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We tested two hypotheses for the mechanism by which xyloglucan-pectin covalent bonds are formed in Arabidopsis cell cultures. Hypothesis 1 proposed hetero-transglycosylation, with xyloglucan as donor substrate and a rhamnogalacturonan-I (RG-I) side-chain as acceptor. We looked for enzyme activities that catalyse this reaction using alpha-(1--5)-L-[(3)H]arabino- or beta-(1--4)-D-[(3)H]galacto-oligosaccharides as model acceptor substrates. The (3)H-oligosaccharides were supplied (with or without added xyloglucans) to living Arabidopsis cell-cultures, permeabilised cells, cell-free extracts, or four authentic XTHs. No hetero-transglycosylation occurred. Therefore, we cannot support hypothesis 1. Hypothesis 2 proposed that some xyloglucan is manufactured de novo as a side-chain on RG-I. To test this, we pulse-labelled Arabidopsis cell-cultures with [(3)H]arabinose and monitored the radiolabelling of anionic (pectin-bonded) xyloglucan, which was resolved from free xyloglucan by ion-exchange chromatography. [(3)H]Xyloglucan-pectin complexes were detectable4 min after [(3)H]arabinose feeding, which is shorter than the transit-time for polysaccharide secretion, indicating that xyloglucan-pectin bonds were formed intra-protoplasmically. Thereafter, the proportion of the wall-bound [(3)H]xyloglucan that was anionic remained almost constant at approximately 50% foror =6 days, showing that the xyloglucan-pectin bond was stable in vivo. Some [(3)H]xyloglucan was rapidly sloughed into the medium instead of becoming wall-bound. Only approximately 30% of the sloughed [(3)H]xyloglucan was anionic, indicating that bonding to pectin promoted the integration of xyloglucan into the wall. We conclude that approximately 50% of xyloglucan in cultured Arabidopsis cells is synthesised on a pectic primer, then secreted into the apoplast, where the xyloglucan-pectin bonds are stable and the pectic moiety aids wall-assembly.

Land plant cell wall glycan epitopes are present in Fucus vesiculosus. RG-I/AG mAbs recognize distinct glycan epitopes in structurally different galactans, and 3-linked glucans are also present in the cell walls. Cell wall-directed monoclonal antibodies (mAbs) have given increased knowledge of fundamental land plant processes but are not extensively used to study seaweeds. We profiled the brown seaweed Fucus vesiculosus glycome employing 155 mAbs that recognize predominantly vascular plant cell wall glycan components. The resulting profile was used to inform in situ labeling studies. Several of the mAbs recognized and bound to epitopes present in different thallus parts of Fucus vesiculosus. Antibodies recognizing arabinogalactan epitopes were divided into four groups based on their immunolocalization patterns. Group 1 bound to the stipe, blade, and receptacles. Group 2 bound to the antheridia, oogonia and paraphyses. Group 3 recognized antheridia cell walls and Group 4 localized on the antheridia inner wall and oogonia mesochite. This study reveals that epitopes present in vascular plant cell walls are also present in brown seaweeds. Furthermore, the diverse in situ localization patterns of the RG-I/AG clade mAbs suggest that these mAbs likely detect distinct epitopes present in structurally different galactans. In addition, 3-linked glucans were also detected throughout the cell walls of the algal tissues, using the β-glucan-directed LAMP mAb. Our results give insights into cell wall evolution, and diversify the available tools for the study of brown seaweed cell walls.

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.