A better in vivo understanding of lignin formation within plant cell walls will contribute to improving the valorization of plant-derived biomass. Although bioorthogonal chemistry provides a promising platform to study the lignification process, methodologies that simultaneously detect multiple chemical reporters in living organisms are still scarce. Here, we have developed an original bioorthogonal labeling imaging sequential strategy (BLISS) to visualize and analyze the incorporation of both p-hydroxyphenyl (H) and guaiacyl (G) units into lignin in vivo with a combination of strain-promoted and copper-catalyzed azide-alkyne cycloadditions. On our path to BLISS, we designed a new azide-tagged monolignol reporter for H units in metabolic lignin engineering and used it in conjunction with an alkyne-tagged G unit surrogate to study lignification dynamics in flax. Here, we show that BLISS provides precise spatial information on the zones of active lignification and reveals polarization in single-cell lignification dynamics.

Reported herein is an in vivo triple labelling strategy to monitor the formation of plant cell walls. Based on a combination of copper‐catalysed alkyne–azide cycloaddition (CuAAC), strain‐promoted azide–alkyne cycloaddition (SPAAC), and Diels–Alder reaction with inverse electronic demand (DARinv), this methodology can be applied to various plant species of interest in research. It allowed detection of the differential incorporation of alkynyl‐, azido‐, and methylcyclopropenyl‐tagged reporters of the three main monolignols into de novo biosynthesized lignin in different tissues, cell types, or cell wall layers. In addition, this triple labelling was implemented with different classes of chemical reporters, using two monolignol reporters in conjunction with alkynylfucose to simultaneously monitor the biosynthesis of lignin and non‐cellulosic polysaccharides. This allowed observation of their deposition occurring contemporaneously in the same cell wall.