Comparative phosphoproteomic analysis of BR-defective mutant reveals a key role of GhSK13 in regulating cotton fiber development

Brassinosteroid (BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1 (pag1) , and its corresponding wild-type (ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage (10 days after pollination (DPA)) and secondary cell wall synthesis stage (20 DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs (GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor Gh_DNF_YB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.