Investigation of Biosynthetic Pathways to Hydroxycoumarins During Post‐Harvest Physiological Deterioration in Cassava Roots by Using Stable Isotope Labelling

Cassava (Manihot esculenta Crantz) is an important starch-rich crop, but the storage roots only have a short shelf-life due to post-harvest physiological deterioration (PPD), which includes the over-production and polymerisation of hydroxycoumarins. Key aspects of coumarin secondary-metabolite biosynthesis remain unresolved. Here we exploit the accumulation of hydroxycoumarins to test alternative pathways for their biosynthesis. Using isotopically labelled intermediates (p-coumarate-2-13C, caffeate-2-13C, ferulate-2-13C, umbelliferone-2-18O and esculetin-2-18O), we show that the major biosynthetic pathway to scopoletin and its glucoside, scopolin, in cassava roots during PPD is through p-coumaric, caffeic and then ferulic acids. An alternate pathway through 2′,4′-dihydroxycinnamate and umbelliferone leads to esculetin and esculin. We have used C18O2-carboxylate-labelled cinnamic and ferulic acids, and feeding experiments under an atmosphere of 18O2, to investigate the o-hydroxylation and cyclisation steps. We demonstrate that the major pathway is through o-hydroxylation and not via a proposed spirolactone-dienone intermediate.