PtoHP6a regulates vascular cambial activity by maintaining cytokinin signaling stability in Populus tomentosa

Cytokinins are predominantly concentrated in the secondary phloem and play a crucial role in regulating vascular cambial activity. HP6, a key negative regulator in the cytokinin signaling pathway, is vital for maintaining cytokinin homeostasis, yet its role in wood development is unclear. In this study, we identified a HP6 gene (PtoHP6a) in Populus tomentosa and confirmed its functional conservation with Arabidopsis AHP6 through mutant phenotype recovery. Expression analysis showed that PtoHP6a is specifically expressed in the secondary phloem. Overexpression of PtoHP6a led to reduced cytokinin signaling and inhibited vascular cambium cell division. Conversely, in transgenic plants with downregulated phloem cytokinin levels, silencing PtoHP6a enhanced cytokinin signaling and restored vascular cambium activity. Notably, PtoHP6-RNAi plants exhibited elevated cytokinin signaling but retained defects in vascular cambium activity, mirroring the phenotypes of transgenic plants overexpressing the AtIPT1 gene, encoding isopentenyl transferases that catalyze the first step of cytokinin biosynthesis. This suggests that excessive cytokinin can negatively affect vascular development. Importantly, PtoHP6a overexpression in PtoCLE41b_pro-AtIPT1 plants mitigated elevated cytokinin signaling, restoring vascular cambium activity. These findings highlight the significance of PtoHP6a as a phloem-specific cytokinin signaling inhibitor in regulating vascular cambium function.