Ammonium-induced increase in NADH-glutamate dehydrogenase activity is caused by de-novo synthesis of the ?-subunit

Loulakakis, Konstantinos A.; Roubelakis‐Angelakis, Kalliopi A.

doi:10.1007/bf00195655

Cited by 35 publications

(19 citation statements)

References 26 publications

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“…The response of V. vinifera suspension cells cultured in the presence of ammonium ions as a sole nitrogen source mirrored the effect of salinity and confirmed our previous results with grapevine calluses grown in the presence of ammonium ions as sole nitrogen source, in which the increase in aminating GDH activity was due to de novo synthesis of the a-GDH polypeptide and the assembly of the anionic GDH isoenzymes (Loulakakis and Roubelakis-Angelakis, 1992). Ammonium treatment resulted in induction of Vv gdh-NAD;A1 transcript, which continued throughout the entire culture period ( Figure 12A); the respective increase was 3.5-, 4.5-, 7-, and 12-fold at 24, 48, 72, and 96 h, respectively.…”

Section: Nhsupporting

confidence: 76%

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

et al. 2006

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Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the a-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the a-subunit of GDH, increase in immunoreactive a-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy-mass spectrometry monitoring of 15 N-Glu, 15 N-Gln, and 15 N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of a-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive a-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces a-GDH subunit expression, and the anionic isoGDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.

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Section: Nhsupporting

confidence: 76%

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

et al. 2006

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“…Such increase in GDH activity as a function of an increase in N level has also been reported in Arabidopsis thaliana by Cammaerts & Jacobs (1985) and in Vitis vinifera by Different lower case letters correspond to significant differences for the same N-source, and different upper case letters correspond to significant differences for the same N concentration (Tukey test, P < 0,05) ( NH 4 + and NH 3 ). Loulakakis & Roubelakis-Angelakis (1992). On the other hand, GDH was not found to be modulated by N concentration in rice (Magalhães & Huber 1991) and Kalanchöe lateriria (Santos & Salema 1992).…”

Section: Resultsmentioning

confidence: 89%

Effects of different ammoniacal nitrogen sources on N-metabolism of the atmospheric bromeliad Tillandsia pohliana Mez

Tamaki

Mercier

2001

Rev. bras. Bot.

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-(Effects of different ammoniacal nitrogen sources on N-metabolism of the atmospheric bromeliad Tillandsia pohliana Mez). Increasing levels of atmospheric ammonia from anthropogenic sources have become a serious problem for natural vegetation. Short-term effects of different ammoniacal sources on the N metabolism of Tillandsia pohliana, an atmospheric bromeliad, were investigated. One-year-old, aseptically grown plants were transferred to a modified Knudson medium lacking N for three weeks. Plants were subsequently transferred to Knudson media supplemented with 0.5, 1.0, or 1.5 mM of N in the forms of NH 3 or NH 4 + as the sole N source. The activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH-NADH) were determined after 40 h. The GS activity was stimulated significantly by increasing the levels of the gaseous form. The GDH-NADH activity increased significantly under increasing N concentrations with NH 3 , while no significant differences were observed with NH 4 + as a N source. These results may reflect a faster NH 3 absorption by T. pohliana compared to NH 4 + uptake. The increased activity of GDH-NADH in NH 3 treatment may play a role in protecting the cells from the toxic effects of increased endogenous level of free ammonium. A raise in the concentration of N, especially in the form of NH 3 , greatly increased the content of free amino acids and soluble proteins. A possible utilisation of T. pohliana to evaluate the changes of atmospheric gaseous ammonia is proposed.RESUMO -(Efeitos de diferentes fontes amoniacais sobre o metabolismo de nitrogênio da bromélia atmosférica Tillandsia pohliana Mez). As fontes antropogênicas de amônia atmosférica têm se tornado um problema sério para a vegetação natural. No presente trabalho foram analisados os efeitos da aplicação de diferentes fontes amoniacais sobre o metabolismo do nitrogênio de Tillandsia pohliana, uma bromélia atmosférica. As sementes foram germinadas in vitro e as plantas cresceram em meio de cultura de Knudson por um ano. Após esse período, elas foram transferidas para o meio de Knudson sem as fontes nitrogenadas originais. Três semanas depois, as plantas foram colocadas nesse mesmo meio de cultura, porém, suplementado com 0,5, 1,0 ou 1,5 mM de N nas formas de NH 3 ou NH 4 + como fontes únicas de nitrogênio. O tempo de exposição a essas fontes foi de 40 h. As atividades enzimáticas da glutamina sintetase (GS) e da glutamato desidrogenase (GDH-NADH) foram analisadas. Os teores endógenos de amônio, de aminoácidos livres e de proteínas solúveis, presentes nos eixos caulinares dessa bromélia, também, foram determinados. As atividades da GS e da GDH-NADH foram estimuladas, significativamente, com o aumento do nitrogênio na forma gasosa. Esses resultados podem refletir a rápida absorção de NH 3 pela planta. Entretanto, a atividade aminante da GDH não apresentou diferença significativa em resposta ao aumento do N na forma de NH 4 + . Provavelmente, o incremento na atividade da GDH-NADH, no tratamento com NH 3 , está relacionado com...

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“…To account for any possible contribution of malate dehydrogenase to the measured in vitro GDH activities, crude extract was precipitated with ammonium sulfate, and the 35% to 70% fraction was collected. The pellet was dissolved in 200 mM Tris-HCl, pH 7.4, containing 14 mM mercaptoethanol and dialyzed (Loulakakis and Roubelakis-Angelakis, 1992). …”

Section: Protein Extraction Enzyme Assays and Electrophoresismentioning

confidence: 99%

“…Extraction of total RNA from plant tissues and northern blotting were performed as described (Loulakakis and Roubelakis-Angelakis, 1992;. The full-length cDNA Vvgdha-NAD;A1 and Slgdh-NAD;B2 (Purnell et al, 1997) were used as probes.…”

Section: Northern-blot Analysismentioning

confidence: 99%

“…Plant GDH has been proposed to be a stressresponsive enzyme (Syntichaki et al, 1996;Restivo, 2004), and consistent with this function, recombinantly produced GDH exhibits considerable thermal stability (Syntichaki et al, 1996). In addition, high intracellular ammonium, provided either externally (Cammaerts and Jacobs, 1985;Srivastava and Singh Rana, 1987;Lea and Ireland, 1999) or as a result of protein hydrolysis (Masclaux et al, 2000;Limami et al, 2002), generally leads to de novo synthesis of the a-subunit, assembly of the anionic isoenzymes, and increased in vitro aminating activity (Loulakakis and Roubelakis-Angelakis, 1992). Furthermore, the gene encoding for the a-subunit is induced in senescing plant organs, accompanied by an increase in immunoreactive protein and aminating in vitro activity (Loulakakis et al, 1994(Loulakakis et al, , 2002.…”

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confidence: 99%

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The Isoenzyme 7 of Tobacco NAD(H)-Dependent Glutamate Dehydrogenase Exhibits High Deaminating and Low Aminating Activities in Vivo

et al. 2007

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Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the b-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the a-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the a-subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of 15 NH 4 or [15 N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities.

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Ammonium-induced increase in NADH-glutamate dehydrogenase activity is caused by de-novo synthesis of the ?-subunit

Cited by 35 publications

References 26 publications

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

Effects of different ammoniacal nitrogen sources on N-metabolism of the atmospheric bromeliad Tillandsia pohliana Mez

The Isoenzyme 7 of Tobacco NAD(H)-Dependent Glutamate Dehydrogenase Exhibits High Deaminating and Low Aminating Activities in Vivo

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