Uncovering miRNA-Mediated Regulation in Phellem Versus Xylem Differentiation in Quercus suber L.

Several regulators of phellem/cork formation have been identified in recent years, using mainly transcriptomic approaches. However, this developmental process, showing parallels to the functioning of vascular cambium, remains poorly understood. The cork oak tree (Quercus suber L.) exhibits a remarkable ability to form a traumatic phellogen after debarking, enabling sustainable cork production. We aimed at uncovering post-transcriptional mechanisms controlled by miRNAs, specifically involved in regulating phellogen functioning and phellem differentiation in cork oak. To achieve this, we conducted a comparative analysis of the small RNA transcriptome between differentiating phellem and xylem, both originating from secondary meristems (phellogen and vascular cambium). In addition to identifying miRNAs exclusive to phellogen/phellem tissues, we discovered 246 differentially expressed miRNAs between the two tissues, of which 74 are conserved. The most abundant miRNA families found in phellem tissues were MIR165/166, MIR167, MIR168 and MIR390. By analysing miRNA predicted targets and their expression in the same tissues, many of the differentially expressed miRNAs were found associated with sequence-specific DNA binding functions. Within these, transcription factor families HD-ZIP III, WRKY, NAC and MYB were highlighted as key in phellem differentiation. Furthermore, hormone-mediated signalling pathways, particularly involving auxin, appeared as an enriched biological process, as several ARF transcripts, among other auxin signalling genes like IAA11, ARF18 and ARF19, were identified as putative targets of conserved or novel miRNAs. Overall, our results provide a comprehensive overview of the miRNA landscape during cork formation, providing valuable knowledge for further functional studies and potential practical applications in forest management.