Hydroxycoumarins in Cassava

Abstract

Manihot esculenta Crantz Euphorbiaceae (cassava) is the sixth most important crop in the world, feeding more than half a billion people around the world and is vital in addressing food security. A longer term aim of this research project is to contribute to the measures to curb PPD. The biosynthetic pathways leading to the production of hydroxycoumarins in cassava were investigated in wildtype and transgenic plants (1x, 2x, 3x) using synthesised stable isotopically-labelled E-cinnamic acids. The transgenic plants were created by knocking down the enzymes F6´H for 1x, F6´H and CCoAOMT for 2x and F6´H, CCoAOMT and COMT for 3x. It was observed in the feeding experiments in the transgenic roots that exogenously applied E-d₇-cinnamic acid could turn the activity of phenylalanine ammonia lyase off resulting in the increased uptake of the labelled substrate, E-caffeic-2-¹³C acid was distributed in the same manner in all the transgenic plants while E-ferulic-2-¹³C acid was not distributed at all in the transgenic plants leading to the conclusion that in the absence of the activity of F6´H, E-ferulate in the plant acts as a sink. The dominant pathway for the synthesis of scopoletin was also confirmed to be pathway 3, via F6´H. The possibility of cichoriin as an intermediate in the pathways, a possible precursor for scopolin biosynthesis was also investigated. The use of four novel E-cinnamic acids designed to study the E-Z-isomerisation mechanism and the lactonisation step confirmed the consistent loss of a deuterium atom from C-2 in the hydroxycoumarins. The E-Z-isomerisation mechanism was also probed further to determine the source of the proton replacing the deuterium atom at C-2 by lyophilisation and rehydration of cassava chips in deuterium oxide. The absence of d₃-peaks is very suggestive of the deuterium atom being replaced by a proton from water in the tissues of the chips.

Date of Award	16 Sept 2020
Original language	English
Awarding Institution	University of Bath
Supervisor	Ian Blagbrough (Supervisor) & Michael Rowan (Supervisor)