Phytochemistry of hydroxycoumarins from Manihot esculenta Euphorbiaceae (cassava)

Lidia Alhalaseh

Research output: ThesisDoctoral Thesis

Abstract

This is an interdisciplinary research project on cassava (Manihot esculenta Crantz, Euphorbiaceae) ultimately working towards producing cassava roots which are long-lasting, free of post-harvest physiological deterioration (PPD). It aims to contribute to ensuring food security. In cassava, scopoletin and its -glycoside scopolin are considered phytoanticipants, not phytoalexins, due to their increasing accumulation during the PPD process compared to their barely detectable levels in fresh roots.
Starting with a focussed literature review on the potential of cassava, contrasted with its limitations on harvesting due to PPD, and biosynthesis along the phenylpropanoid pathway of key hydroxycoumarins, e.g. scopoletin and esculetin, the associated gaps in our current knowledge have been set out. Whether scopoletin is biosynthesized de novo from L-phenylalanine in response to stress, or whether stress prompts its release from the corresponding glycoside is unknown. Therefore, assessing hydroxycoumarin biosynthesis and quantifying their accumulation patterns have been undertaken in wild-type and transgenic plants in order to elucidate the divergence in scopoletin biosynthetic pathways. The identification of key genes on each pathway leading to scopoletin in cassava, and then exploring their functional identities using the model plant A. thaliana and genetically engineered E. coli, where the genes were isolated, cloned, and expressed, were also undertaken. Transgenic A. thaliana lines with no activity of the key enzymes on the proposed pathway, namely F6ʹH1, CCoAOMT, and EOMT, were developed. Competition feeding experiments using stable isotopically labelled potential biosynthetic intermediates showed the incorporation of labelled ferulate into scopoletin in transgenic A.t-F6´H1 and M.e-F6´H. This confirmed the activity of other hydroxylase enzymes rather than F6´H1 in the ortho-hydroxylation steps. The hydroxycoumarins of interest were isolated, characterized, and quantified in the wild type and mutant lines using chromatographic and spectroscopic techniques, mainly NMR, HR-MS, and LC-MS.
Taken together, a significant contribution to knowledge about hydroxycoumarin biosynthesis has been made.
LanguageEnglish
QualificationPh.D.
Awarding Institution
  • University of Bath
Supervisors/Advisors
  • Blagbrough, Ian, Supervisor
  • Beeching, John, Supervisor
  • Rowan, Michael G, Supervisor, External person
Award date16 Oct 2017
StatusPublished - 16 Oct 2017

Fingerprint

scopoletin
plant biochemistry
Manihot esculenta
Euphorbiaceae
cassava
deterioration
biosynthesis
glycosides
genetically modified organisms
taxonomic keys
interdisciplinary research
phytoalexins
hydroxylation
phenylpropanoids
enzymes
research projects
food security
phenylalanine
biochemical pathways
transgenic plants

Keywords

  • Phytochemistry
  • Cassava
  • hydroxycoumarins

Cite this

Phytochemistry of hydroxycoumarins from Manihot esculenta Euphorbiaceae (cassava). / Alhalaseh, Lidia.

2017. 205 p.

Research output: ThesisDoctoral Thesis

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N2 - This is an interdisciplinary research project on cassava (Manihot esculenta Crantz, Euphorbiaceae) ultimately working towards producing cassava roots which are long-lasting, free of post-harvest physiological deterioration (PPD). It aims to contribute to ensuring food security. In cassava, scopoletin and its -glycoside scopolin are considered phytoanticipants, not phytoalexins, due to their increasing accumulation during the PPD process compared to their barely detectable levels in fresh roots.Starting with a focussed literature review on the potential of cassava, contrasted with its limitations on harvesting due to PPD, and biosynthesis along the phenylpropanoid pathway of key hydroxycoumarins, e.g. scopoletin and esculetin, the associated gaps in our current knowledge have been set out. Whether scopoletin is biosynthesized de novo from L-phenylalanine in response to stress, or whether stress prompts its release from the corresponding glycoside is unknown. Therefore, assessing hydroxycoumarin biosynthesis and quantifying their accumulation patterns have been undertaken in wild-type and transgenic plants in order to elucidate the divergence in scopoletin biosynthetic pathways. The identification of key genes on each pathway leading to scopoletin in cassava, and then exploring their functional identities using the model plant A. thaliana and genetically engineered E. coli, where the genes were isolated, cloned, and expressed, were also undertaken. Transgenic A. thaliana lines with no activity of the key enzymes on the proposed pathway, namely F6ʹH1, CCoAOMT, and EOMT, were developed. Competition feeding experiments using stable isotopically labelled potential biosynthetic intermediates showed the incorporation of labelled ferulate into scopoletin in transgenic A.t-F6´H1 and M.e-F6´H. This confirmed the activity of other hydroxylase enzymes rather than F6´H1 in the ortho-hydroxylation steps. The hydroxycoumarins of interest were isolated, characterized, and quantified in the wild type and mutant lines using chromatographic and spectroscopic techniques, mainly NMR, HR-MS, and LC-MS.Taken together, a significant contribution to knowledge about hydroxycoumarin biosynthesis has been made.

AB - This is an interdisciplinary research project on cassava (Manihot esculenta Crantz, Euphorbiaceae) ultimately working towards producing cassava roots which are long-lasting, free of post-harvest physiological deterioration (PPD). It aims to contribute to ensuring food security. In cassava, scopoletin and its -glycoside scopolin are considered phytoanticipants, not phytoalexins, due to their increasing accumulation during the PPD process compared to their barely detectable levels in fresh roots.Starting with a focussed literature review on the potential of cassava, contrasted with its limitations on harvesting due to PPD, and biosynthesis along the phenylpropanoid pathway of key hydroxycoumarins, e.g. scopoletin and esculetin, the associated gaps in our current knowledge have been set out. Whether scopoletin is biosynthesized de novo from L-phenylalanine in response to stress, or whether stress prompts its release from the corresponding glycoside is unknown. Therefore, assessing hydroxycoumarin biosynthesis and quantifying their accumulation patterns have been undertaken in wild-type and transgenic plants in order to elucidate the divergence in scopoletin biosynthetic pathways. The identification of key genes on each pathway leading to scopoletin in cassava, and then exploring their functional identities using the model plant A. thaliana and genetically engineered E. coli, where the genes were isolated, cloned, and expressed, were also undertaken. Transgenic A. thaliana lines with no activity of the key enzymes on the proposed pathway, namely F6ʹH1, CCoAOMT, and EOMT, were developed. Competition feeding experiments using stable isotopically labelled potential biosynthetic intermediates showed the incorporation of labelled ferulate into scopoletin in transgenic A.t-F6´H1 and M.e-F6´H. This confirmed the activity of other hydroxylase enzymes rather than F6´H1 in the ortho-hydroxylation steps. The hydroxycoumarins of interest were isolated, characterized, and quantified in the wild type and mutant lines using chromatographic and spectroscopic techniques, mainly NMR, HR-MS, and LC-MS.Taken together, a significant contribution to knowledge about hydroxycoumarin biosynthesis has been made.

KW - Phytochemistry

KW - Cassava

KW - hydroxycoumarins

M3 - Doctoral Thesis

ER -