Cassava (Manihot esculenta Crantz Family Euphorbiaceae) is an important tropical food crop. However, harvested cassava roots have a shelf-life of only days due to post-harvest physiological deterioration (PPD). Within 1-3 days of harvesting, the roots show blue-black vascular streaking and are unpalatable. PPD includes altered gene expression and the accumulation of hydroxycoumarin secondary metabolites, e.g. scopoletin and esculetin, and their respective glucosides scopolin and esculin. In this research several important aspects of the biosynthesis of these phytochemically important hydroxycoumarins were resolved.
Stable isotopically labelled intermediates on the postulated biosynthetic pathways of scopoletin were fed to cassava cubes and PPD was allowed to occur. Ethanolic extracts of these deteriorated roots were separated (HPLC) and analysed (HRESI-MS). Incorporation (in both scopoletin and scopolin) of only 3 deuterons from E-cinnamic-2,3,2',3',4',5',6'-d7 and E-cinnamic-3,2',3',4',5',6'-d6 is strong support that the E-Zisomerisation step is enzymatic and not photochemical. There are three hypothetical pathways for the biosynthesis of scopoletin via: 2',4'-dihydroxycinnamate, caffeate, or ferulate. High incorporation of label from p-coumaric-2-13C, caffeic-2-13C and ferulic-2-13C acids was observed into labelled scopoletin and scopolin while there was only a small incorporation from 18O-umbelliferone and 18O-esculetin. We conclude that the major biosynthetic pathway to scopoletin and scopolin is via ferulic acid.
C18O2-enrichment of E-cinnamic and ferulic acids and feeding gave scopoletin containing only one 18O-labelled oxygen atom. Therefore the lactonisation step is through o-hydroxylation and not via a postulated spirolactone-dienone intermediate. These results were confirmed by feeding experiments in an atmosphere of 18O2-air which showed that the major isotopic peak was 18O3-enriched scopoletin.
Three glucosyltransferases were isolated and identified from a cassava PPDrelated cDNA library. These genes are expressed in the cassava storage root during PPD and they are also expressed in the fresh root. While one of these glucosyltransferases was novel, two had previously been isolated from cassava cotyledons.
|Date of Award||1 Oct 2008|
|Supervisor||Ian Blagbrough (Supervisor), Michael Rowan (Supervisor) & John Beeching (Supervisor)|
- post-harvest physiological deterioration
- isotopic labelling