Changes in primary metabolism in connection with alkaloid biosynthesis in solanaceous cell suspensions: a13C-NMR study

Marty, Danielle; Mesnard, François; Gillet-Manceau, F.; Fliniaux, Marc‐André; Monti, Jean‐Pierre

doi:10.1016/s0168-9452(96)04534-7

Cited by 12 publications

(4 citation statements)

References 33 publications

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“…Labelled precursors of amino acids can also be used to introduce 13 C-label into the pathways of amino acid metabolism for NMR analysis, and the most commonly used precursors are 13 C-labelled glucose Marty et al 1997;Edwards et al 1998;Martin et al 1998;Glawischnig et al 2000Glawischnig et al , 2001 and acetate (Ashworth et al 1987;Fan et al 2003;Glawischnig et al 2000Glawischnig et al , 2001. In this approach the redistribution of the label is not confined to the pathways of amino acid metabolism and this allows conclusions to be drawn about the interactions between the pathways of carbon and nitrogen metabolism.…”

Section: Amino Acid Metabolismmentioning

confidence: 99%

NMR analysis of plant nitrogen metabolism

Mesnard¹,

Ratcliffe

2005

Photosynth Res

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The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of (1)H, (2)H, (13)C, (14)N and (15)N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with (2)H-, (13)C- and (15)N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.

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Section: Amino Acid Metabolismmentioning

confidence: 99%

NMR analysis of plant nitrogen metabolism

Mesnard¹,

Ratcliffe

2005

Photosynth Res

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“…cell-suspension culture was initiated and maintained as described previously (Manceau et al 1989;Marty et al 1997). The growth medium (FMD) contained 87.6 mM sucrose, 0.55 mM myo-inositol, 20.6 mM ammonium nitrate, 18.8 mM potassium nitrate, 3.0 mM calcium chloride, 1.5 mM magnesium sulphate, 1.2 mM KH 2 PO 4 , 0.1 mM manganese sulphate, 0.1 mM boric acid, 37 lM zinc sulphate, 5 lM potassium iodide, 1.2 lM sodium molybdate, 0.1 lM cupric sulphate, 0.1 lM cobalt chloride, 0.1 mM iron sulphate, 0.1 mM sodium EDTA, 4 lM nicotinic acid, 2.4 lM pyridoxine-HCl, 1.4 lM thiamine-HCl, 27 lM glycine, 0.9 lM 2,4-dichlorophenoxyacetic acid, 0.4 lM 6-benzylaminopurine.…”

Section: Plant Materialsmentioning

confidence: 99%

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

Mesnard

Roscher

Garlick

et al. 2002

Planta

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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. Label was also identified in a cysteinyl derivative and in several methoxylated compounds, but no evidence was obtained with either NMR or ion-trap mass spectrometry for the presence of any intermediate between nicotine and nornicotine. However, experiments with (R,S)-[14C- methyl]nicotine indicated that 70-75% of the metabolised label was released as carbon dioxide. These results are consistent with a pathway in which the oxidative hydrolysis of the nicotine methyl produces an unstable intermediate, N'-hydroxymethylnornicotine, that breaks down spontaneously to nornicotine and formaldehyde, with the formaldehyde being metabolised either directly to formate and carbon dioxide, or through the tetrahydrofolate-mediated pathways of one-carbon metabolism. However since the key intermediate, N-hydroxymethylnornicotine, could not be detected, the possibility of a direct methyl group transfer to tetrahydrofolate cannot be excluded.

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“…However, high-field NMR spectrometers (>600 MHz) equipped with a cryogenically cooled probe could dramatically enhance the sensitivity [11] . The spectral signature arising from predominant metabolite signals can be further enhanced using 13 C-labeled compounds, which could provide new insights into the metabolic flux of the organism [12] .…”

Section: Introductionmentioning

confidence: 99%

Acetate and Bicarbonate Assimilation and Metabolite Formation in Chlamydomonas reinhardtii: A 13C-NMR Study

et al. 2014

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Cellular metabolite analyses by 13C-NMR showed that C. reinhardtii cells assimilate acetate at a faster rate in heterotrophy than in mixotrophy. While heterotrophic cells produced bicarbonate and CO2 aq, mixotrophy cells produced bicarbonate alone as predominant metabolite. Experiments with singly 13C-labelled acetate (13CH3-COOH or CH3-13COOH) supported that both the 13C nuclei give rise to bicarbonate and CO2 aq. The observed metabolite(s) upon further incubation led to the production of starch and triacylglycerol (TAG) in mixotrophy, whereas in heterotrophy the TAG production was minimal with substantial accumulation of glycerol and starch. Prolonged incubation up to eight days, without the addition of fresh acetate, led to an increased TAG production at the expense of bicarbonate, akin to that of nitrogen-starvation. However, such TAG production was substantially high in mixotrophy as compared to that in heterotrophy. Addition of mitochondrial un-coupler blocked the formation of bicarbonate and CO2 aq in heterotrophic cells, even though acetate uptake ensued. Addition of PSII-inhibitor to mixotrophic cells resulted in partial conversion of bicarbonate into CO2 aq, which were found to be in equilibrium. In an independent experiment, we have monitored assimilation of bicarbonate via photoautotrophy and found that the cells indeed produce starch and TAG at a much faster rate as compared to that in mixotrophy and heterotrophy. Further, we noticed that the accumulation of starch is relatively more as compared to TAG. Based on these observations, we suggest that acetate assimilation in C. reinhardtii does not directly lead to TAG formation but via bicarbonate/CO2 aq pathways. Photoautotrophic mode is found to be the best growth condition for the production of starch and TAG and starch in C. reinhardtii.

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Changes in primary metabolism in connection with alkaloid biosynthesis in solanaceous cell suspensions: a13C-NMR study

Cited by 12 publications

References 33 publications

NMR analysis of plant nitrogen metabolism

NMR analysis of plant nitrogen metabolism

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

Acetate and Bicarbonate Assimilation and Metabolite Formation in Chlamydomonas reinhardtii: A 13C-NMR Study

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