Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

Sun, Qiang; Sun, Yuliang; Juzenas, Kevin

doi:10.1093/jxb/erx103

Cited by 19 publications

(14 citation statements)

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“…The use of BSE detection for enhanced detection of nanogold in FESEM imaging has been reported before (Goldberg and Fiserova, ) and our results extend this approach to cell wall analysis. A recent study (Sun et al ., ) used immunogold labelling of xylem cell wall surfaces, followed by silver enhancement, which was necessary because their SEM could not resolve nanogold particles (FESEM has higher resolution than standard SEM). Sun et al .…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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“…The current study used a different technique, immunogold-SEM technique (Sun et al, 2017), to analyze polysaccharide composition of intervessel PMs. This technique also takes advantage of the high sensitivity and specificity of cell wall mAbs in detecting pectic and hemicellulosic polysaccharides but uses SEM to localize target polysaccharides over a large or the whole surface area of cell wall structures.…”

Section: Discussionmentioning

confidence: 99%

“…These techniques have revealed important information about cell wall chemical composition but showed major limitations when comprehensive analyses on the structures and polysaccharide compositions of intervessel PMs are needed (to be described in detail in the Discussion). In this study, we used an immunogold-scanning electron microscopy (SEM) protocol (Sun et al, 2017), which combines an immunocytochemical technique with SEM. The SEM can reveal, at different degrees of resolution, surface structures (intact surface and exposed underlayers) of intervessel PMs as well as those on sections.…”

Section: Introductionmentioning

confidence: 99%

“…The SEM can reveal, at different degrees of resolution, surface structures (intact surface and exposed underlayers) of intervessel PMs as well as those on sections. This immunogold-SEM protocol has been proved powerful in exploring the structures, polysaccharide compositions and functions of cell walls all together (Sun et al, 2017). With an expanding list of monoclonal antibodies (mAbs) and carbohydrate-binding modules (CBMs) for cell wall polysaccharide detection, this technique offers an excellent approach to visualize a large variety of polysaccharides (Willats et al, 2001; McCartney et al, 2004; Pattathil et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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Structural Variation and Polysaccharide Profiling of Intervessel Pit Membranes

Sun

2021

Preprint

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Functional roles of intervessel pit membrane (PM) depend on its structure and polysaccharide composition, which are mostly unknown or lack of accurate information. This study uses grapevine as a model plant and an immunogold-scanning electron microscopy technique to simultaneously analyze structures and polysaccharide compositions of intervessel PMs in relation to their functions. Intervessel PMs with different structural integrity were found in functional xylem with about 90 % of them being intact with a smooth or relatively smooth surface and the rest 10 % with progressively degraded structures. The results also elucidated details of the removal process of wall materials from surface toward its depth during the natural intervessel PM degradation. Four groups of pectic and hemicellulosic polysaccharides were present in intervessel PMs but displayed differential spatial distributions and quantities: weakly methyl-esterified homogalacturonans abundant in the surficial layers, heavily methyl-esterified homogalacturonans and xylans mostly in deep layers, and fucosylated xyloglucans relatively uniform in presence at different depths of an intervessel PM. This information is crucial to reveal the polysaccharide profiling of primary cell wall and to understand intervessel PM's roles in the safety and regulation of water transport as well as the plant susceptibility to vascular diseases.

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“…Yet, direct assessments of their composition were based on low-spatial-resolution methods, such as ultraviolet microspectrophotometry (Bauch and Berndt, 1973;Sano and Fukuzawa, 1994;Schmitz et al, 2008) and x-ray spectromicroscopy (Boyce et al, 2004). Indirect methods employed electron microscopy based on staining techniques (Fineran, 1997;Fromm et al, 2003;Schenk et al, 2017) and immunocytochemistry (Sun et al, 2011(Sun et al, , 2013(Sun et al, , 2017Kim and Daniel, 2013;Herbette et al, 2015;Klepsch et al, 2016). During cell apoptosis, hydrolytic enzymes have been suggested to remove noncellulosic polysaccharides such as hemicellulose and pectin (O'Brien, 1970;Kim and Daniel, 2013;Herbette et al, 2015;Klepsch et al, 2016), although there are contradictory results about noncellulosic compounds (Bamber, 1961;Sano and Fukuzawa, 1994;Fineran, 1997;Donaldson, 2001) such as lignin (Fromm et al, 2003;Boyce et al, 2004;Schmitz et al, 2008Schmitz et al, , 2012Herbette et al, 2015).…”

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confidence: 99%

Infrared Nanospectroscopy Reveals the Chemical Nature of Pit Membranes in Water-Conducting Cells of the Plant Xylem

et al. 2018

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In the xylem of angiosperm plants, microscopic pits through the secondary cell walls connect the water-conducting vessels. Cellulosic meshes originated from primary walls, and middle lamella between adjacent vessels, called the pit membrane, separates one conduit from another. The intricate structure of the nano-sized pores in pit membranes enables the passage of water under negative pressure without hydraulic failure due to obstruction by gas bubbles (i.e. embolism) under normal conditions or mild drought stress. Since the chemical composition of pit membranes affects embolism formation and bubble behavior, we directly measured pit membrane composition in wood. Here, we characterized the chemical composition of cell wall structures by synchrotron infrared nanospectroscopy and atomic force microscopy-infrared nanospectroscopy with high spatial resolution. Characteristic peaks of cellulose, phenolic compounds, and proteins were found in the intervessel pit membranes of wood. In addition, the vessel to parenchyma pit membranes and developing cell walls of the vascular cambium showed clear signals of cellulose, proteins, and pectin. We did not find a distinct peak of lignin and other compounds in these structures. Our investigation of the complex chemical composition of intervessel pit membranes furthers our understanding of the flow of water and bubbles between neighboring conduits. The advances presented here pave the way for further label-free studies related to the nanochemistry of plant cell components.

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Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

Abstract: HighlightA versatile immunogold scanning electron microscopy technique helps reveal the polysaccharide composition and architecture of cell walls of xylem elements.

Cited by 19 publications

References 69 publications

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

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

Structural Variation and Polysaccharide Profiling of Intervessel Pit Membranes

Infrared Nanospectroscopy Reveals the Chemical Nature of Pit Membranes in Water-Conducting Cells of the Plant Xylem

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