PhD defense by Martina Picmanova

Martina Picmanova

Title: Endogenous turnover of cyanogenic glycosides in plants

Principal academic advisor:
Birger Lindberg Møller

Co-advisors:
Raquel Sánchez Pérez
Nanna Bjarnholt
Kirsten Jørgensen 

PhD defense at 13:00 in the Festival Auditorium at Bülowsvej 17, Frederiksberg 
Reception in room M117 afterwards - everybody is welcome!

Turnover of cyanogenic glycosides - click picture to enlarge

Summary:
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 well-established 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 non-cyanogenic 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.