Functional Characterization of a Root-Preferential and Stress-Inducible Promoter of Eca-miR482f in Eucalyptus camaldulensis

MicroRNAs (miRNAs) act as pivotal post-transcriptional regulators of gene expression in plant stress responses. However, the transcriptional regulation mechanisms governing miRNA genes themselves remain insufficiently characterized. This study focuses on Eca-miR482f, a previously identified cold-responsive miRNA from <i>Eucalyptus camaldulensis</i> that targets <i>EcaSIZ1</i>-a key component of the ICE1-CBFs-CORs cold signaling pathway. We first investigated the expression pattern of Eca-miR482f and found it exhibited root-preferential accumulation in <i>E. camaldulensis</i>. Under cold stress, it displayed divergent organ-specific responses: strong induction in roots and suppression in aerial tissues. To elucidate its transcriptional regulation, we cloned a 1938 bp promoter sequence upstream of the Eca-miR482f precursor. Bioinformatics analysis revealed that this promoter was highly conserved within the <i>Eucalyptus</i> genus and enriched with multiple cis-acting elements associated with stress responses-including a low-temperature-responsive element (LTR)-as well as hormone signaling, such as abscisic acid (ABA) and methyl jasmonate (MeJA)-responsive motifs. A series of 5'-deletion fragments were generated to delineate the functional regions within the promoter. Through transgenic approaches in both tobacco and <i>Arabidopsis</i>, we demonstrated that this promoter drove strong, root-preferential expression. Furthermore, it exhibited significant inducibility under cold and MeJA treatments. Systematic truncation analysis delineated specific promoter regions essential for maintaining this organ specificity and stress responsiveness, thus identifying potential functional modules. Briefly, our findings provide crucial insights into the transcriptional regulation of Eca-miR482f and uncover a valuable genetic tool for future biotechnological engineering of stress-tolerant woody plants via precise spatiotemporal modulation of gene expression.