The PtoTCP20-miR396d-PtoGRF15 module regulates secondary vascular development in Populus

Secondary vascular development is a key biological characteristic of woody plants and the basis of wood formation. Our understanding of gene expression regulation and dynamic changes in microRNAs (miRNAs) during secondary vascular development is still limited. Here we present an integrated analysis of the miRNA and mRNA transcriptome of six phase-specific tissues-the shoot apex, procambium, primary vascular tissue, cambium, secondary phloem, and secondary xylem-in Populus tomentosa. Several novel regulatory modules, including the PtoTCP20-miR396d-PtoGRF15 module, were identified during secondary vascular development in Populus. A series of biochemical and molecular experiments confirmed that PtoTCP20 activated transcription of the miR396d precursor gene and that miR396d targeted PtoGRF15 to downregulate its expression. Plants overexpressing miR396d (35S:miR396d) showed enhanced secondary growth and increased xylem production. Conversely, during the transition from primary to secondary vascular development, plants with downregulated PtoTCP20expression (PtoTCP20-SRDX), downregulated miR396 expression (35S:STTM396), and PtoGRF15 overexpression (35S:PtoGRF15) showed delayed secondary growth. Novel regulatory modules were identified by integrated analysis of the miRNA and mRNA transcriptome, and the regulatory role of the PtoTCP20-miR396d-PtoGRF15 signaling cascade in secondary vascular development was validated in Populus, providing information to support improvements in forest cultivation and wood properties.