PtrSHR1 coordinates vascular cambium proliferation and xylem lignin biosynthesis to regulate wood formation in...

Vascular cambium produces cells to form xylem, or wood, in tree stems. Here, we explored transregulatory pathways in this cell lineage development. We tested 20 of the 95 putative vascular cambium-specific (VCS) transcription factor genes through gain-of-function transgenesis in Black cottonwood (Populus trichocarpa) and found that a SHORT-ROOT (PtrSHR1) gene induced some of the most prominent phenotypes. PtrSHR1 transcripts are in the vascular cambium (VC) but not in the stem-differentiating xylem (SDX), whereas PtrSHR1 proteins are in both VC and SDX. Constitutive overexpression or endonuclease-deficient Cas9 (dCas9)-mediated loci-specific gene activation of PtrSHR1 revealed an activated PtrSHR1-PtrWRKY25-PtrVCS2-PtrWOX4a/b (WUSCHEL-related homeobox 4a/b) regulatory path for an abbreviated VC zone and a smaller stem diameter. CRISPR PtrSHR1 mutation reduced the PtrSHR1-PtrWRKY25-PtrVCS2-PtrWOX4a/b regulation resulting in an expanded VC zone and a larger stem diameter. In SDX, PtrSHR1-PtrWRKY94 activated specifically 3 (Ptr4CL3, 4-coumarate:CoA ligase 3; PtrC3H3, p-coumarate 3-hydroxylase 3; and PtrC4H1, cinnamate 4-hydroxylase 1) of the 22 monolignol biosynthetic pathway genes to control lignin content and structure, corroborated by chemical degradation and 2-dimensional (2D) nuclear magnetic resonance analyses. Thus, PtrSHR1 mediates the proliferation of VC and diffuses into SDX to control lignin properties and wood production, unveiling potential strategies for creating advantageous wood feedstock for materials and energy.