Abstract

The initial number of fibers and fiber length affect fiber yield and quality in cotton (Gossypium hirsutum). The mechanism of fiber initiation centers on the transcription factor GhMYB25-like, and the involvement of other regulatory factors remains unclear. Here, we established a GhMYB25-like-based interaction network that regulates fiber initiation, and focused on the interacting protein AGAMOUS-like 4 (GhAGL4), which is a MIKC-MADS transcription factor, and other MIKC-MADS family members. Genome-wide identification of the cotton MIKC-MADS family members identified two highly co-expressed genes, GhAGL1 and GhAGL4, which are predominantly expressed in ovules at 0 days post-anthesis (DPA). CRISPR/Cas9-mediated loss of function of GhAGL1 or GhAGL4 led to a decrease in fiber initials and impeded fiber elongation, with the Ghagl1 Ghagl4 double mutant exhibiting a stronger phenotype. GhAGL1 and GhAGL4 interact in vitro and in vivo. DNA affinity purification and sequencing (DAP-seq) combined with RNA sequencing identified CCCH zinc finger 1 (GhCZF1) as a downstream gene of GhAGL1 and GhAGL4. Interestingly, GhMYB25-like enhances the ability of GhAGL1-GhAGL4 heterodimers to activate downstream genes. During the early fiber elongation stage, GhCZF1 activates the expression of the xyloglucan endotransglycosylase/hydrolase (XTH) genes GhXTH16 and GhXTH23 to regulate fiber elongation. Our findings reveal a molecular mechanism involved in fiber initiation and early elongation, providing a foundation for enhancing cotton fiber yield and quality.