An in situ study of the cell wall utilising atomic force microscopy

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NO FULL TEXT AVAILABLE. Access is restricted indefinitely. ----- The cell wall is a complex structurally dynamic component of the plant cell partaking in cellular events by undergoing controlled structural modifications. An understanding of the role (or response) of the cell wall is limited by our knowledge of its architecture. Most cell wall studies have been conducted on isolated cell walls, which could be very different to the ‘real thing’, as these samples are inactive metabolically, an essential requirement for cell wall modifications. An important step in understanding the cell wall is therefore to conduct in situ studies. The most direct way to study the cell wall is via a high resolution microscopy. We have utilised the advantages of atomic force microscopy (AFM) to provide a unique structural study of in situ cell walls which were compared with data from isolated cell wall samples. We measured changes in the cell wall elasticity, Eᴡ, using the AFM’s nanomechanical probing ability and also studied the response of the cell wall during a cellular event in real-time. The objective of this work was to further progress our understanding of the cell wall network and its response by conducting in situ studies. The studies concentrated on the cell wall’s response during hypo-osmotic shock. We limited potential complications by studying the cell wall at the single cell level, and on a species which has been well characterised in its response to hypo-osmotic shock using other techniques. Our study sample was the green alga Ventricaria ventricosa (V. ventricosa) Olsen West. In this study of the cell wall architecture, the AFM images revealed that the native cell wall of V. ventricosa was abundant in matrix polysaccharides, not readily observed on isolated cell wall samples. We found that the V. ventricosa cell wall was cross-fibrillar throughout, unlike what has been found on its close relative Valonia macrophysa. This may suggest differences in the evolutionary lineages of these two species. The images revealed that the matrix polysaccharides of the native wall could exist in different phases from a glutinous fibrillar phase to a solidified fibrillar phase. A novel finding in this study was the thin fibrillar structures that formed bands across the surface of the cellulose microfibrils (CMF). These structures are possibly xyloglucan-like polymers that are seen in the cell walls of plant cells. As these structures were not seen in other studies, it suggests that they are susceptible to cell wall isolation and sample preparation techniques. In these studies, it was shown that the live cell surface type played a significant role in the cell’s response to hypo-osmotic shock; whether it was with or without mucilage, a hardened or pliable wall, all types responded differently. The structural modifications seen in the cell wall network, along with the corresponding changes in the cell wall mechanical properties provided evidence for the onset of turgor driven expansion followed by cell wall hardening. These studies were able to identify different phases of cell wall modifications, suggesting that the cell wall responded independently of the plasmalemma’s mechanical status.
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