TY - BOOK

T1 - Endogenous Turnover of Cyanogenic Glycosides in Plants

AU - Picmanova, Martina

PY - 2016

Y1 - 2016

N2 - Amongst the vast multitude of plant secondary metabolites, cyanogenic glycosides (CNglcs)occupy an important place as sophisticated defence compounds, releasing toxic hydrogencyanide (HCN) upon herbivore attack – a process known as cyanogenesis. Beside this wellestablishedfunction, there is strong evidence that CNglcs serve a no less significant purpose asa transport and storage form of reduced nitrogen which may be remobilized and recycled tobalance the needs of primary metabolism during certain developmental events. Reducednitrogen from CNglcs may be recovered either via HCN refixation through the formation of β-cyanoalanine, or, potentially, in an alternative endogenous turnover pathway circumventing therelease 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 noncyanogeniccompounds glaringly resembling their structures. In order to examine the spatial aswell 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 fromCNglcs. Based on results common to the three phylogenetically unrelated plant species, arecycling endogenous turnover pathway for CNglcs was suggested in which reduced nitrogenand 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 revolutionaryanalytical tool for sequencing the monosaccharide units of the glycosides studied, including theconfiguration of the glycosidic linkages.The first attempts to prove the metabolic relationship between CNglcs and their putativeturnover products are presented in this thesis. The preliminary data obtained from feedingexperiments with radio-labelled precursors and enzyme assays with plant homogenates, supportthe existence of the proposed catabolic route, even though the enzymes involved remainelusive. Testing of heterologously expressed and purified nitrilases and nitrilase-related enzymesfrom cassava and almond, including NIT4s, glutamine-dependent NAD+ synthetase (QNS) and anewly discovered nucleus-localized nitrilase with an N-terminal TCP domain, showed that theseenzymes do not recognize CNglcs as their substrates. However, the activity of NIT4s on a nitrileglucoside indicates that the enzymatic formation of glycosylated turnover products might be anintegral 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 ourunderstanding of the multiple roles CNglcs play in plants, and thus also for the futureproduction of safe but healthy and resistant crops

AB - Amongst the vast multitude of plant secondary metabolites, cyanogenic glycosides (CNglcs)occupy an important place as sophisticated defence compounds, releasing toxic hydrogencyanide (HCN) upon herbivore attack – a process known as cyanogenesis. Beside this wellestablishedfunction, there is strong evidence that CNglcs serve a no less significant purpose asa transport and storage form of reduced nitrogen which may be remobilized and recycled tobalance the needs of primary metabolism during certain developmental events. Reducednitrogen from CNglcs may be recovered either via HCN refixation through the formation of β-cyanoalanine, or, potentially, in an alternative endogenous turnover pathway circumventing therelease 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 noncyanogeniccompounds glaringly resembling their structures. In order to examine the spatial aswell 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 fromCNglcs. Based on results common to the three phylogenetically unrelated plant species, arecycling endogenous turnover pathway for CNglcs was suggested in which reduced nitrogenand 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 revolutionaryanalytical tool for sequencing the monosaccharide units of the glycosides studied, including theconfiguration of the glycosidic linkages.The first attempts to prove the metabolic relationship between CNglcs and their putativeturnover products are presented in this thesis. The preliminary data obtained from feedingexperiments with radio-labelled precursors and enzyme assays with plant homogenates, supportthe existence of the proposed catabolic route, even though the enzymes involved remainelusive. Testing of heterologously expressed and purified nitrilases and nitrilase-related enzymesfrom cassava and almond, including NIT4s, glutamine-dependent NAD+ synthetase (QNS) and anewly discovered nucleus-localized nitrilase with an N-terminal TCP domain, showed that theseenzymes do not recognize CNglcs as their substrates. However, the activity of NIT4s on a nitrileglucoside indicates that the enzymatic formation of glycosylated turnover products might be anintegral 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 ourunderstanding of the multiple roles CNglcs play in plants, and thus also for the futureproduction of safe but healthy and resistant crops

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122663462005763

M3 - Ph.D. thesis

BT - Endogenous Turnover of Cyanogenic Glycosides in Plants

PB - Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen

ER -