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

Abstract

SUMMARYIn plants, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N 3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N 3 demonstrated that incorporation of Kdo-N 3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N 3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N 3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.

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

“…[11] Since lignin properties are influenced by monomer ratio and the nature of inter-unit bonds,improved knowledge of lignification would contribute to better understanding of plant physiology and factors affecting biomass quality. Although there are various reports of dual labelling methods that combine two biorthogonal reactions both in vitro and in vivo, [6,7,[13][14][15][16][17][18][19] the only instance where three such reactions were used concurrently within the same sample was based on asequence of CuAAC, DAR inv ,a nd Staudinger-Bertozzi ligation in an activity-based protein profiling assay in solution. [13] Theuse of two different cycloaddition reactions is necessary for dual lignin labelling as the monolignol mimics 4 and 5 are incorporated into the same polymer.B ecause of their spatial proximity,u sing consecutive CuAACl igations indeed leads to cross-linking reactions that compete with the desired probe ligation.…”

“…To develop at riple labelling strategy for the three main monolignols,wetested whether the Diels-Alder reaction with inverse electronic demand (DAR inv ) could be used to label the lignin S-unit and successfully combined with SPAACa nd CuAAC. Although there are various reports of dual labelling methods that combine two biorthogonal reactions both in vitro and in vivo, [6,7,[13][14][15][16][17][18][19] the only instance where three such reactions were used concurrently within the same sample was based on asequence of CuAAC, DAR inv ,a nd Staudinger-Bertozzi ligation in an activity-based protein profiling assay in solution. [20] Although three-color imaging using compatible reactions seems intuitively possible,tothe best of our knowledge it has never been applied to track three distinct molecules within asingle living sample.H erein, we demonstrate the feasibility of triple biorthogonal labelling in vivo by sequentially exploiting the major triad of biorthogonal reactions (DAR inv ,S PA AC,a nd CuAAC) to track the three main lignin monomers ( Figure 1).…”

The sialic acid transporter NanT is necessary and sufficient for uptake of 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo) and its azido analog in Escherichia coli

“…In addition to gut microbiome‐derived Kdo, there are dietary sources of Kdo. Plants produce and use Kdo sugars in the conserved and essential cell wall pectin, rhamnogalacturonan‐II (RG‐II) (Smyth and Marchant, ; M. Dumont et al, ) and in LPS‐like molecules (Li et al, ). If one or both gut sources of Kdo lead to free Kdo, it could be scavenged by NanT‐containing pathogens allowing them to survive inhibition of Kdo biosynthesis and perhaps contribute to antibiotic resistance.…”

“…In addition to gut microbiome-derived Kdo, there are dietary sources of Kdo. Plants produce and use Kdo sugars in the conserved and essential cell wall pectin, rhamnogalacturonan-II (RG-II) (Smyth and Marchant, 2013;M. Dumont et al, 2016) and in LPS-like molecules (Li et al, 2011).…”

“…In nearly all Gram‐negative bacteria, the first core sugar attached to lipid A is 3‐deoxy‐ d ‐ manno ‐oct‐2‐ulosonic acid (Kdo) (Raetz and Whitfield, ). Kdo is also biosynthesized by plants where it is found in the conserved and essential cell wall pectin, rhamnogalacturonan‐II (RG‐II) (Smyth and Marchant, ; M. Dumont et al, ), and may also be found in LPS‐like molecules, e.g. in Arabidopsis (Li et al, ).…”