I have developed a method which allows the existence of an intermolecular diferuloyl linkage to be tested. Plant cell-suspension cultures were fed a radiolabelled ferulic acid precursor, (E)-[U-<sup>l4</sup>C]cinnamic acid, and maize was shown to have the <sup>14</sup>C-incorporation of into polymer-esterified groups. More [<sup>14</sup>C]diferulate formed in the cell wall of older cultures than in young cultures, where intraprotoplasmic coupling was prevalent. I was also able to detect a putative [<sup>14</sup>C]hydroxycinnamoyl-CoA, a donor molecule for feruloylation, in 2-d-old maize cell-suspension cultures up to 8 min after [<sup>14</sup>C]cinnamic acid feeding. Iodide diminished dimerisation taking both intraprotoplasmically and within the cell wall. The effect of KI or NaI could be reversed and dimerisation increased with its removal and addition of H<sub>2</sub>O<sub>2. </sub><sup>14</sup>C-Feruloylated polysaccharides secreted in the presence of NaI or KI passed through the cell wall directly into the culture medium, where they accumulated as soluble extracellular polysaccharides. Inhibition of diferulate formation may prevent the integration of newly synthesised arabinoxylans into the cell wall as diferulate cross-links cannot form to integrate the polysaccharides into the wall, suggesting that an intermolecular diferuloyl linkage may act as an anchorage point. Feeding cells with [U-<sup>13</sup>C]glucose in the presence of NaI or KI for 24 h allows the formation of a <sup>12</sup>C/<sup>13</sup>C interface within the cell wall. Removal of the inhibitor and addition of H<sub>2</sub>O<sub>2</sub> would allow the formation of diferulate groups across the <sup>12</sup>C/<sup> 13</sup> interface, if intermolecular dimerisation is possible <i>in vivo.</i> I have developed a method of extraction and purification of fragments of the isotopically labelled cell wall, namely feruloyl-arabinose and diferulic acids, which allow the existence of an intermolecular diferuloyl linkage to be tested. To monitor the incorporation of carbon from exogenous glucose, I fed [U-<sup>14</sup>C]glucose to cell-suspension cultures. Autoradiography and scintillation counting of the isolated cell wall fragments have shown the incorporation of radioactivity into the groups of interest, namely feruloyl-arabinose and diferulate, confirming that glucose was a suitable compound in which to deliver the <sup>13</sup>C. Preliminary results from this method with both [<sup>14</sup>C]glucose and [<sup>13</sup>C]glucose gave some evidence, such as the secretion of <sup>l4</sup>C-feruloylated polysaccharides in the presence of an H<sub>2</sub>O<sub>2 </sub>scavenger and the detection of cell wall fragments into which <sup>13</sup>C<sup> </sup>had been incorporated, for an intermolecular diferuloyl linkage to exist and that my approach is a suitable method to detect such linkages.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:653923 |
| Date | January 2006 |
| Creators | Lindsay, Shona Elaine |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15218 |
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