Poison hemlock (Conium maculatum L.) is a poisonous plant which is speculated to have been the main ingredient of the toxic potion given to the philosopher Socrates. The plant's main compound of the thirteen known piperidine alkaloids is coniine. The compound is also present in twelve Aloe species and an unrelated carnivorous plant, Sarracenia flava L. Coniine is a toxic alkaloid, the biosynthesis of which is not well understood. A possible route, supported by evidence from labelling experiments, involves a polyketide formed by a condensation reaction of one acetyl-CoA and three malonyl-CoAs catalysed by a polyketide synthase (PKS). This study focused on identification and characterization of PKS-genes involved in coniine formation, induction of callus from plants containing hemlock alkaloids and investigation of the possibility to elicitate the alkaloid pathway in cell culture in order to understand coniine biosynthesis. Plant materials involved in different stages of this study were investigated for their alkaloid content using gas chromatographymass spectrometry (GC-MS). PKS-genes or their fragments were isolated from poison hemlock using the rapid amplification of cDNA ends -method and transcriptome analysis. Three expressed enzymes were characterized by feeding different starter-CoAs in vitro. Based on the results of in vitro experiments, two of the three characterised PKS-genes in poison hemlock are chalcone synthases (CPKS1, CPKS2) and one is a novel type PKS (CPKS5). CPKS5 kinetically favours butyryl-CoA as a starter-CoA in vitro. These results suggest that CPKS5 is responsible for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoAs. In order to induce coniine biosynthesis in poison hemlock cell culture, different elicitors were tested. Alginic acid, cellulase, chitosan, silver nitrate and copper(II) sulphate all induced the production of furanocoumarins. Extracts contained bergapten, columbianetin, isopimpinellin, marmesin, oroselone, psoralen and xanthotoxin, but no piperidine alkaloids. Plant hormones (ethylene, methyl jasmonate, and salicylic acid) were not able to induce furanocoumarin biosynthesis. The relative amount of furanocoumarins was generally higher in the medium than in the cells. Aloe gariepensis and A. viguieri formed callus which did not contain piperidine alkaloids. Micropropagation of A. viguieri was investigated using computer-generated statistical experimental design. Up to five plantlets of good quality were produced from a mother plant. In vitro cultivated A. gariepensis, A. globuligemma and A. viguieri were found to contain coniine, -coniceine and also N-methylconiine, an alkaloid not previously reported from Aloe spp. In order to analyse low concentrations of coniine alkaloids in Sarracenia species, a sensitive GC-MS method was developed. S. flava was confirmed to contain coniine, as were seven other Sarracenia species. These results together represent a single step in the long road to understanding the distribution of coniine in the plant kingdom. The hemlock alkaloids are more widely distributed than was previously believed. Different in vitro techniques, such as cell cultures, will be valuable in the investigation of the coniine biosynthesis route. The identification of the first enzyme (CPKS5) of this route at the gene level will open the door for further discovery with the help of the sequenced transcriptome of poison hemlock's different organs.
|Award date||14 Oct 2016|
|Place of Publication||Espoo|
|Publication status||Published - 2016|
|MoE publication type||G5 Doctoral dissertation (article)|
- Conium maculatum
- polyketide synthase
- secondary metabolites