We present the first characterization of K + optimization of N uptake and metabolism in an NH4
Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.
The disruption of K+ transport and accumulation is symptomatic of NH4+ toxicity in plants. In this study, the influence of K+ supply (0.02–40 mM) and nitrogen source (10 mM NH4+ or NO3–) on root plasma membrane K+ fluxes and cytosolic K+ pools, plant growth, and whole-plant K+ distribution in the NH4+-tolerant plant species rice (Oryza sativa L.) was examined. Using the radiotracer 42K+, tissue mineral analysis, and growth data, it is shown that rice is affected by NH4+ toxicity under high-affinity K+ transport conditions. Substantial recovery of growth was seen as [K+]ext was increased from 0.02 mM to 0.1 mM, and, at 1.5 mM, growth was superior on NH4+. Growth recovery at these concentrations was accompanied by greater influx of K+ into root cells, translocation of K+ to the shoot, and tissue K+. Elevating the K+ supply also resulted in a significant reduction of NH4+ influx, as measured by 13N radiotracing. In the low-affinity K+ transport range, NH4+ stimulated K+ influx relative to NO3– controls. It is concluded that rice, despite its well-known tolerance to NH4+, nevertheless displays considerable growth suppression and disruption of K+ homeostasis under this N regime at low [K+]ext, but displays efficient recovery from NH4+ inhibition, and indeed a stimulation of K+ acquisition, when [K+]ext is increased in the presence of NH4+.
The acid-tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using 14 C-benzoic acid (BA) or [2-14 C]-5,5-dimethyloxazolidine-2,4-dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0-7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO2 uptake and O2 evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO2 uptake. The CO2 compensation concentration of the cells ranged from 9.4 mM at pH 4.5 to 16.2 mM at pH 7.0. An examination of the kinetics of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the Km (CO2) was 16.3 mM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO2, and the expression of external CA at neutral pH values would maintain an equilibrium CO2 concentration at the cell surface. This species does not possess a CO2-concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low Km (CO2) Rubisco of the alga.
The mechanism of inorganic carbon uptake was examined in Euglena gracilis Klebs. and the acidophilic species Euglena mutabilis Schmitz. Both species, whether grown in acidic (pH 3.5) or alkaline (pH 7.5) media lack external carbonic anhydrase. Acid-grown E. gracilis was shown to have no capacity for bicarbonate transport, but transport was induced on transfer to alkaline medium (pH 7.5) in the light over a period of 8 h. In contrast, acid-grown E. mutabilis appears to have no capacity for bicarbonate transport even at neutral pH. The overall internal pH of the cells was determined by equilibration with 14C-labelled benzoic acid over the pH range 3.55.0 and with 14C-labelled 5,5-dimethyloxazolidine-2,4-dione over the range pH 5.57.5. The acidophilic species maintains an internal pH range of 6.66.8 in an external pH range of 3.55.5, whereas the acid-tolerant species E. gracilis maintains a neutral internal pH in an external pH range of 3.57.5. Measurement, by mass spectrometry, of the fluxes of CO2 and O2 in photosynthesizing cells at pH 3.5 demonstrated a rapid uptake of CO2 by both species that was completely blocked by iodoacetamide, an inhibitor of CO2 fixation. Uptake of CO2 by E. gracilis, grown at pH 7.5, was not completely inhibited by iodoacetamide and O2 evolution was sustained when the cells reached the CO2 compensation concentration, indicating a direct uptake of bicarbonate. These data indicate that the acidophilic species, E. mutabilis, takes up CO2 by diffusion, whereas the acid-tolerant species, E. gracilis, takes up CO2 by diffusion at acid pH levels but has some capacity for active bicarbonate uptake when grown at alkaline pH levels.Key words: acidophilic alga, acidotolerant alga, bicarbonate uptake, CO2 uptake, Euglena gracilis, Euglena mutabilis, internal pH.
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