ABA-responsive transcription factors converge on evolutionarily layered MIR loci potentially integrating developmental and stress adaptation programs

Abscisic acid (ABA) is a central regulator of plant drought adaptation, orchestrating extensive transcriptional reprogramming through multiple stress-responsive transcription factors (TFs). Although ABA-associated TF networks regulating protein-coding genes have been extensively studied, how these TFs are associated with microRNA (miRNA) regulation during stress adaptation remains poorly understood. Here, we re-analyzed publicly available ChIP-seq datasets for 21 ABA-responsive TFs in Arabidopsis thaliana to systematically examine TF occupancy at miRNA loci. We identified ten MIR loci co-occupied by at least 18 ABA-associated TFs, including members of the bZIP, NAC, MYB, HD-ZIP, NF-Y, AP2/ERF, and zinc-finger TF families. ChIP-seq peak analysis revealed substantial TF occupancy at several MIR loci, including MIR156D , MIR166A , MIR408 , MIR168A , MIR824 , MIR163 , MIR779 , and MIR170 , whereas MIR399C displayed distal peak localization and was excluded from subsequent analyses. Quantitative RT-PCR analyses further showed that most pri-miRNAs were transcriptionally repressed by ABA and PEG-induced osmotic stress, while pri-miR824 was strongly induced under both treatments. Together, our findings reveal associations between ABA-responsive TF occupancy and MIR loci and provide expression-based evidence for their responsiveness to ABA and osmotic stress. In light of the distinct evolutionary histories previously reported for these MIR families, we propose that ABA-responsive MIR loci constitute an evolutionarily layered regulatory landscape that may link ABA-responsive transcriptional networks with developmental and stress-related processes in plants.