In vivo 15N NMR studies of secondary metabolism in transformed root cultures of Datura stramonium and Nicotiana tabacum

Ford, Y.-Yannick; Fox, G. G.; Ratcliffe, George; Robins, Richard J.

doi:10.1016/s0031-9422(00)97071-7

Cited by 30 publications

(18 citation statements)

References 21 publications

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“…Transformed roots were harvested from three or four¯asks. The roots were suspended in a 20-mm-diameter NMR tube and oxygenated by an airlift system (Fox et al 1989) as described previously (Ford 1995;Ford et al 1994).…”

Section: Methodsmentioning

confidence: 99%

“…The 15 N-and 31 P-NMR spectra were recorded at 30.42 and 121.49 MHz respectively, as described previously (Ford et al 1994 …”

Section: Methodsmentioning

confidence: 99%

“…Metabolic relationships between polyamines and alkaloids in transformed D. stramonium roots. ACC, 1-aminocyclopropane-1-carboxylic acid; CHA, cyclohexylamine; DecSAM, decarboxy-Sadenosylmethionine; MGBG, methylglyoxal-bis(guanyl)hydrazone; MPO, N-methylputrescine oxidase; SAM, S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; SPDsyn, spermidine synthase; SPMsyn, spermine synthase; TRI, tropinone reductase I; TRII, tropinone reductase II monitoring complex metabolic events in plants (Ratclie 1994), and recently we have shown that in-vivo 15 N-NMR can be used in studies of nitrogenous secondaryproduct metabolism (Ford 1995;Ford et al 1994Ford et al , 1996a. The advantage of NMR in this context is that several metabolic pools can be monitored simultaneously, allowing time-dependent correlations to be established between metabolites during the response of a tissue to a physiological perturbation.…”

Section: Introductionmentioning

confidence: 99%

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In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

Ford

Ratcliffe

Robins

1998

Planta

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The utilisation and accumulation of 15 Nlabeled metabolites by a 15 N-labeled transformed root culture of Datura stramonium L. was investigated by in vivo 15 N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [ 15 N]glutamate and [ 15 N]glutamine were rapidly depleted and there was an increase in the 15 N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of 15 Nlabel in the hydroxy-cinnamoylputrescines was reduced when the de-dierentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing a-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-dierentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, DL-a-di¯uor-omethylarginine and DL-a-di¯uoromethylornithine. Invivo 15 N-NMR spectra of roots grown in 15 N-enriched medium supplemented with these inhibitors showed that the 15 N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. DL-a-di¯uoro-methylarginine also prevented the phytohormone induced de-dierentiation of the root cultures, and this eect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-dierentiation of the root culture.

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Section: Methodsmentioning

confidence: 99%

“…The 15 N-and 31 P-NMR spectra were recorded at 30.42 and 121.49 MHz respectively, as described previously (Ford et al 1994 …”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

Ford

Ratcliffe

Robins

1998

Planta

Self Cite

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“…Therefore, the ®rst in-vivo NMR spectroscopic investigation on the biosynthesis of 15 N-labelled secondary compounds was appropriately set at a branching point of primary and secondary metabolism and dealt with the incorporation of inorganic nitrogen into amino acids, polyamine conjugates, and alkaloids (Ford et al 1994). Transformed root cultures of Datura stramonium and Nicotiana tabacum grown in 15 N-enriched medium were analyzed by in-vivo 15 N-NMR.…”

Section: Metabolism Of Secondary Nitrogen Compoundsmentioning

confidence: 99%

In-vivo nuclear magnetic resonance spectroscopy of low-molecular-weight compounds in plant cells

Schneider

1997

Planta

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“…15 N NMR spectroscopy was previously used frequently for the study of metabolic processes in bacteria, fungi, and alga (Lapidot and Irving, 1978;Martin et al, 1986;Kanamori et al, 1988;Callies et al, 1992). Recently it has been applied successfully also in the study of metabolism in higher plants (Neeman et al, 1985;Monselise et al, 1987;Kost, 1989, 1993; Thorpe et al, 1989;Lee and Ratcliffe, 1991;Robinson et al, 1991;Amancio and Santos, 1992;Ford et al, 1994Ford et al, , 1996Lee and Lewis, 1994). It allows the study of cellular metabolism in plants avoiding tedious isolation and purification steps.…”

Section: Introductionmentioning

confidence: 99%

15n NMR Spectroscopic Study of Ammonium Ion Assimilation by Spirodela Oligorrhiza—lemnaceae—as Affected by Light and Carbon Supply in Green and Extoliated Plants

Monselise

Kost

1998

Israel J Plant Sci

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This article is dedicated to the memory of my father, Professor Shaul Paul Monselise, a decade after his departure.ABSTRACT Nitrogen assimilation and amino acid production in Spirodela oligorrhiza plants exposed to 30 mM 15 NHFI was studied using 15 N NMR spectroscopy. Green and etiolated plants were studied under different light regimes and in the presence of added carbon, either as sucrose or as a-ketoglutarate. Etiolated plants are capable of ammonium assimilation and, as in green plants, this occurs via the glutamine synthetase/glutamine oxoglutarate amine transferase (GS/GOGAT) and the aspartate aminotransferase/asparagine synthetase pathways. The major assimilation products in both etiolated and green plants were glutamine and asparagine. Thus our results confirm that N-amides are key detoxification products when plants are exposed to external ammonium ion, and act as storage reservoirs or sinks for assimilated ammonium. In plants grown under continuous light, ammonium ion was taken up and assimilated to completion. L-methionine DL-sulfoximine, a GS inhibitor, inhibited ammonium ion assimilation but not its uptake. Addition of azaserine, a GOGA T inhibitor, resulted in the disappearance of a-amino signals, and 15 N incorporation into the glutamine amide-N position only. This is evidence for the operation of the GS/GOGAT pathway, as opposed to the glutamate dehydrogenase (GDH) pathway, in both green and etiolated plants. Even in the dark and under various stress conditions, no sign of ammonium ion assimilation via the GDH pathway could be detected.The amount of amino acid metabolites strongly depended on the light regime and the extent of external carbon supply. Supply of a-ketoglutarate to the etiolated plants increased ammonium ion uptake and assimilation. Ornithine and arginine were also formed, consistent with the operation of the ornithine cycle.

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In vivo 15N NMR studies of secondary metabolism in transformed root cultures of Datura stramonium and Nicotiana tabacum

Cited by 30 publications

References 21 publications

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

In-vivo nuclear magnetic resonance spectroscopy of low-molecular-weight compounds in plant cells

15n NMR Spectroscopic Study of Ammonium Ion Assimilation by Spirodela Oligorrhiza—lemnaceae—as Affected by Light and Carbon Supply in Green and Extoliated Plants

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