Comprehensive analysis of RALF family genes and their expression patterns in barley

Rapid alkalinization factors (RALFs) are plant-derived cysteine-rich peptides that have been widely studied in Arabidopsis, where they regulate pollen tube growth, root development, and stress responses. Barley ( Hordeum vulgare L.) is the fourth most important cereal crop worldwide, after wheat, maize, and rice. However, the RALF gene family has not yet been systematically investigated in barley. In this study, we performed a comprehensive bioinformatics analysis to identify and characterize the barley RALF gene family. 11 HvRALF genes were identified and categorized into three subfamilies, and motif analysis revealed ten conserved motifs. Based on chromosomal localization analysis, 11 HvRALF genes were almost evenly distributed on chromosomes 1 to 5, but the RALF gene was not found on chromosomes 6 to 7. Furthermore, analysis of cis-acting elements in the promoters identified a variety of elements, including ABRE, ARE, AuxRR-core, MBS, LTR, and TC-rich, which are important in responses to hormones as well as abiotic and biotic stresses. Our analysis predicted interactions between four miRNAs and HvRALF genes, suggesting a regulatory mechanism that fine-tunes HvRALF-mediated signaling pathways and enhances plant adaptation to environmental stresses. Overall, these findings advance our understanding of HvRALF functions in barley stress responses and provide a basis for future functional characterization.