Kinetics of ¹⁵NH₄⁺ Assimilation in Zea mays

Abstract

In higher plants it is now generally considered that glutamate dehydrogenase (GDH) plays only a small or negligible role in ammonia assimilation. To test this specific point, comparative studies of 15NH4+ assimilation were undertaken with a GDH1-null mutant of Zea mays and a related (but not strictly isogenic) GDHIpositive wild type from which this mutant was derived. The kinetics of 15NH4+ assimilation into free amino acids and total reduced nitrogen were monitored in both roots and shoots of 2-week-old seedlings supplied with 5 millimolar 99% (15NH4)2S04 via the aerated root medium in hydroponic culture over a 24-h period. The GDHI-null mutant, with a 10-to 15-fold lower total root GDH activity in comparison to the wild type, was found to exhibit a 40 to 50% lower rate of 15NH4+ assimilation into total reduced nitrogen. Observed rates of root ammonium assimilation were 5.9 and 3.1 micromoles per hour per gram fresh weight for the wild type and mutant, respectively. The lower rate of 15NH4+ assimilation in the mutant was associated with lower rates of labeling of several free amino acids (including glutamate, glutamine-amino N, aspartate, asparagine-amino N, and alanine) in both roots and shoots of the mutant in comparison to the wild type. Qualitatively, these labeling kinetics appear consistent with a reduced flux of 15N via glutamate in the GDHI-null mutant.However, the responses of the two genotypes to the potent inhibitor of glutamine synthetase, methionine sulfoximine, and differences in morphology of the two genotypes (particularly a lower shoot:root ratio in the GDH1-null mutant) urge caution in concluding that GDH1 is solely responsible for these differences in ammonia assimilation rate.

Tobacco Isoenzyme 1 of NAD(H)-Dependent Glutamate Dehydrogenase Catabolizes Glutamate in Vivo

“…However, [ 15 N]Glu uptake by mutant roots was only 25% of that of wild-type roots, making direct comparison difficult (Stewart et al, 1995). Besides the results of Magalhaes et al (1990) and Stewart et al (1995) being apparently conflicting, the interpretation of these results is complicated by a lack of an isogenic control, making it unclear whether the altered morphology and [ 15 N]Glu uptake rate of the maize GDH mutant are pleiotropic effects of the mutation or rather merely a function of plant variety.…”

“…Assimilation of [ 15 N]NH 4 1 supplied to roots was decreased 40% to 50% in the mutant, suggesting that GDH isoenzyme 1 assimilates NH 4 1 in roots (Magalhaes et al, 1990). However, the authors urged caution in making this conclusion, because the mutant had an approximately 20% smaller shoot-to-root ratio (due to a greater root mass), and in the presence of the potent GS inhibitor, Met sulfoximine (MSX), NH 4 1 assimilation was prevented.…”

“…Metabolic studies with a maize (Zea mays) b-subunit deficit mutant, which has a 10-to 15-fold decrease in root GDH activity (Magalhaes et al, 1990;Pryor, 1990), have provided the best evidence for the in vivo direction of the GDH isoenzyme 1 reaction. Assimilation of [ 15 N]NH 4 1 supplied to roots was decreased 40% to 50% in the mutant, suggesting that GDH isoenzyme 1 assimilates NH 4 1 in roots (Magalhaes et al, 1990).…”

“…b 6 [isoenzyme 1], b 5 a 1 .b 1 a 5 , a 6 [isoenzyme 7]; Cammaerts and Jacobs, 1983;Loulakakis and Roubelakis-Angelakis, 1990;Magalhaes et al, 1990). The GDH isoenzyme profile of a particular organ can change during development (e.g.…”

Glutamate deamination by glutamate dehydrogenase plays a central role in amino acid catabolism in plants

“…5,6,[8][9][10] In particular, all previous gdh mutants contained mutations only in one of the two GDH genes. [8][9][10] It was possible that the effects of the loss of one functional GDH were masked by the redundant function of the other GDH in previous studies. Recently, we have isolated mutants for both of the two known Arabidopsis GDH genes and obtained a gdh double mutant (gdh1-2/gdh2-1) from crossing gdh1 and gdh2 mutants.…”