A Cytokinin Response Factor PtCRF1 is involved in the regulation of wood formation in poplar.

Wood formation is a complex developmental process under the control of multiple levels of regulatory transcriptional network and hormone signals in trees. It is well known that cytokinin signaling plays an important role in maintaining the activity of the vascular cambium. Cytokinin Response Factors (CRFs) encoding a subgroup of AP2 transcription factors have been identified to mediate the cytokinin-dependent regulation in different plant developmental processes. However, the functions of CRFs in wood development remain unclear. Here, we characterized the function of PtCRF1, a CRF transcription factor isolated from poplar, in the process of wood formation. PtCRF1 is preferentially expressed in secondary vasculature, especially in vascular cambium and secondary phloem, and encodes a transcriptional activator. Overexpression of PtCRF1 in transgenic poplar plants led to a significant reduction in the cell layer number of vascular cambium. The development of wood tissue was largely promoted in the PtCRF1-overexpressing lines, while significantly compromised in the CRISPR/Cas9-generated double mutant plants of PtCRF1 and its closest homolog PtCRF2. RNA-seq and RT-qPCR analyses showed that PtCRF1 repressed the expression of the typical cytokinin responsive genes. Furthermore, bimolecular fluorescence complementation assays revealed that PtCRF1 competitively inhibits the direct interactions between histidine phosphotransfer proteins (PtHPs) and type-B Response Regulator (RR) by binding to HP protein. Collectively, these results indicate that PtCRF1 negatively regulates cytokinin signaling and is required for woody cell differentiation in poplar.