The gland localized CGP1 controls gland pigmentation and gossypol accumulation in cotton

Pigment glands, also known as black glands or gossypol glands, are specific for Gossypium spp. These glands strictly confine large amounts of secondary metabolites to the lysigenous cavity, leading to the glands' intense colour and providing defence against pests and pathogens. This study performed a comparative transcriptome analysis of glanded versus glandless cotton cultivars. Twenty-two transcription factors showed expression patterns associated with pigment glands and were characterized. Phenotypic screening of the genes, via virus-induced gene silencing, showed an apparent disappearance of pigmented glands after the silencing of a pair of homologous MYB-encoding genes in the A and D genomes (designated as CGP1). Further study showed that CGP1a encodes an active transcription factor, which is specifically expressed in the gland structure, while CGP1d encodes a non-functional protein due to a fragment deletion, which causes premature termination. RNAi-mediated silencing and CRISPR knockout of CGP1 in glanded cotton cultivars generated a glandless-like phenotype, similar to the dominant glandless mutant Gl₂^e . Microscopic analysis showed that CGP1 knockout did not affect gland structure or density, but affected gland pigmentation. The levels of gossypol and related terpenoids were significantly decreased in cgp1 mutants, and a number of gossypol biosynthetic genes were strongly down-regulated. CGP1 is located in the nucleus where it interacts with GoPGF, a critical transcription factor for gland development and gossypol synthesis. Our data suggest that CGP1 and GoPGF form heterodimers to control the synthesis of gossypol and other secondary metabolites in cotton.