Cotton fiber elongation is a critical growth phase that affects final fiber length. Morphological analysis indicated an asynchronous fiber elongation pattern between two cotton varieties J7-1 and J14-1. Through phosphoproteomic analysis, total 89 differentially-phosphorylated proteins (DPPs) were identified in elongating fibers between J7-1 and J14-1. Gene ontology (GO) analysis showed that these DPPs were mainly enriched in sucrose synthase activity, transferase activity and UDP-glycosyltransferase activity. In J14-1, the phosphorylation level of GhSUS2, a key sucrose synthase in sucrose metabolism pathway, is significantly higher than that in J7-1. We further revealed that GhSUS2 positively regulates fiber elongation, and GhSUS2-silenced transgenic cotton displayed the phenotype of “short fibers” compared with the controls. During fiber development, the residue Ser11 in GhSUS2 protein is phosphorylated by Ca 2+-dependent protein kinases GhCPK84 and GhCPK93. The phosphorylated GhSUS2 is localized in the cytoplasm, while unphosphorylated GhSUS2 is localized in the plasma membrane. Moreover, abscisic acid (ABA) could promote the transcription and translation of GhCPK84 and GhCPK93, thereby enhancing the phosphorylation of GhSUS2 to impede fiber elongation. Thus, our data demonstrated that GhSUS2 plays a positive role in fiber development, but its phosphorylation by GhCPK84 and GhCPK93 hinders fiber elongation of cotton.

Phytohormones play indispensable roles in plant growth and development. However, the molecular mechanisms underlying phytohormone-mediated regulation of fiber secondary cell wall (SCW) formation in cotton (Gossypium hirsutum) remain largely underexplored. Here, we provide mechanistic evidence for functional interplay between the AP2/ERF transcription factor GhERF108 and auxin response factors GhARF7-1 and GhARF7-2 in dictating the ethylene-auxin signaling crosstalk that regulates fiber SCW biosynthesis. Specifically, in vitro cotton ovule culture revealed that ethylene and auxin promote fiber SCW deposition. GhERF108 RNAi cotton displayed remarkably reduced cell wall thickness compared with controls. GhERF108 interacted with GhARF7-1 and GhARF7-2 to enhance the activation of the MYB transcription factor gene GhMYBL1 (MYB domain like protein 1) in fibers. GhARF7-1 and GhARF7-2 respond to auxin signals that promote fiber SCW thickening. GhMYBL1 RNAi and, GhARF7-1 and GhARF7-2 VIGS cotton displayed similar defects in fiber SCW formation as GhERF108 RNAi cotton. Moreover, the ethylene and auxin responses were reduced in GhMYBL1 RNAi plants. GhMYBL1 directly binds to the promoters of GhCesA4-1, GhCesA4-2, and GhCesA8-1 and activates their expression to promote cellulose biosynthesis, thereby boosting fiber SCW formation. Collectively, our findings demonstrate that the collaboration between GhERF108 and GhARF7-1 or GhARF7-2 establishes ethylene-auxin signaling crosstalk to activate GhMYBL1, ultimately leading to the activation of fiber SCW biosynthesis.