator, exhibited gradual upregulation corresponding to color deepening. Functional characterization through qRT-PCR demonstrated GhMYB5's potential regulation of key flavonoid pathway genes GhCHS1 (Ghir_A10G012390), GhCHI3 (Ghir_A05G041560), and GhF3H (Ghir_D11G018670). Protein-DNA interaction assays (DAP-seq), yeast one-hybrid validation and LUC analysis confirmed direct binding of GhMYB5 to the promoter region of GhCHS1, establishing a regulatory node in proanthocyanidin biosynthesis.

Conclusions: Our findings reveal that GhMYB5-mediated transcriptional activation of GhCHS1 promotes proanthocyanidin accumulation in brown cotton fibers, providing a molecular explanation for color intensification. The identified MYB-CHS regulatory module offers potential targets for molecular breeding of naturally colored cotton varieties with enhanced pigmentation properties. This study advances our understanding of plant pigment biosynthesis and supports sustainable textile production through engineering of natural fiber coloration.