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

Abstract 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‐ 13 C, caffeate‐2‐ 13 C, ferulate‐2‐ 13 C, umbelliferone‐2‐ 18 O and esculetin‐2‐ 18 O), 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 C 18 O 2 ‐carboxylate‐labelled cinnamic and ferulic acids, and feeding experiments under an atmosphere of 18 O 2 , 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.