Bioorthogonal Tracking of Spatiotemporal Lignification Dynamics in Plant Cell Walls Using Alkyne-Tagged Glycosylated Monomers

Lignin, a major component of plant cell walls, plays a critical role in structural support and stress resistance. Despite its importance, the transport and deposition dynamics of the glycosylated lignin monomer during lignification in living cells remain poorly understood, hindering advances in biomass utilization. To address this challenge, alkyne-labeled glycosylated lignin precursors (pGCA_ALK and CF_ALK) were synthesized by introducing propargyl groups at the ortho position of aromatic rings. These precursors were successfully incorporated into lignin polymers in flax (a herbaceous plant) and ginkgo (a gymnosperm), enabling the real-time tracking of lignification via fluorescent click chemistry. Quantitative imaging revealed that lignification initiates at cell corners and the middle lamella and then progressively extends into secondary cell walls. Distinct deposition patterns were observed: parenchyma cells exhibited continuous lignin accumulation, whereas fiber tracheids underwent rapid lignification, followed by cell death. Specialized pit structures displayed "tunnel-like" lignin deposition in longitudinal pits and unilateral patterns in transverse pits. In vitro synthesis of dehydrogenation polymers (DHP) and extraction of the cellulolytic enzyme lignin (CEL) from ginkgo confirmed the biocompatibility of labeled monomers. LC-MS analysis further demonstrated that alkynyl groups formed oxygen-containing cyclic structures without disrupting natural β-O-4 and β-5 lignin linkages. Application of this labeling method in biomass utilization indicated that lower overall fluorescence intensity correlates with more efficient lignin removal during pretreatment. These results provide new insights into the spatiotemporal dynamics of lignification and establish a bioorthogonal platform for lignin research, offering promising strategies for optimizing plant biomass in industrial applications.