Systematic identification and functional characterization of EPF/EPFL peptides involved in drought stress responses in tobacco

: Members of the Epidermal Patterning Factor/EPF-Like (EPF/EPFL) gene family encode cysteine-rich small peptides that participate in plant development and environmental adaptation. To investigate the potential roles of EPF/EPFL peptides in drought responses, a comprehensive bioinformatics analysis was performed to identify NtEPF/EPFL genes in tobacco ( Nicotiana tabacum L.). A total of 23 NtEPF/EPFL proteins were identified and classified into two major groups based on their conserved EPF/EPFL domains. Expression analysis by qRT-PCR showed that several NtEPF/EPFL genes responded to abscisic acid (ABA), low temperature, osmotic stress, and salt stress. Exogenous application of synthetic NtEPF/EPFL peptides alleviated mannitol-induced osmotic damage and was associated with improved drought-related phenotypes in tobacco and Arabidopsis thaliana . In particular, NtEPF3 and NtEPFL6 positively regulated drought tolerance in tobacco, as overexpression lines showed enhanced drought tolerance whereas the corresponding CRISPR/Cas9 mutants were more drought sensitive. NtEPF3 and NtEPFL6 were also associated with changes in stomatal aperture, water loss, and gas-exchange parameters under drought stress. In addition, drought-enhanced phosphorylation of NtSAPK2-L, a putative tobacco ortholog of OST1, was observed following NtEPF3 or NtEPFL6 peptide treatment and in the corresponding overexpression backgrounds, whereas weaker phosphorylation was detected in the mutant backgrounds. Parallel changes in Mitogen-Activated Protein Kinase (MAPK)-like phosphorylation were also observed. These findings indicate that NtEPF3 and NtEPFL6 may participate in tobacco drought responses through stomatal regulation and drought-responsive kinase signaling.