Aktive und inaktive Phosphataufnahme in Blattzellen von Elodea densa bei hohen Phosphat-Außenkonzentrationen

Summary The phosphate uptake in the leaf cells of Elodea densa shows multiple isotherms in the range [S]>1 mmole P/l to 100 mmoles P/l. In the dark the uptake isotherms contain three distinct parts (II/1, II/2 and II/3); the first two obey Michaelis-Menten kinetics, whereas the third is exponential. In the light the phosphate uptake curve consists only of two parts (II/1 and II/2) agreeing with Michaelis-Menten kinetics, the exponential part being absent.Cellular phosphate content was found to be 45 mmoles/l. Data concerning the membrane potential E for Elodea densa were obtained from Jeschke (1970). In accordance with the Nernst equation a change from the hyperbolic curve to an exponential one was expected at a concentration of about 60 mmoles P/l in the dark and at above 100 mmoles P/l in the light. The results obtained agree with these theoretical calculations: in the dark, the change from the hyperbolic to the exponential curve was observed at [S]=50 mmoles P/l, which is in electro-chemical equilibrium with the cellular orthophosphate content of about 35 mmoles/l (inorganic P content amounting to 80 per cent of total phosphate). In the light no change towards an exponential curve was noticed.The effect of the uncoupler CCCP in the light and in the dark was examined in order to elucidate its influenc on ³²P incorporation into the fractions of inorganic, organic and acid-insoluble phosphates, the inorganic fraction representing phosphate uptake. The inhibition of the uptake into the inorganic part decreases with an increasing inactive component of total uptake, while the fixation in the organic fraction is severely curtailed at all concentrations tested. The acid-insoluble fraction remains unaffected.

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Abkürzungen und Symbole CCCP Carbonylcyanid m-Chlorphenylhydrazon - Du Dunkel FG Frischgewicht - GP Gesamtphosphat - [H₂PO₄ ^-]_i Innenkonzentration - [H₂PO₄ ^-]_o Außenkonzentration - Ko Kontrolle - Li Licht - P Phosphat - P_a anorganisches TCE-lösliches Phosphat - P_o organisches TCE-lösliches Phosphat - P_u TCE-unlösliches Phosphat - P_gl TCE-gesamtlösliches Phosphat - [S] Außenkonzentration des H₂PO₄ ^--Ions - TCE Trichloressigsäure     

Der Einfluß von Natrium- und Kaliumionen auf die Phosphataufnahme bei Ankistrodesmus braunii

Summary The uptake of phosphate as influenced by sodium and potassium ions was investigated in the light and in the dark. It was found to be a function of the external phosphate concentration. At a low concentration (up to 10^–5 mol/l) in the presence of Na⁺ phosphate is quickly absorbed and hence phosphate is the limiting factor for further labelling. In the presence of K⁺ phosphate uptake is constant over a long period.The enhancement of phosphate uptake by Na⁺ is also found when the external concentration of P is raised up to 10^–4 mol/l. Then the gross uptake proceeds over six hours, with the greatest Na⁺-dependent increase occurring in the label of the TCA-insoluble phosphate fraction (P_u).The phosphate uptake is strongly dependent on the pH of the reaction mixture. In the presence of Na⁺ it is highest between pH 5.6 and 7. As the uptake in the presence of K⁺ parallels the dissociation curve of the dihydrogen form H₂PO ₄ ^– , the Na⁺-enhancement is optimal in the alkaline pH range (pH 8).On the basis of a comparison between the pH-dependence of phosphate uptake and the dependence of the uptake on the external phosphate concentration analysed by a method of enzyme kinetics, it is suggested that Ankistrodesmus metabolically transports H₂PO ₄ ^– but not HPO ₄ ⁼ . Moreover, it is concluded from the absence of light stimulation and the weak inhibition of the uptake by DCMU or CCCP in the presence of K⁺ that at low P-concentrations the diffusion is limiting the uptake. Only at higher concentrations is an active phosphate uptake measured.Furthermore it is concluded that the observed Na⁺-stimulation of the ³²P-labelling of the TCA-soluble and insoluble compounds inside the cell is indirect and depends only on the action of Na⁺ and K⁺ ions at the first transport site in the plasmalemma.     

Der Einfluß von Natrium- und Kalium-Ionen auf die Photophosphorylierung bei Ankistrodesmus braunii

Summary During short-time experiments (30 sec to 60 min) sodium ions stimulate the phosphate uptake and especially the ³²P-labelling of the organic TCA-soluble phosphate compounds up to 1,500% (K⁺=100%). The labelling is maximally stimulated in the light and in the dark at concentrations of about 5×10^-3 mol/l Na⁺ and at pH 8. Lithium ions stimulate ³²P-labelling in a similar but less effective way. In comparison, in the presence of potassium ions the ³²P-label decreases.It was investigated whether sodium ions specifically stimulate the ATP-synthesis or some reaction of the photosynthetic carbon reduction cycle or whether they only enhance the ³²P-labelling of phosphorylated compounds.Separation by thin-layer chromatography of the MCF-soluble phosphate fraction showed that labelling of all compounds investigated was stimulated by Na⁺ to a similar extent.Experiments performed in red and far-red light (683 and 712 nm) under nitrogen and in the presence of various DCMU-concentrations, as well as in the presence of antimycin A and CCCP showed that Na⁺ exerts no specific influence either on the cyclic or on the non-cyclic photophosphorylation in vivo.ATP-dependent reactions such as ¹⁴CO₂-fixation or glucose uptake are not influenced by Na⁺.Since Na⁺ does not change the size of phosphate pools in a different way from K⁺, there is no evidence for the assumption that the Na⁺-dependent increase in the ³²P-labelling is due to its action on the chloroplast membrane in increasing its permeability to orthophosphate ions. This is supported by the lack of any effect of sodium plus phosphate ions on the CO₂-fixation.Therefore the results give no evidence that sodium acts directly on phosphorus metabolism inside the cell. It is suggested that its action is localised at the phosphate-transporting site of the plasmalemma.     

Utilization of nitrite and nitrate by dwarf bean

The uptake and conversion of NO ₂ ^- and the effect of NO ₂ ^- on the uptake and reduction of NO ₃ ^- were examined in N-depleted Phaseolus vulgaris L. Nitrite uptake at 0.1 mmol dm^-3 was against an electrochemical gradient and became constant after one or two initial phases. Steadystate uptake declined with increasing ambient NO ₂ ^- concentration (0–0.7 mmol dm^-3). In this concentration range root oxygen consumption was unaffected by NO ₂ ^- , indicating that the decrease of NO ₂ ^- uptake was not related to respiration. After 6 h NO ₂ ^- supply, about one-third of the absorbed NO ₂ ^- had accumulated, mainly in the root system. Oxidation of NO ₂ ^- to NO ₃ ^- was not observed. The apparent induction period for NO ₃ ^- uptake was about 6 h in control plants and 3.5 h in plants that were pretreated for 18 h with NO ₂ ^- . In contrast, the time course of NO ₂ ^- uptake was unaffected by pretreatment with NO ₃ ^- . Steadystate NO ₃ ^- uptake was less affected by NO ₂ ^- than was steady-state NO ₂ ^- uptake by NO ₃ ^- . Nitrate reductase activity (NRA) in leaves and roots was induced by both NO ₃ ^- and NO ₂ ^- . In roots, induction with NO ₂ ^- was faster than with NO ₃ ^- , but there was no difference in NRA after 5 h. Nitrite inhibited NRA in the roots of NO ₃ ^- -induced plants and thus seems to stimulate the induction, but not the activity of induced nitrate reductase. In view of the observed differences in time course and mutual competition, a common uptake mechanism for NO ₂ ^- and NO ₃ ^- seems unlikely. Expression of the NO ₂ ^- effect on the induction of NO ₃ ^- uptake required more time than the induction itself. We therefore conclude that NO ₂ ^- is not the physiological inducer of NO ₃ ^- uptake.Abbreviations NR(A) nitrate reductase (activity) - BM basal medium     

Kinetics of ammonium and nitrate uptake among wild and cultivated tomatoes

Summary Concentration dependence of net ammonium and nitrate uptake was monitored for a cultivar of tomato, Lycopersicon esculentum, and two accessions of a neotropical wild relative, L. hirsutum. The kinetics of net NH ₄ ⁺ uptake differed among these taxa and were not dependent on the ionic composition of the nutrient solution. The kinetics of net NO ₃ ^- uptake were dependent on the composition of the nutrient solution; the presence of NH ₄ ⁺ or Cl^- enhanced net NO ₃ ^- uptake for the cultivated species and for a highland accession of the wild species. The capacity for net NO ₃ ^- uptake was greater than the capacity for net NH ₄ ⁺ uptake in all three taxa; the proportion of NO ₃ ^- to NH ₄ ⁺ absorbed was much greater for the wild taxa. Our data suggest that NO ₃ ^- may be a more important source of mineral nitrogen than NH ₄ ⁺ for these tropical taxa.     

Distribution of reducing power between photosynthetic carbon and nitrogen assimilation in Scenedesmus

Fixation of CO₂ and N assimilation were studied in synchronous cultures of Scenedesmus obtusiusculus Chod. under saturating and limiting light. Within the photon-flux range studied, the cells maintained C to N assimilation ratios of 7–10 with either NO ₃ ^- , NO ₂ ⁺ or NH ₄ ⁺ as the N source. Competitive interactions between C and N assimilation were pronounced under light limitation and were proportional to the oxidation status of the N source. Fixation of CO₂ at saturating light was also slightly reduced by NO ₂ ^- and NH ₄ ⁺ . In the absence of CO₂, NO ₃ ^- uptake and reduction was light-saturated at a comparatively low photon flux, whereas NO ₂ ^- uptake and reduction was considerably faster in the absence of CO₂ than in its presence. The pools of reduced pyridine nucleotides (NADPH and NADH) were largely unaffected by the presence or absence of the different N sources. The regulatory influences of CO₂ fixation on N assimilation are discussed in terms of coupling between the rates of CO₂ fixation and NH ₄ ⁺ assimilation, as well as the existance of control mechanisms for NO ₃ ^- uptake and reduction.Abbreviations Chl chlorophyll - PF photon flux     

Fate of 15N labelled urine on four soil types

A field lysimeter experiment was conducted over a 406 day period to determine the effect of different soil types on the fate of synthetic urinary nitrogen (N). Soil types included a sandy loam, silty loam, clay and peat. Synthetic urine was applied at 1000 kg N ha^-1, during a winter season, to intact soil cores in lysimeters. Leaching losses, nitrous oxide (N₂O) emissions, and plant uptake of N were monitored, with soil ¹⁵N content determined upon destructive sampling of the lysimeters. Plant uptake of urine-N ranged from 21.6 to 31.4%. Soil type influenced timing and form of inorganic-N leaching. Macropore flow occurred in the structured silt and clay soils resulting in the leaching of urea. Ammonium (NH ₄ ⁺ –N), nitrite (NO ₂ ^- –N) and nitrate (NO₃ ^-–N) all occurred in the leachates with maximum concentrations, varying with soil type and ranging from 2.3–31.4 g NH ₄ ⁺ –N mL^-1, 2.4–35.6 g NO ₂ ^- –N mL^-1, and 62–102 g NO ₃ ^- –N mL^-1, respectively. Leachates from the peat and clay soils contained high concentrations of NO ₂ ^- –N. Gaseous losses of N₂O were low (<2% of N applied) over a 112 day measurement period. An associated experiment showed the ratio of N₂–N:N₂O–N ranged from 6.2 to 33.2. Unrecovered ¹⁵N was presumed to have been lost predominantly as gaseous N₂. It is postulated that the high levels of NO ₂ ^- –N could have contributed to chemodenitrification mechanisms in the peat soil.     

Energy transduction efficiencies in nitrogenous oxide respirations of Azospirillum brasilense Sp7

For Azospirillum brasilense Sp7, the energy transformation efficiencies were measured in anaerobic respirations with either nitrate, nitrite or nitrous oxide as respiratory electron acceptors by determining the maximal molar growth yields and the H⁺-translocations using the oxidant pulse method. In continuous cultures grown with malate limiting, the maximal molar growth yields (Y _s ^max -values) were essentially the same with O₂ or N₂O but were 1/3 and 2/3 lower with NO ₂ ^- or NO ₃ ^- , respectively, as respiratory electron acceptors. Both the maximal molar growth yields and the maintenance energy coefficients were surprisingly high when Azospirillum was grown with nitrite as the sole electron acceptor and source for N-assimilation. Growth under N₂-fixing conditions drastically reduced the Y _s ^max -values in the N₂O and O₂-respiring cells. In the H⁺-translocation measurements, the /oxidant ratios were 5.6 for O₂→H₂O, 2.5–2.8 for NO ₃ ^- →NO ₂ ^- , 2.2 for NO ₂ ^- →N₂O and 3.1 for N₂O→N₂ respirations when the cells were preincubated with valinomycin and K⁺. All the values were enhanced when the experiments were performed with valinomycin plus methyltriphenylphosphonium (=TPMP⁺) cation. The uncoupler carbonyl cyanide-m-chlorophenyl-hydrazone diminished the H⁺-excretion indicating that this translocation was due to vectorial flow across the membrane. In the absence of any ionophore, nitrate and nitrite respirations were accompanied by a H⁺-uptake . Any significant H⁺-translocation could not be detected in N₂O- and O₂-respirations under these conditions. It is concluded that nitrate reduction proceeds inside the cytoplasmic membrane, whereas nitrite is reduced extramembraneously. The data are not conclusive for the location of nitrous oxide reductase. The maximal molar growth yield determinations and the absence of any H⁺-uptake in untreated cells indicate a cytoplasmic orientation of the enzyme similar to the terminal cytochrome oxidase of respiration. The low H⁺-extrusion values for N₂O-respiration compared to O₂-respiration in cells treated with valinomycin plus TPMP⁺ are, however, not in accord with such an interpretation.     

Denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Rhodopseudomonas sphaeroides f. denitrificans grown photosynthetically with NO ₃ ^- under anaerobic conditions accumulated NO ₂ ^- in the culture medium. In washed cells succinate, lactate, fumarate, citrate and malate, were effective electron donors for the reduction of NO ₃ ^- , NO ₂ ^- and N₂O to N₂ gas. Nitrate reductase was inhibited by amytal and potassium thocyanate. Nitrite reductase activity was severely restricted by potassium cyanide, sodium diethyldithiocarbamate, Amytal and 2-n-heptyl-4-hydroxyquinoline-N-oxide whereas N₂O reductase was inhibited by NaN₃, C₂H₂ and KCNS. Cells incubated with either K¹⁵NO₃ or K¹⁵NO₂ produced ¹⁵N₂O and ¹⁵N₂. A stoichiometry of 2:1 was recorded for the reduction of either NO ₃ ^- or NO ₂ ^- to N₂O and N₂ and for N₂O to N₂ it was 1:1.Abbreviations BVH reduced benzyl viologen - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - CCCP carbonyl cyanide-m-chlorophenyl-hydrazone - DIECA diethyl dithiocarbamate - KCN potassium cyanide     

Effect of HCO - 3 concentration in the absorption solution on the energetic coupling of H+-cotransports in roots of Zea mays L.

The effect of HCO ₃ ^- on ion absorption by young corn roots was studied in conditions allowing the independent control of both the pH of uptake solution and the CO₂ partial pressure in air bubbled through the solution. The surface pH shift in the vicinity of the outer surface of the plasmalemma induced by active H⁺ excretion was estimated using the initial uptake rate of acetic acid as a pH probe (Sentenac and Grignon (1987) Plant Physiol. 84, 1367). Acetic acid and orthophosphate uptake rates and NO ₃ ^- accumulation were slowed down, while ⁸⁶Rb⁺ uptake and K⁺ accumulation rates were increased by HCO ₃ ^- . These effects were similar to those induced by 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid/2-amino-2-(hydroxymethyl)-1,3-propanediol (Hepes-Tris). They were more pronounced when the H⁺ excretion was strong, were rapidly reversible and were not additive to those of Hepes-Tris. The hypothesis is advanced that the buffering system CO₂/H₂CO₃/HCO ₃ ^- accelerated the diffusion of equivalent H⁺ inside the cell wall towards the medium. This attenuated the surface pH shift in the vicinity the plasma membrane and affected the coupling between the proton pump and cotransport systems.Abbreviations FW fresh weight - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - J_aa acetic acid influx - J_K ⁺ K⁺ influx - J_Pi orthophosphate influx - Mes 2-(N-morpholino)ethanesulfonic acid - pCO₂ CO₂ partial pressure - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Temperature dependence of inorganic nitrogen uptake and assimilation in Antarctic sea-ice microalgae

Summary The influence of temperature on NO ₃ ^- and NH ₄ ⁺ uptake, and the activity of the assimilatory enzyme NO ₃ ^- reductase (NR) was compared to inorganic C uptake (photosynthesis) in natural assemblages of Antarctic sea-ice microalgae. NO ₃ ^- and NH ₄ ⁺ uptake reached a maximum between 0.5°–2.0°C and 2.0°–3.0°C, respectively, which was close to that for photosynthesis (2.5°–3.0°C). NR showed a distinctly higher temperature maximum (10.0°–12.0°C) and a lower Q₁₀ value than inorganic N and C transport. Our data imply that, owing to differential temperature characteristics between N transport and N assimilation at in situ temperature (-1.9°C), the incorporation of extracellular NO ₃ ^- into cellular macromolecules, may be limited by transport of NO ₃ ^- into the cell rather than the intracellular reduction of NO ₃ ^- to NH ₄ ⁺ . Despite differences in temperature maxima between N transport and N assimilation, the overall low temperature maxima of inorganic N metabolism characterizes Antarctic sea-ice microalgae as psychrophilic. Our study is the first to examine the temperature dependence of inorganic N uptake and assimilation in sea-ice microbial communities.     

Untersuchungen über die Raten der Polyphosphatsynthese durch die Photophosphorylierung bei Ankistrodesmus braunii

Summary Short-time experiments with ³²P-labelled phosphate and chase experiments with equally labelled cells were carried out with synchronized algae under conditions of optimum phosphate uptake. In short-time experiments, in the presence as in the absence of CO₂, orthophosphate and organic phosphates are rapidly labelled, but their time curves show saturation behaviour after 10 to 20 min. Labelling of polyphosphates proceeds at a constant rate after a short lag period of about 5 min. In equally labelled algae ³²P-labelling correspondingly decreases in orthophosphate and in organic phosphates, but increases by about the same amount in the fraction of acid-insoluble polyphosphates. In the presence of external phosphate and in the light, polyphosphates show no visible decay within the 20 min of the chase experiments.A comparison of the two kinds of experiments suggests that polyphosphates are secondary products of photophosphorylation following only after orthophosphate and organic phosphates, probably after ATP. The rates of photophosphorylation are certainly much higher than the rates of labelling in organic phosphates because of the limiting phosphate uptake. Since the polyphosphates show no decay during the time of the experiments their turnover is low and the rates of polyphosphate labelling after a phosphate starvation period, and after the short lag period, can be regarded as approximate rates of polyphosphate synthesis. These rates are lower than the rates of phosphate uptake.In young cells of the synchronous culture phosphate replenishment after a 5-h starvation requires 2 to 3 h. After replenishment or in a culture undisturbed by phosphate starvation, the rates of polyphosphate accumulation, like the rates of phosphate uptake are much lower. In the presence of CO₂ they are constant for several hours, if related to culture volume with constant cell number. Polyphosphate accumulation is proportional to phosphate uptake under these conditions amounting to about one third. In the absence of CO₂, the rates decrease after 2 to 4 h of CO₂-starvation and, like in short-time experiments a large proportion of the phosphate taken up is used for polyphosphate accumulation. The low rates of long-time experiments may represent a steady state between formation and decay of polyphosphates. Since the cells kept in the absence of CO₂ are prevented from growing they actually accumulate more polyphosphates per cell volume, per chlorophyll, and per dry weight than the cells in the presence of CO₂.The rates of polyphosphate formation are discussed with respect to their turnover in the light observed by other investigators. They are regarded to be a result of competition for ATP together with the orthophosphate pool of the cells, and of the compartmentation. The rates of polyphosphate formation are rather low compared with the probable rates of ATP formation under various conditions of photophosphorylation. Therefore, the formation of polyphosphates is regarded as a process of secondary order of magnitude in the energy metabolism of algal cells.

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Im Text verwendete Abkürzungen P₁ Trichloressigsäure lösliche Phosphate - davon P_i Orthophosphat - P_o organisches Phosphat - P_ul Hydrolyse-labiles TCE-unlösliches Phosphat - P_us Hydrolyse-stabiles TCE-unlösliches Phosphat - P_ges Gesamtphosphat, bei kurzzeitiger ³²P-Markierung Phosphataufnahme - Chl Chlorophyll     

Über die Aufnahme von Phosphat-und Sulfationen Durch Blätter von Elodea Densa und Ihre Beeinflussung Durch Licht,Temperatur und Aussenkonzentration

Zusammenfassung An Elodea densa wurde die Aufnahme von Phosphat-und Sulfationen im Konzentrationsbereich zwischen 10^-8 und 5×10^-3 m bei Temperaturen zwischen 2 und 18°C im Licht und im Dunkeln untersucht. Die Phosphataufnahme wurde in den Fraktionen anorganisches Phosphat (P_i), TCA-lösliche labile (P_lo) sowie stabile (P_so) organische Phosphorsäureverbindungen und TCA-unlösliche Verbindungen (P_u) untersucht. Die Hauptmenge des aufgenommenen Phosphates findet sich unter allen Bedingungen, besonders aber bei niedrigem pH, tiefen Temperaturen oder hohen Phosphatkonzentrationen in der P_i-Fraktion.Licht fördert die H₂PO ₄ ^- -Aufnahme und den Einbau von Phosphat in die einzelnen Fraktionen, am stärksten bei den Fraktionen P_u und P_so, weniger bei P_lo, am wenigsten bei P_i. Die Lichtförderung der H₂PO ₄ ^- -Aufnahme hat ein Maximum bei mittleren Konzentrationen, sie nimmt mit fallender Temperatur ab.Licht fördert dienfalls die SO ₄ ^- -Aufnahme, auch hier tritt ein Maximum bei mittleren Konzentrationen auf, mit fallender Temperatur wird die Lichtförderung jedoch größer.Die Konzentrationsabhängigkeit der Aufnahme der beiden Anionen weist komplexe Isothermen auf, die einander sehr ähnlich sind. Die Isothermenform bei höherer und tiefer Temperatur ist weitgehend gleich.Die Temperaturabhängigkeit gehorcht innerhalb eines beschränkten Temperaturbereiches einer Arrheniusbeziehung. Die Aktivierungsenergien der Phosphat-und Sulfationenaufnahme zeigen ein Maximum bei mittleren Konzentrationen, ihre Werte sind hier etwa 15,8±2,9 bzw. 24,8±2,8 kcal/mol. Die Aktivierungsenergie des Phosphateinbaues ist bei den Fraktionen P_u und P_so am größten, kleiner bei P_lo und P_i.Energetischen Betrachtungen zufolge kann die Nettoaufnahme der beiden Anionen im gesamten Konzentrationsbereich nur endergonisch und somit aktiv erfolgen.Aufgrund der Veränderung der Lichtwirkung und der Aktivierungsenergien mit der Konzentration werden drei Bereiche der Konzentrationsabhängigkeit unterschieden, in denen folgende Vorgänge als geschwindigkeitsbestimmend bei der Bruttoaufnahme angesehen werden: bei niedrigen Konzentrationen Diffusion in der nichtgerührten Schicht (Filmkinetik), bei mittleren Konzentrationen die aktive Aufnahme und bei hohen Konzentrationen die aktive sowie eine passive Aufnahme durch Diffusion.Dieser Interpretation zufolge werden bei einer Konzentration von 5×10^-3 m die Bruttoaufnahme in ihre Anteile aufgeteilt und die Werte des passiven und des aktiven Influxes sowie der Permeabilitätskoeffizienten abgeschätzt. Die Aktivierungsenergie des passiven Transportes ergibt sich dann zu 5 bzw. 8 kcal/mol bei H₂PO ₄ ^- bzw. SO ₄ ^-- .Die Übereinstimmung der Aktivierungsenergien der Aufnahme von Phosphat in die P_lo-und die P_i-Fraktion wird als ein Hinweis dafür interpretiert, daß möglicherweise ATP an der Phosphationenaufnahme beteiligt ist.

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On the uptake of phosphate and sulphate ions by leaves of Elodea densa as influenced by light, temperature and external concentrations

Summary Using ³²P and ³⁵S as tracers the uptake of phosphate and sulphate anions by Elodea densa has been studied in external concentrations of 10^-8 to 5×10^-3 m at varying temperatures between 2 and 18°C and under different conditions of light. The uptake of phosphate was investigated in the fractions inogranic phosphate (P_i), TCA-soluble acid-labile organic phosphate (P_lo), TCA-soluble stabile organic phosphate (P_so) and TCA-insoluble phosphate (P_u). The main portion of the phosphate taken up was found in the P_i fraction under all conditions, but especially under conditions of low pH, low temperatures and high phosphate concentration.In the case of phosphate light was found to increase the overall uptake at intermediary concentrations, the stimulation being greatest in the fractions P_so and P_u, less in P_lo and least in P_i. At high and low concentrations the stimulation by light disappeared. At low temperatures the stimulation was considerably smaller, but it showed the same dependence on external concentration and even became negative at low concentrations.Light stimulates the uptake of sulphate, too, the stimulation being dependent on external concentration in the same way as it is with phosphate. However, at low temperatures this stimulation of sulphate uptake by light is considerably higher than at high temperatures.Variation of external concentrations of phosphate and sulphate ions results in similar isotherms of uptake for both anions. The isotherms exhibit a linear dependence at low concentrations, a saturation curve at intermediary concentrations and a further rise at high concentrations. Using enzyme kinetics one obtains hyperbolic curves indicating that at least two different processes are taking place.The dependence on temperature within a limited range can be approximated by an Arrhenius equation and is described by the apparent energies of activation E _app which are found to be 15.8±2.9 and 24.8±2.8 kcal/mol for the phosphate and sulphate uptake respectively at intermediary concentrations. In the case of phosphate E _app is higher for the incorporation into P_u and P_so and considerably lower but very similar for both the incorporation into P_lo and P_i. In this respect E _app parallels the stimulation by light.The energies of activation of uptake for both anions are strongly dependent on external concentration and reach a maximum at intermediary concentrations.Considering the energetics with respect to internal and external concentrations and the value of the membrane potential, net uptake of both ions must occur with an expenditure of free energy, thus representing an active process in the whole range of external concentrations.However, from the concomitant changes in the stimulation by light and the energies of activation, the isotherms of uptake are interpreted as being composed of three concentration ranges with the following steps being rate-limiting: the diffusion in the unstirred layer at low concentrations, the active uptake at intermediary concentrations and a considerable contribution of diffusive influx at high external concentrations. This diffusive uptake can occur with tracer flux (influx) only, not with net uptake. Thus it is not contradictory to the energetics of anion uptake.On the basis of this interpretation active and passive fluxes at high concentration are separated and permeability coefficients are estimated from the passive fluxes. According to this sparation the energies of activation of diffusive transport in the cellular membrane are calculated as 5 and 8 kcal/mol (Q ₁₀=1.35 or 1.6) for phosphate and sulphate respectively. The phosphate fluxes are considerably higher than the sulphate fluxes.From the values of E _app it is deduced that a major component of P_lo, possibly ATP, might participate in active phosphate uptake.The isotherms compare well with isotherms of anion uptake by other species so that the results may have some bearing on the interpretation of anion uptake in general.

     

The effect of different N substrates on biological N2O production from forest and agricultural light textured soils

N₂O emissions from two slightly alkaline sandy soils, from arable land and a woodland, were determined in a laboratory experiment in which the soils were incubated with different sources of nitrogen, with or without glucose, and with 0, 1 and 100 mL C₂H₂ L^-1. Large differences in the rate of N₂O production were observed between the two soils and between the different N treatments. The arable soil showed very low N₂O emissions derived from reduced forms of N as compared with the N₂O which was produced when the soil was provided with NO ₂ ^- or NO ₃ ^- and a C source, suggesting a very active denitrifier population. In contrast, the woodland soil showed a very low denitrification activity and a much higher N₂O production derived from the oxidation of NH ₄ ⁺ and reduction of NO ₂ ^- by some processes probably mediated by autotrophic or heterotrophic nitrifiers or dissimilatory NO ₂ ^- reducers. In both soils, the highest N₂O emissions were induced by NO ₂ ^- addition. Those emissions were demonstrated to have a biological origin, as no significant N₂O emissions were measured when the soil was autoclaved.     

Denitrification, Dissimilatory Reduction of Nitrate to Ammonium, and Nitrification in a Bioturbated Estuarine Sediment as Measured with 15N and Microsensor Techniques

Nitrogen and oxygen transformations were studied in a bioturbated (reworked by animals) estuarine sediment (Norsminde Fjord, Denmark) by using a combination of ¹⁵N isotope (NO₃^-), specific inhibitor (C₂H₂), and microsensor (N₂O and O₂) techniques in a continuous-flow core system. The estuarine water was NO₃^- rich (125 to 600 μM), and NO₃^- was consistently taken up by the sediment on the four occasions studied. Total NO₃^- uptake (3.6 to 34.0 mmol of N m^-2 day^-1) corresponded closely to N₂ production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, NO₃^- reduction to NH₄⁺ was insignificant. When C₂H₂ was applied in the flow system, denitrification measured as N₂O production was often less (58 to 100%) than the NO₃^- uptake because of incomplete inhibition of N₂O reduction. The NO₃^- formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from ¹⁵NO₃^- dilution and from changes in NO₃^- uptake before and after C₂H₂ addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m^-2 day^-1 on the four occasions. Attempts to measure total nitrification activity by the difference between NH₄⁺ fluxes before and after C₂H₂ addition failed because of non-steady-state NH₄⁺ fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N₂O and O₂ microelectrodes. The N₂O profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N₂O after C₂H₂ was added was much too fast to be described by a simple diffusion model. Only bioturbation by a dense population of infauna could explain these observations. This was corroborated by the relationship between diffusive and total fluxes, which showed that only 19 to 36 and 29 to 62% of the total O₂ uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.     

Induktion von Tumoren und Produktion des Phagen PS8 durch Agrobacterium

Summary Isolated, intact spinach chloroplasts incubated in light in the presence of HCO ₃ ^- and NO ₂ ^- have the greater proportion of their NADP present in the reduced form. The steady state concentration of NADPH in light in these chloroplasts is significantly higher in the presence of NO₂ than in its absence. These results invalidate earlier conclusions (Grant and Canvin, 1970) that NO ₂ ^- inhibits photosynthesis by preventing NADP reduction.This work was supported by the National Research Council of Canada.     

Active transport of proline into washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans grown with nitrate

Washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, prepared from cultures grown anaerobically in light with NO ₃ ^- as the terminal acceptor, readily incorporated [¹⁴C]-proline both in light and in the dark. The proline uptake was coupled to the reduction of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. Light stimulated the accumulation of proline in these cells. The addition of NO ₃ ^- to washed cells in light decreased the K _m for proline from 40 M to 5.7 M. Proline transport was inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide both in light and in the dark with nitrate indicating that electron transfer from both denitrification and photosynthesis are involved in this uptake. Inhibition by carbonyl cyanide-m-chlorophenyl hydrazone and 2.4-dinitrophenol indicate that proline transport is energy dependent. The H⁺/proline stoichiometry increased from 1 to 2.5 when the external pH was increased from 6.0 to 8.0. Under these conditions pro increased but p decreased markedly above pH 7.0.Abbreviations TPP⁺ Tetraphenylphosphonium bromide - EDTA ethylenediamine-tetra-acetic acid - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - DBMIB dibromo-methyl-isopropyl-p-benzoquinone - DCCD N,N-dicyclohexylcarbodiimide     

Inhibitory Effect of Several Nitric Oxide-Generating Compounds on Purified Na+,K+-ATPase Activity from Porcine Cerebral Cortex

Abstract: Nitric oxide (NO)-generating compounds (NO donors) such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, S-nitroso-l -glutathione, 3-morpholinosyndnonimine (SIN-1), (dl )-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide, and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene inhibited the Na⁺,K⁺-ATPase activity purified from porcine cerebral cortex. NO-reducing or -scavenging agents, such as superoxide dismutase or N-(dithiocarbamate)-N-methyl-d -glucamine sodium salt, l -ascorbic acid, and sulfhydryl (SH) compounds, such as dithiothreitol or the reduced form of glutathione, but not α-tocopherol, prevented the inhibition of the enzyme activity by all NO donors except sodium nitroprusside. Enzyme inhibition could also be reversed by these SH compounds, but not by superoxide dismutase, l -ascorbic acid, and α-tocopherol. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (PTIO), which is able to scavenge NO radicals and generate nitrogen dioxide radicals (^?NO₂), potentiated the inhibition of this enzyme activity induced by all NO donors (except SIN-1). PTIO did not potentiate, but rather attenuated, the SIN-1-induced inhibition. SIN-1 has been reported to release both NO and superoxide and thereby to rapidly form peroxynitrite (ONOO^?). These potentiated and attenuated inhibitions of the enzyme activity induced by PTIO plus all of the NO donors except sodium nitroprusside were prevented by SH compounds, but not by superoxide dismutase, l -ascorbic acid, and α-tocopherol. These results suggest that NO donors may release NO or NO-derived products, presumably ^?NO₂ and ONOO^?, and may inhibit the Na⁺,K⁺-ATPase activity by interacting with a SH group at the active site of the enzyme.     

A field method of determining NH 4 + and NO 3 - uptake kinetics in intact roots: Effects of CO2 enrichment on trees and crop species

Models describing plant and ecosystem N cycles require an accurate assessment of root physiological uptake capacity for NH ₄ ⁺ and NO ₃ ^- under field conditions. Traditionally, rates of ion uptake in field-grown plants are determined by using excised root segments incubated for a short period in an assay solution containing N either as a radioactive or stable isotope tracer (e.g., ³⁶ClO₃ as a NH ₄ ⁺ analogue, ¹⁴CH₃NH₃ as an NO ₃ ^- analogue or ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^- ). Although reliable, this method has several drawbacks. For example, in addition to radioactive safety issues, purchase and analysis of radioactive and stable isotopes is relatively expensive and can be a major limitation. More importantly, because excision effectively interrupts exchange of compounds between root and shoot (e.g., carbohydrate supply to root and N transport to shoot), the assay must be conducted quickly to avoid such complications. Here we present a novel field method for simultaneous measurements of NH ₄ ⁺ and NO ₃ ^- uptake kinetics in intact root systems. The application of this method is demonstrated using two tree species; red maple (Acer rubrum) and sugar maple (Acer saccharum) and two crop species soybean (Glycine max) and sorghum (Sorghum bicolor). Plants were grown in open-top chambers at either ambient or elevated levels of atmospheric CO₂ at two separate US national sites involved in CO₂ research. Absolute values of net uptake rates and the kinetic parameters determined by our method were found to be in agreement with the literature reports. Roots of the crop species exhibited a greater uptake capacity for both N forms relative to tree species. Elevated CO₂ did not significantly affect kinetics of N uptake in species tested except in red maple where it increased root uptake capacity, V, for NH ₄ ⁺ . The application, reliability, advantages and disadvantages of the method are discussed in detail. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP