Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.651773
Title: Apoplastic ascorbate metabolism in rose cell suspension cultures
Author: Green, Martha Alexandra
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Abstract:
Endogenous intraprotoplasmic ascorbate in rose cell suspension cultures as a model system ranged from 0.05 mmol kg-1 in 0-d-old cultures to 1.1 mmol kg-1 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-[1-14C]ascorbate was almost completely consumed (metabolised and/or taken up) by rose cultures within 8 hours of administration. Total 14C 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 [1-14C]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 14C-metabolites were identified as dehydroascorbate, diketogulonate and oxalate. Other acidic 14C-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. 14C-F was converted to [14C]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. [14C]Oxalate was produced from 14C-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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.651773  DOI: Not available
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