TurboID‐Based Proximity Labelling Identifies MED25 as an Activator of GoPGF‐Mediated Gland Formation and Gossypol Biosynthesis in Cotton

The pigment glands of cotton synthesize the toxic gossypol that contributes to cotton's defence, but also limits the usage of cottonseed as a food source. Previous studies have shown that the gland-localized GoPGF is the master transcription factor controlling the gland morphogenesis and gossypol biosynthesis in cotton. However, the precise mechanism underlying the GoPGF-mediated transcription of downstream genes remains unclear. In this study, TurboID-based proximity labelling was established in cotton using a transient expression system to identify the components of GoPGF-complex. A total of 48 high-confidence GoPGF-proximal proteins were identified, and a mediator subunit, MED25, was shown to control gland formation through phenotypic studies. Several lines of evidence indicate that GhMED25 physically interacts with GoPGF in the nucleus, and GhMED25-GoPGF complex regulates the expression of GhJUB1 related to gland morphogenesis, and several well-characterized gossypol biosynthetic genes. ChIP-qPCR further revealed that GhMED25 acts as an enhancer of the GoPGF-mediated transcriptional output. Our study shows the value and reliability of the proximity labelling system in studying the low-abundance and cell-type-specific proteins in crops, adding a new role to the multifunctionality of MED25 in plants, and provides new insights into the intricate signalling network governing gland formation and terpene production in cotton.