Cytological studies on mitochondria-induced cytoplasmic transformation in yeasts

In Saccharomyces cerevisiae, protoplasts from respiratory-deficient(rho^–) cytoplasmic mutant cells were transformed intorespiratory-sufficient (rho⁺) cells by incubation with mitochondriaprepared from rho⁺ cells in the presence of polyethylene glycoland CaCl₂. Mitochondria prepared from different species, Hansenulawingei and Schizosaccharomyces pombe, also caused the transformationof S. cerevisiae rho^– protoplasts into the rho⁺ cellsas previously reported (14) The obtained transformants wereconfirmed to contain one nucleus and several mitochondrial DNAsby fluorescent staining of DNA. The transformants clearly restoredcytochromes a and b while untransformed recipient cells lackedthe cytochromes. In order to know the mechanism of the transformation,physiological measurement of endocytotic activity of protoplastsand cytological examination of mitochondria-protoplast aggregatesunder the transforming condition were performed. Protoplastshad significant endocytotic activity under this condition. Onthe other hand, fluorescence and electron microscopic observationsindicated that mitochondria forming aggregates with protoplastswere subsequently integrated into recipient protoplasts throughfusion rather than endocytosis. However, the possibility ofendocytosis could not be completely excluded when the low frequencyof the transformation (about 10^–6 to 10^–7) was takeninto account. This is discussed in this paper. A new convenientmethod for measuring endocytosis is also presented. (Received September 27, 1979; )     

Permeability of Lipophilic Cations

The permeability (P) of a lipophilic cation, triphenylmethylphosphonium(TPMP⁺) which is frequently used as a membrane potential probe,has been measured in Chara australis (Charophyceae). P_TPMP+across biological membranes is usually thought to be very highbut this is not the case across the plasmalemma of Chara. Thepermeability of TPMP⁺ across the plasmalemma was found to betypical of inorganic cations, about 1.0 nm s^–1. Estimateswere made of the permeability of lipophilic cations across someother cell membranes, based on previously published work. Thepermeability of TPMP⁺ across the plasma membranes of the redalga, Griffithsia monilis and the blue-green alga, Anabaenavariabilis was about 2–5 nm s^–1. The permeabilityof TPMP⁺ across the plasma membranes of eukaryotes and prokaryotesappears to be similar. The permeability of lipophilic cationsacross the cristae of isolated mitochondria are exceptionallyhigh, about 170 nm s^–1. TPMP⁺ did not behave as a thiamineanalogue in Chara, unlike in the case of yeast. The means ofentry of TPMP⁺ into the Chara cell, driven by the electrochemicalgradient across the plasmalemma, has not been identified. Thepresence of a second lipophilic cation probe, DDA⁺ (dibenzyldimethylammonium),caused a decrease in the uptake flux of TPMP⁺; this suggeststhat the two lipophilic cations compete for the same site atthe surface of the plasmalemma. Key words: Chara australis, TPMP⁺, Permeability, Lipophilic cation     

Further Characteristics of Salt-Dependent Bicarbonate Use by the Seagrass Zostera muelleri

Millhouse, J. and Strother, S. 1987. Further characteristicsof salt-dependent bicarbonate use by the seagrass Zostera muelleri.—J.exp. Bot. 38: 1055–1068. The contribution of HCO^–₃to photosynthetic O₂ evolutionin the seagrass Zostera muelleri Irmisch ex Aschers. increasedwith increasing salinity of the bathing seawater when the inorganiccarbon concentration was kept constant. K_1/2 (seawater salts)for HCO^–₃ -dependent photosynthesis was 66% of seawatersalinity. Both short- and long-term pretreatment at low salinitiesstimulated photosynthesis in full strength seawater. Twentyfour hours pre-incubation of seagrass plants in 3·0 molm^–3 NaHCO₃ resulted in increased photosynthesis at allsalinities, apparently due to stimulation of HCO^–₃ use(K_1/2 (seawater salts) = 26%). V_max (HCO^–₃) was not affectedby low salinity pretreatment. The kinetics of HCO^–₃ stimulationby the major seawater cations was investigated. Ca²⁺ was themost effective cation with the highest V_max (HCO^–₃) andwith K_1/2(Ca²⁺) = 14 mol m^–3. Mg²⁺ was also very effectiveat less than 50 mol m^–3 but higher concentrations wereinhibitory. This inhibition cannot be accounted for solely byprecipitation of MgCO₃. Na⁺ and K⁺ were both capable of stimulatingHCO^–₃ use. Stimulation was in two distinct parts. Up to500 mol m^–3, both citrate and chloride salts gave similarresults (K_1/2(Na⁺) 81 mol m^–3, V_max(HCO^–₃) 0·26µmol O₂ mg^–1 chl min^–1), but use of citratesalts above 500 mol m^–2 caused a second stimulation ofHCO^–₃ use (K_1/2(Na⁺) 830 mol m^–3, V_max(HCO^–₃)0·68 µmol O₂ mg^–1 chl min^–1). V_max(HCO^–₃)for the second-phase Na⁺ or K⁺ stimulation was of the same orderas for Ca²⁺-stimulated HCO^–₃ use. To further characterizesalt-dependent HCO^–₃ use, the sensitivity of photosynthesisto Tris and TES buffers was investigated. The effects of Trisappear to be due to the action of Tris⁺ causing stimulationof HCO^–₃ -dependent photosynthesis in the absence of salt,but inhibition of HCO^–₃ use in saline media. TES has noeffect on photosynthesis. External carbonic anhydrase, althoughimplicated in salt-dependent HCO^–₃ use in Z. muelleri,could not be detected in whole leaves. Key words: Zostera muelleri, HCO^–₃ use, salinity     

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     

Photosynthetic Nitrogen Metabolism in High and Low CO2-adapted Scenedesmus: II. EFFECT OF AMMONIUM AND METHIONINE SULPHOXIMINE ON NITRATE UTILIZATION

Larsson, C.-M., Larsson, M. and Guerrero, M. G. 1985. Photosyntheticnitrogen metabolism in high and low CO²-adapted Scenedesmus.II. Effect of ammonium and methionine sulphoximine on nitrateutilization.—J. exp. Bot. 36: 1387–1395 In 3% CO²-grown Scenedesmus obtusiusculus Chod. utilizing NO₃^–J as the N source, NH₄⁺ addition caused a prompt inhibitionof NO₃^– utilization. Nitrate reductase (NR) activity declinedrapidly in response to the presence of NO₄⁺, but the cessationof NO₃^– utilization was too rapid to be accounted forby the loss in NR activity. The first site of NO₄⁺ inhibitionin these cells seems to be the entrance of NO₃^– into thecells. Upon exhaustion of NO₄⁺ from the medium, NO₃^– utilizationwas rapidly restored and NR activity increased. Air-grown cellswere much less sensitive to the effect of NO₄⁺, more than 30min being required for added NO₄⁺ to cause complete inhibitionof NO₃^– utilization. In these cells, NO₃^– uptakeand NR activity decreased in parallel in response to NO₄⁺ addition.In 3% CO²-grown cells simultaneously subjected to NO₄⁺ and air-levelof CO², NO₄⁺ initially inhibited NO₃^– utilization completely,but a slight recovery took place after approximately 20 min The glutamine synthetase (GS) inhibitor L-methionine D, L-sulphoximine(MSO) behaved as a potent inhibitor of NO₃^– uptake in3% CO²-grown cells, but had considerably less effect in air-growncells, although the time-course of the MSO-induced inhibitionof GS was the same in both cases Key words: Ammonium, nitrate utilization, Scenedesmus     

Potassium-Ammonium Uptake Interactions in Tobacco Seedlings

Short-term (< 12 h) uptake experiments were conducted with6–7-week-old tobacco (Nicotiana tabacum L. cv. Ky 14)seedlings to determine absorption interactions between K⁺ andNH₄⁺. At equal solution concentrations (0.5 mol m^–3) netK⁺ uptake was inhibited 30–35% by NH₄⁺ and NH₄⁺ uptakewas decreased 9–24%. Removal of NH₄⁺ resulted in completerecovery in K⁺ uptake rate, but NH⁴⁺ uptake rate did not recoverwhen K⁺ was removed. In both cases, inhibition of the uptakerate of one cation saturated as the concentration of the othercation was increased up to 0.5 mol m^–3. The relative effectof K⁺-NH₄⁺ interactions was not altered when Cl- was replacedwith SO₄^2–, but the magnitudes of the uptake rates wereless in the absence of Cl-. The V_max for NH₄⁺ uptake was reducedfrom 128 to 105 µmol g^–1 dry wt. h^–1 in thepresence of 0.5 mol m^–3 K⁺ and the K_m for NH₄⁺ doubledfrom 12 to 27 mmol m^–3 in the presence of K⁺. The resultsof these K⁺-NH₄⁺ experiments are interpreted as mixed-noncompetitiveinteractions. However, an enhanced efflux of K⁺ coupled to NH₄⁺influx via an antiporter cannot be ruled out as contributingto the decrease in net K⁺ uptake. Key words: Nicotiana tabacum, K⁺, NH₄⁺, Uptake interactions     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Nitrogen utilization by plant species from acid heathland soils: II. Growth and shoot/root partitioning of NO3- assimilation at constant low pH and varying NO3-/NH4+ ratio

The growth of four heathland species, two grasses (D. flexuosa,M. caerulea) and two dwarf shrubs (C. vulgaris, E. tetralix),was tested in solution culture at pH 4.0 with 2 mol m^–3N, varying the N0₃^–/NH₄⁺ ratio up to 40% nitrate. In addition,measurements of NRA, plant chemical composition, and biomassallocation were carried out on a complete N0₃^–/NH₄⁺ replacementseries up to 100% nitrate. With the exception of M. caerulea, the partial replacement ofNH₄⁺ by NO₃^– tended to enhance the plant's growth ratewhen compared to NH₄⁺ only. In contrast to the other species,D. flexuosa showed a very flexible response in biomass allocation:a gradual increase in the root weight ratio (RWR) with NO₃^–increasing from 0 to 100%. In the presence of NH₄⁺, grassesreduced nitrate in the shoot only; roots did not become involvedin the reduction of nitrate until zero ambient NH₄⁺. The dwarfshrubs, being species that assimilate N exclusively in theirroots, displayed an enhanced root NRA in the presence of nitrate;in contrast to the steady increase with increasing NO₃^–in Calluna roots, enzyme activity in Erica roots followed arather irregular pattern. Free nitrate accumulated in the tissuesof grasses only, and particularly in D. flexuosa. The relative uptake ratio for NO₃^– [(proportion of nitratein N uptake)/(proportion of nitrate in N supply)] was lowestin M. caerulea and highest in D. flexuosa. Whereas M. caeruleaand the dwarf shrubs always absorbed ammonium highly preferentially(relative uptake ratio for NO₃^– <0.20), D. flexuosashowed a strong preference for NO₃^– at low external nitrate(the relative uptake ratio for N0₃^– reaching a value of2.0 at 10% NO₃^–). The ecological significance of thisprominent high preference for NO₃^– at low NO₃^–/NH₄⁺ratio by D. flexuosa and its consequences for soil acidificationare briefly discussed. Key words: Ammonium, heathland lants, N0₃^–/NH₄⁺ ratio, nitrate, nitrate reductase activity, soil acidification, specific absorption rate     

Efficiency of Potassium Utilization by Barley Varieties: The Role of Subcellular Compartmentation

Memon, A. R., Saccomani, M. and Glass, A. D. M. 1985. Efficiencyof potassium utilization by barley varieties: The role of subcellularcompartmentation.?J. exp. Bot. 36: 1860–1876. The subcellulardistributions of K⁺ in roots of three barley (Hordeum vulgareL.) varieties, grown at 10 and 100 mmol m^–3 external K⁺([K⁺]_o) were estimated by compartmental analyses. In general,increased [K⁺]_o caused a 2–3 fold increase in vacuolar[K⁺], but cytoplasmic [K⁺] increased only slightly. Nevertheless,the three varieties, which had been selected for study on thebasis of their different rates of K⁺ utilization, showed distinctdifferences in the allocation of K⁺ between cytoplasm and vacuole.At 10 mmol m^–3 [K⁺]_o var. Betzes exhibited typical K⁺deficiency symptoms while var. Fergus and var. Compana did not,even though Betzes had higher [K⁺] in shoots and roots. Theinefficient utilization of K⁺ in this variety appears to beassociated with a failure to mobilize vacuolar K⁺ into the cytoplasmiccompartment (the ratio of vacuolar: cytoplasmic K⁺ contentsfor Betzes was 4.1 compared to 2.7 and 2.5, respectively, forFergus and Compana). Fergus and Betzes, which demonstrate pronouncedgrowth responses to increased [K⁺]₀ between 10 and 100 mmolm^–3, showed significant increases of cytoplasmic [K⁺]in this range of [K⁺]_o. By contrast, cytoplasmic [K⁺] in Compana,a variety whose growth is not stimulated by increased [K⁺]₀(from 10 to 100 mmol m^–3) showed virtually no increase.It is suggested that the efficiency of K⁺ utilization and thegrowth response to [K⁺]₀ in these varieties are functions ofthe subcellular distribution of this ion between cytoplasm andvacuole. Key words: Barley varieties, K⁺ subcellular compartmentation, utilization efficiency     

Changes in the growth rates of saline-lake diatoms in response to variation in salinity, brine type and nitrogen form

The growth rates of four saline-lake diatom taxa were measuredunder varying conditions of salinity (5, 8 and 11), brine type(sulfate- versus bicarbonate-dominated) and nitrogen form (NH₄⁺versus NO₃^–), using a full factorial design. With NO₃^–as the nitrogen source, Cyclotella quillensis, Cymbella pusillaand Anomoeoneis costata exhibited lower growth rates in thesulfate versus bicarbonate media. The strain of Chaetoceroselmorei used in these experiments, isolated from a sulfate-dominatedlake, was unable to grow on NO₃^– alone. In the NH₄⁺ treatments,neither salinity nor brine type affected the growth rates ofC.quillensis or C.elmorei. When supplied with NH₄⁺, C.pusillaand A.costata had higher growth rates in the bicarbonate versussulfate media, although for C.pusilla the difference on NH₄⁺was not as great as on NO₃^–. The impact of brine typeon NO₃^– use is consistent with the theory that sulfateinhibits molybdate uptake, as molybdenum is required for NO₃^–use but not NH₄⁺. Cymbella pusilla was the only taxon affectedby changes in salinity. The four taxa used in these experimentsare frequently found in saline lakes and saline-lake sediments,hence they are used in paleoclimate reconstructions; the resultspresented here provide additional information that may enhancethese diatom-based reconstructions.     

Involvement of anion channel(s) in the modulation of the transient outward K(+) channel in rat ventricular myocytes

The cardiac Ca²⁺-independent transient outward K⁺ current (I_to), a major repolarizing ionic current, is markedly affected by Cl^– substitution and anion channel blockers. We reexplored the mechanism of the action of anions on I_to by using whole cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by 4-aminopyridine (4-AP) and was abolished by Cs⁺ substitution for intracellular K⁺. Replacement of most of the extracellular Cl^– with less permeant anions, aspartate (Asp^–) and glutamate (Glu^–), markedly suppressed the current. Removal of external Na⁺ or stabilization of F-actin with phalloidin did not significantly affect the inhibitory action of less permeant anions on I_to. In contrast, the permeant Cl^– substitute Br^– did not markedly affect the current, whereas F^– substitution for Cl^– induced a slight inhibition. The I_to elicited during Br^– substitution for Cl^– was also sensitive to blockade by 4-AP. The ability of Cl^– substitutes to induce rightward shifts of the steady-state inactivation curve of I_to was in the following sequence: NO₃^– > Cl^– Br^– > gluconate^– > Glu^– > Asp^–. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of I_to similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F-actin, whereas Asp^– substitution for Cl^– was without significant effect on the intensity. These results suggest that the I_to channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated. transient outward potassium current; anion channel; actin cytoskeleton; myocyte; potassium ion     

Nitrate Transport into Chara corallina Cells using 36ClO-3 as an Analogue for Nitrate: I. INTERACTION BETWEEN 36ClO-3 AND NO-3 AND CHARACTERIZATION OF 36CIO-3/NO-3 INFLUX

The use of chlorate as an analogue for NO^–₃ during nitrateuptake into Chara corallina cells has been investigated. NO^–₃inhibits ³⁶C1O^–₃ influx into Chara over the concentrationrange 0–1000 mmol m^–3. Lineweaver-Burke plots ofthe data are characteristic of competitive inhibition by NO^–3in the low concentration range (0–300 mmol m^–3 ClO^–₃)and apparent K_INO3 is 140 mmol m^–3 which is of a similarorder of magnitude as apparent K_mCIO3- 180 mmol m^–3. Athigher substrate concentrations the inhibition by NO^–₃was not characteristic of competitive or uncompetitive inhibition. ³⁶C1O^–₃/NO^–₃ influx was dependent on K⁺ and Ca²⁺in the external medium and inhibited by FCCP. NO^–₃ pretreatmentor N starvation increased subsequent ³⁶C1O^–₃/NO^–₃influx into Chara. A comparison between rates of net NO^–₃uptake and ³⁶C1O^–₃/NO^–₃ influx supported the previoushypothesis that NO^–₃ efflux is an important componentin the determination of overall uptake rates. Key words: Nitrate, Chara, ³⁶CIO^–₃     

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)     

Chloride Transport in Chara: I. KINETICS AND CURRENT-VOLTAGE CURVES FOR A PROBABLE PROTON SYMPORT

Chara cells show an inward positive electric current acrossthe plasmalemma when exposed to Cl^– under voltage-clampconditions. The rapid rise of this current suggests that itis directly associated with the inward transport of Cl^–.The dependence of the current on Cl^– concentration showssaturation, the data fitting the Michaelis-Menten equation withV_m up to 100 nmol m^–2 s^–1 (for Cl^–starvedcells) with K_M 10–20 µM, and with some allowancefor an unstirred layer of water adjacent to the membrane. Theeffects on the current of clamp potential, illumination, withdrawalof alkali metal cations, and addition of amine were also investigated.These results suggest that the mechanism is the symport of 2H⁺ with each Cl^–, and that the actions of light, externalK⁺, and amine in stimulating Cl^–, influx are indirect.     

Salinity-induced Malate Accumulation in Chara

Ion absorption by Chara corallina from solutions containingpredominantly KC1 or RbCl at up to 100 mol m^–3 resultedin accumulation of salts and turgor regulation. Turgor regulationdid not occur in solutions containing Na⁺ or Li⁺salts. Duringion absorption from various salts of K⁺ and Rb⁺ vacuolar cationconcentration exceeded Cl^– concentration. This differencewas shown to be balanced by the synthesis and accumulation ofmalate. Vacuolar malate concentration reached 48 mol m₃^–,with accumulation occurring at rates of up to 0.45 mol m^–3h^–1. Malate accumulation was inhibited by low externalpH and was dependent upon external HCO₃^– concentration.The synthesis of malic acid and its subsequent dissociationimposed a severe acid load on the cell. Biophysical regulationof cellular pH was achieved by a H⁺efflux at a rate of about40 nmol m^–2 s^–1from the cell. The results presentedargue against cytoplasmic Cl^–, HCO₃^– or pH regulatingmalate accumulation in Chara and it is suggested that malatetransport across the tonoplast may regulate malate accumulation. Key words: Malate, Chara corallina, pH regulation, salinity     

Efficiency of K+ Utilization by Barley Varieties: Activation of Pyruvate Kinase

Barley (Hordeum vulgare L.) varieties differed in their raponseto [K⁺]₀, in terms of their utilization efficiencies (UE = freshweight. concentration of [K⁺]₁^–1). At low [K⁺]₀, Compana,an efficient-non-responder demonstrated superior utilizationof absorbed K⁺. On the other hand, at high [K⁺]₀, Fergus (anefficient responder) and BT 334 (an inefficient responder) hadhigher UE values for K⁺ than Compana which performed poorlyat this [K⁺]₀. Kinetic parameters for K⁺ activation of the enzyme pyruvatekinase from 12 barley varieties, representing a range of UEvalues, were determined. Varieties showed substantial differencesin their V_max values (P<0·01). Compana, an efficientvariety, had the highest V_max (31 µmol g^–1 freshwt. h^–1) which was about 50% higher than that of Mingo,an inefficient variety. By contrast, K_m values for the enzymeswere not significantly different among varieties The mean valuesfor all varieties (3·9±0·15 mol m^–3K⁺) is far below the estimated cytoplasmic [K⁺] (100-200 molm^–3). It is, therefore, unlikely that differences in theutilization of K⁺ by these varieties can be explained on thebasis of differential requirements for (K⁺) activation of theseenzymes. Alternative possibilities for differences in the utilizationof K⁺ are discussed. Key words: K⁺ utilization efficiency, Pyruvate kinase, Barley varieties     

Quantitative Analysis of ATP-Dependent H+ Efflux and Pump Current Driven by an Electrogenic Pump in Nitellopsis obtusa

Internodal cells of Nitellopsis were made tonoplast-free byperfusion with a medium containing EGTA. Cytoplasmic concentrationsof solutes were controlled by a second perfusion with mediaof known composition. The electrogenic pump current (I_p), whichwas calculated from electrical data obtained from cells withand without ATP, was compared with the current carried by H⁺(I_H⁺) across the plasma membrane. A close correlation betweenI_p and I_H⁺ was found under various internal and external conditions.(1) I_p and I_H⁺ depended on the internal ATP and showed Michaelis-Mententype saturation curves. For I_p, K_m was 120 µM and themaximum current V_max was 15.1 mA m^–2, while for I_H⁺, K_mwas 160 µM and V_max was 16.6 mA m^–2. (2) I_p andI_H⁺ showed almost the same I_H²⁺ dependence. The Mg²⁺-dependentI_p was 19.5 mA m^–2, while the Mg²⁺-dependent I_H²⁺ was17.7 mA m^–2. (3) I_H²⁺ was maximal at an external pH of8 and decreased both in acidic and alkaline pH ranges. I_p wasnearly equal to I_H⁺ in the pH range between 8 and 5. (4) I_H⁺became maximal at an internal pH of 7.3, which is nearly thesame as the pH for maximal electrogenecity found by Mimura andTazawa (1984). All these facts support the idea proposed in our previous paper(Takeshige et al. 1985) that the electrogenic ion pump locatedin the plasma membrane of Nitellopsis is the H⁺ pump. ¹ Dedicated to Professor Dr. Erwin Bünning on the occasionof his 80th birthday. (Received June 21, 1985; Accepted December 20, 1985)     

Regulation of the Cytoplasmic pH of Chara corallina in the Absence of External Ca2+: Its Significance in Relation to the Activity and Control of the H+ Pump

Effects of removal of external Ca²⁺ on the cytoplasmic pH (pH_c)of Chara corallina have been measured with the weak acid 5,5-dimethyl-oxazolidine-2,4-dione(DMO) as a function of external pH (pH₀) and of the externalconcentration of K⁺. Removal of Ca²⁺ always decreased pH_c whenpH₀ was below about 6.0; the decrease was about 0.2–0.4units at pH₀ 5.0, increasing to about 0.5 units at pH₀ 4.3.When pH₀ was 6.0 or higher the removal of Ca²⁺ had little orno effect on pH_c. This situation was not altered by changingthe concentration of K⁺, though in some experiments at pH₀ 5.0–5.2there was a slight decrease in pH₀ (about 0.2 units) when K⁺was increased from 0.2 to 2.0 mol m^–3, an effect apparentlyreversed when K⁺ was higher (5.0 or 10.0 mol m^–3). Theresults suggest that H⁺ transport continues in the absence ofexternal Ca²⁺, despite previous suggestions to the contrary,and that the H⁺ pump does not necessarily run near thermodynamicequilibrium with its chemical driving reaction. They indicate,rather, that the H⁺ pump is under kinetic control and providefurther evidence for the inadequacy of present models for theoperation of the H⁺ pump in charophyte cells, especially inrelation to its proposed role in regulating pH_c. Key words: Chara corallina, Cytoplasmic pH, Calcium     

Photosynthetic Nitrogen Metabolism in High and Low CO2-adapted Scenedesmus: I. INORGANIC CARBON-DEPENDENT O2 EVOLUTION, NITRATE UTILIZATION AND NITROGEN RECYCLING

Larsson, M., Larsson, C.-M. and Guerrero, M. G. 1985. Photosyntheticnitrogen metabolism in high and low CO₂-adapted Scenedesmus.I. Inorganic carbon-dependent O2 evolution, nitrate utilizationand nitrogen recycling.—J. exp Bot. 36: 1373–1386 Scenedesmus obtusiusculus Chod. was grown on an inorganic mediumflushed with either air or air supplemented with 3% CO₂. Inair-grown cells, O₂ evolution dependent on low, but not high,HCO^–₃ concentrations was strongly inhibited by the carbonicanhydrase inhibitor acetazolamide. Cells grown with 3% CO² exhibitedlow rates of O2 evolution at low external inorganic C; however,after 30 min in air O2 evolution rates at low inorganic C approachedthose of air-grown cells. These results are compatible withthe view that Scenedesmus develops a ‘CO² concentratingmechanism’ in air, with carbonic anhydrase as an importantconstituent When 3% CO²-grown cells were subjected to air-level of CO²,just a transient decline in NO^–₃ utilization was observed,but in the presence of acetazolamide the rate of the processdecreased drastically in response to the decrease in the CO²level. In CO²-free air NO₃^– was taken up at high ratesbut in a deregulated manner, leading to release of NH₄⁺. A portionof the NO₃^– taken up in the absence of CO² was apparentlyassimilated Cellular nitrate reductase (NR) activity initially decreasedbut subsequently recovered after a transition from 3% CO² toair. In the presence of acetazolamide, a persistent decreasein NR activity was observed. Cellular glutamine synthetase (GS)activity increased after transition from 3% CO² to air, theactivity increase being unaffected by acetazolamide. NH₄⁺ releaseto the medium in the presence of L-methionine-D, L-sulphoximine(MSO) transiently increased in 3% CO²-grown cells in responseto a transfer to air. MSO-induced NH₄⁺ release was in fact higherin air-grown cells than in 3% CO²-grown cells. Glycollate wasinitially released after transition from 3% CO² to air, butthere was no difference in glycollate release between MSO-treatedand untreated cells. In air-adapted Scenedesmus, N recyclingseems to be of minor importance in comparison to primary N assimilation Key words: CO²-fixation, N recycling, nitrate uptake, Scenedesmus     

Changes in the Rate of Photosynthesis during Grain Filling and the Enzymatic Activities Associated with the Photosynthetic Carbon Metabolism in Rice Panicles

Photosynthesis is known to occur in rice panicles, but littlehas been reported about the photosynthetic or biochemical characteristicsof such panicles. The estimated gross amount of photo-syntheticallyassimilated CO₂ in a panicle is 30% of that in a flag leaf.This result and the good light-intercepting characteristicsof the panicle in the canopy suggest that photosynthesis inthe panicle may contribute significantly to grain filling. Therice panicle is composed of spikelets and of rachis-branchesincluding rachis which have estimated gross rates of photosynthesisduring the 30-day period after anthesis of 130 to 180 and 50to 100 µmol CO₂.(mg Chl)^–1.h^–1, respectively.The corresponding rate for the flag leaf is 180 to 230 µmolCO₂.(mg Chl)^–.h^–. On the basis of Chl, spikeletshave a high photosynthetic capability which is similar to thatof the flag leaf. The activities of ribulose-l,5-bisphosphate carboxylase (RuBPCase),phosphoenolpyruvate carboxylase (PEPCase), and pyruvate.P_i dikinase(PPDK) in spikelets were 129, 220, and 87 µmol.(mg Chl)^–.h^–,respectively. The activities of PEPCase and PPDK in spikeletswere considerably higher than those in the flag leaf or rachis-branches.Oxygen-insensitive photosynthesis was found only in spikelets.The K_m of NaHCO₃ for photosynthesis by slices of spikelets inan aqueous solution (0.6 mM) was considerably lower than thatfor slices of flag leaf (4.2 mM). All these results indicatethat spikelets have different photosynthetic characteristicsfrom those of the flag leaf and rachis-branches. The possibilityof C₃–C₄ intermediate photosynthesis or C₄-like photosynthesisin spikelets is discussed. ⁴Present address: Department of Biochemistry, Faculty of Science,Saitama University, Urawa, 338 Japan (Received February 14, 1990; Accepted June 12, 1990)