Cell Wall Invertase 4 Governs Sucrose-Hexose Homeostasis in the Apoplast to Regulate Wood Development in Poplar.

In perennial trees, wood development is a carbon-demanding process, pivotal for secondary cell wall (SCW) formation and xylem development. Sugars, functioning both as carbon substrates and signaling molecules, orchestrate cambial proliferation and xylem differentiation. However, few molecular candidates involved in the sugar-mediated regulation of wood development have been characterized. Cell wall invertases (CWINs), a subclass of the invertase enzyme family localized in the apoplastic space, catalyze the irreversible hydrolysis of sucrose into glucose and fructose, thereby governing carbon allocation in sink tissues. Here, PtoCWIN4 shows preferential expression in the stem of Populus tomentosa and has a high efficiency in sucrose cleavage activity. We demonstrated that the knockout of PtoCWIN4 results in stunted growth, aberrant branching patterns, and compromised secondary xylem formation. In contrast, mutant lines displayed enhanced SCW thickness accompanied by elevated cellulose and hemicellulose accumulation. Following this, the knockout of PtoCWIN4 led to impaired carbon partitioning from sucrose to hexose metabolites during wood development, corroborating the enzyme's role in sustaining sucrose hydrolysis. Collectively, these findings establish PtoCWIN4 as a master regulator of sucrose-to-hexose conversion, a metabolic gateway critical for balancing structural biomass production and developmental growth during wood formation.