Xyloglucan in the primary cell wall: assessment by FESEM, selective enzyme digestions and nanogold affinity tags

Abstract

SUMMARYXyloglucan has been hypothesized to bind extensively to cellulose microfibril surfaces and to tether microfibrils into a load-bearing network, thereby playing a central role in wall mechanics and growth, but this view is challenged by newer results. Here we combined high-resolution imaging by field emission scanning electron microscopy (FESEM) with nanogold affinity tags and selective endoglucanase treatments to assess the spatial location and conformation of xyloglucan in onion cell walls. FESEM imaging of xyloglucanase-digested cell walls revealed an altered microfibril organization but did not yield clear evidence of xyloglucan conformations. Backscattered electron detection provided excellent detection of nanogold affinity tags in the context of wall fibrillar organization. Labelling with xyloglucan-specific CBM76 conjugated with nanogold showed that xyloglucans were associated with fibril surfaces in both extended and coiled conformations, but tethered configurations were not observed. Labelling with nanogold-conjugated CBM3, which binds the hydrophobic surface of crystalline cellulose, was infrequent until the wall was predigested with xyloglucanase, whereupon microfibril labelling was extensive. When tamarind xyloglucan was allowed to bind to xyloglucan-depleted onion walls, CBM76 labelling gave positive evidence for xyloglucans in both extended and coiled conformations, yet xyloglucan chains were not directly visible by FESEM. These results indicate that an appreciable, but still small, surface of cellulose microfibrils in the onion wall is tightly bound with extended xyloglucan chains and that some of the xyloglucan has a coiled conformation.

Diffuse Growth of Plant Cell Walls

“…Computational studies indicate that xyloglucan likewise preferentially binds to the hydrophobic face of cellulose (Zhao et al, 2014). This computational result is supported by a recent field emission scanning electron microscopy (FESEM)-based study showing that xyloglucan indeed covers the hydrophobic faces of cellulose microfibrils in onion (Allium cepa) cell walls (Zheng et al, 2017b). Complementing these results, recent experimental and computational studies indicate that substituted xylans in secondary cell walls may bind Figure 2.…”

“…Cellulose microfibrils are represented as thick rods with hydrophobic (blue) and hydrophilic faces (orange). Xyloglucan (green) is found in solvated, coiled conformations and in extended conformations bound to the hydrophobic faces of cellulose, based on Zheng et al (2017b). It is also depicted as entrapped between microfibrils.…”

Disentangling loosening from softening: insights into primary cell wall structure

“…Moreover, during AFM imaging wall samples can be kept fully hydrated, avoiding dehydration or freezing artefacts. In the future it may be possible to characterize the 3D organization of matrix polymers as well as cellulose microfibrils by use of multichannel nanomechanical mapping (Zhang et al ., , ) or high resolution scanning electron microscopy combined with gold‐labeled affinity tags (Zheng et al ., ).…”

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

“…However, this would not be expected for soluble polysaccharides, such as XyG, especially under dilute assay conditions in vitro. In the plant cell wall, XyG associates with crystalline cellulose microfibrils and other matrix glycans in an amorphous, hydrated state (63)(64)(65).…”