Research techniques of miRNA and its regulatory network under biotic and abiotic stresses in plants

• The expression patterns of microRNAs (miRNAs) under stress conditions are complex. • There are several key technologies for exploring the mechanism of action of miRNA. • miRNA enhances the resistance of plants by regulating multiple signaling pathways. Biological and abiotic stresses exert adverse effects on plant productivity. Consequently, elucidating the molecular mechanisms underlying responses to these stresses is essential. The expression patterns of microRNAs (miRNAs) under biological and abiotic stress conditions exhibit significant diversity and complexity. However, the mechanism by which miRNAs participate in plant stress resistance responses and regulate associated target genes requires more comprehensive and systematic elucidation. This review systematically states biosynthesis mechanisms of miRNAs, we describe Pol II-mediated primary miRNA transcript generation and DCL1 complex-mediated stepwise processing. At the technical level, classic detection methods such as Northern blotting and Real-time qPCR are detailed, while omics techniques including next-generation bioinformatics platforms and degradome analysis are integrated. Moreover, review systematically introduces the dynamic regulatory networks of plant miRNAs under biotic and abiotic stress. In terms of miRNA stress response mechanisms, novel insights have emerged regarding the enhancement of plant stress resistance through regulation of key antioxidant enzymes (SOD, POD), activation of plant hormone signaling pathways (ABA, JA), and direct regulation of defense-related genes. Despite the critical role of miRNAs in environmental adaptation, plant growth and development, and the regulation of various biological metabolic processes, many of their functions need yet to be fully explored. It is expected that this paper can contribute to further in-depth studies addressing the analysis of miRNA-regulated stress responses in plants.