Lipid and Sterol Composition of Plasma Membranes Isolated from Stele and Cortex of Maize Roots

The phospholipid and sterol contents of plasma membranes isolatedfrom stele and cortex of maize roots were compared. The majorsterol present in both tissues was stigmasterol which was foundin significantly higher quantities in the cortex (27·4µg mg^–1 membrane protein) compared to the stele(17·4 µg mg^–1). Other sterols detected includedsitosterol, campesterol and small quantities of cholesterol.The major phospholipids found were phosphatidylethanolamine(PE) and phosphatidylcholine (PC), with PC more abundant inthe stele. The fatty acid composition indicated that the majorfatty acid in both stele and cortex was 16:0 (palmitic), withothers found in lesser amounts. Key words: Zea mays, cortex, phospholipids, plasma membrane, stele, sterols     

Characterization of NADH-dependent Fe3+-chelate reductases of maize roots

Iron-deficient maize seedlings exhibit a starvation syndromecharacterized by an increase in different parameters such asroot fresh weight (+ 30%), protein (+ 25%) and plasma membrane-associatedNADH Fe³⁺ –EDTA reductase (NFR; +45%). NFR activity wasfound associated with 9 000g (20 min) and 110 000 g (1 h) sediments,purified plasma membrane and 110000 g supernatants. No differenceswere observed between the properties of reductases from Fe-starvedversus Fe-sufficient roots. The characterization of NFR wasundertaken. Low Mr forms (46 and 28 kDa, as detected by size-exclusionchromatography) were present in all fractions whereas 210 and110 kDa forms were unique in membranes and 110 000 g supernatants,respectively. The 210 kDa form was solubilized from microsomes and characterized.The enzyme is cetone-resistant and appears to be comprised largelyif not totally of the low Mr forms (46 and 28 kDa, correspondingto 30 and 32 kDa bands, respectively, in SDS-PAGE). The 210kDa form tended to break down to subunits following dilution,and the effect could be prevented by addition of 10% (v/v) glycerol.A three-step purification procedure for microsomal NFR was devised,consisting of acetone fractionation of lysophosphatidycholinesolubilized microsomes, Blue Sepharose CL-6B affinity chromatographyand a final size exclusion chromatography in the absence ofdetergent, resulting in a 700-fold purification of the 28 kDaprotein. The best electron acceptor for the purified 28 kDaform was ferricyanide (400µmol min^–1mg^–1 protein)followed by Fe³⁺–chelates (up to 200µol min^–1mg^–1 protein) and other compounds to a lesser extent (cytc, DCPIP).The 46 kDa form, on the other hand, had high ferricyanidereductase activity (about 300µmol min^–1mg^–1protein) and relatively low Fe³⁺–chelate reductase activity.The properties of NFR (high M, active forms, donor and acceptorspecificity, purification behaviour, large hydrophilic domains,size of subunits) suggest a relationship with the NADH-cyt b5reductase family of FAD-containing proteins. None of the latterflavoproteins is a transmembrane enzyme. Key words: Maize roots, Fe³⁺–reductase, ferricyanide reductase, iron     

Radial Turgor Pressure Profiles in Growing and Mature Zones of Wheat Roots--A Modification of the Pressure Probe

A modification of the pressure probe is described which allowsaccurate routine recording of the turgor pressure of singlecells at measured depth within a tissue. Measurements of radial profiles of turgor pressure in wheatroots grown in some simple salt solutions (0.5 mol m^–3CaCl₂, 0.5 mol m^–3 CaCI₂ plus 10 mol m^–3 NaCl, and0.5 mol m^–3 CaCl₂ plus 10 mol m^–3 KCI), are described.Turgor pressure was constant (approximately, 0.65 MPa) alonga radius within the elongation zone irrespective of the natureof the bathing solution. In mature root tissue turgor pressurein the cortex was lower than that of the growing zone in alltreatments and the pressure of the stele was on average 0.22MPa higher than that of the cortex. Potassium in the mediumbathing the root increased the turgor pressure in mature root(both cortex and stele) relative to low salt and sodium treatments. The results are discussed in relation to both root growth andion accumulation. Key words: Pressure probe, wheat roots, salt solution     

The Distribution of some NADP-Linked Dehydrogenases in Photosynthetic Tissues5

Mesophyll protoplasts of one-month-old maize leaves were separatedenzymatically from bundle sheath strands, and purified by centrifugationthrough a Percoll layer. The protoplasts and BS strands wereessentially pure as judged by microscopy, chl a/b ratios, andlevels of enzyme markers (PEP carboxylase and NADP-malic enzyme).Chioroplasts were obtained from the protoplasts and from homogenates,and purified through Percoll. The distribution of four NAD P-linked dehydrogenases in tissuesand organdies was examined. NADP-triose phosphate dehydrogenasc,used as a chloroplast marker, shows high and comparable specificactivities in both main tissues. Glucose 6-phosphate dehydrogenaseis located mainly in the mesophyll (at a specific activity of15.1 µmol h^–1 mg^–1chl in protoplasts) andis exclusively cytosolic. 6-Phosphogluconate dehydrogenase,also present in both tissue types, has a higher activity inthe BS (12.6 in purified strands versus 7.3 µmol h^–1mg^–1 chl in protoplasts). It is a cytosolic enzyme, althoughplastids may contain a low activity. Glyceraldehyde 3-phosphate:NADP reductasc is entirely in the mcsophyll cytoplasm (11.2µmol h^–1 mg^–1 chl). It is suggested that thecytoplasm of mcsophyll cells is a site of diversion of sugarphosphates for production of NADPH, at rates, however, compatiblewith the operation of the triose phosphate shuttle to bundlesheath cells for the synthesis of starch. Key words: Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dchydrogenase, glyceralde-hyde-3-phosphate : NADP reductase, Zea mays     

H+ Efflux and Trans-Root Potential Measured while Increasing the Temperature of Solutions Bathing Excised Roots of Zea mays

Kennedy, C. D. and Gonsalves, F. A. N. 1988. H⁺ efflux and trans-rootpotential measured while increasing the temperature of solutionsbathing excised roots of Zea mays.—J. exp. Bot. 39: 37–49. Novel temperature-ramp procedures have been used to measureH⁺ efflux and trans-root potential of excised roots of Zea mays(var. Fronica). Two types of experiment were performed: (1),increasing temperature from 17°C, and (2), pre-cooling theroots to 1°C before starting the temperature ramp. The ratesof increase of temperature for H⁺ efflux and trans-root potentialexperiments were 0·5 and 2·1°C min^–1respectively The H⁺ scans revealed strong sharp maxima at 30°C and 32°C,for non-pre-cooled and pre-cooled roots respectively, the latterbeing significantly smaller. The trans-root potential scansfor the pre-cooled roots showed a corresponding maximum at 30°C,which was inhibited by KCN (1-0 mmol dm^–3) with or withoutSHAM (10 mmol dm^–3), or Hg²⁺ (1, 10, 100 µmol dm^–3)in the bathing solutions. Some of the evidence suggests thatthese maxima are associated with electrogenic H⁺ pumping, mediatedby a plasma membrane-bound ATPase. However, no correspondingmaximum was observed in the trans-root potential scans for non-pre-cooledroots, the potential remaining at about — 75 m V from20°C to 35°C. As there is a 7-fold increase in H⁺ effluxbetween 20°C and 30°C, the relationship between netH+ efflux and electrogenic proton pumping in these roots isby no means clear. Some possibilities are considered here. Pre-cooled and non-pre-cooled roots show clear maxima in thetrans-root potential scans at about 46°C, at which temperaturethere is a slight net H⁺ influx. This, and other less prominentfeatures observed, are briefly discussed. Key words: H⁺ efflux, trans-root potential, temperature-ramp procedure, Zea mays, roots     

Enzyme Distribution in Potato Mitochondria

Enzyme distribution in potato mitochondria was investigatedby selectively disrupting the outer and inner membranes withdigitonin. Antimycin-insensitive NADH-cytochrome c reductase,an outer membrane marker, was released at low digitonin concentrations(0.1 mg mg^–1 mitochondrial protein). Soluble matrix enzymes,fumarase and malate dehydrogenase were released at 0.3–0.4mg digitonin mg^–1 protein, as the inner membrane ruptured.Very little (about 10%) cytochrome oxidase activity was released,even at higher digitonin concentrations, in accord with thisenzyme being an integral inner membrane protein. By this criterionadenylate kinase is also firmly bound to the inner membrane.Evidence indicates that it faces the intermembrane space. Malic enzyme activity was released by the same digitonin concentrationthat released fumarase and malate dehydrogenase, indicatingthat malic enzyme is a soluble matrix enzyme. No activity wasreleased at low digitonin concentrations which selectively breakthe outer membrane, showing that malic enzyme is not presentin the intermembrane space. Considerable catalase activity (20—40 µmol O₂ min^–1mg^–1 protein) was associated with washed mitochondrialpreparations, but 95% of this was lost upon purification ofmitochondria. The remaining activity was firmly bound to themitochondrial membranes.     

Sodium Exclusion from the Shoots by Roots of Zea mays (cv. LG 11) and its Breakdown with Oxygen Deficiency

The effects of sodium chloride salinity and root oxygen deficiency(anoxia) were studied in 11-12d old maize plants (Zea mays L.cv. LG 11) in nutrient solution culture. Transport of ²²Na bythe roots to the shoot in 24 h was markedly increased by anoxiawhen the external concentration of NaCl was in the range 0·1-10·9mol m^–3. Anoxia severely inhibited uptake of ⁴²K by rootsand its transport to the shoot, so that the ratio of Na⁺/K⁺moving into the shoot was increased by a factor of approximately10. When the external concentration of NaCl was increased to2.4 mol m^–3, the roots showed much less ability to excludeNa⁺ under aerobic conditions, and anoxia caused no further increasein the movement of Na⁺ to the shoot. It is concluded that atthe higher concentration the ability of the roots to excludeNa⁺, presumably through an active mechanism in the xylem parenchymacells or in the root cortex and transporting Na⁺ to the outersolution, is saturated by excessive inward diffusion of Na⁺.The ratio of Na⁺/K⁺ transported to the shoot increased by afactor of 600 when the concentration of NaCl was increased from2·4 mol m^–3 to 40 mol m^–3 and roots weremade anoxic. Such imbalances in the supply of cations to theshoot, particularly when roots are oxygen-deficient, may contributeto salinity damage. Key words: Anaerobic, Anoxic, Oxygen deficiency, Roots, Salinity, Salt stress, Sodium chloride, Zea mays     

Flexible Coupling of Phosphate Uptake in Dunaliella acidophila at Extremely Low pH Values

The acidophilic alga Dunaliella acidophila exhibits optimalgrowth at pH 1. We have investigated the regulation of phosphateuptake by this alga using tracer techniques and by performingintracellular phosphate measurements under different growthconditions including phosphate limitation. In batch culturewith 2·2 mol m^–3 phosphate in the medium the uptakeof phosphate at micromolar phosphate concentrations followeda linear time dependence in the range of minutes and rates werein the range of 1 µmol phosphate mg^–1 chl h^–1,only. However, under discontinuous phosphate-limited growthconditions, tracer influx revealed a biphasic pattern at micromolarphosphate concentrations: An initial burst phase resulted ina 10⁴-fold internal phosphate accumulation and levelled offafter about 10 s. A double reciprocal plot of the initial influxrates obtained for phosphate-limited and unlimited algae exhibitedMichaelis-Menten kinetics. Phosphate limitation caused a significantactivation of the maximum velocity of uptake, yielding V_maxup to 1 mmol mg^–1 chl h^–1 as compared to valuesin the order of 50 µmol phosphate mg^–1 chl h^–1for the second phase (this magnitude is also representativefor non-limited batch cultures). Concomitantly the Michaelisconstant was altered from 4 mmol m^–3 to 0·7 mmolm^–3. The rapid uptake of phosphate was inhibited by arsenateand FCCP and was not stimulated by Na⁺. The pH dependence oftracer accumulation and measurements of the intracellular phosphatepool under different growth conditions indicate that at lowpH and low external phosphate concentrations the high protongradient present under these conditions is utilized for a H₃PO₄uptake or a H⁺/H₂PO^–4 cotransport. However, when the externalphosphate concentration was increased to levels sufficientlyhigh for transport to be driven by the positive membrane potential(10 mol m^–3 phosphate), the pH dependence of phosphateuptake was more complex, but could be explained by the uptakeof H₃PO₄ or a H⁺/H₂PO^–₄-cotransport at low pH and a differenttype H₂PO^–4-transport (with unknown type of ion coupling)at high pH-values. It is suggested that this flexible couplingof phosphate transport is of essential importance for the acidresistance of Dunaliella acidophila. Key words: Acid resistance, Dunaliella acidophila, phosphate cotransport, phosphate limitation, plasma membrane, sodium     

Active vanadate-sensitive H+ translocation in corn roots membrane vesicles and proteoliposomes

A member fraction from corn roots which contains a vanadate-sensitive ATPase activity has been prepared. The specific activity at 38°C is between 3 and mol 12 μmol · min⁻¹ · mg⁻¹, depending on the age of roots. Addition of ATP promotes a very rapid quenching of the fluorescence of 9-amino-6-chloro-3-methoxy-acridin (ACMA). Proton pumping exhibits a delayed sensitivity to vanadate but is strongly and instantaneously inhibited by the new inhibitor SW 26. Both proton pumping, measured by the initial quenching rate, and ATP hydrolysis show maximum activities at ATP concentrations in the millimolar range, but the apparent K_m-value for hydrolysis is higher than that observed for proton pumping. This is interpreted as being due to the presence of two populations of ATPases, one of them hydrolyzing ATP without creating a pH-gradient. The vanadate-sensitive ATP hydrolysis and H⁺-pumping activity may be solubilized with lysolecithin and reconstituted into liposomes either by a freeze-thawing-sonication or an octylglucoside dilution procedure. Both methods yield proteoliposomes exhibiting very effecient proton pumping, which is more sensitive to vanadate (I₅₀ = 2 μM) or to SW 26 (I₅₀ = 0.5 μM) than that of the original membrane fractions.     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Plasma Membrane-associated Adenosine Triphosphatase Activity of Isolated Cortex and Stele from Corn Roots

The plasma membrane fractions from separated cortex and stele of primary roots of corn (Zea mays L. WF9 × M14) contained cation ATPase activity at similar levels but with somewhat different properties. ATPase activity from cortex was optimum at pH 6.5, showed a simple Michaelis-Menten saturation with increasing ATP·Mg, and showed complex kinetic data for K⁺ stimulation similar in character to the kinetic data for K⁺-ATPase and K⁺ influx in primary roots. The results for cortex indicate that homogenates of primary roots are dominated by membranes from cortical cells.

ATPase activity from stele was optimum at pH 6.5 and showed another maximum at pH 9. At pH 6.5, activity from stele had properties similar to that from cortex except that the kinetics of K⁺ stimulation closely approached that expected for a Michaelis-Menten enzyme. At pH 9, the enzyme activity from stele was inhibited by 5 μg/ml oligomycin, suggesting that a significant portion of the activity was of mitochondrial origin. Sucrose density gradient analysis indicated some contamination of mitochondrial membranes in the plasma membrane fraction from stele. The results for stele are consistent with the view that stelar parenchyma cells are not deficient in ion pumps.

     

Bafilomycin A1 is a non-competitive inhibitor of the tonoplast H+-ATPase of maize coleoptiles

When microsomal membranes from maize (Zea mays L. cv. Clipper)coleoptiles were separated by isopyc-nic centrifugation on acontinuous 10–45% sucrose gradient, bafilomycin A1-inhibitedATPase activity co-localized with the activities of the tonoplastmarker-enzymes, nitrate-Inhibited ATPase and K⁺-dependent pyrophosphatase.Thus, bafilomycin A1 is a specific inhibitor of the vacuolarH⁺-ATPase of maize coleoptiles. Inhibition of the vacuolar H⁺-ATPaseby bafilomycin A1 was strictly dependent upon the concentrationof the enzyme present in the assay medium, suggesting a stoichiometricassociation between bafilomycin A1 and the vacuolar H⁺-ATPase.In tonoplast-enriched preparations, half-maximal inhibitionwas obtained at 43 pmol bafilomycin A1 mg^–1 protein. BafilomycinA1 inhibited the vacuolar H⁺-ATPase in a simple non-competitivemanner: increasing bafilomycin A1 concentrations reduced theV_max, of the H+ -ATPase, but had no effect on its K_m towardsATP. Key words: Bafilomycin A1, coleoptile, H⁺-ATPase (vacuolar), maize, Zea mays L     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H⁺ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H⁺-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90–100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase     

Proton Fluxes and the Activity of a Stelar Proton Pump in Onion Roots

The xylem vessels of excised adventitious roots of onion, Alliumcepa, were perfused with unbuffered nutrient solution adjustedinitially to either pH 9·3 or 3·9; the pH of thesolution after passage through the xylem, at rates not lessthan 2 xylem volume changes min^–1, was close to pH 6·5in both instances. The flux of H⁺ across the xylem/symplastboundary into mildly alkaline, phosphate-buffered solutionsperfusing the vessels could be increased greatly with increasingbuffer strength, up to a maximum value between 0·5–1·0pmol H⁺ mm^–2 s^–1. The apparent neutralization ofacidic malic acid buffers had a slightly lower maximum capacity,equivalent to –0·3 to –0·5 pmol H⁺mm^–2 s^–1. The addition of 5·0 pmol m^–3fusicoccin (FC) to the xylem perfusion solution stimulated theentry of H⁺ into the xylem; in unbuffered perfusion solutionsthe pH fell to pH 3·6 after a lag of 25–35 min.FC additions to phosphate-buffered solutions also stimulatedthe H⁺ flux to an extent similar to that in unbuffered solution,viz. 0·2–0·4 pmol mm^–2 s^–1. The release of K⁺ (³⁶Rb-labelled) into xylem sap transientlyincreased as the [K⁺] in weakly buffered perfusion solutionswas raised stepwise; a very marked increase being seen whenthe concentration was raised to 100 mol m^–3 from 40 molm^–3. The addition of 5·0 mmol m^–3 FC to theperfusing solution containing 100 mol m^–3 K⁺ rapidly decreasedthe K⁺ flux to the xylem as the H⁺ flux increased. Fusicoccinalso inhibited the flux of K⁺ into unbuffered perfusion solutionsbut the effect appeared reversible. Addition of 10 mmol m^–3abscisic acid (ABA) to the perfusion solution quickly producedtransient increases in both K⁺ and H⁺ fluxes into the xylem.In this and other experiments using weakly phosphate-bufferedperfusing solutions, H⁺ fluxes were comparable in size to thoseof K⁺ The results are consistent with the idea that the stele of onionroots contains a proton trarislocating ATPase whose activityresponds to the pH of the xylem sap. It is evident that theactivity of the proton secreting and proton neutralizing mechanismsin the xylem parenchyma control the movement of other ions acrossthe xylem/symplast boundary. Key words: Xylem perfusion, fusicoccin, abscisic acid, pH gradient     

The Effects of Vitamin K3 and Dicumarol on the Plasma Membrane Redox System and H+ Pumping Activity of Zea mays L Roots Measured over a Long Time Scale

The effects of vitamin K₃ or dicumarol on plasma membrane boundhexacyanoferrate (III) and hexabromoiridate (IV) reductase activityand on the H⁺ pumping rate were investigated. Incubation withvitamin K₃ followed by intense rinsing stimulated the subsequentreduction of hexabromoiridate (IV) and hexacyanoferrate (III)as well as proton secretion induced by external electron-acceptors,while pretreatment with dicumarol inhibited proton secretioninduced by redox activity and hexacyanoferrate (III) reductionrate, but not the effects of hexabromoiridate (IV). A 30 minincubation in 0·2 mM K₃ or dicumarol, followed by rinsing,inhibited H⁺ secretion for about 2 d. Incubation for more than12 h in 0·1 mM dicumarol or 0·2 mM K₃ caused lethalinjury to the root cells. Key words: Vitamin K.₃, dicumarol, plasmalemma redox system, Zea mays L., proton pump     

Orthophosphate Relations of Root: NO-3 Effects on Orthophosphate Influx, Accumulation and Secretion into the Xylem

Lamaze, T., Sentenac, H. and Grignon, C. 1987. Orthophosphaterelations of root: NO^–₃effects on orthophosphate influx,accumulation and secretion into the xylem.—J. exp. Bot.38: 923–934. Orthophosphate (Pi) accumulation by barley (Hordeum vulgareL.) roots was specifically inhibited by NO^–₃ as comparedto Cl^– and SO₄^{2 –}, and Pi secretion into the xylemwas stimulated. The inhibition of Pi accumulation by NO^–₃was also observed in roots of intact photosynthesizing horsebean(Vicia faba L.), rice (Oryza sativa L.) and soybean (Glycinemax L.) plants. NO^–₃ effects on Pi transport by rootswere more thoroughly investigated with corn (Zea mays L.). Theywere due to intracellular NO^–₃. Pi secretion was stillstimulated by NO^–₃ after Pi withdrawal from the absorptionsolution. ³²Pi influx decreased during Pi accumulation, supportingthe hypothesis that this ion allosterically regulated its owntransport system by feedback control. This control was modulatedby other anions: the decrease was more pronounced in the presenceof nitrate. Chronologically, the depressive effect of NO^–₃on ³²Pi influx appeared after the inhibition of Pi accumulation.Furthermore, under conditions where Pi accumulation was notaffected by NO^–₃, ³²Pi influx and Pi secretion into thexylem became insensitive to the presence of nitrate. Our hypothesisis that the stimulative effect of NO^–₃ on Pi secretionand the depressive one on ³²Pi influx are the repercussionsof an increase in the Pi cytosolic concentration due to an NO^–₃-induced decrease in Pi uptake by the vacuoles. Key words: Root, orthophosphate fluxes, orthophosphate accumulation, nitrate, ionic interaction     

Ascorbate enhancement of H1 histamine receptor sensitivity coincides with ascorbate oxidation inhibition by histamine receptors

Ascorbate has previously been shown to enhance both ₁- and ₂-adrenergic activity. This activity is mediated by ascorbate binding to the extracellular domain of the adrenergic receptor, which also decreases the oxidation rate of ascorbate. H1 histamine receptors have extracellular agonist or ascorbate binding sites with strong similarities to _1- and ₂-adrenergic receptors. Physiological concentrations of ascorbate (50 µM) significantly enhanced histamine contractions of rabbit aorta on the lower half of the histamine dose-response curve, increasing contractions of 0.1, 0.2, and 0.3 µM histamine by two- to threefold. Increases in ascorbate concentration significantly enhanced 0.2 µM histamine (5–500 µM ascorbate) and 0.3 µM histamine (15–500 µM ascorbate) in a dose-dependent manner. Histamine does not measurably oxidize over 20 h in oxygenated PSS at 37°C. Thus the ascorbate enhancement is independent of ascorbate's antioxidant effects. Ascorbate in solution oxidizes rapidly. Transfected histamine receptor membrane suspension with protein concentration at >3.1 µg/ml (56 nM maximum histamine receptor) decreases the oxidation rate of 392 µM ascorbate, and virtually no ascorbate oxidation occurs at >0.0004 mol histamine receptor/mol ascorbate. Histamine receptor membrane had an initial ascorbate oxidation inhibition rate of 0.094 min·µg protein^–1·ml^–1, compared with rates for transfected ANG II membrane (0.055 min·µg protein^–1·ml^–1), untransfected membrane (0.052 min·µg protein^–1·ml^–1), creatine kinase (0.0082 min·µg protein^–1·ml^–1), keyhole limpet hemocyanin (0.00092 min·µg protein^–1·ml^–1), and osmotically lysed aortic rings (0.00057 min·µg wet weight^–1·ml^–1). Ascorbate enhancement of seven-transmembrane-spanning membrane receptor activity occurs in both adrenergic and histaminergic receptors. These receptors may play a significant role in maintaining extracellular ascorbate in a reduced state. molecular complementarity; vitamin C; seven-transmembrane-spanning membrane receptors     

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     

Measurement of the Apoplastic Activity of K+ and Cl- in the Leaf Epidermis of Commelina communis in Relation to Stomatal Activity

Bowling, D. J. F. 1987. Measurement of the apoplastic activityof K⁺ and Cl^– in the leaf epidermis of Commelina communisin relation to stomatal activity.–J. exp. Bot. 38: 1351–1355. Ionic activity of K⁺ and Cl^– in the apoplast of the lowerepidermis of the leaf of Commelina communis was measured usingion selective micro-electrodes. Large gradients across the stomatalcomplex were observed which were related to stomatal aperture.On stomatal closure the activity of K⁺ and Cl^– in theapoplast of the guard cell rose from 3·0 mol m^–3to 100 mol m^–3 and 33 mol m^–3 respectively. It wasconcluded that the apoplast is an important pathway for iontransport between the cells. Key words: Stomata, ionic activity, leaves, apoplast     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)