Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Abstract

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

One, Two, Three: A Bioorthogonal Triple Labelling Strategy for Studying the Dynamics of Plant Cell Wall Formation In Vivo

“…Subsequent confocal fluorescence microscopy observation indicates that S CP 6 is incorporated in a time‐ and dose‐dependent manner into the lignifying walls of differentiating secondary xylem cells but is not/poorly incorporated into already lignified mature cell walls (Figure ). This labelling pattern is in agreement with our previous observations of the alkyne‐ and azide‐tagged G‐ and H‐unit reporters 4 and 5 , respectively . Additional experiments showed that S CP incorporation is 1) abolished by competition with the natural S monolignol 3 and 2) almost completely eliminated in samples when the enzymatic machinery is heat denatured prior to incubation, indicating that S CP incorporation is under enzymatic control (see Figures S3 and S4).…”

“…Results showed that DAR inv is orthogonal to the other two reactions as S CP is fully converted into the corresponding dihydropyridazine cycloadduct while H AZ and G ALK remain untouched. After full conversion of S CP , specific SPAAC cycloaddition between H AZ and DBCO‐PEG 4 ‐Rhodamine Green occurs without degradation of either the S CP cycloadduct or of the remaining G ALK reporter in the mixture, and can in turn be linked to TAMRA‐azide by CuAAC as previously reported . Based on these results we developed an in vivo, sequential triple‐labelling protocol to visualize lignification in flax plant cell walls.…”

“…As expected, the CuAAC labelling of G ALK units must be performed last, after removal of the excess cyclooctyne‐ and tetrazine‐activated probes from the biological sample. This protocol avoids possible competitive G ALK ‐ H AZ cross‐linking that may result in signal loss, as well as side‐reactions of the tetrazine‐functionalized probe in the presence of copper sulfate that may result in strong background . Our results (Figure ; see Figure S6) show that the triple‐labelling protocol can be successfully used to visualize H‐, G‐, and S‐monolignol reporter incorporation into actively lignifying cell walls in differentiating flax secondary xylem using the two experimental systems we previously reported (i.e., flax stem sections as an easily implemented model, or entire flax stems as a relevant in vivo system).…”

UDP-GLYCOSYLTRANSFERASE 72E3 Plays a Role in Lignification of Secondary Cell Walls in Arabidopsis

“…Chemical reporters H az , G alk and S cp were synthesized as previously described [ 36 , 61 , 62 ]. Natural and tagged monolignols were metabolically incorporated in the plant samples at a final concentration of 5 µM for 20 h ( Figure S5 ).…”

Enzymatic Preparation and Characterization of Spherical Microparticles Composed of Artificial Lignin and TEMPO-Oxidized Cellulose Nanofiber

“…Meanwhile, lignin is an aromatic polymer formed via enzymatic radical coupling of several tautomeric isomers of phenylpropanoid monolignols [ 18 ]. Lignin in wood absorbs ultraviolet light and endows the wood architecture with hydrophobicity through accumulation between the cell walls, known as lignification [ 18 , 19 ]. Lignin is also enzymatically degraded by the action of white-rot fungi [ 20 , 21 ].…”