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The Edinburgh Cell Wall Group |
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The Edinburgh Cell Wall Group's objective is to study the biochemistry and physiology of plant cell walls, and associated metabolism, from several points of view, including the following:
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Changes in the mechanical properties of primary cell walls ('loosening' and 'tightening') are evoked by auxins, gibberellins, ethylene and other hormones. These changes play a major role in governing the rate and pattern of plant growth and development. The Edinburgh Cell Wall Group is investigating the chemical basis of these physical changes in the cell wall. We discovered an interesting new enzyme activity, xyloglucan endotransglycosylase (XET), which breaks and re-forms glycosidic bonds in the backbone of xyloglucan, a structural polysaccharide of primary cell walls. XET activity is now widely regarded as a prime candidate for playing a key role in wall loosening during cell expansion and fruit ripening. The properties of this enzyme, and its role in reversibly loosening the wall, are under intensive investigation. Following our discovery of XET, molecular biological studies in other labs have led to the discovery of at least 33 different XET-related genes in Arabidopsis. Edinburgh Cell Wall Group is now particularly interested in understanding the enzymological properties of these isoenzymes of XET and in monitoring qualitatively and quantitatively the reactions catalysed by XETs in the walls of living plant cells. The latter undertaking is a particular challenge because the substrates of XET are often chemically indistinguishable from the products; our approach is to use sophisticated 13C/2H/3H-pulse-chase experiments so that we can detect XET action by physical changes (in buoyant density).
Within this subject area, we are investigating the mode of action of oxaziclomefone, a novel herbicide that appears to act by specifically inhibiting cell expansion.
Opinions on how plants loosen their walls have until recently focused exclusively on molecular modifications catalysed by wall-bound proteins. In a new departure, our work in this area has recently advanced to include a consideration of the possible contribution of non-enzymic scission of polysaccharides in the walls of living cells. This study emerged from our finding that the major cell wall polysaccharides are highly susceptible to scission by hydroxyl radicals (•OH) and that •OH is readily produced under physiological conditions of temperature and pH by reactions involving apoplastic solutes. A major new focus of the Edinburgh Cell Wall Group is therefore on determining whether •OH is generated in the apoplast in vivo and on whether wall-bound polysaccharides are thereby degraded.
A possible mechanism whereby hydroxyl radicals are generated in the apoplast in vivo is by the non-enzymic oxidation of ascorbate to form Cu+ and H2O2, which together generate •OH radicals via the Fenton reaction. We are therefore also monitoring the metabolism of ascorbate in the apoplast in vivo. This work involves the use of radiolabelled ascorbate and of its precursors.
The Edinburgh Cell Wall Group is investigating the synthesis and assembly of the primary cell wall, i.e. the dynamic process by which newly secreted polysaccharides and glycoproteins become integrated into the growing plant cell wall. Molecules of non-cellulosic polymers become held together by cross-links, which we are attempting to identify. Our work concerns both covalent and non-covalent cross-links.
Work on non-covalent bonding centres on the hypothesis that xyloglucan chains act as molecular tethers between neighbouring cellulosic microfibrils within the cell wall. Our discovery of XET (see above) has suggested an interesting new means (interpolymeric transglycosylation) by which newly secreted xyloglucan chains could become integrated into the cell wall. We are also using 13C/2H/3H-pulse-chase experiments to test this.
Work on covalent cross-links is based on the hypothesis that some or all of the following bonds contribute to the coherency of the primary cell wall:
Our work on cross-links formed by oxidative phenolic coupling has recently progressed to a study of the in vivo site and kinetics of biosynthesis. This work is revolutionising thinking about this topic by showing that under certain physiological circumstances the cross-linking reaction occurs principally in the endo-membrane system (probably Golgi bodies) rather than in the cell wall itself; thus the polysaccharides are secreted into the cell wall already cross-linked.
For the discovery of novel cross-links, our general approach for each putative covalent cross-link is to synthesise chemically a range of model compounds of the type under investigation, characterise these (especially with respect to their chromatographic properties and resistance to chemical and enzymic treatments), and then to look for the occurrence of these and related substances in enzymic digests of plant cell walls. The chemical characterisation of these novel (synthetic and natural) compounds has recently been promoted by 2-dimensional NMR analysis made possible by a productive collaboration with Dr I.H. Sadler, Dept of Chemistry.
The Edinburgh Cell Wall Group has recently begun a study of the in vivo inter-conversion of the precursors of wall polysaccharide biosynthesis. There are several examples where two or more putative pathways lead to the same important metabolite (e.g. UDP-glucuronate and GDP-mannose); we are employing techniques of in-vivo radiolabelling to dissect which of these apparently competing pathways predominate in the living cell. Attempts to solve this problem by use of gene knock-outs are confused by the remarkable ability of plant cells to compensate for the lack of one pathway by exploiting an alternative pathway - one which does not necessarily occur to any appreciable extent in the normal cell.
Specific oligosaccharides, produced from cell wall polysaccharides e.g. pectins and xyloglucan, are known to exert diverse and potent physiological effects on living plant cells. Such oligosaccharides are termed oligosaccharins. our work in this area focuses on the synthesis, degradation and transport of oligosaccharins in vivo as well as their biological effects. Recent biosynthetic studies have revealed the natural production of a novel oligosaccharin that appears to be derived from glycosphingolipids, a poorly understood group of glycolipids. This has opened an interest in these particular glycolipids - their occurrence, structure and biological significance.
Work has begun to trace the changes in primary cell wall composition that have occurred during the evolution of land plants (embryophytes) from their hypothetical algal ancestors (charophytes) and during the diversification of land plants from bryophyte-like ancestors. Our recent results show that major events in plant evolution were often accompanied by remarkable changes in cell wall chemistry, indicating a special role for cell wall modification in adapting plants to their newly colonised environments.