GhFIR_A08: An uncharacterized regulator promotes cotton fiber initiation by integrating into a GhWRKY16-GhRL6 transcriptional hierarchy.

INTRODUCTION Cotton is an important global economic crop, and enhancing yield remains a primary breeding objective. Fiber development is controlled by a complex regulatory network, with the initiation stage being pivotal because it determines fiber number and ultimately yield. However, most studies have focused on upstream transcription factors, leaving downstream functional genes largely uncharacterized.

Objectives

To explore the gene function and regulatory network of the unannotated gene GhFIR_A08 (Fiber Initiation Regulator on chromosome A08), which was identified based on previous structural-variant analyses and highly expressed during fiber initiation.

Methods

Currently high-quality Gossypium genome assemblies were compared to investigate the origin of the FIRs gene family and the mechanism of transposon-mediated copy number variation. Gene function was evaluated by generating stable homozygous transgenic lines (both knockout and overexpression) in the recipient backgrounds. Interacting proteins were screened and verified by MBP pull-down combined with mass spectrometry, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays. The regulatory network was explored by yeast one-hybrid, dual-luciferase reporter, electrophoretic mobility shift assays, RNA-seq, and measurements of related metabolites.

Results

Alongside the evolutionary origin and polyploidization-driven expansion of this gene family in Gossypium, a transposon-mediated, haplotype-specific loss of GhFIR_A08 was uncovered. Overexpressing GhFIR_A08 increased the number of ovule protrusions and enhanced lint percentage (LP), whereas knockout produced the opposite phenotype. An upstream regulatory hierarchy was further uncovered in which GhWRKY16 induces another activator, GhRL6; both GhWRKY16 and GhRL6 bind the respective sites of GhFIR_A08 promoter, forming a coherent GhWRKY16-GhRL6-GhFIR_A08 module to activate GhFIR_A08. Functionally, GhFIR_A08 cooperates with Gh14-3-3, GhHSP70, GhSPL13, and GhPPR to regulate cotton fiber initiation by modulating hormone signaling, protein homeostasis, and redox processes.

Conclusion

The uncharacterized gene, GhFIR_A08 is revealed to positively regulate fiber initiation to increase LP, and provides valuable genetic resources for molecular breeding aimed at improving cotton yield.