Genome-wide analysis of Phaseolus vulgaris L. miR172 gene family members under heavy metal stress

Phaseolus vulgaris L. is a vital source of protein, vitamins, and minerals, significantly addressing nutritional deficiencies. P. vulgaris L., like other plant species, demonstrates considerable susceptibility to heavy metal stress, a significant environmental challenge. MicroRNAs (miRNAs) and their target genes play a crucial role in enabling plant responses to stress. This study investigated the role of the miR172 gene family, which is essential for growth and development and is among the first miRNAs identified in plants under heavy metal stress in common bean plants. By employing bioinformatics and experimental methods, we sought to gain insights into its regulatory mechanisms and potential roles in enhancing stress tolerance. Bioinformatics analysis identified six miR172 genes in the P. vulgaris L. genome on five different chromosomes. The precursor sequences of Pvul-miR172 exhibited a characteristic root-loop structure, and the 19-nucleotide (nt) mature sequences were conserved across various species, categorizing them into three distinct phylogenetic classes. Elements responsive to stress were identified in the promoter regions of Pvul-miR172. Gene Ontology (GO) analysis revealed that eight target genes associated with Pvul-miR172 are involved in carrier activity and binding, which is crucial for stress response. Measured parameters included root and shoot length, cell membrane integrity, relative water content (RWC), and chlorophyll levels in two common bean genotypes. Exposure to heavy metal stress increased antioxidant enzyme activity and elevated proline (Pro), hydrogen peroxide (H2O2), and malondialdehyde (MDA) levels. The analysis of expression profiles for Pvul-miR172s and their target genes under heavy metal stress revealed diverse regulatory patterns. Principal Component Analysis (PCA) showed positive and negative correlations between Pvul-miR172s and their predicted target genes, indicating a complex regulatory interaction under heavy metal stress. The findings suggest that Pvul-miR172s contribute to increased tolerance to heavy metal stress, with effects differing by genotype. Our findings suggest that members of the Pvul-miR172 gene family may play important roles in the common bean’s response to heavy metal stress. Expression levels of these genes and their targets varied depending on the type of heavy metal and genotype, with the G68 (tolerant) genotype being particularly prominent in stress tolerance. Stress-related cis-regulatory elements and differential expression profiles under stress conditions suggest that this gene family may be involved in regulatory networks associated with the stress response. These results provide fundamental information for developing transgenic lines with stress tolerance through Pvul-miR172s and their targets using miRNA-based approaches such as amiRNA, STTM, and miPEP.