Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

Ammonia uptake and retention in some cyanobacteria

The internal pool of ammonia in strains of unicellular and filamentous cyanobacteria was found to be 6–12 nmol·mg^-1 protein. In nitrate grown Anacystis nidulans R-2 the pool size averaged 12 nmol·mg^-1 protein, which corresponds to 2.3 mM, and was little affected by N-source or medium pH during growth. Cells from NH ₄ ⁺ -limited continuous culture contained comparable pools, and cell yield was independent of medium pH (7.2–8.5). The internal pool was not bound to macromolecules. The pool fell transiently to about one-third within 2 h on shifting cells to N-free medium, but was slowly regenerated over 24 h.Added ammonia was removed from solution by illuminated cell suspensions at a linear rate, adequate to supply biosynthetic needs, to residual concentrations less than 5 M. An apparent K _m of less than 1 M can be inferred. Uptake rates were independent of N-source during growth, and of assay pH over the range 6.2–8.7. Bicarbonate was needed for uptake, but the rate of uptake was not influenced by the simultaneous presence of NaNO₃ (10 mM) or CH₃NH₃Cl (0.15 mM). Uptake was energydependent, and was eliminated in dark, anaerobic conditions or by the addition of protonophores. Uptake was also strongly inhibited by dicyclohexylearbodiimide, an ATPase inhibitor, by — SH reagents and methionine sulfoximine, suggesting that interference with energy supply or with ammonia metabolism prevented further entry into the cells.Non-standard abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DCMU dichlorophenyl dimethylurea - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoate - MSX L-methionine Dl-sulfoximine     

Transport of sugars across the plasma membrane of beetroot protoplasts

Protoplasts isolated from beetroot tissue took up glucose preferentially whereas sucrose was transported more slowly. The ¹⁴C-label from [¹⁴C]glucose and [¹⁴C]sucrose taken up by the cells could be detected rapidly in phosphate esters and, after feeding of [¹⁴C]glucose was found also in sucrose. The temperature-dependent uptake process (activation energy E_A about 50 kJ · mol^–1) seems to be carrier mediated as indicated by its substrate saturation and, for glucose, by competition experiments which revealed positions C₁, C₅ and C₆ of the D-glucose molecule as important for effective uptake. The apparent K_m(20° C) for glucose (3-O-methylglucose) was about 1 mM whereas for sucrose a significantly lower apparent affinity was determined (K_m about 10 mM). When higher concentrations of glucose (5 mM) or sucrose (20 mM) were administered, the uptake process followed first-order kinetics. Carrier-mediated transport was inhibited by N,N-dicyclohexylcarbodiimide, Na-orthovanadate, p–chloromercuribenzenesulfonic acid, and by uncouplers and ionophores. The uptake system exhibited a distinct pH optimum at pH 5.0. The results indicate that generation of a proton gradient is a prerequisite for sugar uptake across the plasma membrane. Protoplasts from the bundle regions in the hypocotyl take up glucose at higher rates than those derived from bundle-free regions. The results favour the idea that apoplastic transport of assimilates en route of unloading might be restricted to distinct areas within the storage organ (i.e. the bundle region) whereas distribution in the storage parenchyma is symplastic.Abbreviations CCCP Carbonylcyanide m–chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DOG deoxyglucose - Mes 2-(N-morpholino)ethanesulfonic acid - 3-OMG 3-O-methylglucose - PCMBS p–chloromercuribenzenesulfonic acid - SDS Sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Analysis of valine uptake by Commelina mesophyll cells in a biphasic active and a diffusional component

Valine uptake by isolated Commelina benghalensis L. mesophyll cells was measured over a wide concentration range (10^-6–4·10^-2 mol l^-1). The uptake data were subjected to iterative fitting. Experiments with carbonyl cyanide mchlorophenyl hydrazone (CCCP), diethylstilbestrol (DES), and p-chloromercuriphenylsulphonic acid (PCMBS) provided evidence that the biphasic uptake kinetics of valine consists of a diffusional component and a biphasic active uptake. The data from the control experiments, were also best fitted to one diffusional component and two Michaelis-Menten systems. The presence of two carrier systems in the plasmalemma, however, was considered to be virtual for the following reasons: (1) Both phases of active uptake were equally decreased by high concentrations of K⁺-ions. (2) Fusicoccin stimulated the active uptake in both phases to the same extent. (3) Inhibitors of the proton-driven uptake (CCCP, DES, PCMBS) similarly inhibited the active uptake at all concentrations. (4) The active uptake equally responded in both phases to changes in the pH. (5) Light also promoted the active uptake over the whole concentration range. These results strongly indicate that, despite its biphasic character, the active uptake is due to one proton-driven carrier system.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DES diethylstilbestrol - FC fusicoccin - MES 2-(N-morpholino)ethanesulphonic acid monohydrate - PCMBS p-chloromercuriphenylsulphonic acid - v uptake velocity - S substrate concentration - K _m1 and K _m2 Michaelis constants of the apparent high- and low-affinity system, respectively - V _m1 and V _m2 maximal uptake velocities of the apparent high- and low-affinity system - k linear uptake constant     

Calcium influx at the plasmalemma of isolated guard cells of Commelina communis

The influx of ⁴⁵Ca into isolated guard cells of Commelina communis L. has been measured, using short uptake times, and washing in ice-cold La³⁺-containing solutions to remove extracellular tracer after the loading period. Over 0.5–4 min the uptake was linear with time, through the origin. Over 20–200M external Ca²⁺ the influx measured with 10–20 mM external KCl was in the range 0.3–2.3 pmol·cm^-2·s^-1 (on the basis of estimated guard-cell area); with only 1 mM KCl externally the ⁴⁵Ca influx was significantly reduced, in the range 0.3–1.1 pmol·cm^-2·s^-1 for external Ca²⁺ of 50–100 M. The results indicate that the Ca-channel is voltage-sensitive, opening with depolarisation. No consistent effect of the addition of abscisic acid could be found. In different experiments, on the addition of 0.1 mM abscisic acid the Ca²⁺ influx was sometimes stimulated by 28–79%, was sometimes unaffected, and was sometimes inhibited by 16–29%. The results rule out a long-lasting stimulation of ⁴⁵Ca influx by ABA, but they do not rule out a transient stimulation followed by inhibition, perphaps as a consequence of down-regulation of Ca²⁺ influx by increasing cytoplasmic Ca²⁺. The hypothesis that ABA may act via an action on Ca²⁺ influx, increasing cytoplasmic Ca²⁺, with consequent effects on voltage-dependent and Ca²⁺-dependent ion channels in both plasmalemma and tonoplast, is neither proved nor disproved by these results.Abbreviations ABA abscisic acid - Ca_o, K_o external Ca and K concentrations     

Uptake of iron byGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum (isolate 1–9) pathogenic on citrus fruits, appears to lack siderophore production. Iron uptake byG. candidum is mediated by two distinct iron-regulated, energy-and temperature-dependent transport systems that require sulfhydryl groups. One system exhibits specificity for either ferric or ferrous iron, whereas the other exhibits specificity for ferrioxamine-B-mediated iron uptake and presumably other hydroxamate siderophores. Radioactive iron uptake from⁵⁹FeCl₃ showed an optimum at pH 6 and 35° C, and Michaelis-Menten kinetics (apparentK _m = 3 m,V _max = 0.054 nmol · mg^–1 · min^–1). The maximal rate of Fe²⁺ uptake was higher than Fe³⁺ (V _max = 0.25 nmol · mg^–1 · min^–1) but theK _m was identical. Reduction of ferric to ferrous iron prior to transport could not be detected. The ferrioxamine B system exhibits an optimum at pH 6 and 40° C and saturation kinetics (K _m = 2 M,V _max = 0.22 nmol · mg^–1 · min^–1). The two systems were distinguished as two separate entities by negative reciprocal competition, and on the basis of differential response to temperature and phenazine methosulfate. Mössbauer studies revealed that cells fed with either⁵⁷FeCl₃ or⁵⁷FeCl₂ accumulated unknown ferric and ferrous binding metabolites.     

The effect of endogenous and externally supplied nitrate on nitrate uptake and reduction in sugarbeet seedlings

The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and -amino-N. These N-reserves enable an autonomous development of the seedling for 8–10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake system (v_max = 0.8 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (v_max = 3.4 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates of the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (t_1/2 of v_max = 5 d) probably caused by the decay of carrier molecules. Small differences in K_s but significant differences in v_max indicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules.Abbreviations NR nitrate reductase - pFPA para-fluorophenylalanine This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Nitrate and nitrite uptake and reduction by intact sunflower plants

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO ₃ ^– or NO ₂ ^– upon initial exposure to these anions. Ability of the plants to take up NO ₃ ^– and NO ₂ ^– at high rates from the beginning was induced by a pretreatment with NO ₃ ^– . Nitrite also acted as inducer of the NO ₂ ^– -uptake system. The presence of cycloheximide during NO ₃ ^– -pretreatment prevented the subsequent uptake of NO ₃ ^– and NO ₂ ^– , indicating that both uptake systems are synthesized de novo when plants are exposed to NO ₃ ^– . Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO ₃ ^– and NO ₂ ^– . Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO ₃ ^– and NO ₂ ^– uptake as a function of external anion concentration exhibited saturation kinetics. The calculated K_m values for NO ₃ ^– and NO ₂ ^– uptake were 45 and 23 M, respectively. Rates of NO ₃ ^– uptake were four to six times higher than NO ₃ ^– -reduction rates in roots. In contrast, NO ₂ ^– -uptake rates, found to be very similar to NO ₃ ^– -uptake rates, were much lower (about 30 times) than NO ₂ ^– -reduction rates. Removal of oxygen from the external solution drastically suppressed NO ₃ ^– and NO ₂ ^– uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO ₃ ^– and NO ₂ ^– into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.Abbreviations CHI cycloheximide - NEM N-ethylmaleimide - NiR nitrite reductase - NR nitrate reductase - pHME p-hydroxymercuribenzoate This research was supported by grant PB86-0232 from the Dirección General de Investigatión Científica y Técnica (Spain). One of us (E.A.) thanks the Consejeria de Educación y Ciencia de la Junta de Andalucia for the tenure of a fellowship. We thank Miss G. Alcalá and Miss C. Santos for their valuable technical and secretarial assistance.     

Therlt11 andraec1 mutants ofArabidopsis thaliana lack the activity of a basic-amino-acid transporter

The concentration dependence of the influx ofl-lysine in excised roots ofArabidopsis thaliana seedlings was analyzed for the wild-type (WT) and two mutants,rlt11 andraec1, which had been selected as resistant to lysine plus threonine, and to S-2-aminoethyl-l-cysteine, respectively. In the WT three components were resolved: (i) a high-affinity, low-capacity component [K _m = 2.2 M;V _max = 23 nmol·(g FW)^–1·h^–1]; (ii) a low-affinity, high-capacity component [K _m = 159 M;V _max = 742 nmol·(g FW)^–1·h^–1]; (iii) a component which is proportional to the external concentration, with a constant of proportionalityk = 104 nmol·(g FW)^–1 h^–1];·mM^–1. The influx ofl-lysine in the mutants was lower than in the WT, notably in the concentration range 0.1–0.4 mM, where it was only 7% of that in the WT. In both mutants the reduced influx could be fully attributed to the absence of the low-affinity (high-K _m ) component. This component most likely represents the activity of a specific basic-amino-acid transporter, since it was inhibited by several other basic amino acids (arginine, ornithine, hydroxylysine, aminoethylcysteine) but not byl-valine. The high-affinity uptake ofl-lysine may be due to the activity of at least two general amino acid transporters, as it was inhibitable byl-valine, and could be further dissected into two components with a high affinity (K _i = 1–5 M; and a low affinity (K _i = 0.5–1mM) forl-valine, respectively. Therlt11 andraecl mutant have the same phenotype and the corresponding loci were mapped on chromosome 1, but it is not yet clear whether they are allelic.Abbreviations AEC S-2-aminoethyl-l-cysteine - K _i equilibrium constant - WT wild-type     

Ca2+ transport by opercular epithelium of the fresh water adapted euryhaline teleost,Fundulus heteroclitus

We examined transepithelial transport of Ca²⁺ across the isolated opercular epithelium of the euryhaline killifish adapted to fresh water. The opercular epithelium, mounted in vitro with saline on the serosal side and fresh water (0.1 mmol·l^–1 Ca²⁺) bathing the mucosal side, actively transported Ca²⁺ in the uptake direction; net flux averaged 20–30 nmol·cm^–2·h^–1. The rate of Ca²⁺ uptake varied linearly with the density of mitochondria-rich cells in the preparations. Ca²⁺ uptake was saturable, apparent K _1/2 of 0.348 mmol·l^–1, indicative of a multistep transcellular pathway. Ca²⁺ uptake was inhibited partially by apically added 0.1 mmol·l^–1 La³⁺ and 1.0 mmol·l^–1 Mg²⁺. Addition of dibutyryl-cyclic adenosine monophosphate (0.5 mmol·l^–1)+0.1 mmol·l^–1 3-isobutyl-l-methylxanthine inhibited Ca²⁺ uptake by 54%, but epinephrine, clonidine and isoproterenol were without effect. Agents that increase intracellular Ca²⁺, thapsigargin (1.0 mol·l^–1, serosal side), ionomycin (1.0 mol·l^–1, serosal side) and the calmodulin blocker trifluoperazine (50 mol·l^–1, mucosal side) all partially inhibited Ca²⁺ uptake. In contrast, apically added ionomycin increased mucosal to serosal unidirectional Ca²⁺ flux, indicating Ca²⁺ entry across the apical membrane is rate limiting in the transport. Verapamil (10–100 mol·l^–1, mucosal side), a Ca²⁺ channel blocker, had no effect. Results are consistent with a model of Ca²⁺ uptake by mitochondria rich cells that involves passive Ca²⁺ entry across the apical membrane via verapamil-insensitive Ca²⁺ channels, intracellular complexing of Ca²⁺ by calmodulin and basolateral exit via an active transport process. Increases in intracellular Ca²⁺ invoke a downregulation of transcellular Ca²⁺ transport, implicating Ca²⁺ as a homeostatic mediator of its own transport.Abbreviations DASPEI 2-(4-dimethylaminostyryl)-N-ethylpyridinium iodide - db-cAMP dibutyryl-cyclic adenosine monophosphate - FW fresh water - G _t transepithelial conductance - I _sc short-circuit current - IBMX 3-isobutyl-1-methylxanthine - SW sea water - TFP trifluoperazine - V _t transepithelial potential     

In situ measurements of uptake of inorganic carbon and nitrogen by the aquatic liverworts Jungermannia vulcanicola Steph. and Scapania undulata (L.) Dum. in an acid stream,Kashiranashigawa, Japan

In situ uptake of inorganic carbon and nitrogen by the aquatic liverworts Jungermannia vulcanicola Steph. and Scapania undulata (L.) Dum. was measured in an acid stream, Kashiranashigawa, Japan. The uptake activities were similar in the both species. The activities were highest at the tip of shoots, and decreased gradually towards the base. Carbon uptake at the tip in the light was 10.4 × 10^–4 for J. vulcanicola and 8.1 × 10^–4 g C g dry wt^–1 h^–1 1 for S. undulata. Ammonium was effectively incorporated into the shoots, and the uptake activity at the tip was between 1.9 × 10^–5 and 5.8 × 10^–5 g N g dry wt^–1 h^–1. Nitrate uptake was smaller than ammonium uptake. The ratio of dark to light uptake in ammonium uptake experiments was larger than that in carbon uptake experiments. These results suggest that the liverworts use ammonium as a major nitrogen source, and that ammonium uptake was less dependent on light than carbon uptake.     

Ammonium and nitrate uptake in gap,generalist and understory species of the genus Piper

Summary We studied root net uptake of ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ^– ) in species of the genus Piper (Piperaceae) under high, intermediate and low photosynthetically active photon flux densities (PFD). Plants were grown hydroponically, and then transferred to temperature controlled (25° C) root cuvettes for nutrient uptake determinations. Uptake solutions provided NH ₄ ⁺ and NO ₃ ^– simultaneously (both) or separately (single). In the first experiment, seven species of Piper, from a broad range of rainforest light habitats ranging from gap to understory, were screened for mineral nitrogen preference (100 M NH ₄ ⁺ and/or 100 M NO ₃ ^– ) at intermediate PFD (100 mol m^–2 s^–1). Preference for NH ₄ ⁺ relative to NO ₃ ^– , defined as the ratio of NH ₄ ⁺ (both):NO ₃ ^– (both) net uptake, was higher in understory species than in gap species. Ammonium repression of NO ₃ ^– uptake, defined as the ratio of NO ₃ ^– (single): NO ₃ ^– (both) net uptake, was also higher in understory species as compared to gap species. In a second set of experiments, we examined the effect of nitrogen concentration (equimolar, 10 to 1000 M) on NH ₄ ⁺ preference and NH ₄ ⁺ repression of NO ₃ ^– net uptake at high (500 mol m^–2 s^–1) and low (50 mol m^–2 s^–1) PFD in a gap (P. auritum), generalist (P. hispidum) and understory species (P. aequale). All species exhibited negligible NH ₄ ⁺ repression of NO ₃ ^– net uptake at high PFD. At low PFD, NH ₄ ⁺ preference and repression of NO ₃ ^– net uptake occurred in all species (understory > generalist > gap), but only at intermediate nitrogen concentrations, i.e. between 10 and 200 M. Ammonium repression of net NO ₃ ^– uptake decreased or increased rapidly (in < 48 h) after transitions from low to high or from high to low PFD respectively. No significant diurnal patterns in NO ₃ ^– or NH ₄ ⁺ net uptake were observed.CIWDPB publication # 1130     

Selenite uptake and incorporation by Selenomonas ruminatium

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhbit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent K _m of 1.28 mM and a V _max of 148 ng Se min^-1 mg^-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chloropromazine (CPZ), N,N-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating ⁷⁵[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. ⁷⁵[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium.     

Outdoor pond cultivation of the subtropical marine red algaGracilaria tenuistipitata in brackish water in Sweden. Growth,nutrient uptake,co-cultivation with rainbow trout and epiphyte control

Pond cultivation of the subtropical, euryhaline macroscopic red algaGracilaria tenuisipitata var.liui Zhanget Xia was carried out in brackish seawater (6–7) in the Gryt archipelago on the east coast of Sweden, using four outdoor tanks of 30–40 m³. Growth rate and nutrient uptake in batch culture were measured with the aim of estimating the water purification capacity ofG. tenuisipitata in outdoor conditions. Its ability to withstand epiphytic infections was also studied. An average growth rate of 4 biomass increase per day was recorded during two seasons with a maximum growth rate of 9 d^–1. The initial biomass was usually 1 kgFW m^–3 (FW, fresh weight). The nutrient uptake capacity was on average ca. 1 g N_i kgFW^–1 d^–1 and 0.08 g P_i kgFW^–1 d^–1 and the uptake rates for NH₄ ⁺-N were higher than those for NO₃ ^–-N. Both the growth rate and the nutrient uptake rate were highest at the highest water temperature. Co-cultivation with rainbow trout (Oncorhynchus mykiss) was tested: with trout fodder as the only nutrient inputG. tenuistipitata could grow and maintain low levels of N_i and P_i with optimum efficiency at a trout: alga ratio of 1:1 (w:w). Epiphytic growth of filamentous green and brown algae was limited, probably as a result of the high pH values caused by inorganic carbon uptake byG. tenuistipitata. The growth ofEnteromorpha intestinalis, the only significant epiphyte, was completely inhibited and the majority of plants died by a few days treatment with 100 µg 1^–1 Cu²⁺, a concentration that did not severely affectG. tenuistipitata. We conclude thatG. tenuistipitata can be cultivated in outdoor ponds in southern Sweden during 5–6 months of the year using aerated or unaerated batch cultures and that wastewater from trout cultivation may be used as a nutrient source, resulting in purification with respect to N and P.     

Chromate tolerance in strains of Rhodosporidium toruloides modulated by thiosulfate and sulfur amino acids

Cr(VI) tolerance was studied in four strains of Rhodosporidium toruloides and compared with that of a fifth strain, DBVPG 6662, isolated from metallurgical wastes and known to be Cr(VI) resistant. Tolerance was studied in relation to different species of sulfur (sulfates, thiosulfates, methionine, cysteine) at different concentrations. Djenkolic acid, a poor source of sulfur and an activator of sulfate transport, was also considered. In synthetic medium all strains except the Cr(VI)-resistant one started to be inhibited by 10 g ml^– (0.2 mm) Cr(VI) as K₂Cr₂O₇. DBVPG 6662 was inhibited by 100 g ml^– (2.0 mm) Cr(VI). In Yeast Nitrogen Base without amino acids (minimal medium), supplemented with varying concentrations of chromate, all Cr(VI)-sensitive strains accumulated concentrations of total chromium (from 0.8 to 1.0 g mg^– cell dry wt) after 18 h of incubation at 28 °C. In minimal medium supplemented with 10 g ml^– Cr(VI), the addition of sulfate did not significantly improve the yeast growth. Cysteine at m levels increased tolerance up to 10 g ml^–, whereas methionine only reduced the Cr(VI) toxicity in the strain DBVPG 6739. Additions of djenkolic acid resulted in increased Cr(VI) sensitivity in all strains. The best inorganic sulfur species for conferring high tolerance was thiosulfate at concentrations up to 1 mm. In all cases increased Cr(VI) tolerance was due to a significantly reduced uptake in the oxyanion by the cells and not to the chemical reduction of Cr(VI) to Cr(III) by sulfur compounds.     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.     

Calcium influx at the plasmalemma of Chara corallina

Influx of ⁴⁵Ca into internodal cells of Chara corallina has been measured, using short uptake times, and a wash in ice-cold La³⁺-containing pondwater after the labelling period to overcome the difficulty of distinguishing extracellular tracer from that in the cell. Over 5–15 min the uptake was linear with time, through the origin. The basal influx from 0.1 mM Ca²⁺ externally was 0.25–0.5 pmol·cm^-2·s^-1, but some batches of cells showed higher fluxes. The influx was markedly stimulated by depolarisation in pondwater containing 20 mM K⁺. In cells in which the control flux was less than about 0.5 pmol·cm^-2·s^-1 there was no effect of 50 M nifedipine. In cells in which the control flux was greater than about 0.5 pmol·cm^-2·s^-1 (whether by natural variability, pretreatment, or by depolarisation in 20 mM K⁺), the flux was reduced by 50 M nifedipine to a value in the range 0.25–0.59 pmol·cm^-2·s^-1. It is suggested that two types of Ca-channel are probably involved, both opening on depolarisation, but only one sensitive to nifedipine. The flux was inhibited by 10 M BAY K 8644, which in animal cells more commonly opens Ca-channels. The apparent influx measured over long uptake times was much reduced, and the kinetics indicated filling a pool of apparent size about 1.45 nmol·cm^-2 with a halftime of about 38 min, probably representing cytoplasmic stores. It is argued that in spite of the very small pool of (free+bound) cytoplasmic Ca²⁺ the measured influx is a reasonable estimate of the influx at the plasmalemma.Abbreviations 0.4K-APW6 artificial pondwater, pH 6, containing 0.4 mM KCl - 20 K-APW6 artificial pondwater, pH 6, containing 20 mM KCl - Ca_o external Ca²⁺     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

Nitrite inhibition of nitrogenase from soybean bacteroids

Nitrogenase from soybean bacteroids was purified and used to study NO ₂ ^– effects either as unfractionated enzyme or as reconstituted enzyme from separated nitrogenase components I and II. Partially purified enzyme was strongly inhibited by nitrite at concentrations less than 0.1 mM. This inhibition was typically referred to as competitive with an inhibition constant (K _i) for NO ₂ ^– which was 5.2 mM. Kinetics studies showed an abnormally low apparent constant of association between enzyme and NO ₂ ^– (k _a=60 M^-1·s^-1). Nitrite appeared to bind to the MoFe protein, without any effect on Fe component, giving a completely reversible inhibition. Nitrite was found not to be an alternative substrate for nitrogenase.Abbreviations TES N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid - PPG Polypropylene glycol     

Presence of a Na+/H+ exchanger in brush border membranes isolated from the kidney of the spiny dogfish,Squalus acanthias

Summary A membrane fraction, rich in brush border membranes, was prepared from renal proximal tubules of the spiny dogfish,Squalus acanthias, and the sodium-proton exchange mechanism in these membrane vesicles was investigated by both a rapid filtration technique and the fluorescence quenching of acridine organe.²²Na⁺ uptake was stimulated by an outwardly directed H⁺ gradient, and was inhibited by amiloride at a single inhibitory site with an apparentK _i of approximately 1.7×10^–5 M. In the presence of an H _i ⁺ >H _o ⁺ gradient, the of the Na⁺/H⁺ exchanger were 9.7±0.8 mM and 48.0±12.0 nmol·mg protein^–1·min^–1, respectively. The uptake of Na⁺ was electroneutral in the presence of a H⁺ gradient, indicating a stoichiometry of 1. In the fluorescence studies, quenching of acridine orange occurred in the presence of an outwardly directed Na⁺ gradient which was inhibited by amiloride. Thus, an electroneutral Na⁺/H⁺ exchanger with properties similar to those found in the mammalian kidney is also present in the spiny dogfish and may contribute to the urinary acidification of this marine animal.