Lineage-Specific Duplication and Functional Analysis of TT8L in Cotton Proanthocyanidin Biosynthesis

Cotton ( Gossypium spp.) is a globally vital fiber crop. Naturally colored cotton (NCC) offers an alternative for the textile industry due to its pigmented fibers. However, the commercial potential of NCC is extensively constrained by the limited and unstable fiber colors. Deciphering the molecular basis of fiber coloration is therefore crucial for the genetic improvement of NCC. In brown cotton, proanthocyanidins (PAs) and their derivatives serve as the primary pigments. Although TRANSPARENT TESTA 8 (TT8) has been known as a central regulator of PA biosynthesis, its functional evolution remains unexplored. Here, we identified a lineage-specific duplicate of TT8, designated TT8L, which originated from gene duplication in Gossypium ancestors. Functional assays showed that silencing TT8L in two G. hirsutum L. accessions strongly inhibited PA biosynthesis. Conversely, heterologous expression of cotton TT8L in Arabidopsis thaliana significantly enhanced PA accumulation. Furthermore, our study showed that TT8L physically interacts with TT2, a core component of the MBW transcriptional complex governing PA synthesis. In summary, our study demonstrates TT8L functions in regulating PA biosynthesis, with its role mediated by the conserved MBW complex. These findings elucidate the plasticity of the PA regulatory network and propose TT8L as a prime target for developing novel NCC varieties. • TT8L is a lineage-specific duplicate of TT8 in Gossypium species. • TT8L is involved in regulating proanthocyanidin biosynthesis. • TT8L might have undergone subfunctionalization.