Wood formation is a complex process that involves cell differentiation, cell expansion, secondary wall deposition, and programmed cell death. We constructed a 4-layer wood formation transcriptional regulatory network (TRN) in black cottonwood (Populus trichocarpa) that has four Secondary wall-associated NAC-Domain 1 (PtrSND1) transcription factor (TF) family members as the top-layer regulators. We characterized the function of a MYB (PtrMYB161) TF in this PtrSND1-TRN, using transgenic P. trichocarpa cells and whole plants. PtrMYB161 is a third-layer regulator that directly transactivates five wood formation genes. Overexpression of PtrMYB161 in P. trichocarpa (OE-PtrMYB161s) led to reduced wood, altered cell type proportions, and inhibited growth. Integrative analysis of wood cell-based chromatin binding assays with OE-PtrMYB161s transcriptomics revealed a feedback regulation system in the PtrSND1-TRN, where PtrMYB161 represses all four top-layer regulators and one secondlayer regulator, PtrMYB021, possibly affecting many downstream TFs in, and likely beyond, the TRN, to generate the observed phenotypic changes. Our data also suggested that the PtrMYB161's repressor function operates through interaction of the base PtrMYB161-target binding system with gene silencing co-factors. PtrMYB161 protein does not contain any known negative regulatory domains. CRISPR-based mutants of PtrMYB161 in P. trichocarpa exhibited phenotypes similar to the wildtype, suggesting PtrMYB161's activator functions are redundant among many TFs. Our work demonstrated that PtrMYB161 binds to multiple sets of target genes, a feature that allows it to function as an activator as well as a repressor. The balance of the two functions may be important to the establishment of regulatory homeostasis for normal growth and development.

Stem vascular cambium cells in forest trees produce wood for materials and energy. WOX4 affects the proliferation of such cells in Populus. Here we show that PtrWOX4a is the most highly expressed stem vascular-cambium-specific (VCS) gene in P. trichocarpa, and its expression is controlled by the product of the second most highly expressed VCS gene, PtrVCS2, encoding a zinc finger protein. PtrVCS2 binds to the PtrWOX4a promoter as part of a PtrWOX13a–PtrVCS2–PtrGCN5-1–PtrADA2b-3 protein tetramer. PtrVCS2 prevented the interaction between PtrGCN5-1 and PtrADA2b-3, resulting in H3K9, H3K14 and H3K27 hypoacetylation at the PtrWOX4a promoter, which led to fewer cambium cell layers. These effects on cambium cell proliferation were consistent across more than 20 sets of transgenic lines overexpressing individual genes, gene-edited mutants and RNA interference lines in P. trichocarpa. We propose that the tetramer–PtrWOX4a system may coordinate genetic and epigenetic regulation to maintain normal vascular cambium development for wood formation.