EPF/EPFL Peptide Signaling as a Regulatory Node Linking Stomatal Development to Drought and Heat Tolerance in Tomato (Solanum lycopersicum L.)

Stomatal development and function are tightly regulated by signaling peptides with distinct, overlapping, or antagonistic roles in gas exchange and water balance. EPIDERMAL PATTERNING FACTOR (EPF) / EPF-LIKE (EPF/EPFL) family is a founding member of the secreted peptide family that regulates stomatal patterning. The roles of EPF/EPFL genes in tomato responses to drought, heat, and combined drought-heat stress remain largely unexplored. To characterize the EPF/EPFL gene family and elucidate its potential regulatory roles in stomatal development, 15 family members were identified in S. lycopersicum L. All 15 candidate SlEPF proteins contained an EPF-type domain. Of these, 13 exhibited specific matches to the EPF domain, whereas SlEPF6 was classified within the EPF superfamily and SlEPF11 within the STOMAGEN-like superfamily. Phylogenetic analysis grouped EPF genes from diverse plant species into two major clades (A and B), each comprising distinct evolutionary subgroups. GO annotation indicated that most SlEPF genes were involved in stomatal complex development and guard cell differentiation, with molecular functions associated with MAP kinase activity, receptor serine/threonine kinase binding, and receptor ligand activity. Promoter analysis of SlEPF genes identified diverse cis-regulatory elements potentially involved in the regulation of environmental stress responses and plant development. Expression analysis of five putative stress-responsive genes (SlEPF5, SlEPF8, SlEPF9, SlEPF10, and SlEPF13) revealed distinct transcriptional patterns in the heat-tolerant drought-sensitive Money Maker (MM) and drought-tolerant Ailsa Craig (AC) tomato genotypes under drought, heat, and combined drought-heat stress. In MM, SlEPF5 and SlEPF13 were consistently downregulated across all stress conditions, whereas SlEPF8 was strongly induced under drought and combined stress. In contrast, AC showed broader transcriptional activation, particularly under heat and combined stress. Overall, these contrasting expression profiles suggest genotype- and stress-dependent regulation of SlEPF genes, highlighting their potential involvement in tomato abiotic stress responses.