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 low-alkaloid 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.

Figure 1.

Score plots for unsupervised, pareto-scaled three-dimensional PCA of NMR spectral data obtained for aqueous or chloroform extracts of opium poppy latex and root tissues. NMR spectra for aqueous and chloroform extracts were obtained in D₂O and CDCl₃, respectively. Samples representing six different varieties (L, M, P, T, 11, and 40) are color coded as indicated.

Figure 2.

Score and loading plots for unsupervised, pareto-scaled two-dimensional PCA of NMR spectral data obtained for aqueous or chloroform extracts of opium poppy latex. NMR spectra for aqueous and chloroform extracts were obtained in D₂O and CDCl₃, respectively. For score plots, samples representing six plant varieties are color coded as follows: L, red; M, orange; P, blue; T, green; 11, yellow; 40, pink. For loading plots, bin numbers corresponding to those listed in Table I (aqueous) and Supplemental Table S1 (chloroform) are indicated.

Figure 3.

Metabolite linkage map representing primary and secondary plant metabolism in opium poppy. The circles associated with each metabolite indicate whether the metabolite was detected (green), not detected (red), or masked (yellow) in root and latex extracts (first and second circles, respectively). Data could not be obtained for metabolites shown in gray because information regarding their standard ¹H NMR spectra was not available.

Figure 4.

Individual metabolite quantities in latex as determined by ¹H NMR analysis for six varieties of opium poppy. Plant variety abbreviations are the same as in Figure 1. Data are given as means ± se, which were calculated using at least three biological replicates. Letters above the bars indicate pair-wise differences identified by Tukey-Kramer multiple comparison tests. Bars lacking letters were not significantly different. Quantification was achieved using Chenomx NMR Suite software with DSS as the internal standard.

Figure 5.

¹H NMR spectra of latex aqueous extracts for six varieties of opium poppy. The spectral region corresponding to C-ring protons is shown, with proton resonances for morphine (Mo), codeine (C), thebaine (Th), and oripavine (O) indicated. Comparisons of relative alkaloid abundance may be made between plant varieties, whose abbreviations are the same as in Figure 1.

Figure 6.

Quantification of alkaloids determined by HPLC analysis for six varieties of opium poppy. Plant variety abbreviations are the same as in Figure 1. The different alkaloids are color coded as indicated. Data are given as means ± se, which were calculated using six biological replicates. Compounds were identified by comparing retention times and UV spectra with those of authentic standards. LC-MS analysis of each sample was also performed for further confirmation. Quantification was achieved using standard curves developed for each alkaloid using authentic standards.

Figure 7.

Quantitative real-time RT-PCR expression analysis of 30 primary metabolic and 12 alkaloid biosynthetic genes in flower buds of six opium poppy varieties. Plant variety abbreviations are the same as in Figure 1. Each bar represents the average of six measurements (two technical replicates on each of three independent biological samples) ± sd. Letters above bars indicate pair-wise differences identified by Tukey-Kramer multiple comparison tests. Bars lacking letters were not significantly different. BBE, Berberine bridge enzyme; CYP80B3, N-methylcoclaurine 3′-hydroxylase; DAHP, deoxy-d-arabino-heptosonate-7-phosphate; EPSP, 5-enolpyruvylshikimate-3-phosphate; NCS1, norcoclaurine synthase; 7OMT, reticuline 7-O-methyltransferase; SAM, S-adenosyl-Met; TNMT, tetrahydroprotoberberine-cis-N-methyltransferase.