In Situ Nuclear Magnetic Resonance of <sup>15</sup>N Pulse Labels Monitors Different Routes for Nitrogen Assimilation

Callies, R.; Altenburger, Rolf; Abarzua, Sibylle; Mayer, A.; Grimme, L. Horst; Leibfritz, Dieter

doi:10.1104/pp.100.3.1584

Cited by 16 publications

(5 citation statements)

References 19 publications

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“…'^N-NMR spectroscopy has proved to have adequate sensitivity and resolution for N assimilation studies in a number of micro-organisms and plant cells (Legerton et al, 1981;Martin, 1985;Robinson et al, 1991 ;Amancio & Santos, 1992;Callies et al, 1992;Fox et al, 1992;Monselise & Kost, 1993). It permits direct monitoring of incorporation of'NH^î nto key metabolites such as glutamate and glutamine, and the incorporation of '''N into the [amino-'•'N]-and [am/(io-'''N]groups of glutamine can be clearly distinguished.…”

Section: Discussionmentioning

confidence: 99%

NH₄⁺ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a ¹⁵N‐NMR study

Martin

Côté

Canet³

1994

New Phytologist

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summary Nuclear magnetic resonance spectroscopy was used to monitor 15NH4+ assimilation and amino acid biosynthesis in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton. (strain S238). In mycelium growing rapidly on 15NH4+, [amido‐15N]glutamine was the major 15N‐labelled species. When 15N‐labelled mycelium was transferred into medium containing 14NH4+, the resonance for [amino‐15N]glutamine decreased with a half‐life of about 3.0 h, whereas the resonance for [amino‐15N]glutamine remained unchanged. Such behaviour is consistent with glutamine synthetase (GS) being the major route of 15NH4+ assimilation. However, the higher accumulation of [15N]alanine observed in the presence of the GS inhibitor, methionine sulfoximine, indicated that a part of the glutamate pool was formed by the glutamate dehydrogenase (GDH) pathway. When the mycelium was in stationary phase (i.e. low extracellular NH4+), the intramolecular 15N labelling of glutamine suggested that the GDH and GS pathways were simultaneously assimilating NH4+. The N supply and the growth stage, therefore, influence the expression of the activities of GDH and GS. The current isotopic data identify other fates of absorbed 15N: glutamate decarboxylation gives rise to γ‐aminobutyrate; transamination between glutamate and pyruvate yields alanine; and arginine accumulates. It is concluded that GS is the main pathway of primary assimilation of NH4+ in L. bicolor, but GDH may also contribute significantly to this process.

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Section: Discussionmentioning

confidence: 99%

NH₄⁺ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a ¹⁵N‐NMR study

Martin

Côté

Canet³

1994

New Phytologist

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“…Fortunately, NH4 cati be detected relatively easily in vivo in plant tissues by '•'N nuclear magnetic resonance (NMR) spectroscopy (Belton etal, 1985, Lee andRatcliffe 1991), '"N and '*N NMR spectroscopy has been used to investigate nitrogen metabolism in piants, and it has been suggested that NHJ has to be assimilated by the etizymes glutamine synthetase (GS) and glutamate synthase (GO-GAT) to avoid metabolic disorders (Thorpe et al, 1989. Robinson et al, 1991, Amancio and Santos 1992, Callies et al, 1992, Monseiise and Kost 1993, We have used in vivo ' "' N and '"N NMR for both quantitative and qualitative detenninations of tissue ammonium during the course of nitrogen assimilation in Norway spruce seedlings. The sensitivity of the '•'N ammonium NMR line shape to pH (Lee and Ratcliffe 1991) means that we were able estimate the pH of the vacuoiar NHj storage pool.…”

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confidence: 99%

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo ¹⁴N and ¹⁵N NMR spectroscopy

Aarnes¹,

Eriksen²,

Southon³

1995

Physiologia Plantarum

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S. Metabolism of nitrate and anunonium in seedlings of Norway spruce (Picea ahies) measured by in vivo "N and ' In vivo "N and '*N nuclear magnetic resonance spectroscopy was used to investigate the assimilation of nitrate and ammonium in seedlings of Norway spruce (Picea ahies [L.] Karst,), The main objective was to study accumulation of free NHJ and examine to what extent the nitrogen source affects the composition of the free amino acid pools in roots, stems and needles, NHJ concentrations in plants growing in the presence of 0,5-50 mM ammonium were quantified using ' "^N NMR. The NH:I values in tissues ranged from 6 to 46;(mol (g fresh weight)'', with highest concentrations in roots and needles. The tissue NHl peaked at 5,0 mAf NHJ in the medium, and failed to increase when NH4 in the medium was increased to 50 mM, indicating metabolic control of the concentration of this cation in tissues. The ^""N NMR spectra were used to estimate pH of the NH4 storage pools. Based on the pH sensitivity of the quintet of "NHJ resonance, we suggest that the pH of the ammonium storage compartments in the roots and stems should be 3,7-3.8, and in needles 3.4-3.5, representing extremely low pH values of the tissue, "N from nitrate or ammonium was first incorporated into the amide group of glutamine and then into a-amino groups, confirming that the glutamine synthetase/ glutamate synthase cycle is the major route of nitrogen assimilation into amino acids and thus plays a role in lowering the levels of NH* in the eytoplasm. NHJ can also be assimilated in roots in piants growing in darkness. The main "N-labelled amino acids were glutamine, arginine and alanine. Almost no ''N signals from needles were observed. Double labelling (dN + (o,a>N) of arginine is consistent with the operation of the omithine cycle, and enrichment indicates that this cycle is a major sink of newly assimilated nitrogen. Nitrogen assimilation in roots in the presence of added methionine sulphoximine and glutamate indicated the catabolic action of glutamate dehydrogenase. The ' •'N NMR spectra of plants grown on '^N-urea showed a marked increase in the labelling of ammonium and glutamine, indicating high urease activity. Amino acids were also quantified using high pressure liquid chromatography. Arginine was found to be an important transport form cf nitrogen in the stem.

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“…The PEM culture contained several "N-labeled compounds over a protracted period in inorganic nitrogen (Fig, 1), In addition to the amide of Gin, a-amino ni-trogen and Ala, resonances for His, Arg, urea, Ser, aliphatic amines and several unknowns were also present. Short-term studies with "KH4 showed that the amide of Gin was the first compound to contain inorgatiic nitrogen, as has been found in Zea mays, Chloreltafusca, Microcystis firma, Picea glauca and Lemna gibba (Monselise et al, 1987, TTiorpe et al, 1989, Amancio and Satitos 1992, Callies et al, 1992, Use of the inhibitors AZA and MSO supported this finding in al! of these studies.…”

Section: Discussionmentioning

confidence: 81%

Stage‐specific nitrogen metabolism in developing carrot somatic embryos

Joy

McLntyre

Vogel

et al. 1996

Physiologia Plantarum

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The physiology of individual somatic embryo developmental stages otDaucus carota L. was examined by in vivo nuclear magnetic resonance (NMR) spectroscopy, amino acid analysis and 14C‐labeling. 15N NMR spectroscopy was used to examine the uptake and incorporation of 15N isotopically labeled inorganic nitrogen sources. NMR spectra of proembryogenic masses (PEMs) contained resonances for histidine, amino sugars, glutamine, arginine, urea, alanine. α‐amino nitrogen, serine, aliphatic amines and several unknowns. Similar resonances were found in various embryo developmental stages. However, resonances for arginine and aliphatic amines peaked during globular and torpedo stages and substantially decreased in germinating stage embryos. The dominant resonances observed in non‐embryogenic cells and germinating embryos were glutamine and α‐amino nitrogen. Amino acid analysis of the various embryo stages showed that glutamate, glutamine and arginine were the major contributors to the soluble amino acid profiles. During development, glutamate and glutamine continued to increase in concentration whereas arginine and its related metabolites (i.e. ornithine and y‐aminobutyric acid [GABA]) were biphasic; increasing in globular and torpedo stage embryos and decreasing in germinating embryos. Carbon‐14 labeling indicated that labeled glutamine pools in non‐embryogenic and germinating embryos were greatest compared to other embryo stages, whereas labeled GABA pools were greatest in globular and torpedo stage embryos. Taken together, these data indicate that the physiology of each embryo developmental stage is distinct. They also suggest that during somatic embryo development, a switch takes place in metabolism whereby the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway is predominant in non‐embryogenic cells and germinating stage embryos. Furthermore, during early to mid‐embryo development (PEMs, globular and torpedo stage embryos), metabolism utilizing the omithine cycle is enhanced and predominant.

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In Situ Nuclear Magnetic Resonance of ¹⁵N Pulse Labels Monitors Different Routes for Nitrogen Assimilation

Cited by 16 publications

References 19 publications

NH₄⁺ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a ¹⁵N‐NMR study

NH₄⁺ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a ¹⁵N‐NMR study

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo ¹⁴N and ¹⁵N NMR spectroscopy

Stage‐specific nitrogen metabolism in developing carrot somatic embryos

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In Situ Nuclear Magnetic Resonance of 15N Pulse Labels Monitors Different Routes for Nitrogen Assimilation

Cited by 16 publications

References 19 publications

NH4+ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a 15N‐NMR study

NH4+ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a 15N‐NMR study

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo 14N and 15N NMR spectroscopy

Stage‐specific nitrogen metabolism in developing carrot somatic embryos

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In Situ Nuclear Magnetic Resonance of ¹⁵N Pulse Labels Monitors Different Routes for Nitrogen Assimilation

NH₄⁺ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a ¹⁵N‐NMR study

NH₄⁺ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a ¹⁵N‐NMR study

Metabolism of nitrate and ammonium in seedlings of Norway spruce (Picea abies) measured by in vivo ¹⁴N and ¹⁵N NMR spectroscopy