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.