Dynamic imaging of cell wall polysaccharides by metabolic click‐mediated labeling of pectins in living elongating cells

Abstract

Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.

Metabolic glycoengineering – exploring glycosylation with bioorthogonal chemistry

“…MGE is a suitable tool for imaging plant structures, even in living plants. 231 First, FucAl was successfully incorporated into Arabidopsis roots. 232 This gave new insights into the development of pectin structures.…”

Plant cytokinesis and the construction of new cell wall

“…The generation of probes that allow live imaging of the cell wall [118], such as Pontamine Fast Scarlet 4B for cellulose [119], aniline blue fluorochrome for callose, fluorophore-functionalized chitosan oligosaccharides (COS) and metabolic click-mediated labelling for pectin [120][121][122], are critical to better understand polysaccharide deposition and assembly in the cell wall. Overcoming the major bottleneck of live polysaccharide imaging along with the biosynthetic enzymes during cytokinesis can help understand the complex regulation of polysaccharide transport, deposition and assembly into the cell plate and new cell wall.…”

Exploring Lignification Complexity in Plant Cell Walls with Airyscan Super-resolution Microscopy and Bioorthogonal Chemistry

“…Coined bioorthogonal reactions, these ligations often fullfill the criteria of click chemistry as they are high-yielding and exhibit fast kinetics, and mostly belong to the cycloaddition family. Bioorthogonal chemistry has been applied successfully to diverse biological models over the past 20 years − and has greatly facilitated in vivo , ex vivo , and in situ studies of the main classes of biomolecules such as glycans, , lipids, nucleic acids, and proteins exploiting a wide range of bioanalytical and bioimaging readout techniques. , Although many notable examples have been described in mammalian living systems, , there are surprisingly few reports in plants, − despite the fact that they represent 82% of all living matter on earth and make up the first economic resource in terms of nutrition, renewable materials and renewable energies. Lignin is one of the main constituents of plant cell walls, with cellulose.…”