Abstract: Tension wood (TW), a type of reaction wood that develops in angiosperm trees in response to gravistimulation, serves as an ideal model for investigating the regulatory mechanisms underlying xylem cell differentiation and cell wall deposition. The initial biological signals that induce the formation of reaction wood in response to gravitational stimuli remain poorly understood. In this study, we utilized pharmacological and genetic approaches to modulate Ca²⁺ levels in hybrid white poplar (Populus alba × P. glandulosa) and examine the role of calcium signaling during the early stages of gravitropic responses. Our findings revealed differential cytosolic Ca²⁺ signal distribution in gravistimulated stems during the early phase of gravity induction, characterized by lower Ca²⁺ levels on the upper side (where TW forms) and higher Ca²⁺ levels on the lower side (where opposite wood forms). Consistent with this hypothesis, plants treated with LaCl₃ and those with genetically disrupted calcium channels (PagGLR3.3 knockout using the CRISPR/Cas9 system) showed reduced Ca²⁺ signals and developed characteristic TW features. These results suggest that decreased Ca²⁺ levels induce the formation of TW. Furthermore, PagGLR3.3 knockout plants with TW-like stems displayed diminished sensitivity to gravistimulation. Transcriptomic analysis revealed that the knockout of PagGLR3.3 resulted in the upregulation of genes associated with TW formation and reactive oxygen species (ROS) production. Notably, superoxide anion (O₂^·-) levels were significantly elevated in the cambium zone of stems subjected to gravistimulation, LaCl₃ treatment, or PagGLR3.3 knockout, indicating that reduced Ca²⁺ levels promote TW formation through increased O₂^·- accumulation. This study offers novel insights into the critical role of Ca²⁺ in gravitropism and TW induction in poplar.

Key words: Ca²⁺, gravistimulation, O₂^·-, PagGLR3.3, Populus, tension wood