Endogenous intraprotoplasmic ascorbate in rose cell suspension cultures as a model system ranged from 0.05 mmol kg<sup>-1</sup> in 0-d-old cultures to 1.1 mmol kg<sup>-1</sup> in 5-d-old cultures. Apoplastic ascorbate was estimated as 0.5 and 8 μM in 0- and 5-d-old cultures respectively, indicating that ascorbate is endogenous to, and may be metabolised within, the apoplast. Exogenous (apoplastic) 1 mM L-[1-<sup>14</sup>C]ascorbate was almost completely consumed (metabolised and/or taken up) by rose cultures within 8 hours of administration. Total <sup>14</sup>C was removed from medium but slower than ascorbate. The calculated concentration of metabolites of ascorbate showed that metabolites were formed in the medium and then removed from the medium in 5-d-old cultures. Removal of metabolites could be due to either uptake by or binding to cells. The nature of the metabolites of 0.5 mM [1-<sup>14</sup>C]ascorbate was examined in 5-d-old rose culture and spent medium by electrophoresis at pH 6.5. Ascorbate was metabolised both enzymically in spent medium and non-enzymically in boiled spent medium. Three <sup>14</sup>C-metabolites were identified as dehydroascorbate, diketogulonate and oxalate. Other acidic <sup>14</sup>C-metabolites (C, D, E and F) have not as yet been identified. F is highly mobile during electrophoresis at pH 2.0, showing that it has a low pK. C, D and E are also mobile at pH 2.0 but less so than F. E and C are interconvertible non-enzymically during storage and can E be regenerated by treatment with NaOH, suggesting that C is a lactone of E. <sup>14</sup>C-F was converted to [<sup>14</sup>C]oxalate by whole culture and by spent medium but not by boiled spent medium, indicating an enzyme-catalysed reaction. The enzyme was partially inhibited by 100 mM azide but not by antioxidants. [<sup>14</sup>C]Oxalate was produced from<sup> 14</sup>C-F by alkali hydrolysis indicating the presence of an oxalyl ester group. The metabolism of apoplastic ascorbate, described in this thesis, is very different from its intraprotoplasmic metabolism. I have identified novel metabolites and propose a novel pathway for the metabolism of apoplastic ascorbate.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:651773 |
| Date | January 2003 |
| Creators | Green, Martha Alexandra |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14944 |
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