Correlated induction of nitrate uptake and membrane polypeptides in corn roots

Induction of corn (Zea mays L.) seedling root membrane polypeptides was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis in relation to induction of nitrate uptake. When nitrate uptake was studied using freshly harvested roots from 4-day old corn seedlings, a steady state rate of uptake was achieved after a lag of 2 to 3 hours. The plasma membrane fraction from freshly harvested roots (uninduced) and roots pretreated in 5 millimolar nitrate for 2.5 or 5 hours (induced) showed no differences in the major polypeptides with Coomassie blue staining. Autoradiography of the ³⁵S-methionine labeled proteins, however, showed four polypeptides with approximate molecular masses of 165, 95, 70, and 40 kilodaltons as being induced by both 2.5 and 5-hour pretreatment in 5 millimolar nitrate. All four polypeptides appeared to be integral membrane proteins as shown by Triton X-114 (octylphenoxypolyethoxyethanol) washing of the membrane vesicles. Autoradiography of the two-dimensional gels revealed that several additional low molecular weight proteins were induced. A 5-hour pretreatment in 5 millimolar chloride also induced several of the low molecular weight polypeptides, although a polypeptide of about 30 kilodaltons and a group of polypeptides around 40 kilodaltons appeared to be specifically induced by nitrate. The results are discussed in relation to the possibility that some of the polypeptides induced by nitrate treatment may be directly involved in nitrate transport through the plasma membrane.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.     

Ascorbate transport from the apoplast to the symplast in intact leaves

Infiltration of reduced ascorbate (ASC) into the leaves of Betula pendula Roth and subsequent measurement of its loss therein after incubation allowed us to follow ascorbate transport from apoplast to symplast in intact leaves. All of the ascorbate extracted from the native apoplast was in fully oxidized form, dehydroascorbate (DHA). When 5 m M of ASC was infiltrated into the leaves, its intense decay occurred, but only 55% of ASC lost was recovered in apoplast as DHA. When ASC was added to the freshly extracted intercellular washing fluid (IWF), ASC oxidation occurred as well. However, all oxidized ASC was recovered as DHA, indicating that further decomposition of DHA did not occur. Similarly, all of the ASC infiltrated into the leaves was found therein either as ASC or DHA after incubation of leaves for up to 60 min. On this base the ascorbate infiltrated into the leaves and not recovered in the IWF was interpreted as ascorbate taken up into the symplast. The calculated uptake rates of ascorbate at different ASC concentrations followed saturation kinetics with the maximum uptake rate of 300 nmol m⁻² plasma membrane (PM) area min⁻¹ and Michaelis constant of 12.8 m M . The uptake of ascorbate was significantly inhibited by the addition of dithiothreitol or by PM H⁺ ATPase inhibitor erythrosin B. Thus, our results support the previous observations that DHA is preferably transported from the apoplastic to the cytoplasmic side of the membrane and show that this process is dependent upon PM proton gradient.     

Kinetics of efflux of K+ from cultured rose cells treated with ultraviolet light

Irradiation of cultured rose ( Rosa damascena Mill. cv. Gloire de Guilan) cells with ultraviolet light caused of loss of K⁺, which occurred with sigmoid kinetics. The kinetics of loss of K⁺ were not changed when the extracellular concentration of K⁺ was held constant during the period of efflux. Furthermore, the rate of loss of K⁺ was approximately the same even though the K⁺ concentration in the medium was increased from 0.1 to 10 m M . The kinetics of uptake of the lipophilic methyltriphenylphosphonium cation, an indicator of the plasma membrane potential, were linear throughout the period of K⁺ efflux, suggesting that the starting and stopping of K⁺ efflux do not reflect a passive response to changes in the membrane potential of the cells. The results are interpreted in terms of activation and inactivation of an efflux channel or pump for K⁺.     

The effects of light on induction, time courses, and kinetic patterns of net nitrate uptake in barley

Barley seedlings ( Hordeum vulgare L.) were grown hydroponically with (induced) or without (uninduced) nitrate in a light/dark cycle with high photon flux density to determine the effects of light on time courses, induction and kinetics of net nitrate uptake. Nitrate uptake was induced by external nitrate in both light and dark and was prevented by 1 mol m^–3 p-fluorophenylalanine. In high light, nitrate uptake was about 2-fold higher than in low light. During time course experiments the uptake rates oscillated due to daily light–dark changes. Rates of nitrate uptake also increased at about 2200 h during continuous darkness. This increase coincided approximately with the time at which the dark period started during the previous culture of the plants, indicating that it was due to a mechanism associated with an endogenous diurnal rhythm. When calculating the kinetics of nitrate uptake, a model with two saturable systems, including a high-affinity system (HATS) and a low-affinity system (LATS), gave the best fit to data in all treatments. The apparent affinity of the HATS ranged from 7·7 to 12·2 mmol m^–3 in induced plants in all light conditions. The effect of light on the HATS was mainly an increase of apparent V _max in the step from low to high light. In uninduced plants the HATS operated at a very low activity which was strongly enhanced during induction. Interpretation of the calculated kinetics of the LATS was much more difficult on the basis of net uptake data. The apparent affinity of the LATS increased from 24·3 mol m^–3 in low light up to 0·17 mol m^–3 after acceleration in high light. These extreme changes in apparent affinity of the LATS could not be explained satisfactorily, and the nature of this system is also discussed with respect to the method used.     

Response of wheat seedlings to short-term drought stress with particular respect to nitrate utilization

Abstract. The effect of short-term changes in the water potential (from 0 to – 2.5 MPa) by addition of PEG 4000 to the nutrient solution was investigated with respect to nitrate uptake and reduction in 3-week-old wheat plants ( Triticum aestivum , cv Fidel). Plants were harvested at the end of 12-h treatments in the dark. The water potential of the mature leaves was similar to that of the medium down to – 0.8 MPa and was maintained at this level even though the external water potential was much lower. The medium water potential of 0.8 was a threshold level below which elongation of the youngest leaf was inhibited. Increase of the PEG concentration in the medium brought about a decrease of evapotranspiration and enhancement of nitrate uptake. No difference in the rate of nitrate reduction was observed, although the in vitro nitrate reductase activity was lowered. Nitrate accumulation in the shoot was ascribed both to the stimulation of net uptake from the medium, and to the mobilization and translocation of nitrate from the root. It is suggested that increase in the storage pool of nitrate in shoots was related to the role of NO₃⁻ as an osmoticum.     

Disruption of Ca2+ Homeostasis In Trypanosoma Cruzi By Crystal Violet

ABSTRACT. We have demonstrated previously that crystal violet induces a rapid, dose-related collapse of the inner mitochondrial membrane potential of Trypanosoma cruzi epimastigotes. In this work, we show that crystal violet-induced dissipation of the membrane potential was accompanied by an efflux of Ca²⁺ from the mitochondria. In addition, crystal violet inhibited the ATP-dependent, oligomycin-, and antimycin A-insensitive Ca²⁺ uptake by digitonin-permeabilired epimastigotes. Crystal violet also induced Ca²⁺ release from the mitochondria and endoplasmic reticulum of digitonin-permeabilized trypomastigotes. Furthermore, crystal violet inhibited Ca²⁺ uptake and the (Ca²⁺-Mg²⁺)ATPase of a highly enriched plasma membrane fraction of epimastigotes, thus indicating an inhibition of other calcium transport mechanisms of the cells. Disruption of Ca²⁺ homeostasis by crystal violet may be a key process leading to trypanosome cell injury by this drug.     

Relationship of combined nitrogen sources to salt tolerance in freshwater cyanobacterium Anabaena doliolum

Higher concentrations of NaCl inhibit the growth and reduce the specific growth rate of the freshwater cyanobacterium Anabaena doliolum. Among the nitrogen sources tried, nitrate protected the cyanobacterial cells most from salt toxicity. However, supplementing of medium with nitrate could increase the adaptability of the cells at sublethal doses but it would not permit growth at otherwise lethal doses of 300 mmol 1^-1 NaCl. Nitrate uptake was proportionally related to the NaCl level in the medium. The uptake of sodium was minimum when nitrate was simultaneously available to the cells, indicating the interaction of nitrate with the Na⁺ carrier. Na⁺ efflux was maximum in N², ammonia, urea and nitrate in decreasing order. This led to the conclusion that Na⁺ influx plays the critical role in salt tolerance, rather than its efflux.     

Vitamin D3 affects growth and Ca2+ uptake by Phaseolus vulgaris roots cultured in vitro

Vitamin D₃ at low concentration (10⁻⁹ M) inhibited the growth of Phaseolus vulgaris L. (cv. Contrancha) roots in vitro as measured by elongation (14 h) and [³H]-leucine incorporation into protein (2 h), and increased their labelling with ⁴⁵Ca²⁺ (2 h). Cycloheximide and puromycin (50 u.M) blocked vitamin D₃ stimulation of root ⁴⁵Ca²⁺ labelling, indicating that it is mediated by de novo protein synthesis. The calcium ionophore X-537A (10⁻⁵JW) induced similar changes both in root elongation and ⁴⁵Ca²⁺ uptake (14 h). This may indicate that the inhibitory effects of the sterol on root growth are mediated by changes in Ca²⁺ fluxes. However, this interpretation should be further strengthened by additional studies as the ionophore may have acted on root growth, affecting physiological processes other than Ca²⁺ transport.     

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg I⁻¹), amphotericin (10 mg I⁻¹) and teicoplanin (4 mg I⁻¹) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0·65 μm filters and a 150-fold reduction using the 0·45 μm filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10⁴ cfu ml⁻¹). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0·65 μm) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.     

Control of Nitrate Uptake by Phloem-Translocated Glutamine in Zea mays L. Seedlings

Abstract: The putative role of glutamine, exported from leaves to roots, as a negative feedback signal for nitrate uptake was investigated in Zea mays L. seedlings. Glutamine (Gln) was supplied by immersion of the tip-cut leaves in a concentrated solution. Nitrate (NO₃⁻) uptake was measured by its depletion in amino acid-free medium. The treatment with Gln resulted in a strong inhibition of nitrate uptake rate, accompanied by a significant enrichment of amino compounds in root tissue. The effect of N-availability on NO₃⁻ uptake was determined in split-root cultures. The plants were subjected to complete or localized N supply. Inducible NO₃⁻ uptake systems were also induced in N-deprived roots when the opposite side of the root system was supplied with KNO₃. The inhibitory effect of Gln was unaffected by localized N supply on one side of the split-root. The potential role of Gln in the shoot-to-root control of NO₃⁻ uptake is discussed.     

Short-term aluminium uptake by tobacco cells: Growth dependence and evidence for internalization in a discrete peripheral region

Short-term uptake and initial localization of aluminium (Al) were investigated in cultured cells of Nicotiana tabacum L. cv. BY-2. Graphite furnace atomic absorption spectrometry and an in vivo Al-sensitive fluorometric assay, employing morin, yielded similar results in all experiments. Aluminium uptake was critically dependent on cell growth. As opposed to negligible uptake in stationary-phase cells, Al uptake (20 μ M AlCl₃, pH 4.5, 23°C) by actively growing cells was detectable within 5 min, with an initial rate of 16 nmol Al (10⁶ cells)⁻¹ h⁻¹. Increased CaCl₂ levels (up to 20 m M ), low temperature (4°C), and pre-chelation of Al to citrate greatly reduced Al uptake (by 75–90%). A pH-associated permeabilization of cells at pH 4.5, as monitored by trypan blue, was observed in some growing cells. Although permeability to trypan blue was not a requirement for Al uptake, enhanced membrane permeability at pH 4.5, relative to pH 5.6, may contribute to Al uptake. Aluminium was observed to localize mainly in a pronounced and discrete fluorescent zone at the cell periphery (2–30 μm wide), presumably in the cortical cytosol and/or the adjoining plasma membrane section, although the possibility cannot be excluded that some Al resided in the cell wall apposing this discrete region. However, as judged by the Al-morin assay, there were no detectable Al levels in the remaining, larger portion of the cell wall. The potential of the Al-morin method in Al toxicity studies is illustrated.     

Photooxidative inhibition of nitrate reductase during chilling

The effect of a temperature close to the freezing point (chilling) on the nitrate reductase system of leaf discs of Cucumis sativus L. cv. Kleine Groene Scherpe was determined in the absence and presence of light. The capacity of leaf discs in the light (250 μE m⁻²s⁻¹) at 20°C to increase in vivo and in vitro nitrate reductase activity, was unaffected by chilling pretreatment in the dark, but 4 h of chilling pretreatment in the light (250 μE m⁻²s⁻¹) decreased the capacity to less than 50% of the unchilled control. The chilling inhibition of the capacity to increase nitrate reductase activity was of a photooxidative nature since it only occurred in the presence of light and oxygen. Plants grown at a low light intensity (65 μE m⁻²s⁻¹) lost 95% of their capacity to increase nitrate reductase activity, while plants grown at 195 μE m⁻²s⁻¹ retained 80% of their nitrate reducing capacity after 6 h chilling pretreatment in the 250 μE m⁻²s⁻¹ light. Previously induced nitrate reductase activity was also affected by light during chilling. A lag phase of 7 h preceded a fast phase of decrease in activity. Both in vivo and in vitro activity decreased to 15% of the control value after 18 h of chilling in the light. It is concluded that the induction mechanism of nitrate reductase is primarily affected by photooxidation during chilling. The decrease in nitrate reductase activity is attributed to a decrease in the amount of activity enzyme.     

Oxygen and nitrate respiration in a reed swamp sediment from a eutrophic lake

Seasonal measurements of the oxygen and nitrate uptake by a reed swamp sediment were carried out in a shallow, eutrophic Danish lake, Arreskov Sø. The oxidation of organic carbon in the sediment by aerobic and nitrate respiration was 290 and 188 g C m⁻² yr⁻¹ respectively. During winter, nitrate respiration amounted to 94% of the total carbon oxidation, whereas it was zero during summer. On an annual basis nitrate respiration constituted 39% of total respiration. Sediment nitrate uptake was correlated to nitrate concentration. In consequence of this the nitrate uptake rates varied during the year from zero in summer to 55 mg N m⁻² d⁻¹ in spring.
Oxygen uptake rates varied from 30 to 250 mg O₂ m⁻² h⁻¹ during the year, with a maximum uptake in August. The oxygen uptake per year was calculated to 860 g O₂ m⁻². The oxygen uptake rate was correlated to lake temperature and Kjeldahl nitrogen content of the sediment. The oxygen uptake rate, however, showed no correlation with loss on ignition of the sediment. A Q₁₀-value of 2.2 was found for lake measurements in the temperature interval of 5–15°C. The corresponding O₁₀-value in the laboratory was 2.6. A high microbial biomass indicated by the maximum content of Kjeldahl nitrogen and the lowest ratio of loss on ignition on Kjeldahl nitrogen appeared in late August, when the maximum oxygen uptake occurred. The oxygen uptake rate increased during the time interval from sampling to the start of the experiments.     

Development of pH sensitivity of sucrose uptake during ageing of Vicia faba leaf discs

Uptake of [U-¹⁴C]-sucrose (40 m M ) by fresh and aged peeled leaf discs of broad bean ( Vicia faba L. cv. Aguadulce) has been studied. In fresh discs, uptake was nearly insensitive to external pH, whereas the pH response of absorption in discs aged for 12 h was bell-shaped, with an optimum between pH 5 and 6. At this pH, uptake was nearly twice that in fresh tissue. The passive (insensitive to carbonyl cyanide m -chlorophenylhydrazone and to cold treatment) uptake was the same in fresh or aged discs. The development of pH sensitivity of absorption did not appear when ageing was performed in the presence of 10^−H M cycloheximide or 5.7 × 10⁻⁵ M actinomycin D. Similarly, when the tissues were treated with 10⁻³ M spermidine for 2 h after excision and then aged for 10 h, the development of the pH-sensitive uptake system was inhibited. Ca²⁺ (10⁻² M ) supplied together with spermidine prevented the inhibiting effect of spermidine. The appearance of the pH-sensitive system was also markedly reduced if ageing took place in the presence of 10⁻³ M aminoethoxyvinylglycine. Autoradiographs from fresh discs and from discs aged with or without the inhibitors suggest that pH sensitivity developed more intensively in the parenchyma than in the veins.
The results suggest some caution when using excised leaf discs for studies on sucrose uptake and phloem loading. Development of pH sensitivity of uptake may require the synthesis of both DNA-dependent RNA and protein and could be related to ethylene metabolism.     

Net uptake and partitioning of nitrogen and potassium in cultivars of barley during ageing

Experiments were carried out with barley cultivars ( Hordeum vulgare L.) grown in both pot- and water-culture. Net uptake of NO3₋ and K⁺ in the roots was followed in two barley cultivars grown on water-culture for 85 days. After an initial period of low net uptake of both ions, uptake increased until a maximum was reached after 30 to 45 days. Thereafter, net uptake of NO₃⁻ and K⁺ steadily decreased. In the pot experiments, effects of different mineral supply on day 4 to 18 upon the development of five barley cultivars of various earliness were investigated. The effect of earliness on fresh weight production was largest when mineral supply on day 4 to 18 was limited. The influence of limited mineral supply on day 4 to 18 on K-economy was independent of earliness of the cultivars. The maximal N-content was reached at the same time as maximal fresh and dry weight in fairly late cultivars; in early cultivars maximum N-level was reached later than maximum fresh and dry weight. Overall, maximal N-content was higher in the fairly late cultivars than in the early cultivars. The highest rate of ¹⁵N-transport was attained later in two of three fairly late cultivars than in early cultivars. Partitioning of dry weight, N and K in the shoots changed during ageing, ears being an important sink. Varietal differences in partitioning depended on the earliness of the cultivars. The largest fraction of recently supplied ¹⁵N, supplied as nitrate, and K⁺ (⁸⁶Rb) were found in the stems. In the oldest plants of the early cultivars the transport to the ears of these isotopes was gradually impaired, reflecting the decreasing function of the long distance transport system.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.     

The membrane potential is the driving force for siderophore iron transport in fungi

Abstract Respiratory inhibitors and uncouplers severely impair [⁵⁵Fe]ferricrocin uptake by Neurospora crassa . parallel measurements of ATP decay and ferricrocin uptake, however, disprove the idea that direct input of metabolic energy in the form of ATP is required for transmembrane movement of siderophores. The role of the membrane potential for siderophore uptake was demonstrated using iron-deficient cells, which were derepressed in the glucose-II uptake system. Addition of high amounts of glucose (1 mM) to glu-II-derepressed cells leads to a membrane depolarization of about 120 mV, followed by a significant inhibition of ferricrocin uptake, which recovered after some minutes. Full transport inhibition occurred after membrane depolarization in the presence of plasma membrane ATP-ase inhibitors (DCCD or DES), indicating that the membrane potential is essential for siderophore transport in fungi.     

MODIFICATION OF PHYTOPLANKTON GROWTH BY EXCRETED COMPOUNDS IN LOW-DENSITY POPULATIONS1

Conditioned seawater, removed by filtration from an exponentially growing mixed phytoplankton population, inhibited the growth of a small inoculum of cells from the same source without reducing the lag period. When the 2 populations, one in exponential growth and the other freshly inoculated, were separated by a filter membrane allowing passage of excreted compounds, the growth rate of the freshly inoculated cells was again depressed but the lag phase was reduced almost as effectively as by the addition of the chelator, EDTA. Thus both inhibitory and stimulatory compounds appear to be excreted by the cells during exponential growth, the stimulatory group, apparently involved with trace metal metabolism, being more labile to degradation. A test was made to determine if the population could be induced to utilize unconditioned water. Even after repeated transfers there was no reduction in the lag period, indicating that the initiation of exponential growth represents a response to excreted conditioning agents (possibly chelating compounds) rather than an adjustment by the population to use unconditioned water.     

L-Aspartate Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na⁺ gradient ([Na⁺] outside > [Na⁺] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na⁺ and Cl⁻ ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m -chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na⁺+ K⁺)-ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing K _m and V _max values of similar order to those found in slightly "cruder" preparations. Inhibition of the l -aspartate uptake by d -aspartate and d - and l -glutamate indicates that a common carrier is involved in the process, this being stereospecific for the d - and l -glutamate stereoisomers.