Coordinated Auxin–Cytokinin–Nitrogen Signaling Orchestrates Root Suckering in Populus

Root suckering is a key mode of clonal propagation in white poplar group, such as aspens (<i>Populus</i> section <i>Leuce</i>), enabling rapid vegetative spread, yet the molecular triggers remain elusive. Here, we developed a rapid protocol that produces abundant root suckers with the root cutting of white poplar (<i>Populus davidiana</i> × <i>P. bolleana</i>) roots in greenhouse. Anatomical analyses and daily resolution transcriptomes resolved three sequential developmental stages: primordium initiation (Days 0-1), SAM (shoot apical meristem) establishment (Days 1-4), and organ differentiation/growth (Days 4-6). Weighted gene co-expression network analysis revealed that auxin- and cytokinin-mediated signaling, integrated with nitrogen metabolism, orchestrates SAM formation and maintenance. Exogenous application of 0.5-1.0 mg L^-1 NAA suppressed sucker emergence by 48-60%, whereas inhibition of cytokinin biosynthesis with lovastatin reduced initiation by 60%. These data establish that auxin negatively regulates and cytokinin is indispensable for de novo shoot apical meristem establishment during poplar root-suckering, underscoring that a precise auxin-cytokinin balance governs the timing and extent of this developmental process. Cambial regulators <i>WUSCHEL</i>-Related Homeobox 4-1/2 (<i>WOX4-1/2</i>), together with core meristem regulators <i>WUSCHEL</i> (<i>WUS</i>) and SHOOT MERISTEMLESS (STM), were specifically induced during SAM establishment that underpin vascular integration between the nascent shoot and the parental root. These results uncover the molecular pathway controlling root suckering and provide potential targets for molecular breeding to either enhance or suppress root suckering in <i>Populus</i>.