To navigate the different challenges faced, plants produce a large array of bioactive natural compounds. These specialized metabolites defend the plants against herbivores and pathogens, attract beneficial insects and enable communication between plants. Cyanogenic glucosides (CNglcs) are produced from amino acids and are present throughout the plant kingdom. CNglcs were originally thought to only be involved in herbivore defense via the bioactivation pathway, which release hydrogen cyanide (HCN) from the sequential action of endogenous β-glucosidases (BGD) and α-hydroxynitrile lyases. Recent research has revealed that they may also be involved in scavenging of reactive oxygen species, defense against fungal pathogens, function as transporters and metabolic buffers of nitrogen and glucose. In Sorghum bicolor (L.) Moench, the release of reduced nitrogen from CNglcs has been hypothesized to occur via two endogenous turnover pathways, which avoids the toxic intermediates from the bioactivation- and detoxification pathways. So far, only a single enzyme complex from one of these pathways has been characterized. To investigate the dynamic roles of CNglcs and their metabolism in cyanogenic plants, the developing grain of sorghum was chosen as the main model system.
The research presented in this PhD project demonstrates the accumulation and turnover
of dhurrin in the developing grain of sorghum by the two hypothesized pathways. The investigation of these pathways was facilitated by the absence of competing BGD mediated hydrolysis of dhurrin and concomitant HCN release and verified by the absence of the two dhurrinase transcripts. The results obtained, point to one pathway being involved in a continuous turnover, while the other primarily degrade dhurrin to release dhurrin acid and ammonia during the grain maturation phase. The released ammonia may be utilized as a reduced source of nitrogen to fuel grain development and the production of storage proteins and starch. Combined, all results suggest a possible evolutionary progression of dhurrin from being a defense compound to additionally function as a nitrogen storage/buffer compound in the developing grain. The combination of transcriptomic-, phylogenetic- and cluster analyses revealed gene candidates for all enzymatic steps in the pathway involving the heteromeric nitrilase complex. Novel nitrilases were also discovered, which could be putatively involved in catalyzing the formation of dhurrin amide and acid, but none of these where ideal candidates.
To investigate the possible release of the aldoxime intermediate from the dhurrin biosynthesis as a fungal defense compound, an experimental setup was designed using the scent from the orchid Angraecum sesquipedale. From the aldoximes and intermediates thereof detected and the knowledge from cyanogenic plants, pathways for the aldoxime metabolism were proposed. The knowledge gained from this project was applied to the investigation of aldoxime release from germinating grain of sorghum. No aldoximes or other plant emitted volatiles were registered in the headspace. The absence of volatiles was primarily attributed to a low sensitivity of the static headspace sampling technique and the lack of a sorghum specific fungal pathogen capable of eliciting a plant response.
The results obtained in this PhD thesis, represents a significant advance in the research of the putative endogenous turnover pathways of CNglcs hypothesized to function in cyanogenic plants and furthermore contribute to the growing amount of evidence describing the diversified role of CNglcs as more than just herbivore defense compounds.