Nitrite reductase in Dunaliella tertiolecta: Isolation and properties

1. A soluble nitrite reductase has been isolated from cell-freepreparations of Dunaliella tertiolecta and purified fifty fold. 2. The enzyme resembles nitrite reductases isolated from higherplants in that it is a ferredoxin-nitrite reductase, but differsin that it will not accept electrons from either NADH or NADPHeven if exogenous diaphorase is added. 3. The Km value for nitrite is 1.1 x 10^–4 M and the molecularweight as determined by chromatography on G-200 Sephadex is70,000. 4. The rates of nitrite reduction obtained in vitro, using thedithionite-viologen electron donor system are sufficient toaccount for the in vivo rates of nitrate and nitrite assimilationobserved in this species. (Received July 4, 1969; )     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Purification and some properties of polyphenol oxidase from root-forming carrot callus tissues

1. Polyphenol oxidase (o-diphenol : O₂ oxidoreductase; E.C.1.10.3.1 [EC] ) was isolated from the other phenolases which werepresent in root-forming carrot callus, and its properties wereexamined. 2. The enzyme was purified about 45-fold over crudeextracts (precipitates between 40–70% saturation widiammonium sulfate) by a combination of Bio-gel filtration, protein-bagfiltration, and carboxymethyl cellulose chromatography. Thepurified oxidase was homogeneous according to polyacrylamidegel electrophoresis and Sephadex gel filtration. It was confirmedby CM-cellulose chromatography that the enzyme was absent incallus tissues without accompanying redifferentiation. 3. Themolecular weight of this oxidase was estimated to be 110,000-120,000 from molecular weight-mobility profiles on polyacrylamidegels containing sodium dodecyl sulfate and molecular size-elutionvolume correlations on Sephadex G-150 columns. 4. The enzymeoxidized o-diphenols but showed no detectable activity againstmonophenols. Pyrocatechol, dopamine, caffeic acid, and chlorogenicacid were effectual substrates of the enzyme with K_m valuesranging from 10^–3 M to 10^–5M. The enzyme effectivelycatalyzed the oxidation of o-diphenols over the range of pH6.0 to 7.0 and was readily inactivated by heating. The enzymeactivity was slightly influenced by increasing ionic strength.The initial rate of the enzymic reaction was enhanced by additionof Cu²⁺, Co²⁺ and Mn²⁺ ions, and was reduced in the presenceof DTT, PCMPS, glycylglycine, and DIECA. (Received June 17, 1978; )     

Purification and Characterization of Assimilatory Nitrate Reductase from the Cyanobacterium Plectonema boryanum

Assimilatory nitrate reductase (NR) was solubilized by acetonetreatment from Plectonema boryanum and was purified 7,700-foldby heat treatment, ammonium sulfate fractionation and chromatographyon DEAE-Sephacel and Sephadex G-150. Purified NR had a specificactivity of 85 µmol NO₂^– formed min^–1 mg^–1protein. The enzyme retained both ferredoxin (Fd)- and methylviologen (MV)-linked NR activities throughout the purificationprocedure. Molecular weight was 80,000. The pH optimum was 10.5in the MV-assay and 8.5 when assayed with enzymatically reducedFd as the electron donor. Apparent Km values for nitrate andMV were 700 µM and 2,500µM in the MVassay and 55µM and 75 µM for nitrate and Fd in the Fd-assay.The enzyme was inhibited by thiol reagents and metal-chelatingreagents. (Received October 1, 1982; Accepted March 8, 1983)     

Isolation, Purification and Some Properties of Cytochrome c-551 from Chromatium vinosum

Cytochrome c-551 was isolated and purified from a photosyntheticbacterium Chromatium vinosum by ammonium sulfate fractionation,ion-exchange chromatography and gel filtration. The cytochromehad absorption maxima at 280, 407 and 523–524 nm in theoxidized form, and 416, 521 and 549.5 nm in the reduced form.The reduced-minusoxidized difference millimolar absorption coefficientwas 9.90 mM^–1cm^–1 for the wavelength pair, 550.5minus 540 nm. The molecular weight of the cytochrome was 16,000by gel filtration on Sephadex G-100 and 15,500 by sodium dodecylsulfate-polyacrylamidegel electrophoresis. The midpoint redox potential was +240 mVat pH 8.0. Cytochrome c-551 was released from bacterial cells when spheroplastswere produced but EDTA and lysozyme treatments. The releasedcytochrome had the same properties as those of the cytochromepreparation obtained by disruption of cells through a Frenchpressure cell. This confirms the earlier suggestion that cytochromec-551 is located in the periplasmic space of cells. (Received August 21, 1982; Accepted October 28, 1982)     

Purification and properties of nitrite reductase from the blue-green alga Anabaena cylindrical

Nitrite reductase was purified about 40-fold from the blue-greenalga Anabaena cylindrica by acetone precipitation and chromatographyon DEAE-cellulose columns. The nitrite reductase had its pHoptima at about 7.6 with Tris-HCl and at about 7.4 with phosphatewhen reduced methyl viologen was used as an electron donor.The Km's for nitrite, methyl viologen and ferredoxin were 510^–55,210^–4 and 510^–6M, respectively. A stoichiometryof one molecule of ammonia formation per one molecule of nitritedisappearance was confirmed. Ferredoxin which had been reducedeither chemically with dithionite or enzymatically with NADPHin the presence of diaphorase was active as an electron donor.Dithionite-reduced FAD and FMN were inactive. NADPH could notgive electrons directly to nitrite reductase. Hydroxylaminereductase was segregated from nitrite reductase by DEAE-cellulosecolumn chromatography. Purified nitrite reductase showed noactivity for sulfite reduction. A molecular weight of 68,000was estimated for nitrite reductase using a calibrated SephadexG-200 column. ¹This work was supported by grants 4090 and 955008 from theMinistry of Education. ²This work was supported by grants 4090 and 955008 from theMinistry of Education. 2 Present address: Department of Botany,Faculty of Science, University of Tokyo, Tokyo.     

Time-Resolved Spectroscopy of Chlorophyll-a like Electron Acceptor in the Reaction Center Complex of the Green Sulfur Bacterium Chlorobium tepidum

The absorption changes of chlorophyll (Chl) a-like pigments(C670) were studied by ns-ms laser spectroscopy at 77 K in theuntreated and urea-treated homodimeric reaction center (RC)complex of the green sulfur bacterium Chlorobium tepidum. Theuntreated RC complex contained 9 molecules of C670 in additionto 41 molecules of Bchl a and 0.9 molecules of menaquinone-7per one primary electron donor Bchl a dimer (P840). Upon photo-oxidationof P840, C670 showed an absorption change of a red-shift withan isosbestic wavelength at 668 nm. The absorption change ofP840 decayed with time constants (t_1/e) of 55 and 37 ms at 283and 77 K, respectively, and was assigned to represent the chargerecombination between P840⁺ and FeS^–. In the urea-treatedRC complex, a bleach peaking at 670 nm with a shoulder peakat 662 nm, which is ascribable to the reduced primary electronacceptor A₀^–, was detected after the laser excitationin addition to the shift at 668 nm indicating the formationof the P840⁺A₀^– state. The P840⁺A₀^– state decayedwith a t_1/e of 43 ns at 77 K and produced a triplet state p840^Tdue to the suppression of the forward electron transfer. Theseresults indicate the two different types of C670 species inthe RC complex; the one peaking at 670 nm functions as A⁰, whilethe other peaking at 668 nm shows the electrochromic shift,which presumably functions as the accessory pigment locatedin the close vicinity of P840. (Received May 17, 1999; Accepted July 14, 1999)     

Photosynthetic Electron Transport under Phosphorylating Conditions as Influenced by Different Concentrations of Various Salts

Osman, M. E-A. H. and El-Shentenawy, F. 1988. Photosyntheticelectron transport under phosphorylating conditions as influencedby different concentrations of various salts.—J. exp.Bot. 39: 859–863. The rate of light-induced electron transport by isolated spinachthylakoids under phosphorylating conditions, as affected bydifferent concentrations of Br^–, Cl^–, NO₃^–,HCO₃^–, SO₄^2– and CO₃^2– has been investigated.The results show that both low and high concentrations of HCO₃^2–stimulated the oxygen evolution capacity under phosphorylatingconditions, whereas only low concentrations of CO₃^2–,SO₄^2– and Cl^– stimulated the oxygen evolution capacity.However, irrespective of concentration, both Br^– and NO₃^–reduced this capacity. The rate of photosynthetic electron transportwas generally stimulated by addition of ADP, even in cases whereelectron transport was inhibited by addition of bromide andnitrate. The different concentrations of these anions also causedreduction of the power generated by proton pumping and usedfor phosphorylation. The greatest level of reduction was observedin the presence of high concentrations of Cl^– and HCO₃^–. Key words: Spinach thylakoids, photosynthetic electron transport, phosphorylation     

Specific Inhibition of Electron Transfer for Nitrate Reduction by a Low Concentration of Cyanide in Rhodobacter sphaeroides f. s. denitrificans

The effects of cyanide on the electron flow in NO₃^– andNO₂^– reductions and photosynthetic electron transfer wereinvestigated with intact cells of a photodenitrifier, Rhodobactersphaeroides f. s. denitrificans. In the presence of 30 µMKCN, electron transfer for NO₃^– reduction was inhibitedby about 70% and the concomitant H translocation was completelyinhibited. However, neither NO₂^– reduction nor photosyntheticcyclic electron transfer was affected at 30 µM. Theseresults suggested that the electron transfer pathway to NO₃^–has, in addition to a b-type cytochrome and the nitratereductase,a component sensitive to a low concenration of cyanide whichis not involvedin the cytochrome bc₁ complex. (Received April 13, 1987; Accepted July 23, 1987)     

Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium

Corneal transparency and hydration control are dependent on HCO₃^– transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na⁺-3HCO₃^– cotransporter (NBC1) in addition to a basolateral 1Na⁺-2HCO₃^– cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO₃^– permeability and whether the cotransporter participates in transendothelial net HCO₃^– flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5–15 nM siRNA decreased NBC1 expression by 80–95%, 4 days posttransfection. Apical and basolateral HCO₃^– permeabilities were determined by measuring the rate of pH_i change when HCO₃^– was removed from the bath under constant pH or constant CO₂ conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO₃^– permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO₃^– permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO₃^– flux was 0.707 ± 0.009 mM·min^–1·cm² in the basolateral-to-apical direction and increased to 1.74 ± 0.15 when cells were stimulated with 2 µM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO₃^– flux to 0.236 ± 0.002. NBC1 siRNA treatment or 100 µM ouabain also eliminated steady-state HCO₃^– flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO₃^– permeability, basolateral-to-apical fluxes, and net HCO₃^– flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO₃^– flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Proton Translocation Associated with Denitrification in a Photodenitrifier, Rhodopseudomonas sphaeroides forma sp. denitrificans

H⁺ translocation driven by NO₃^–, NO₂^– and N₂O reductionswith endogenous substrates in cells of Rhodopseudomonas sphaeroidesforma sp. denitrificans was investigated by the oxidant pulsemethod. Upon injection of nitrogenous oxides to anaerobic cellsin darkness, an alkaline transient in the external medium wasobserved, followed by acidification. The alkaline transientwas enhanced by carbonyl cyanide m-chlorophenylhydrazone. When a viologen dye was used as an electron donor in the presenceof 1 mM Af-ethylmaleimide and 0.1 mM 2-n-heptyl-4-hydroxyquinoline-N-oxideto preclude respiration-linked H⁺ extrusion, addition of KNO₃,KNO₂ and N₂O caused only a rapid alkalinization. The H⁺ consumptionstoichiometries, H⁺/2e^– ratios for NO₃^– reductionto NO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were –1.90, –3.18 and –2.04, respectively.These values agreed well with the fact that all reductions ofnitrogenous oxides in denitrification occur on the periplasmicside of the cytoplasmic membrane. When corrected for H⁺ consumption in the periplasm, the H⁺ extrusionstoichiometries, H⁺/2e^– ratios with endogenous substratesin the presence of K⁺/valinomycin for NO₃^– reduction toNO₂^–, NO₂^– reduction to 1/2 N₂O and N₂O reductionto N₂ were 4.05, 4.95 and 6.01, respectively. (Received August 4, 1982; Accepted January 13, 1983)     

Ethylene, Nitrate and Nitrite Interactions in the Promotion of Dark Germination of Common Purslane Seeds

Ethylene (10 µ1^–1) caused about one-third of highlydark-dormant seeds of common purslane (Portulaca oleracea L.)to germinate in the dark. Attempts were made to increase germinationin the dark with nitrate and ethylene combinations. When applieddirectly to the seeds, KNO₃ did not stimulate germination andKNO₃ plus ethylene did not increase germination above that ofethylene alone. Pre-incubation of seeds in KNO₃ for 4 to 7 dbefore the ethylene applications significantly increased germination.The effects of the KNO₃ pre-incubation were additive at eachof four ethylene concentrations (0.1–100 µ11^–1).Potassium nitrate was effective only when ethylene followedthe KNO₃ pre-incubation period. Potassium nitrite stimulatedabout 25 per cent of the seeds to germinate without a pre-incubationperiod and without ethylene. Also, ethylene plus KNO₂ enhancedgermination above that achieved by either stimulus alone. Silvernitrate did not block the ethylene promotion of germination,but reversed the typical ethylene inhibition of seedling growthfollowing germination. The results support the views that nitrateexerted its effect via conversion to nitrite within the seedand that the rate of nitrate conversion may be a limiting factorin the dark germination of common purslane seeds. Ethylene mayfacilitate nitrite activity by increasing seed sensitivity tothe stimulus. Common purslane, Portulaca oleracea L., ethylene, nitrate, nitrite, germination, dormancy     

Nitrate reductase from Dunaliella tertiolecta, purification and properties

The purification and properties of a nitrate reductase fromthe green alga Dunaliella tertiolecta are described. The enzymeis soluble, with a molecular weight greater than 500,000 andhas Km values of 0.26, 0.18, 0.10 and 0.06 m for NO₃^–,NADH, NADPH and FADH₂ respectively. Even at the highest specificactivity obtained, (0.86 µmoles NO₃^– reduced min^–1mg protein^–1) the enzyme retains the capacity to acceptelectrons from both NADH and NADPH. Unlike other nitrate reductasesit does not appear to be able to use reduced viologens as electrondonors. Its other properties are consistent with its being amolybdoflavoprotein of high molecular weight, which is alsoable to function as a cytochrome C reductase. ¹ Supported in part by the National Research Council of Canada. (Received June 18, 1972; )     

EFFECTS OF CULTURE CONDITIONS ON HYDROGENASE ACTIVITY OF SCENEDESMUS D3

The effects of culture conditions and of culture ages upon hydrogenaseactivity in Scenedesmus D₃ was investigated with nitrite andbenzoquinone as acceptors. It was noticed that level of ironin the culture medium played an important part in the developmentof hydrogenase activity. The cells grown on a medium of relatively high level of iron(10^–4 M) showed higher activity both in nitrite and benzoquinonereduction than those grown on a medium of relatively low levelof iron (10^–5 M). In the former case, hydrogenase activityappeared even after a short incubation with hydrogen (30 min). When the cells with low hydrogenase activity were transferredto a medium containing 10^–;4 M iron and the activity wasmeasured every 24 hours, the following facts were observed. (a) Nitrite reduction activity increased markedly during thefirst 24 hours and then decreased gradually during further cultivation.Furthermore, a parallelism was observed between nitrite reductionactivity and cell size. (b) On the other hand, the pattern of change in quinone reductionactivity was somewhat different from that of nitrite reduction.This activity appeared only after 3 days' cultivation, reacheda maximum at 4th or 5th day and then decreased. However, thepresence of glucose in the culture medium suppresses the appearanceof quinone reduction activity, while the nitrite reduction activitywas less markedly affected. ¹Present address: Department of Experimental Radiology, Facultyof Medicine, Kyushu University, Fukuoka.     

Purification and general properties of spinach leaf nitrite reductase

Nitrite reductase was isolated from spinach leaves. The enzymewas purified 168-fold by a procedure involving extraction withphosphate buffer, gel filtration on Sephadex G-200, ion-exchangechromtography on DEAE-Sephadex A-50, and adsorption on hydroxyapatite.The preparation was homogeneous in the ultracentrifuge withsedimentation coefficient at infinite dilution (s?_20,w) of 4.57S. Disc electrophoresis revealed some small bands together witha major protein band. The molecular weight of the spinach nitritereductase was estimated to be 60,000 by gel filtration on SephadexG-100 while a molecular weight of 72,000 was obtained from thesedimentation-diffusion coefficients of the protein. Resultsof sodium dodecyl sulfate gel electrophoresis suggested thatthe enzyme molecule consists of two subunits of molecular sizeof 37,000. After close examination of assay systems based onsodium dithioniteviologen dye procedures, we developed a moreelaborate, improved chemical assay method. Some enzymatic propertiesof the purified nitrite reductase were examined. ¹This work was reported in part at the Annual Meeting of JapaneseSociety of Plant Physiologists, April 6–8, 1972. (Received November 16, 1972; )     

Plant Growth in Relation to the Supply and Uptake of NO3-: A Comparison Between Relative Addition Rate and External Concentration as Driving Variables

Two approaches to quantifying relationships between nutrientsupply and plant growth were compared with respect to growth,partitioning, uptake and assimilation of NO₃^– by non-nodulatedpea (Pisum sativum L. cv. Marma). Plants grown in flowing solutionculture were supplied with NO₃^– at relative addition rates(RAR) of 0·03, 0·06, 0·12, and 0·18d^–1, or constant external concentrations ([NO₃^–)of 3, 10, 20, and 100 mmol m^–3 over 19 d. Following acclimation,relative growth rates (RGR)approached the corresponding RARbetween 0·03–0.12 d^-1, although growth was notlimited by N supply at RAR =0.18 d^-1. Growth rates showed littlechange with [NO₃–] between 10–100 mmol m^–3(RGR=0·15 –0·16 d^-1). The absence of growthlimitation over this range was suggested by high unit absorptionrates of NO₃^–, accumulation of NO₃^– in tissues andprogressive increases in shoot: root ratio. Rates of net uptakeof NO₃^– from 1 mol m^–3 solutions were assessed relativeto the growth-related requirement for NO₃^–, showing thatthe relative uptake capacity increased with RGR between 0·03–0·06d^–1 , but decreased thereafter to a theoretical minimumvalue at RGR     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

The processes of NO₃^– uptake and transport and the effectsof NH₄⁺ or L-glutamate on these processes were investigatedwith excised non-mycorrhizal beech (Fagus sylvatica L.) roots.NO₃^– net uptake followed uniphasic Michaelis-Menten kineticsin a concentration range of 10µM to 1 mM with an apparentK_m of 9.2 µM and a V_max of 366 nmol g^–1 FW h^–1.NH₄⁺, when present in excess to NO₃^–, or 10 mM L-glutamateinhibited the net uptake of NO₃^– Apparently, part of NO₃^–taken up was loaded into the xylem. Relative xylem loading ofNO₃^– ranged from 3.21.6 to 6.45.1% of NO₃^– netuptake. It was not affected by treatment with NH₄⁺ or L-glutamate.¹⁶N/¹³N double labelling experiments showed that NO₃^–efflux from roots increased with increasing influx of NO₃^–and, therefore, declined if influx was reduced by NH₄⁺ or L-glutamateexposure. From these results it is concluded that NO₃^–net uptake by non-mycorrhizal beech roots is reduced by NH₄⁺or L-glutamate at the level of influx and not at the level ofefflux. Key words: Nitrate transport, net uptake, influx, efflux, ammonium, Fagus, Fagaceae     

Internal Factors Regulating Nitrate and Chloride Influx in Plant Cells

The primary factor determining the observed decrease in activeC1^– influx during salt accumulation in carrot and barleyroot cells has been shown to be the concentration of C1^–+ NO₃^– in the vacuole. The relationship between C1^– influx and the vacuolar concentrationsof various substances was examined after the tissues had accumulatedions from various salt solutions. After accumulating K⁺ malate,C1^– influx was not reduced, but after accumulating C1^–or NO₃^– salts, C1^– influx was reduced by up to 90per cent. Considering all treatments, C1^– influx was notcorrelated with the vacuolar concentration of K⁺, Na⁺, (K⁺+Na⁺),reducing sugars, malate, C1^–, or NO₃^–, nor withthe cellular osmotic pressure. The correlation coefficient betweenCl^– influx and log (C1^– + NO₃^– concentrationin the vacuole) was highly significant, and accounted for allthe variation in C1^– influx in this experiment. Net NO₃^– influx is similarly reduced by a high C1^–concentration in the vacuole. External Cl^– and NO₃^–have quantitatively different, apparently competitive, effectson C1^– influx. These differ from the apparently negative-feedbackeffects of C1^– and NO₃^– in the vacuole, which arequantitatively similar. Decreasing the internal hydrostatic pressure by raising theexternal osmotic pressure increased active K⁺ influx in Valoniaventricosa, but had no effect on C1^– or K⁺ influx in carrotor maize root cells. Cl^– influx is not related to thereducing sugar concentration during ageing drifts in excisedcarrot root tissue. Acetazolamide did not inhibit C1^– influx to carrot tissue. The implications of this type of negative feedback regulation,and the relationship between C1^– and NO₃^– transportare discussed.