Small RNA sequencing reveals drought-responsive microRNAs and regulatory networks in roots of mungbean genotypes differing in drought tolerance

Drought stress is a major environmental constraint limiting mungbean productivity, particularly in arid and semi-arid regions. However, the molecular mechanisms underlying drought tolerance in mungbean remain poorly understood. MicroRNAs (miRNAs), which function as important post-transcriptional regulators of gene expression, play critical roles in modulating plant responses to abiotic stresses. This study aimed to identify drought-responsive miRNAs and elucidate their regulatory roles in contrasting mungbean genotypes under drought stress. Seven widely cultivated mungbean genotypes were evaluated under controlled drought stress conditions using physiological, molecular, and yield-related parameters. Traits including root and shoot length, biomass accumulation, nodule number, expression of drought-responsive genes under prolonged stress (30 days), and yield attributes were assessed. Based on these analyses, genotype cv. ML267 was identified as drought tolerant, whereas cv. PDM139 was identified as drought susceptible. To investigate the molecular basis of drought tolerance, small RNA sequencing was performed using root tissues of the contrasting genotypes. A total of 61 novel drought-responsive miRNAs and 274 conserved miRNA homologs were identified, showing genotype-specific differential expression under drought stress conditions. Quantitative RT-PCR validation of selected miRNAs and their predicted target genes demonstrated inverse expression patterns, confirming miRNA-mediated post-transcriptional regulation. Functional annotation and network analyses revealed that these miRNA-target regulatory modules are associated with hormone signaling, antioxidant defense, root development, and stress adaptation pathways. The study also revealed complex miRNA-mediated regulatory networks that regulate mungbean gene expression in response to drought. This study offers a thorough understanding of small RNA-mediated regulation associated with drought tolerance in mungbean. The identified drought-responsive miRNAs along with their target genes indicate potential molecular candidates for enhancing drought resistance and could aid in the creation of climate-resilient mungbean varieties through molecular breeding and biotechnological methods.