Integrative RNA sequencing uncovers the miRNA-mRNA landscape of water-deficit response in Setaria italica

In miRNA-mRNA regulatory modules of Setaria italica, the candidate novel miRNAs Sit-miR74-N/APX7 and Sit-miR132-N/PEPC involved in key pathways underlying water-deficit response have been detected. In the first module, APX7 expression was directly associated with water-deficit intensity, whereas in the second module, PEPC activation occurred under moderate water deficit. Drought severely constrains plant growth and productivity, yet the underlying regulatory mechanisms remain incompletely understood. Here, we determined the miRNA-mediated post-transcriptional landscape during water deficit in the panicoid grass Setaria italica. Two independent water-deficit experiments combined with small RNA and degradome sequencing identified 159 miRNAs, including 135 candidate novel sequences, of which 20 were differentially expressed under contrasting watering regimes. In silico analysis predicted 1,612 miRNA-mRNA regulatory modules, representing a broad landscape of potential post-transcriptional regulation. Complementarily, degradome sequencing identified 19 miRNA-mRNA modules, several of which exhibited inverse expression patterns, particularly under severe water-deficit conditions. Functional annotation of target mRNAs uncovered multiple functional processes, including transcriptional regulation, photosynthesis, redox homeostasis, and solute transport. Additionally, transient expression assays validated selected miRNA-mRNA modules, confirming the interaction between candidate novel miRNAs and water deficit-related mRNA targets. These modules are involved in key water deficit-responsive pathways, including oxidative stress regulation (Sit-miR74-N/APX7), photosynthetic carbon metabolism (Sit-miR132-N/PEPC), and flavonoid biosynthetic pathways (Sit-miR39-N/OMT). Together, these findings reveal a complex regulatory network in which miRNAs contribute to the coordination of water-deficit responses in S. italica, with post-transcriptional regulation underlying plant responses to abiotic stress.