Estimation of Ammonium Ion Distribution between Cytoplasm and Vacuole Using Nuclear Magnetic Resonance Spectroscopy

Abstract

Evidence is presented that intracellular ammonium is trapped in vacuoles of maize (Zea mays L.) root tips because of rapid movement of ammonia between cytoplasm and vacuoles. The concentration of cytoplasmic ammonium is estimated to be <15 Mm at extracellular ammonium concentrations up to 1 mm. The implications for pathways of ammonium assimilation are discussed.The concentration of NH4' in the various compartments of plant cells has a direct bearing on the pathway of NH4' assimilation, because the enzymes GDH2 and GS have very different affinities for NH4' (25). Published estimates of the concentration of NH4+ in maize leaf mitochondria (31), root cytoplasm (10), and soybean nodule cytosol (7,29) have been in the millimolar range, similar to the Km of GDH for NH4+. These results suggest that GDH could catalyze significant assimilation of NH4' in plants. However, Streeter (27) estimated the NH4+ concentration in soybean nodule cytosol to be 'essentially nil. ' The cytoplasmic NH4' concentration may also be important with respect to pH gradients between intracellular compartments; millimolar concentrations of NH4' can collapse transmembrane pH gradients in vitro (3). Solutions of NH3 have been shown to rapidly and dramatically increase intracellular pH (15, 21). Biomembranes are highly permeable to NH3 (6). However, the significance of transmembrane fluxes of NH3 in plant cells exposed to solutions in which NH4' predominates over NH3, as occurs under normal physiological' conditions, is unclear (cf. refs. 6, 10, 12, 13, 16). Here, we describe the effects of extracellular NH4+ on cytoplasmic and vacuolar pHs using 3'P and 13C NMR, respectively. We present evidence for rapid equilibration of NH3 between cytoplasm and vacuole and estimate the concentrations of NH4+ in the different intracellular compartments.

Cytosolic Concentrations and Transmembrane Fluxes of NH4 +/NH3. An Evaluation of Recent Proposals

“…Thus, despite a 1,000-fold increase of [ 13 NH 3 ] o , tracer influx actually declined by 40%; a similar decline of 13 NH 4 ϩ fluxes between pH 5 and 7, was observed by Kosegarten et al (1997) Figure 1A, the gradient for NH 3 permeation across the tonoplast is in the opposite direction (from vacuole to cytosol), requiring active transport of NH 3 to the vacuole. The data reported by Lee and Ratcliffe (1991), Roberts and Pang (1992), and Wang et al (1993a) provide [NH 4 ϩ ] v values that are Ͼ1 mm. This leads to the unlikely scenario whereby transport of NH 3 to the vacuole requires an active NH 3 flux, not a passive flux as they propose.…”

“…First, they argue that "it is difficult to reconcile the estimates of cytosolic NH 4 ϩ concentrations made by this group (referring to the data of Wang et al, 1993a) with the high affinity of cytosolic GS for NH 4 ϩ (K m ϭ 10-20 m)." Second, they cite the findings of a single NMR study by Roberts and Pang (1992) (2000) go on to suggest that both influx and efflux of NH 3 are mediated by passive diffusion through a reversible low-affinity transport system (LATS), previously considered to be an NH 4 ϩ transporter. Finally, they suggest that NH 4 ϩ entry to the vacuole is via passive permeation of NH 3 and acid trapping of NH 4 ϩ .…”

“…Hexokinase activity is not regulated by the available Glc but as a result of allosteric inhibition from its product Glc-6-phosphate, with the result that large changes of Glc concentration hardly alter the rate of glycolysis (Fersht, 1985). GS is also known to be a highly regulated enzyme, located at a pivotal biochemical position to regulate plant nitrogen assimilation (Eisenberg et al, 2000), and available data indicate that [NH 4 ϩ ] c can vary considerably even in the same system under different conditions of [NH 4 ϩ ] o supply (Lee and Ratcliffe, 1991;Roberts and Pang, 1992;Wang et al, 1993a;Kronzucker et al, 1995). The rationale that an enzyme's K m should necessarily track ambient substrate concentration to optimize reaction rate, therefore, fails to apply to such enzymes.…”

“…The rationale that an enzyme's K m should necessarily track ambient substrate concentration to optimize reaction rate, therefore, fails to apply to such enzymes. Roberts and Pang (1992), cited by Howitt and Udvardi (2000) in support of micromolar values of [NH 4 ϩ ] c , incubated excised maize root tips in solutions containing from 0 to 10 mm NH 4 ϩ , and used NMR signals from 31 P and 13 C to estimate cytosolic and vacuolar pH. [NH 4 ϩ ] c was then calculated from total root NH 4 ϩ to be from 2 to 438 m (the upper limit being already Ն20 times higher than the reported GS K m for NH 4 ϩ ), on the presumption that NH 3 rapidly equilibrated across the tonoplast and was trapped as NH 4 ϩ according to cytosolic and vacuolar pH values.…”

“…1, A and B), assuming [NH 4 ϩ ] c of 10 m (Howitt and Udvardi, 2000) or 10,000 m (Wang et al, 1993a). In this model, [NH 4 ϩ ] o was varied from 10 to 10,000 m, pH values were set at 5, 7, and 5, respectively, for external solution, cytosol, and vacuole, ⌬ values of Ϫ100 and ϩ10 mV were selected for plasma membrane and tonoplast, respectively, (Wang et al, 1994), and [NH 4 ϩ ] v was set at 20,000 m. This value was selected to represent a midpoint of a quite wide range of literature values (Lee and Ratcliffe, 1991;Roberts and Pang, 1992;Wang et al, 1993a;Wells and Miller, 2000). The model predicts that when [NH 4 ϩ ] c is 10 m (Fig.…”