In Vivo Use of 1D and 2D 1H NMR to Examine the Glycosylation of Scopoletin in Duboisia myoporoides Cell Suspensions.

Cell suspensions initiated from -a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids-previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an NMR approach was developed using a perfusion system in an 8-mm NMR tube and H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling.

Kinetics of the incorporation of the main phenolic compounds into the lignan macromolecule during flaxseed development.

The main flax lignan, secoisolariciresinol diglucoside, is stored in a macromolecule containing other ester-bound phenolic compounds. In this study, NMR and HPLC-UV analyses were performed on flaxseeds harvested at different developmental stages to identify and quantify the main phenolic compounds produced during seed development. Extraction was carried out with or without alkaline hydrolysis to determine if these molecules accumulate in the lignan macromolecule and/or in a free form.

Microwave-assisted extraction of herbacetin diglucoside from flax (Linum usitatissimum L.) seed cakes and its quantification using an RP-HPLC-UV system.

Flax (Linum usitatissimum L.) seeds are widely used for oil extraction and the cold-pressed flaxseed (or linseed) cakes obtained during this process constitute a valuable by-product. The flavonol herbacetin diglucoside (HDG) has been previously reported as a constituent of the flaxseed lignan macromolecule linked through ester bonds to the linker molecule hydroxymethylglutaric acid.

In vivo NMR for ¹³C Metabolic Flux Analysis.

The use of in vivo NMR within the framework of Metabolic Flux Analysis in plants is presented. In vivo NMR allows to visualize the active metabolic network, to determine metabolic and isotopic steady state and to measure metabolic fluxes which are not necessarily accessible by isotopic steady state (stationary) Metabolic Flux Analysis. The kinetic data can be used as input for dynamic (nonstationary) Metabolic Flux Analysis.

Quantifying ¹³C-labeling in free sugars and starch by GC-MS.

We describe an approach to extract (13)C-labeled sugars (glucose, fructose, maltose, sucrose, myo-inositol as well as glucose from starch) from plant tissues and to analyze their isotopomer distribution by gas chromatography-mass spectrometry (GC-MS). Sugars are derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) into their Si(CH3)3 derivatives. Electronic and chemical ionizations are used to obtain suitable fragments for metabolic flux analysis (MFA).

Concentration kinetics of secoisolariciresinol diglucoside and its biosynthetic precursor coniferin in developing flaxseed.

Introduction: In the plant kingdom, flaxseed (Linum usitatissimum L.) is the richest source of secoisolariciresinol diglucoside (SDG), which is of great interest because of its potential health benefits for human beings. The information about the kinetics of SDG formation during flaxseed development is rare and incomplete.

Gas chromatography-mass spectrometry analysis of 13C labeling in sugars for metabolic flux analysis.

Metabolic flux analysis, using 13C labeled substrates, has become a powerful methodology for quantifying intracellular fluxes. Most often, analysis is restricted to nuclear magnetic resonance or mass spectrometry measurement of 13C label incorporation into protein amino acids. However, amino acid isotopomer distribution insufficiently covers the entire network of central metabolism, especially in plant cells with highly compartmented metabolism, and analysis of other metabolites is required.

Resolving the role of plant glutamate dehydrogenase. I. In vivo real time nuclear magnetic resonance spectroscopy experiments.

In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme.

In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos.

The dynamics of developing linseed embryo metabolism was investigated using (13)C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools - such as sucrose or lipids - to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6h for the sucrose pool.

Nicotine demethylation in Nicotiana cell suspension cultures: N'-formylnornicotine is not involved.

Nicotine or nornicotine enriched with stable isotopes in either the N'-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography-mass spectroscopy (GC-MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N'-demethylation product.

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures.

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates.

Evidence for the involvement of tetrahydrofolate in the demethylation of nicotine by Nicotiana plumbaginifolia cell-suspension cultures.

The conversion of nicotine to nornicotine by Nicotiana plumbaginifolia Viv. cells was investigated by analysing the redistribution of label during feeding experiments with (R,S)-[2H- methyl]nicotine, (R,S)-[13C- methyl]nicotine and (R,S)-[14C- methyl]nicotine, and the results show that the N-methyl group of nicotine can be recycled into primary metabolism. Nuclear magnetic resonance (NMR) analysis of ethanolic extracts of cells grown in the presence of (R,S)-[13C- methyl]nicotine, using 1H-13C correlation spectroscopy (HMQC, HMBC), revealed the presence of [3-13C]serine and [13C- methyl]methionine.

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells.

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media.