C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba

Nitrogen assimilation was studied in the deciduous, perennial climber Clematis vitalba. When solely supplied with NO₃^– in a hydroponic system, growth and N-assimilation characteristics were similar to those reported for a range of other species. When solely supplied with NH₄⁺, however, nitrate reductase (NR) activity dramatically increased in shoot tissue, and particularly leaf tissue, to up to three times the maximum level achieved in NO₃^– supplied plants. NO₃^– was not detected in plant material that had been solely supplied with NH₄⁺, there was no NO₃^– contamination of the hydroponic system, and the NH₄⁺-induced activity did not occur in tobacco or barley grown under similar conditions. Western Blot analysis revealed that the induction of NR activity, either by NO₃^– or NH₄⁺, was matched by NR and nitrite reductase protein synthesis, but this was not the case for the ammonium assimilation enzyme glutamine synthetase. Exposure of leaf disks to N revealed that NO₃^– assimilation was induced in leaves directly by NO₃^– and NH₄⁺ but not glutamine. Our results suggest that the NH₄⁺-induced potential for NO₃^– assimilation occurs when externally sourced NH₄⁺ is assimilated in the absence of any NO₃^– assimilation. These data show that the potential for nitrate assimilation in C. vitalba is induced by a nitrogenous compound in the absence of its substrate and suggest that NO₃^– assimilation in C. vitalba may have a significant role beyond the supply of reduced N for growth.     

Light-dependent reduction of pyruvate by pea chloroplasts in the presence of glutamate dehydrogenase and C5-dicarboxylic acids

Illuminated pea chloroplasts supported (glutamine plus α-oxoglutarate (α-OG)) and (NH₃ plus α-OG)-dependent O₂ evolution. The properties of these reactions were consistent with light-coupled glutamate synthase and glutamine synthetase activities. In the presence of a glutamate-oxidizing system (component C) comprised of NAD-specific glutamate dehydrogenase (NAD-GDH), lactate dehydrogenase (LDH), 4 mM pyruvate and 0.2 mM NAD, illuminated chloroplasts supported O₂ evolution in the presence of glutamine. The reaction did not proceed in the absence of any one of the constituents of component C and the properties of O₂ evolution were consistent with light-coupled glutamate synthase activity. In the presence of component C, chloroplasts also catalysed O₂ evolution in the presence of catalytic concentrations of glutamate. Studies of O₂ evolution and metabolism of [¹⁴C]-glutamate in the presence of the inhibitors methionine sulphoximine (MSO) and azaserine suggest that O₂ evolution was dependent on the synthesis of glutamine from the products of glutamate oxidation. This was supported by polarographic studies using α-OG and NH₃ instead of glutamate.The results are consistent with a C₅-dicarboxylic acid shuttlemechanism for the export of reducing equivalents from illuminated chloroplasts (glutamate) and recycling of the oxidation products (α-OG and NH₃).     

Responses of soybean to ammonium and nitrate supplied in combination to the whole root system or separately in a split-root system

To address the questions of whether allocation of carbohydrates to roots is influenced by ionic form of nitrogen absorbed and whether allocation of carbohydrates to roots in turn influences proportionality between NH₄⁺ and NO₃^? uptake from mixed sources, NH₄⁺ and NO₃^? were supplied separately to halves of a split-root hydroponic system and were supplied in combination to a whole-root system. Dry matter accumulation in the split-root system was 18% less in the NH₄⁺-fed axis than in the NO₃^?-fed axis. This, however, does not indicate that partitioning of carbohydrate between the two axes was different. Most of the reduction in dry matter accumulation in the NH₄⁺-fed axis can be accounted for by the retransport of CH₂O equivalents from the root back to the shoot with amino acids produced by NH₄⁺ assimilation. Uptake of NH₄⁺ or NO₃^? by the respective halves of the split-root system was proportional to the estimated allocation of carbohydrate to that half. When NH₄⁺ and NO₃^? were supplied to separate halves of the split-root system, the cumulative NH₄⁺ to NO₃^? uptake ratio was 0.81. When supplied in combination to the whole-root system, the cumulative NH₄⁺ to NO₃^? uptake ratio was 1.67. Thus, while the shoot may affect total nitrogen uptake through the export of carbohydrates to roots, the shoot (common for halves of the split-root system) apparently does not exert a direct effect on proportionality of NH₄⁺ and NO₃^? uptake by roots. For whole roots supplied with both NH₄⁺ and NO₃^?, the restriction in uptake of NO₃^? may involve a stimulation of NO₃^? efflux rather than an inhibition of NO₃^? influx. While only the net uptake of NH₄⁺ and NO₃^? was measured by ion chromatography, monitoring at approximately hourly intervals during the first 3 days of treatment revealed irregularly occurring intervals of both depletion (net influx) and enrichment (net efflux) in solutions. In the case of NH₄⁺, numbers of net efflux events were similar (21 to 24 out of 65 sequential sampling intervals) whether NH₄⁺ was supplied with NO₃^? to whole-root systems or separately to an axis of the split-root system. In the case of NO₃^?, however, the number of net efflux events increased from 8 when NO₃^? was supplied to a separate axis of the split-root system to between 19 and 24 when NO₃^? was supplied with NH₄⁺ to whole-root systems.     

Substrates regulate the phosphorylation status of nitrate reductase

The effect of substrates on the phosphorylation status of nitrate reductase (NR; EC 1.6.6.1) was studied. The enzyme was obtained from the first leaf of 7-day-old oat (Avena sativa L. cv. Suregrain) plants, grown in the light. When desalted crude extracts were incubated with ATP, NR was strongly phosphorylated, as evidenced by the inhibition of the enzyme's activity in the presence of Mg²⁺. NR sensitivity to Mg²⁺ remained unchanged when 10 mM nitrate was added to crude extracts after ATP. Addition of nitrate before or simultaneously with ATP slightly decreased Mg²⁺ inhibition of NR, which was strongly diminished in the presence of 10 mM NO₃^?+ 100 µM NADH. Incubation with NADH alone did not affect the enzyme's susceptibility to Mg²⁺ inhibition. When ammonium sulfate was added to crude extracts, NR was recovered in a 0-40% saturation fraction (F1). After incubation of F1 with ATP, the sensitivity of the enzyme to Mg²⁺ inhibition remained low, but it strongly increased after mixing F1 with a 45-60% saturation fraction (F2) suggesting that also in oats an additional factor (inactivating protein, IP), which probably binds to phospho-NR, would be required to keep the phosphorylated enzyme inactive in a +Mg²⁺ medium. Addition of 10 mM NO₃^?+ 100 µM NADH together with desalted F2 did not prevent Mg²⁺ inhibition suggesting that NO₃^? did not interfere with IP binding to phospho-NR. Again, incubation of F1 with both substrates during in vitro phosphorylation kept the enzyme active after adding F2, even in the presence of Mg²⁺, After in vitro phosphorylation, NR in crude extract was hardly reactivated when incubated alone or in the presence of 10 mM NO₃^? at 30°C. On the other hand, a strong and very rapid reactivation was found when the extract was incubated with both nitrate and NADH. Microcystine, an inhibitor of types 1 and 2A phosphoprotein phosphatases, inhibited the reactivation of phospho-NR induced by the substrates. The results presented here show that the substrates could prevent NR phosphorylation and induce the enzyme's dephosphorylation, but they were effective only after their binding to the NR protein. Thereby, they seemed to affect the NR protein itself and not the phosphatase- or the kinase-proteins. It has been reported that nitrate binding to the enzyme's active site induces conformational changes in the NR protein. We propose that this conformational change would prevent NR phosphorylation, by converting the enzyme into a form in which the site recognized by the protein kinase is no longer accessible, and, simultaneously, stimulate NR dephophorylation by allowing the specific phosphatases to recognize NR.     

Photosynthetic energy supply for NO3− assimilation in Scenedesmus

NO₃^?-dependent O₂ in synchronous Scenedesmus obtusiusculus Chod. in the absence of CO₂ is stoichiometric with NH₄⁺ excretion, indicating a close coupling of NO₃^? reduction to non-cyclic electron flow. Also in the presence of CO₂, NO₃^? stimulates O₂ evolution as manifested by an increase in the O₂/CO₂ ratio from 0.96 to 1.11. This quotient was increased to 1.36 by addition of NO₂^?, without competitive interaction with CO₂ fixation, indicating that the capacity for non-cyclic electron transport at saturating light is non-limiting for simultaneous reduction of NO₃^? and CO₂ at high rates. During incubation with NO₃^?+ CO₂, no NH₄⁺ is released to the outer medium, whereas during incubation with NO₂^?+ CO₂, excess NH₄⁺ is formed and excreted. NO₃^? uptake is stimulated by CO₂, and this stimulation is also significant when the cellular energy metabolism is restricted by moderate concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, whereas NO₃^? uptake in the absence of CO₂ is severely inhibited by the uncoupler. Also under energy-restricted conditions NO₃^? uptake is not competitive with CO₂ fixation. Antimycin A is inhibitory for NO₃^? uptake in the absence of CO₂, and there is no enhancement of NO₃^? uptake by CO₂ in the presence of antimycin A. It is assumed that the energy demand for NO₃^? uptake is met by energy fixed as triosephosphates in the Calvin cycle. Antimycin A possibly affects the transfer of reduced triose phosphates from the chloroplast to the cytoplasm. Active carbon metabolism also seems to exert a control effect on NO₃^? assimilation, inducing complete incorporation of all NO₃^? taken up into amino acids. This control effect is not functional when NO₂^? is the nitrogen source. Active carbon metabolism thus seems to be essential both for provision of energy for NO₃^? uptake and for regulation of the process.     

Effects of increasing inorganic carbon supply to roots on net nitrate uptake and assimilation in tomato seedlings

We investigated the influence of an increased inorganic carbon supply in the root medium on NO^?₃ uptake and assimilation in seedlings of Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were pre-grown for 4 to 7 days with 0 or 100 mM NaCl in hydroponic culture using 0.2 mM NO^?₃ (group A) or 0.2 mM NH⁺₄ (group B) as nitrogen source. The nutrient solution for group A plants was aerated with air or with air containing 4 800 μumol mol^?1 CO₂. Nitrate uptake rate and root and leaf malate contents in these plants were determined. The plants of group B were subdivided into two sets. Plants of one set were transferred either to N-free solution containing 0 or 5 mM NaHCO₃, or to a medium containing 2 mM NO^?₃ and 5 mM NaHCO₃. Both sets of group B plants were grown for 12 h in darkness prior to 2 h of illumination, and were assayed for malate content and NO^?₃ uptake rate (only for plants grown in N-free solution). The second set of group B plants was labeled with ¹⁴C by a 1-h pulse of H¹⁴CO^?₃ which was added to a 5 mM NaHCO₃ solution containing 0 or 100 mM NaCl and 0 or 2 mM NO^?₃, and ¹⁴C-assimilates were extracted and fractionated. The roots of group B plants growing in carbonated medium accumulated twice as much malate as did control plants. This malate was accumulated only when NO^?₃ was absent from the root medium. Both a high level of root malate and aeration with CO₂-enriched air stimulated NO^?₃ uptake. Analysis of ¹⁴C-assimilates indicated that with no NO^?₃ in the medium, the ¹⁴C was present mainly in organic acids, whereas with NO^?₃, a large proportion of ¹⁴C was incorporated into amino acids. Transport of root-incorporated ¹⁴C to the shoot was enhanced by NO^?₃, while the amino acid fraction was the major ¹⁴C-assimilates in the shoot. It is concluded that inorganic carbon fixed through phosphoenolpyruvate carboxylase (EC 4.1.1.31) in roots of tomato plants may have two fates: (a) as a carbon skeleton for amino acid synthesis; and (b) to accumulate, mainly as malate, in the roots, in the absence of a demand for the carbon skeleton. Inorganic carbon fixation in the root provides carbon skeletons for the assimilation of the NH⁺₄ resulting from NO₃ reduction, and the subsequent removal of amino acids through the xylem. This ‘removal’ of NO^?₃ from the cytoplasm of the root cells may in turn increase NO^?₃ uptake.     

Nitrate reductase and nitrate accumulation in relation to nitrate toxicity in Boronia megastigma

Moderate levels of N were toxic to the native Australian plant boronia (Boronia megastigma Nees). As NO^-₃ is the major N form available for plants under cultivated conditions, NO^-₃ reduction and accumulation patterns in boronia were examined following the supply of various levels of NO^-₃ to understand the physiological basis of this toxicity. At a low level of supplied NO^-₃ [15 mmol (plant)^-1], NO^-₃ was reduced without any detectable accumulation and without nitrate reductase activity (NRA) reaching its maximum capacity. When higher NO^-₃ levels [≥25 mmol (plant)^-1] were supplied, both NRA and NO^-₃ accumulation increased further. However, NRA increased to a maximum of ca 500 nmol NO^-₃ (g fresh weight)^-1 h^-1, both in the roots and leaves, irrespective of a 4-fold difference in the levels of supplied NO^-₃, whereas NO^-₃ continued to accumulate in proportion to the level of supplied NO^-₃. Chlorotic toxicity symptoms appeared on the leaves at an accumulation of ca 32 μmol NO^-₃ (g fresh weight)^-1. High endogenous NO^-₃ concentrations inhibited NRA. The low level of NRA in boronia was not limited by NO^-₃ or electron donor availability. It is concluded that the low NR enzyme activity is a genetic adaptation to the low NO^-₃ availability in the native soils of boronia. Thus, when NO^-₃ supply is high, the plat cannot reduce it at high rates, leading to large and toxic accumulations of the ion in the leaf tissues.     

The effect of some ammonium salts on nitrate reductase level,onin vivo nitrate reduction and on nitrate content in excisedpisum sativum roots

The effect of some ammonium salts on nitrate reductase (NR) level, onin vivo nitrate reduction and on nitrate content was followed in the presence of nitrate in the medium, under changing experimental conditions, in excisedPisum sativum roots, and their effect was compared with that of KNO₃, Ca(NO₃)₂ and NaNO₃ at 15 mM NO₃ ^- concentration, i.e. at a concentration which considerably exceeded the level of saturation with nitrate with respect to nitrate reductase. The effect of ammonium salts on NR level is indirect and changes from a positive one to a strongly negative one which is dependent on the time of action of the salt, on the presence of other cations, on pH of the solution of the ammonium salt and on the nature of the anion of the ammonium salt. A positive effect on the enzyme level can be observed in the presence of other cations than NH₄ ⁺ at suitable concentrations of those ammonium salts, the solutions of which have their pH values in the acid region (i.e. NH₄H₂PO₄, (NH₄)₂SO₄ and NH₄NO₃). However their positive effect is independent of the presence of NH₄ ⁺ ions, and it is obviously the result of an increased concentration of H⁺ ions. A clear-cut negative effect on NR level can be observed after 24 h in one-salt NH₄NO₃ solution where NH₄ ⁺ is not balanced with other cations and thus certainly can adversely influence many metabolic processes, and in the solutions containing neutral (pH 6.2) and dibasic ammonium phosphates in which dissolved undissociated ammonia [(NH₃). (H₂O) which can also affect many metabolic processes incl. proteosynthesis] probably has a toxic influence. Thein vivo nitrate reduction is always depressed in excised pea roots in the presence of ammonium salts in the medium, regardless of the level of nitrate reductase. Under the described conditions, no relationship could be established between the enzyme level and the so-called metabolic NO₃ ^- pool (i.e. NO₂ ^- production under anaerobic conditions), nor between NR level and the total nitrate content in the roots. One-salt solutions of NaNO₃, Ca(NO₃)₂ and KNO₃ exert different effects on the level of nitrate reductase and on the content of NO₃ ^- in the roots, but the in vivo NO₃ ^- reduction shows the same trend as NR level in the roots influenced by these salts. Cl^- ions, supplied in NH₄C1, depress both NR level and NO₃ ^- content in the roots at higher concentrations, but they do not significantly affect the in vivo nitrate reduction in comparison with other ammonium salts. These results indicate that NR level,in vivo nitrate reduction, and nitrate uptake can be regulated in pea roots independently of each other.     

Reduction of Nitrate via a Dicarboxylate Shuttle in a Reconstituted System of Supernatant and Mitochondria from Spinach Leaves

Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD⁺, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO₃⁻ with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO₃⁻ reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD⁺, and NO₃⁻ in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain.     

Dark induction of nitrate reductase in the halophilic alga Dunaliella salina

The effect of nitrogen starvation on the NO₃-dependent induction of nitrate reductase (NR) and nitrite reductases (NIR) has been investigated in the halophilic alga Dunaliella salina. When D. salina cells previously grown in a medium with NH ₄ ⁺ as the only nitrogen source (NH ₄ ⁺ -cells) were transferred into NO ₃ ^? medium, NR was induced in the light. In contrast, when cells previously grown in N-free medium were transferred into a medium containing NO ₃ ^? , NR was induced in light or in darkness. Nitrate-dependent NR induction, in darkness, in D. salina cells previously grown at a photon flux density of 500 umol · m^?2 s^?1 was observed after 4 h preculture in N-free medium, whilst in cells grown at 100 umol · m^?2 s^?1 NR induction was observed after 7–8 h. An inhibitor of mRNA synthesis (6-methylpurine) did not inhibit NO ₃ ^? -induced NR synthesis when the cells, previously grown in NH ₄ ⁺ medium, were transferred into NO ₃ ^? medium (at time 0 h) after 4-h-N starvation. However, when 6-methylpurine was added simultaneously with the transfer of the cells from NH ₄ ⁺ to NO ₃ ^? medium (at time 0 h), NO ₃ ^? induced NR synthesis was completely inhibited. The activity of NIR decreased in N-starved cells and the addition of NO ₃ ^? to those cells greatly stimulated NIR activity in the light. The ability to induce NR in darkness was observed when glutamine synthetase activity reached its maximal level during N starvation. Although cells grown in NO ₃ ^? medium exhibited high NR activity, only 0.33% of the total NR was found in intact chloroplasts. We suggest that the ability, to induce NR in darkness is dependent on the level of N starvation, and that NR in D. salina is located in the cytosol. Light seems to play an indirect regulatory role on NO ₃ ^? uptake and NR induction due to the expression of NR and NO ₃ ^? -transporter mRNAs.     

Effect of inhibitors on ammonia-, 2-oxoglutarate-, and oxaloacetate-dependent o(2) evolution in illuminated chloroplasts

The evolution of O₂ in spinach chloroplasts in the presence of oxaloacetate (OAA) was inhibited by a wide range of dicarboxylates. In contrast, (ammonia, 2-oxoglutarate)-dependent O₂ evolution was stimulated by malate, succinate, fumarate, glutarate, maleiate, and l-tartrate although OAA has little effect. This increase in O₂ evolution was accompanied by a similar increase in ¹⁴C incorporation from [5-¹⁴C]oxoglutarate into amino acids which was sensitive to azaserine inhibition. Glutamate and aspartate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution, but this inhibition was relieved by the addition of succinate, malate, or fumarate. OAA-dependent O₂ evolution also was inhibited by glutamate and aspartate, but succinate, malate, or fumarate had little effect on this inhibition. Phthalonate and n-butyl malonate inhibited (ammonia, 2-oxoglutarate)-dependent O₂ evolution competitively with respect to 2-oxoglutarate and uncompetitively with respect to malate. Both these inhibitors inhibited OAA-dependent O₂ evolution competitively. This evidence suggests that different mechanisms might be involved in the transport of OAA, 2-oxoglutarate, and malate into the chloroplasts.     

Oxygen stress in Salvinia natans: Interactive effects of oxygen availability and nitrogen source

The ability of Salvinia natans (L.) All. to tolerate growth in oxic, hypoxic and anoxic nutrient solutions when supplied with either NH₄⁺ or NO₃^? were studied in the laboratory to test the hypothesis that inorganic N-source affects the response of the plants to O₂ deprivation. The relative growth rate (RGR) was significantly reduced in the anoxic treatment, but in the hypoxic treatment RGR was only slightly affected. The NH₄⁺ fed plants generally had a higher shoot to root ratio than the NO₃^? fed plants, and highest in the anoxic treatment. Plants had more roots and larger leaves when supplied with NH₄⁺ as compared with NO₃^?, particularly in the oxic treatment, and root length was most affected by O₂ deprivation for NO₃^? fed plants. Cell walls in the endodermis, the bundle sheath and the cortex adjacent to endodermis developed thickened sclerenchymatous walls when deprived of O₂, and more so in plants supplied with NO₃^?. Plants lost chlorophylls, had lower rates of photosynthetic electron transport (ETR_max) and lower quantum yields (Fv/Fm ratios) when grown in anoxic solutions, and the negative effects were mildest for NO₃^? fed plants suggesting that NO₃^? may be used as an alternative e^?-acceptor in non-cyclic electron transport in the chloroplasts. Overall S. natans grew best on NH₄⁺, but it also grew well on NO₃^?, and the O₂ stress symptoms differed somewhat between NH₄⁺ fed and NO₃^? fed plants. However, because N-form itself significantly influenced morphology and cell metabolism, it was impossible to conclusively identify the role of N-form for the O₂ stress reactions. S. natans is not well-adapted to grow in O₂ deficient waters and will not tolerate completely anoxic conditions as will prevail in waters receiving high loadings of organic pollutants such as livestock wastewater.     

The influence of enriched rhizosphere CO2 on N uptake and metabolism in wild-type and NR-deficient barley plants

Positive influences of high concentrations of dissolved inorganic carbon (DIC) in the growth medium of salinity-stressed plants are associated with carbon assimilation through phosphoenolpyruvate carboxylase (PEPc) activity in roots; and also in salinity-stressed tomato plants, enriched CO₂ in the rhizosphere increases NO^?₃uptake. In the present study, wild-type and nitrate reductase-deficient plants of barley (Hordeum vulgare L. cv. Steptoe) were used to determine whether the influence of enriched CO₂ on NO^?₃ uptake and metabolism is dependent on the activity of nitrate reductase (NR) in the plant. Plants grown in NH₄⁺and aerated with ambient air, were transferred to either NO₃^? or NH₄⁺ solutions and aerated with air containing between 0 and 6 500 μmol mol^?1 CO₂. Nitrogen uptake and tissue concentrations of NO₃^? and NH₄⁺ were measured as well as activities of NR and PEPc. The uptake of NO^?₃ by the wild-type was increased by increasing CO₂. This was associated with increased in vitro NR activity, but increased uptake of NO₃^? was found also in the NR-deficient genotype when exposed to high CO₂ concentrations; so that the influence of CO₂ on NO₃^? uptake was independent of the reduction of NO₃^? and assimilation into amino acids. The increase in uptake of NO₃^? in wild-type plants with enriched CO₂ was the same at pH 7 as at pH 5, indicating that the relative abundance of HCO₃^? or CO₂ in the medium did not influence NO₃^? uptake. Uptake of NH₄⁺ was decreased by enriched CO₂ in a pH (5 or 7) independent fashion. Thus NO₃^? and NH⁺₄ uptakes are influenced by the CO₂ component of DIC independently of anaplerotic carbon provision for amino acid synthesis, and CO₂ may directly affect the uptake of NO₃^? and NH₄⁺ in ways unrelated to the NR activity in the tissue.     

The nitrogen balance of Raphanus sativus X raphanistrum plants. I. Daily nitrogen use under high nitrate supply

Abstract Growth-chamber cultivated Raphanus plants accumulate nitrate during their vegetative growth. After 25 days of growth at a constant supply to the roots of 1 mol m^?3 (NO^?₃) in a balanced nutrient solution, the oldest leaves (eight-leaf stage) accumulated 2.5% NO^?₃-nitrogen (NO₃-N) in their lamina, and almost 5% NO₃-N in their petioles on a dry weight basis. This is equivalent to approximately 190 and 400 mol^?3 m^?3 concentration of NO^?₃ in the lamina and the petiole, respectively, as calculated on a total tissue water content basis. Measurements were made of root NO^?₃ uptake, NO^?₃ fluxes in the xylem, nitrate uptake by the mesophyll cells, and nitrate reduction as measured by an in vivo test. NO^?₃ uptake by roots and mesophyll cells was greater in the light than in the dark. The NO^?₃ concentration in the xylem fluid was constant with leaf age, but showed a distinct daily variation as a result of the independent fluxes of root uptake, transpiration and mesophyll uptake. NO^?₃ was reduced in the leaf at a higher rate in the light than in the dark. The reduction was inhibited at the high concentrations calculated to exist in the mesophyll vacuoles, but reduction continued at a low rate, even when there was no supply from the incubation medium. Sixty-four per cent of the NO^?₃ influx was turned into organic nitrogen, with the remaining NO^?₃ accumulating in both the light and the dark.     

COMPARISONS OF NITRATE UPTAKE, STORAGE, AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES

Diatoms, but not flagellates, have been shown to increase rates of nitrogen release after a shift from a low growth irradiance to a much higher experimental irradiance. We compared NO₃ ^? uptake kinetics, internal inorganic nitrogen storage, and the temperature dependence of the NO₃ ^? reduction enzymes, nitrate (NR) and nitrite reductase (NiR), in nitrogen‐replete cultures of 3 diatoms (Chaetoceros sp., Skeletonema costatum, Thalassiosira weissflogii) and 3 flagellates (Dunaliella tertiolecta, Pavlova lutheri, Prorocentrum minimum) to provide insight into the differences in nitrogen release patterns observed between these species. At NO₃ ^? concentrations <40 μmol‐N·L ^{? 1}, all the diatom species and the dinoflagellate P. minimum exhibited saturating kinetics, whereas the other flagellates, D. tertiolecta and P. lutheri, did not saturate, leading to very high estimated K _s values. Above ～60 μmol‐N·L ^{? 1}, NO₃ ^? uptake rates of all species tested continued to increase in a linear fashion. Rates of NO₃ ^? uptake at 40 μmol‐N·L ^{? 1}, normalized to cellular nitrogen, carbon, cell number, and surface area, were generally greater for diatoms than flagellates. Diatoms stored significant amounts of NO₃ ^? internally, whereas the flagellate species stored significant amounts of NH₄ ⁺ . Half‐saturation concentrations for NR and NiR were similar between all species, but diatoms had significantly lower temperature optima for NR and NiR than did the flagellates tested in most cases. Relative to calculated biosynthetic demands, diatoms were found to have greater NO₃ ^? uptake and NO₃ ^? reduction rates than flagellates. This enhanced capacity for NO₃ ^? uptake and reduction along with the lower optimum temperature for enzyme activity could explain differences in nitrogen release patterns between diatoms and flagellates after an increase in irradiance.     

Ammonium, nitrate, and proton fluxes along the maize root

Ion-selective microelectrodes were used to measure NH₄⁺, NO₃^– and H⁺ fluxes along the primary root of maize seedlings. Plants were exposed to nutrient solutions containing NH₄⁺, NO₃^– or both ions. Nitrogen fluxes along the root varied substantially among the different treatments. Net NH₄⁺ and NO₃^– uptake and H⁺ extrusion were low at the very apex of the root and generally increased in the more basal regions. In the absence of nitrogen or in the presence of NO₃^– alone, net H⁺ uptake (and root surface alkalinization) occurred at the root tip (0–1 mm), whereas net H⁺ extrusion occurred in all other regions. In the presence of NH₄⁺ alone, a dramatic increase in net H⁺ extrusion was detected in all regions except for the region 6–11 mm from the apex. In contrast, when NO₃^– alone was supplied, net H⁺ extrusion was depressed at all locations except for the tip (0–1 mm). When both NH₄⁺ and NO₃^– were supplied, NO₃^– uptake was suppressed at all locations while net H⁺ extrusion was increased relative to NO₃^– alone. The capacities to absorb NH₄⁺ and NO₃^– at the tip were similar, as indicated by flux rates when NH₄⁺ or NO₃^– were supplied as sole sources, but when supplied together, net NO₃^– uptake was half that of net NH₄⁺ uptake, indicating that NH₄⁺ may satisfy the nitrogen requirements of the poorly vascularized apical tissue in the most energy-efficient way. The high spatial resolution of the measurements enabled us to establish that acidification in the root expansion zone is maintained regardless of nitrogen source.     

Root growth as a function of ammonium and nitrate in the root zone

We examined the effect of soil NH₄⁺ and NO₃^? content upon the root systems of field-grown tomatoes, and the influence of constant, low concentrations of NH₄⁺ or NO₃^? upon root growth in solution culture. In two field experiments, few roots were present in soil zones with low extractable NH₄⁺ or NO₃^?; they increased to a maximum in zones having 2μg-N NO₃^? g^?1 soil and 6 μg-N NO₃⁼ g^?1 soil, but decreased in zones having higher NH₄⁺ or NO₃^? levels. Root branching was relatively insensitive to available mineral nitrogen. Plants maintained in solution culture at constant levels of NH₄⁺ or NO₃^?, had similar shoot biomass, but all root parameters – biomass, length, branching and area – were greater under NH4 nutrition than under NO₃^?. These results suggest that the size of root system depends on a functional equilibrium between roots and shoots (Brouwer 1967) and on the balance between soil NH₄⁺ and NO₃^?.     

Regulation of Photosynthetic Electron Transport in Intact Spinach Chloroplasts: I. INFLUENCE OF EXOGENOUS SALTS ON OXALOACETATE REDUCTION

Relatively high concentrations of monovalent salts (150 millimolar) stimulated light-saturated uncoupled rates of O₂ evolution linked to oxaloacetic acid (OAA) reduction by intact chloroplasts 2-to 3-fold. In contrast, monovalent salts partially inhibited light-saturated rates of O₂ evolution coupled to CO₂ fixation and uncoupled rates of nitrite reduction. In the presence of high salt concentration, light-saturated rates of electron transport were about equivalent for all three terminal electron acceptors. It is inferred that exogenous monovalent salts have at least two effects on photosynthetic electron transport, independent of photophosphorylation and CO₂ metabolism: a partial inhibitory effect common to OAA, NO₂⁻ and CO₂ reduction and a marked stimulatory effect unique to the photoreduction of OAA.