A Versatile Click-Compatible Monolignol Probe to Study Lignin Deposition in Plant Cell Walls

Abstract

Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly.

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

“…In principle, dual labeling studies with our approach can also be extended to other biomolecules by using two distinct modified precursors of plant cell wall polymers - including all three main monolignols or their metabolic precursors as well as various monosaccharides that constitute the polysaccharide matrix. Since its inception, bioorthogonal chemistry has indeed been mainly developed to investigate glycans/polysaccharides through metabolic oligosaccharide engineering (MOE) 4 5 17 28 , but surprisingly there have been only very few applications to plant biology so far 7 8 9 10 11 12 . In terms of compatibility of the reactions, the study of lignin was indeed a complex case to solve as both chemical reporters are incorporated into the same reticulated polymer.…”

“…Despite the fast-growing popularity of this powerful method in bacterial and animal cells, reports describing its use in plant biology are surprisingly few and far between 7 8 9 10 11 12 . We were particularly interested in applying this strategy in plants to study the formation of lignin, one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass.…”

Regulation of gene expression in roots of the pH-sensitive Vaccinium corymbosum and the pH-tolerant Vaccinium arboreum in response to near neutral pH stress using RNA-Seq

“…Primary cell walls are composed of cellulose microfibrils and structural proteins [ 69 ]. Secondary cell walls are less flexible than primary cell walls and contain lignin [ 70 ]. In this experiment, VA and VC exhibited limited DE of genes involved in primary cell wall deposition when grown at higher pH.…”

Plant cell wall imaging by metabolic click‐mediated labelling of rhamnogalacturonan II using azido 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid

“…Click chemistry was recently used to label cells undergoing DNA synthesis while following a Golgi protein marker fused to a fluorescent protein (Bourge et al ., ). Several recent studies have reported the imaging of plant cell wall components using a click chemistry approach and two groups have recently reported the imaging of plant cell wall lignification using different azido‐ and alkynyl‐tagged monolignol analogues (Bukowski et al ., ; Tobimatsu et al ., ; Pandey et al ., ). In addition, an alkyne derivative of L‐fucose (Fuc‐Al) has been used for cell wall labelling in Arabidopsis thaliana (Anderson et al ., ).…”