Pan-genomic diversity of the EPF/EPFL gene family across wild and modern wheat species

BACKGROUND: The epidermal patterning factor/epidermal patterning factor-like (EPF/EPFL) proteins are pivotal regulators of stomatal development and responses to abiotic stress. However, their functions in wheat (Triticum aestivum L.) remain underexplored despite their critical roles in water-use efficiency and drought adaptation. RESULT: This pan-genomic identification study investigated a diverse panel of 13 wheat accessions, including wild relatives and modern cultivars, to analyze the genomic diversity, evolutionary history, and functional characterization of the EPF/EPFL family. A total of 365 non-redundant EPF/EPFL genes were identified, revealing substantial structural and phylogenetic divergence between the EPF (24.9%) and EPFL (75.1%) clades. Comparative analyses demonstrated species-specific chromosomal distributions, conserved motif architectures (e.g., Stomagen domains in EPFL9 homologs), and widespread purifying selection (66% of duplicated pairs), indicating functional constraints. Promoter cis-element profiling revealed enrichment for motifs related to stress responses (ABRE, MBS) and hormonal signaling (auxin, jasmonate), consistent with their roles in drought adaptation. MicroRNA target prediction identified Tae-miR530 and Tae-miR408 as key post-transcriptional regulators of EPF/EPFL genes. Collinearity analyses across Poaceae species emphasized conserved synteny blocks, while RNA-seq and RT-qPCR validation in T. aestivum cv. Chinese Spring revealed tissue and stress-specific expression patterns, with TaCSEPFL5, TaCSEPFL8, and TaCSEPFL9 homologs exhibiting significant upregulation in spikes, leaves, and roots under PEG-induced osmotic stress. Notably, TaCSEPF1-1B and TaCSEPFL2-3 A displayed antagonistic regulatory dynamics, suggesting functional diversification. CONCLUSION: This study provides a genomic atlas of the wheat EPF/EPFL gene family, elucidating their evolutionary trajectories, regulatory networks, and drought-responsive expression landscapes. These insights can benefit wheat breeding by enhancing climate resilience through targeted stomatal optimization and the development of stress-adaptive traits.