Metabolic changes of Brassica rapa transformed with a bacterial isochorismate synthase gene.

Metabolome analysis by 1-dimensional proton nuclear magnetic resonance (¹H NMR) coupled with multivariate data analysis was carried out in Brassica rapa plants transformed with a gene encoding bacterial isochorismate synthase (ICS). Partial least square-discrimination analysis (PLS-DA) on selected signals suggested that the resonances that were dominant in the transgenic plants corresponded to a glucosinolate (neoglucobrassicin), phenylpropanoids (sinapoyl malate, feruloyl malate, caffeoyl malate), organic acids (succinic acid and fumaric acid) and sugars (α- and ß-glucose). In contrast, amino acids alanine threonine, valine, leucine were dominant in the untransformed controls. In addition, HPLC data showed that the transgenic plant accumulated salicylic acid (SA) at significantly higher levels than the control plants, whereas the phylloquinone levels were not affected. The results suggest that the expression of the bacterial isochorismate synthase gene in B. rapa does not affect fluxes into pathways to other groups of secondary metabolites through competition for the same precursor. On the contrary, the biosynthesis of isochorismate-derived products (SA) seems to induce the competitive pathways via phenylalanine (phenylpropanoids) and tryptophan (IAA and indole glucosinolates).

Biosynthesis of salicylic acid in fungus elicited Catharanthus roseus cells.

Feeding experiments using [1-(13)C]-d-glucose to Catharanthus roseus (L.) G.Don cell suspension cultures followed by elicitation with Pythium aphanidermatum extract were performed in order to study the salicylic acid (SA) biosynthetic pathway and that of 2,3-dihydroxybenzoic acid (2,3-DHBA) as a comparison. A strongly labeled C-7 and a symmetrical partitioning of the label between C-2 and C-6 would occur if SA was synthesized from phenylalanine. In case of the isochorismate pathway, a relatively lower incorporation at C-7 and a non-symmetrical incorporation at C-2 and C-6 would be obtained. Relatively, high- and non-symmetrical enrichment ratios at C-2 and C-6, and a lower enrichment ratio at C-7 were observed in both SA and 2,3-DHBA detected by (13)C NMR inverse gated spectrometry leading to the conclusion that the isochorismate pathway is responsible for the biosynthesis of both compounds. However, different enrichment ratios of the labeled carbons in SA and 2,3-DHBA indicate the use of different isochorismate pools, which means that their biosynthesis is separated in time and/or space.

Effect of benzothiadiazole on the metabolome of Arabidopsis thaliana.

Benzothiadiazole (BTH) is a functional analog of the plant endogenous hormone-like compound, salicylic acid (SA), which is required for the induction of plant defense genes leading to systemic acquired resistance (SAR). Previous molecular and genetic studies have suggested that BTH itself might potentiate SAR resulting in the induction of several pathogenesis-related (PR) genes. However, the changes in the metabolome, which occur as a result of BTH-treatment, remain unclear. In this study, metabolic alterations in BTH-treated Arabidopsis thaliana were investigated using nuclear magnetic resonance (NMR) spectroscopy followed by multivariate data analyses such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA). Both PCA and PLS-DA show that increase of glucose, glutamine, inositol, malic acid, sucrose, and threonine as well as BTH and its degraded metabolites contribute to the clear discrimination of the metabolome of BTH-treated Arabidopsis from control plants. However, the levels of phenolic metabolites, which have generally been observed to be induced by other signaling molecules were significantly reduced in BTH-treated Arabidopsis. In addition to these changes due to BTH-treatment, it was also found that the EtOH used as a solvent in this treatment may per se act as an inducer of the accumulation of a flavonoid.

Metabolomic differentiation of Brassica rapa following herbivory by different insect instars using two-dimensional nuclear magnetic resonance spectroscopy.

The metabolic alterations of Brassica rapa (L.) leaves attacked by larvae of the specialist Plutella xylostella L. (Lepidoptera: Yponomeutidae) and the generalist Spodoptera exigua Hubner (Lepidoptera: Noctuidae) were investigated with nuclear magnetic resonance (NMR) spectroscopy, followed by a multivariate data analysis. The principal component analysis (PCA) of (1)H NMR spectra showed that metabolic changes in B. rapa leaves induced by the 2nd and the 4th instars were different from each other. However, the congestion of the one-dimensional (1)H NMR spectrum made it difficult to identify discriminating metabolites. To overcome the spectral complexity, several two-dimensional NMR techniques were applied. Of those evaluated, J-resolved spectroscopy, which affords an additional coupling constant, provided a wide range of structure information on differentiating the metabolites. Based on the J-resolved spectra combined with PCA, the major signals contributing to the discrimination were alanine, threonine, glucose, sucrose, feruloyl malate, sinapoyl malate, and gluconapin.

NMR metabolomics to revisit the tobacco mosaic virus infection in Nicotiana tabacum leaves.

Tobacco mosaic virus (TMV) infection of tobacco is a well-known and extensively studied model system for which a number of genes and proteins involved in the systemic acquired resistance (SAR) have been characterized. Little is known about the metabolic changes connected with the infection and SAR. Here we describe the use of NMR spectroscopy in combination with multivariate data analysis to study the metabolic changes. Particularly 2-D NMR methods, such as 2-D J-resolved spectra and their projected spectra, are shown to be powerful tools in the metabolomic studies. The macroscopic view of the metabolomes obtained by NMR spectroscopy of crude extracts enabled the identification of a series of totally different metabolites that seem connected with resistance, such as the clearly increased 5-caffeoylquinic acid, alpha-linolenic acid analogues, and sesqui- and diterpenoids in the infected plant parts.

Application of two-dimensional nuclear magnetic resonance spectroscopy to quality control of ginseng commercial products.

Ginseng has been used as a powder or a crude extract of the plant roots. The quality control of commercial ginseng preparations is difficult due to the diverse compounds present. Most previous quality control methods using TLC or HPLC-UV (or -MS) cannot be expected to cover a wide range of compounds in the commercial ginseng preparations. In this study, the metabolic fingerprinting of ginseng preparations was performed by (1)H-NMR spectroscopy. Although (1)H-NMR spectroscopy could provide information about the total profile of the compounds present, low resolution and overlapping signals make it difficult for further identification of each compound. For overcoming the problem two-dimensional J-resolved NMR spectra and multivariate data analysis techniques was applied for the analysis. Principal component analysis (PCA) of projected J-resolved NMR spectra shows a clear discrimination among those samples by principal component 1 and principal component 3. The loading plot of PC values obtained from all NMR signals indicates that alanine, arginine, choline, fumaric acid, inositol, sucrose as well as ginsenosides are important metabolites to differentiate the preparations from each other. This method allows an efficient discrimination of a ginseng preparation in less than 15 minutes without any pre-purification steps.

Classification of Ilex species based on metabolomic fingerprinting using nuclear magnetic resonance and multivariate data analysis.

The metabolomic analysis of 11 Ilex species, I. argentina, I. brasiliensis, I. brevicuspis, I. dumosavar. dumosa, I. dumosa var. guaranina, I. integerrima, I. microdonta, I. paraguariensis var. paraguariensis, I. pseudobuxus, I. taubertiana, and I. theezans, was carried out by NMR spectroscopy and multivariate data analysis. The analysis using principal component analysis and classification of the (1)H NMR spectra showed a clear discrimination of those samples based on the metabolites present in the organic and aqueous fractions. The major metabolites that contribute to the discrimination are arbutin, caffeine, phenylpropanoids, and theobromine. Among those metabolites, arbutin, which has not been reported yet as a constituent of Ilex species, was found to be a biomarker for I. argentina,I. brasiliensis, I. brevicuspis, I. integerrima, I. microdonta, I. pseudobuxus, I. taubertiana, and I. theezans. This reliable method based on the determination of a large number of metabolites makes the chemotaxonomical analysis of Ilex species possible.

Metabolic fingerprinting of Ephedra species using 1H-NMR spectroscopy and principal component analysis.

The metabolomic analysis of Ephedra species was performed using 1H-NMR spectroscopy and multivariate data analysis. A broad range of metabolites could be detected by 1H-NMR spectroscopy without any chromatographic separation. The principal component analysis used to reduce the huge data set obtained from the 1H-NMR spectra of the plant extracts clearly discriminated three different Ephedra species. The major differences in Ephedra sinica, Ephedra intermedia and Ephedra distachya var. distachya were found to be due to benzoic acid analogues in the aqueous fraction and ephedrine-type alkaloids in the organic fraction. Based on this metabolomic recognition, one of nine commercial Ephedra materials evaluated was shown to be a mixture of Ephedra species. This method will be a useful tool for chemotaxonomic analysis and authentification of Ephedra species including quality control of plant materials.

NMR assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa.

The complete 1H- and 13C-NMR assignments of the major Cannabis constituents, delta9-tetrahydrocannabinol, tetrahydrocannabinolic acid, delta8-tetrahydrocannabinol, cannabigerol, cannabinol, cannabidiol, cannabidiolic acid, cannflavin A and cannflavin B have been determined on the basis of one- and two-dimensional NMR spectra including 1H- and 13C-NMR, 1H-1H-COSY, HMQC and HMBC. The substitution of carboxylic acid on the cannabinoid nucleus (as in tetrahydrocannabinolic acid and cannabidiolic acid) has a large effect on the chemical shift of H-1" of the C5 side chain and 2'-OH. It was also observed that carboxylic acid substitution reduces intermolecular hydrogen bonding resulting in a sharpening of the H-5' signal in cannabinolic acid in deuterated chloroform. The additional aromaticity of cannabinol causes the two angular methyl groups (H-8 and H-9) to show identical 1H-NMR shifts, which indicates that the two aromatic rings are in one plane in contrast to the other cannabinoids. For the cannabiflavonoids, the unambiguous assignments of C-3' and C-4' of cannflavin A and B were determined by HMBC spectra.

Isolation of the acetylcholinesterase inhibitor ungeremine from Nerine bowdenii by preparative HPLC coupled on-line to a flow assay system.

In an attempt to isolate the active compound while detecting acetylcholinesterase inhibitory activity, we applied a fluorometric flow assay system to an on-line coupled preparative HPLC. The MeOH extract of Nerine bowdenii showed a strong inhibitory peak in the on-line assay, and the active compound was isolated by CPC and HPLC. It was identified as ungeremine by analysis of its (1)H-NMR, 2D-NMR, and NOESY spectra. The assignment of the active N. bowdenii constituent was also confirmed by co-TLC, co-HPLC, and co-(1)H-NMR experiments using an authentic sample of synthetic ungeremine. The IC(50) value of ungeremine was 0.35 microM, showing stronger activity than galanthamine (2.2 microM).

Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis.

A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the (1)H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma.

Metabolomic differentiation of Cannabis sativa cultivars using 1H NMR spectroscopy and principal component analysis.

The metabolomic analysis of 12 Cannabis sativa cultivars was carried out by 1H NMR spectroscopy and multivariate analysis techniques. Principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by principal component 1 (PC1) and principal component 3 (PC3) in cannabinoid fraction. The loading plot of PC value obtained from all 1)H NMR signals shows that Delta9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) are important metabolites to differentiate the cultivars from each other. The discrimination of the cultivars could also be obtained from a water extract containing carbohydrates and amino acids. The level of sucrose, glucose, asparagine, and glutamic acid are found to be major discriminating metabolites of these cultivars. This method allows an efficient differentiation between cannabis cultivars without any prepurification steps.

Metabolic fingerprinting of wild type and transgenic tobacco plants by 1H NMR and multivariate analysis technique.

The metabolomic analysis of wild type and constitutive salicylic acid producing tobacco plants (CSA tobacco, Nicotiana tabacum 'Samsun' NN) plants overexpressing salicylate biosynthetic genes was carried out by 1H NMR spectrometry and multivariate analysis techniques. The principle component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples by PC1 and PC2. The discrimination of non-inoculated, TMV-virus inoculated, and systemic leaves or veins could also be obtained by PCA analysis. Major peaks in 1H NMR spectra contributing to the discrimination were assigned as those of chlorogenic acid, malic acid, and sugars. This method allows an efficient differentiation between wild type and transgenic plants without any pre-purification steps.

Synthetic analogues of the histidine-chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex.

Mg(II)-porphyrin-ligand and (bacterio)chlorophyl-ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. (1)H, (13)C and (15)N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9-5.2 ppm (1)H, 0.2-5.5 ppm (13)C and 2.1-27.2 ppm (15)N coordination shifts were measured for selectively labeled [1,3-(15)N]-Im, [1,3-(15)N,2-(13)C]-Im and [1,3-(15)N,1,2-(13)C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the (1)H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a-histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.

Quantitative analysis of bilobalide and ginkgolides from Ginkgo biloba leaves and Ginkgo products using (1)H-NMR.

1H-NMR spectrometry was applied to the quantitative analysis of the bilobalide, ginkgolides A, B, and C in Ginkgo biloba leaves and six kinds of commercial Ginkgo products without any chromatographic purification. The experiment was performed by the analysis of each singlet H-12, which were well separated in the range of delta 6.0-7.0 in the (1)H-NMR spectrum. However, the H-12 protons of bilobalide and ginkgolides may have overlapped with H-6 or H-8 protons of the Ginkgo flavonoids. Therefore, the optimum (1)H-NMR solvent for the analysis of the compound was selected through the evaluation of solvent effects on the resolution of these signals from the compounds. Acetone-d(6)-benzene-d(6) (50 : 50) was found to be the best one among the solvents evaluated. The quantity of the compounds was calculated by the relative ratio of the intensity of each compound to the known amount of internal standard (25 microgram), phloroglucinol. This method allows rapid and simple quantitation of underivatized bilobalide and ginkgolides in 5 min without any pre-purification steps.

Biosynthesis of anthraquinones in cell cultures of Cinchona 'Robusta' proceeds via the methylerythritol 4-phosphate pathway.

Robustaquinone B was found as a major anthraquinone in cell cultures of Cinchona 'Robusta' after treatment with a fungal elicitor. Anthraquinones in Cinchona are considered to be of the Rubia type, i.e. rings A and B are derived from chorismate and alpha-ketoglutarate, whereas ring C is formed from isopentenyl diphosphate (IPP). To determine the origin of IPP, either formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway, the incorporation of [1-13C]glucose into robustaquinone B was studied. The 13C labeling of robustaquinone B was analyzed by one- and two-dimensional NMR spectroscopy and the labeling pattern was compared with the hypothetical labeling patterns obtained via the different biosynthetic pathways. The results clearly show that the IPP, constituting the ring C of robustaquinone B, is biosynthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway. Moreover, the data also confirm that rings A and B of robustaquinone B are formed from chorismate and alpha-ketoglutarate via o-succinylbenzoate.