TRANSPARENT TESTA 16 collaborates with the MYB-bHLH-WD40 transcriptional complex to produce brown fiber cotton.

Naturally colored cotton (NCC; Gossypium spp.) does not require additional chemical dyeing and is an environmentally friendly textile material with great research potential and applications. Our previous study using linkage and association mapping identified TRANSPARENT TESTA 2 (Gh_TT2) as acting on the proanthocyanin synthesis pathway. However, limited information is available about the genetic regulatory network of NCC. Here, we verified the effectiveness of Gh_TT2 and the roles of Gh_TT2 and red foliated mutant gene (Re) in pigment formation and deposition of brown fiber cotton (BFC). Variations in Gh_TT2 derived from interspecific hybridization between Gossypium barbadense acc. Pima 90-53 and Gossypium hirsutum acc. Handan208 resulted in gene expression differences, thereby causing phenotypic variation. Additionally, the MYB-bHLH-WD complex was found to be negatively modulated by TRANSPARENT TESTA 16/ARABIDOPSIS BSISTER (TT16/ABS). RNA-seq suggested that differential expression of homologous genes of key enzymes in the proanthocyanin synthesis pathway strongly contributes to the color rendering of natural dark brown and light brown cotton. Our study proposes a regulatory model in BFC, which will provide theoretical guidance for the genetic improvement of NCC.