Abstract
Amongst the vast multitude of plant secondary metabolites, cyanogenic glycosides (CNglcs)
occupy an important place as sophisticated defence compounds, releasing toxic hydrogen
cyanide (HCN) upon herbivore attack – a process known as cyanogenesis. Beside this wellestablished
function, there is strong evidence that CNglcs serve a no less significant purpose as
a transport and storage form of reduced nitrogen which may be remobilized and recycled to
balance the needs of primary metabolism during certain developmental events. Reduced
nitrogen from CNglcs may be recovered either via HCN refixation through the formation of β-
cyanoalanine, or, potentially, in an alternative endogenous turnover pathway circumventing the
release of HCN. The investigation of the latter is the primary focus of this PhD project.
Scattered data from the literature indicated that CNglcs co-occur in plants with noncyanogenic
compounds glaringly resembling their structures. In order to examine the spatial as
well as temporal presence and abundance of the putative turnover products of CNglcs,
comparative metabolic profiling using LC-MS/MS was performed in three cyanogenic crops –
cassava, almond and sorghum. This work revealed the formation of glycosides of amides,
carboxylic acids and "anitriles", including their di- and triglycosides, evidently derived from
CNglcs. Based on results common to the three phylogenetically unrelated plant species, a
recycling endogenous turnover pathway for CNglcs was suggested in which reduced nitrogen
and carbon are recovered for primary metabolism, without the release of free HCN. Moreover,
we demonstrated the use of ion-mobility mass spectrometry (IM–MS) as a revolutionary
analytical tool for sequencing the monosaccharide units of the glycosides studied, including the
configuration of the glycosidic linkages.
The first attempts to prove the metabolic relationship between CNglcs and their putative
turnover products are presented in this thesis. The preliminary data obtained from feeding
experiments with radio-labelled precursors and enzyme assays with plant homogenates, support
the existence of the proposed catabolic route, even though the enzymes involved remain
elusive. Testing of heterologously expressed and purified nitrilases and nitrilase-related enzymes
from cassava and almond, including NIT4s, glutamine-dependent NAD+ synthetase (QNS) and a
newly discovered nucleus-localized nitrilase with an N-terminal TCP domain, showed that these
enzymes do not recognize CNglcs as their substrates. However, the activity of NIT4s on a nitrile
glucoside indicates that the enzymatic formation of glycosylated turnover products might be an
integral part of the endogenous turnover of CNglcs.
The elucidation of the suggested turnover pathway of CNglcs by extensive genomic,
transcriptomic and enzymological studies will have important implications for our
understanding of the multiple roles CNglcs play in plants, and thus also for the future
production of safe but healthy and resistant crops
occupy an important place as sophisticated defence compounds, releasing toxic hydrogen
cyanide (HCN) upon herbivore attack – a process known as cyanogenesis. Beside this wellestablished
function, there is strong evidence that CNglcs serve a no less significant purpose as
a transport and storage form of reduced nitrogen which may be remobilized and recycled to
balance the needs of primary metabolism during certain developmental events. Reduced
nitrogen from CNglcs may be recovered either via HCN refixation through the formation of β-
cyanoalanine, or, potentially, in an alternative endogenous turnover pathway circumventing the
release of HCN. The investigation of the latter is the primary focus of this PhD project.
Scattered data from the literature indicated that CNglcs co-occur in plants with noncyanogenic
compounds glaringly resembling their structures. In order to examine the spatial as
well as temporal presence and abundance of the putative turnover products of CNglcs,
comparative metabolic profiling using LC-MS/MS was performed in three cyanogenic crops –
cassava, almond and sorghum. This work revealed the formation of glycosides of amides,
carboxylic acids and "anitriles", including their di- and triglycosides, evidently derived from
CNglcs. Based on results common to the three phylogenetically unrelated plant species, a
recycling endogenous turnover pathway for CNglcs was suggested in which reduced nitrogen
and carbon are recovered for primary metabolism, without the release of free HCN. Moreover,
we demonstrated the use of ion-mobility mass spectrometry (IM–MS) as a revolutionary
analytical tool for sequencing the monosaccharide units of the glycosides studied, including the
configuration of the glycosidic linkages.
The first attempts to prove the metabolic relationship between CNglcs and their putative
turnover products are presented in this thesis. The preliminary data obtained from feeding
experiments with radio-labelled precursors and enzyme assays with plant homogenates, support
the existence of the proposed catabolic route, even though the enzymes involved remain
elusive. Testing of heterologously expressed and purified nitrilases and nitrilase-related enzymes
from cassava and almond, including NIT4s, glutamine-dependent NAD+ synthetase (QNS) and a
newly discovered nucleus-localized nitrilase with an N-terminal TCP domain, showed that these
enzymes do not recognize CNglcs as their substrates. However, the activity of NIT4s on a nitrile
glucoside indicates that the enzymatic formation of glycosylated turnover products might be an
integral part of the endogenous turnover of CNglcs.
The elucidation of the suggested turnover pathway of CNglcs by extensive genomic,
transcriptomic and enzymological studies will have important implications for our
understanding of the multiple roles CNglcs play in plants, and thus also for the future
production of safe but healthy and resistant crops
| Originalsprog | Engelsk |
|---|
| Forlag | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
|---|---|
| Antal sider | 170 |
| Status | Udgivet - 2016 |