Quantitative 1H Nuclear Magnetic Resonance Metabolite Profiling as a Functional Genomics Platform to Investigate Alkaloid Biosynthesis in Opium Poppy  

Abstract

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The plant is widely cultivated as the only commercial source of the narcotic analgesics morphine and codeine. Variations in plant secondary metabolism as a result of genetic diversity are often associated with perturbations in other metabolic pathways. As part of a functional genomics platform, we used 1 H nuclear magnetic resonance (NMR) metabolite profiling for the analysis of primary and secondary metabolism in opium poppy. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principle component analysis of the 1 H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety and a high-thebaine, low-morphine cultivar. Distinction was also made between pharmaceutical-grade opium poppy cultivars and a condiment variety. Such phenotypic differences were not observed in root extracts. Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Quantification of 34 root and 21 latex metabolites, performed using Chenomx NMR Suite version 4.6, showed major differences in the accumulation of specific alkaloids in the latex of the low-alkaloid and high-thebaine, low-morphine varieties. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. Exceptions in the lowalkaloid cultivar included an increased accumulation of the alkaloid precursor tyramine and reduced levels of sucrose, some amino acids, and malate. Real-time polymerase chain reaction analysis of 42 genes involved in primary and secondary metabolism showed differential gene expression mainly associated with alkaloid biosynthesis. Reduced alkaloid levels in the condiment variety were associated with the reduced abundance of transcripts encoding several alkaloid biosynthetic enzymes.

Chemometric analysis of ESIMS and NMR data from Piper species

“…Currently, various experimental techniques have been routinely applied to metabolomic analysis including gas chromatography-mass spectroscopy (GCMS), 1 H nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESIMS), high performance liquid chromatography-mass spectrometry (HPLCMS), center for environmental mass spectrometry (CEMS) or a combination of them. [6][7][8][9][10][11][12] Since the generated data are very large and complex for interpreting, the principal component analysis (PCA) has been frequently used to analyze all types of matrices because the method is capable of extracting relevant information from large collection of samples having large number of variables which together make the manual analysis virtually impossible. [13][14][15] Piperaceae species are very common in the tropics with ca.…”

Genetics, Genomics and Metabolomics

“…Whilst the entire scope of possibilities is beyond what we can enter into here this emergent research field is expertly reviewed by Redestig et al, 2011. Here, we will only highlight specific examples of particular pertinence to understanding gene-metabolite interactions. Several recent studies have illustrated the utility of combining data from metabolomics with that from other genomics platforms to provide new insights into both gene annotation (Goossens et al, 2003;Achnine et al, 2005;Fridman & Pichersky, 2005;Hagel et al, 2008) and regulation in complex biological systems (Urbanczyk-Wochniak et al, 2003;Alba et al, 2005;Hirai et al, 2005). These approaches have resulted in the identification of numerous candidate genes including several in which expression correlates strongly with the levels of metabolites with important nutritional or organoleptic properties.…”

Impact of mutagenesis on cytological behavior in relation to specific alkaloids in Opium Poppy (Papaver somniferumL.)

“…Opium poppy (Familiy papaveraceae, 2n=22), a natural manufacturer of these opioids, has emerged as a versatile model system to study physiological, morphological as well as molecular aspects of alkaloids (HAGEL et al 2008). Yield potential has always been a point of concern for every breeding experiment.…”