Regulatory role of MicroRNA164 in heat and salinity stress responses via candidate target genes during seed germination in petunia

Abstract MicroRNA164 (miR164) is a highly conserved miRNA that targets NAC transcription factors in plants. This study explores its role in regulating stress responses in petunia by generating MIR164 overexpression ( MIR164 -ox) and miR164 suppression (miR164-STTM) lines. Under heat stress, MIR164 -ox lines exhibited enhanced tolerance, while miR164-STTM lines showed reduced tolerance with growth reduction in both shoots and roots. Conversely, under salinity stress, miR164-STTM lines displayed improved tolerance, while MIR164 -ox lines were more sensitive, suggesting that miR164 may have a differential regulatory effect on heat and salinity stress responses. To further investigate the molecular mechanisms underlying these differential stress responses mediated by miR164, we conducted a bioinformatic analysis of the NAC transcription factor family in Petunia, the primary targets of miR164. A genome-wide analysis of the NAC gene family in Petunia identified 80 NAC genes in P. axillaris and 91 in P. inflata . Phylogenetic analysis revealed nine distinct clades, indicating both conserved and divergent functions of NAC proteins. Expression analysis of miR164-targeted NAC genes showed tissue- and stress-specific patterns. Protein–protein interaction (PPI) network and Gene Ontology (GO) enrichment analyses provided insights into the potential functions of NAC proteins in petunia, particularly in secondary cell wall biogenesis and xylem development. Real-time qRT-PCR analysis revealed differential expression of specific NAC genes in miR164 transgenic lines under heat and salinity stress, with NAC06 and NAC39 / NAC54 showing the most pronounced changes, suggesting their potential involvement in heat and salinity stress responses, respectively. Taken together, our findings suggest that miR164 plays a crucial role in regulating NAC gene expression and stress responses in petunia, providing a foundation for further functional characterization of miR164-NAC regulatory modules in plant stress adaptation.