• First functional analysis of antagonistic EPF-like peptides in cotton. • GhEPF2-2 and GhEPFL9-6 show opposite roles via the GhERECTA1 receptor. • Cross-species assays confirm conserved yet divergent stomatal regulation. • GhEPFL9-6 enhances drought tolerance despite increasing stomatal density. • EPF-ERECTA signaling module provides promising targets for cotton breeding. Precise regulation of stomatal development is essential for optimizing water use efficiency and drought adaptation in crops. EPIDERMAL PATTERNING FACTOR (EPF) and EPF-LIKE (EPFL) peptides are well-established regulators of stomatal patterning in model species, yet their functional roles in cotton ( Gossypium hirsutum ) remain insufficiently characterized. Here, we investigate two cotton homologs, GhEPF2-2 and GhEPFL9-6 , and show that they exert contrasting effects on stomatal development and drought-associated physiological responses. Transcript profiling revealed leaf-preferential expression of both genes and opposite transcriptional responses to dehydration. Virus-induced gene silencing (VIGS) of GhEPF2-2 increased stomatal density and aggravated oxidative stress, whereas silencing GhEPFL9-6 reduced stomatal density and improved antioxidant status under PEG-induced dehydration. Heterologous expression in Arabidopsis thaliana further supported their divergent roles: GhEPF2-2 overexpression decreased stomatal density and altered antioxidant profiles, whereas GhEPFL9-6 overexpression increased stomatal density but exhibited improved physiological performance in controlled drought assays, likely through secondary changes in shoot architecture and osmotic adjustment rather than stomatal traits alone. Protein–protein interaction assays indicated that both peptides can interact with the receptor-like kinase GhERECTA1, suggesting their involvement in a shared signaling framework. Together, these findings reveal functional divergence of EPF/EPFL peptides in cotton and provide a basis for further evaluation of their roles in drought adaptation using stable genetic systems.
He et al. (Sun,) studied this question.