Heterodimers of the MADS transcription factors GhAGL1 and GhAGL4 modulate cotton fiber initiation and elongation

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.