Lignin is an important polymer of many plant cell walls providing specific mechanical and chemical properties necessary for the correct biological functioning of different tissues including xylem and sclerenchyma. In Angiosperms, the lignin polymer is usually composed of 3 main monomeric units or monolignols: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol that are enzymatically synthesized by plant cells and give rise, respectively to lignin H (p-hydroxypenyl), G (guaiacyl) and S (syringyl) units. Since the quality of lignocellulose biomass is affected by the quantity and chemical composition of lignin it is important to obtain detailed knowledge about how this polymer is incorporated into the growing cell wall. Fluorescence-tagged monolignols were previously developed for studying lignin formation [1], but present the disadvantage of the tag being larger than the tagged molecule thereby potentially interfering with lignification. More recently, coniferyl alcohol monolignols tagged with clickable azide or alkyne tags have been used in a bioorthogonal chemical reporter strategy on the model species Arabidopsis [2], [3]. In this strategy, an analogue of the biomolecule of interest is modified with a biocompatible chemical function and is metabolically incorporated into the target biomacromolecule where it functions as a ‘chemical reporter’. The incorporated chemical reporter is then visualized by fluorophore tagging initiated by a bioorthogonal chemistry reaction. We have previously [4], [5] 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 (SPAAC/CuAAC). Here we report the successful utilization of the Diels-Alder Reaction with inverse electronic demand (DARinv) to label lignin S-units and its incorporation into a triple labeling strategy that enables us to co-visualize the dynamic incorporation of all of the major lignin monomers into the cell walls of different plant species [6].

One, two three, a triple chemical reporter strategy for studying lignification in plant cell walls

2019 Spring National Meeting

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