The isoenzyme 7 of tobacco NAD(H)-dependent glutamate dehydrogenase exhibits high deaminating and low aminating activities in vivo

Abstract

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 beta-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 alpha-subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the alpha-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.

Figure 1.

Transcript abundance of Vvgdh-NAD;A1 in root and shoot of 30-d-old transgenic tobacco plants grown in half-strength Murashige and Skoog solution. Shoots and roots of wild-type (WT) tobacco and Vvgdh-NAD;A1 transgenic lines S2 and S5.

Figure 2.

Abundance of α- and β-GDH polypeptide and isoenzyme distribution of gdh-NAD;A1 in the shoots and roots of 30-d-old wild-type tobacco plants (WT) and transgenic lines grown in half-strength MS solution. A, α- and β-immunoreactive subunit polypeptides of GDH in wild-type tobacco and Vvgdh-NAD;A1 transgenic lines S2 and S5. B, Isoenzyme profiles of GDH in wild-type tobacco and Vvgdh-NAD;A1 transgenic lines S2 and S5.

Figure 3.

In vitro aminating (NADH) and deaminating (NAD) GDH activities in wild-type and Vvgdh-NAD;A1 tobacco transgenic lines S2 and S5 and NS and in Slgdh-NAD;B2 tobacco transgenic lines S49 and S77. A and B, Enzyme activities were determined in semipurified plant extracts. C and D, Enzyme activities were determined in the same plant extracts following dialysis. Statistical analysis of data was performed by one-way ANOVA. Asterisks indicate that means (n = 4) of tobacco transgenics are significantly different from the means of the corresponding wild-type and NS for either roots or shoots at P < 0.05 (*) and P < 0.01 (**).

Figure 4.

GC/MS analysis to monitor the fate of [¹⁵N]Glu (A and B) and ¹⁵NH₄ (C and D) in wild-type (WT) and Vvgdh-NAD;A1 tobacco transgenic lines S2 and S5 and in NS and Slgdh-NAD;B2 tobacco transgenic lines S49 and S77. A and B, The in vivo deaminating activity was determined as the percent residual [¹⁵N]Glu compared to wild-type/NS values. C and D, The in vivo amination GDH activity was determined as the ¹⁵NH₄ incorporated into [¹⁵N]Glu with MSX, and the percent amination in the transgenic lines was calculated with respect to the wild-type/NS values (insert). Statistical analysis of data was performed by one-way ANOVA. Asterisks indicate that means (n = 4) of tobacco transgenics are significantly different from these of the corresponding wild-type and NS for either roots or shoots at P < 0.05 (*) and P < 0.01 (**).

Figure 5.

Catabolism of ¹⁵N[Glu] into ¹⁵N[NH₄] in roots and shoots of wild-type tobacco (WT) and Vvgdh-NAD;A1 transgenic line S5. A, NMR spectrum of 20 mm ¹⁵N[NH₄Cl] standard. B and C, NMR spectrum of ¹⁵N[NH₄] accumulation at 4 h after ¹⁵N[Glu] treatment in shoots of wild-type tobacco and transgenic line S5. D and E, NMR spectrum of ¹⁵N[NH₄] accumulation at 4 h after ¹⁵N[Glu] treatment in roots of wild-type tobacco and transgenic line S5.