CRISPR/Cas9‐Mediated SiEPF2 Mutagenesis Attenuates Drought Tolerance and Yield in Foxtail Millet (Setaria italica).

Plants employ peptide ligands to coordinate development and integrate environmental signals via dedicated cascades (#ref‐0013). Epidermal patterning factor (EPF), plays a significant role in regulating stomatal density, seed germination and panicle development (#ref‐0008). EPF/EPFL enhance drought tolerance by reducing stomatal density have been reported in multiple species, including Hordeum vulgare, Arabidopsis thaliana, Populus spp., Vitis vinifera, Sorghum bicolor and Brassica napus (#ref‐0001). Our previous study has shown that EPF can mediate drought resistance in foxtail millet by regulating stomatal density (#ref‐0004). Furthermore, evidence suggests that plants can regulate photosynthesis through stomatal modification, ultimately enhancing yield (#ref‐0005). Beyond stomatal density regulation, the EPF/EPFL gene family modulates seed germination through phytohormone signalling and regulates inflorescence development via ligand‐receptor interactions (#ref‐0007). OsEPFLs act as upstream ligands for the OsER1 receptor, activating the MAPK signalling cascade to regulate panicle morphogenesis (#ref‐0002). In this study, we sought to elucidate how SiEPF2 balances drought resistance and yield in foxtail millet by modulating stomatal density and panicle morphology. Our findings not only provide novel insights into SiEPF2's role in abiotic stress responses but also contribute valuable genetic resources for high‐yield breeding programmes in millet crops. Summary statement: Foxtail millet (Setaria italica), a drought‐tolerant crop vital for food security, was studied using CRISPR‐Cas9 to target SiEPF2, yielding two allelic mutants. We found SiEPF2 regulates stomatal density (enhancing drought resistance) and panicle development (affecting yield). Our work reveals how SiEPF2 balances stress tolerance and productivity, offering genetic insights for high‐yield millet breeding. [ABSTRACT FROM AUTHOR]