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Title: The synthesis, wall-binding and breakdown of hemicelluloses in maize cell-suspension cultures
Author: Kerr, Ellen M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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A method was developed to separate protoplasmic contents from the cell wall causing polymer degradation. Alkali treatment (6 M NaOH at 37°C) was shown then to solubilise essentially all remaining (wall-bound) 3H-polymers. Gel-permeation chromatography (GPC) on Sepharose CL-4B was used to size-fractionate the isolated 3H-polymers. Alkali treatments, similar to those employed to extract wall-bound 3H-hemicellulose, were found to minimise aggregation of xylan and xyloglucan caused by sample storage and such treatments were applied to 3H-polymers from each of the cell compartments. GPC-fractionated 3H-polymers was Driselase-digested to diagnostic 3H-fragments; maximisation of [3H]xylan susceptibility to Driselase required an acid pre-treatment. The data presented illustrate the synthesis and subsequent secretion of 3H-polymers from the protoplasm to the cell wall, where they become integrated before subsequent degradation or sloughing to the culture medium. Protoplasmic [3H]xylan and [3H]xyloglucan were polymerised from 40 kDa to 2 MDa before secretion to the cell wall, where the hemicelluloses further increased in size in over 2 MDa. Within the medium, [3H]xylan and [3H]xyloglucan were approximately 1-2 MDa until 2 d after the onset of 3H-labelling, when they dramatically increased in size, probably due to phenolic coupling. The Mr values observed were much higher than reported for rose hemicellulose (Thompson and Fry, 1997). Attempts to ‘disaggregate’ the very high-Mr wall-bound 3H-hemicelluloses, using different GPC eluents, alkali and enzymic digestion, were unsuccessful. Alkali, however, was successful in ‘disaggregating’ the very high-Mr soluble extracellular [3H]xylan and [3H]xyloglucan, suggesting that alkali-labile bonds, more resistant than feruloyl esters, play a significant part in the extracellular cross-linking of xylan and xyloglucan. The in vivo formation of high-Mr complexes of soluble extracellular 3H-polysaccharides xyloglucan. The in vivo formation of high-Mr complexes of soluble extracellular 3H-polysaccharides (SEPs) was delayed by sinapic acid, chlorogenic acid and rutin but promoted by ferulic acid and tyrosine: these data support a role for phenolic groups in the extracellular cross-linking of hemicelluloses.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available