Cassava (Manihot esculenta Crantz) is an important tropical crop which provides a large portion of daily calories intake to hundreds of millions of people in Africa, Latin America, and tropical Asia. Cassava is grown for its starchy storage roots as staple food, as animal feed, and as industrial raw material. The utilisation of cassava is hindered by its characteristic physiological response, the post-harvest physiological deterioration (PPD). The inevitable wounding caused during harvesting and handling will trigger a series of physiological responses within 24 to 48 hours, which causes a blue-black discoloration in the storage roots, rendering these roots unmarketable and unpalatable in a few days. During the PPD response large amount of phenylpropanoid compounds, especially scopoletin and its glycoside, accumulate in the roots. Scopoletin may play an important role in PPD development but little work has been done on the possible relationship. Here we aim to examine the effects of altering scopoletin synthesis in cassava roots on the PPD response. In Arabidopsis thaliana, gene F6’H1 (feruloul CoA 6’-hydroxylase 1) is indispensable in the biosynthesis of scopoletin. Cassava F6’H1 candidate gene family involved in scopoletin synthesis were identified by their ability to functionally complement F6’H1 T-DNA insertion mutation in Arabidopsis thaliana that prevented synthesis of scopoletin. RNAi constructs targeting the identified cassava F6’H1 candidate gene family were designed, under the control of either constitutive CaMV 35S or root-specific StPAT promoters. These were used to transform wild-type cassava to down-regulate the expression of these scopoletin synthetic genes in F6’H1 gene family. The inhibition of cassava F6’H1 candidate gene expression and thus the scopoletin synthesis in transgenic cassava roots were confirmed by qRT-PCR and LC-MS, respectively. The RNAi transgenic cassava lines show less scopoletin accumulation and inhibited F6’H1 candidate genes expression during the PPD response. A reduced PPD discoloration development compared to that of the wild-type was also observed in the RNAi transgenic cassava lines.

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.

The rapid postharvest deterioration of the roots, known as postharvest physiological deterioration (PPD), has been a major problem to the utilisation and development of cassava (Manihot esculenta Crantz) as a food and industrial crop. PPD usually occurs within two to three days after harvest and it is characterised by a blue-black discoloration of the roots, which renders the roots unpalatable and unmarketable. Scopoletin, which is synthesised de novo and released from its glucoside, scopolin, during PPD, plays a central role in this discoloration response. Interconversion of scopoletin and scopolin, which is catalysed by scopoletin-glucosyltransferase (scopoletin-GT) and scopolin-beta-glucosidase (scopolin-BG), regulates homeostasis of scopoletin in the cells. However, how this interconversion contributes to root discoloration development is poorly understood. In the present study, we identified and characterised cassava genes for the enzymes that are responsible for the interconversion of scopoletin and scopolin, subsequently manipulated their expression in transgenic cassava through scopoletin-GT RNAi gene silencing and scopolin-BG overexpression constructs. These approaches would potentially alter scopoletin and scopolin content in the root, thereby affecting PPD response. A BLAST search for homologous cassava genes revealed that scopoletin-GT and scopolin-BG are encoded by multiple genes, most of which belong to glucosyltransferase family-1 (GT1) and glycosyl hydrolase family-1 (GH1), respectively. Scopoletin-GT-down-regulated and scopolin-BG overexpressed transgenic cassava lines showed reduced not only scopolin but surprisingly also scopoletin, and delayed PPD. Additionally, other coumarins esculetin and esculin were also identified and both scopoletin-GT, MeSGT1, and scopolin-BG, BGLU23, were up-regulated during PPD development at day 4 and day 2, respectively. Our study reveals that disrupting the interconversion of scopoletin and scopolin by inhibiting scopoletin-GT and overexpressing scopolin-BG led to the decrease of both scopoletin and scopolin content and delayed PPD in cassava. These findings provide useful insights into the role of interconversion of scopoletin and scopolin in cassava PPD response and may suggest alternative ways to tackle PPD.