Isolation of the Photoactive Reaction Center Complex that Contains Three Types of Fe-S Centers and a Cytochrome c Subunit from the Green Sulfur Bacterium Chlorobium limicola f. thiosulfatophilum, Strain Larsen

The photoactive reaction center (RC) complex from the greensulfur bacterium Chlorobium limicola f. thiosulfatophilum, strainLarsen, was isolated after solubilization and ammonium sulfatefractionation followed by ion-exchange chromatography. The spectrumof the complex was almost identical with that of the similarRC complex isolated by Feiler et al. [(1992) Biochemistry 31:2608–2614] except for the presence of cytochrome c₅₅₁instead of c₅₅₃ in the latter study. A molecular ratio of BChla to P840 of the isolated RC complex was assayed to be 25–35.SDSPAGE analysis revealed that the isolated complex containedthree major polypeptides with apparent molecular masses of 68,41 and 21 kDa, respectively. The 21-kDa polypeptide was identifiedto be a heme-binding protein by staining the gel for peroxidaseactivity. The cytochrome c₅₅₁ was oxidized by flash light ina biphasic manner with half times of 90 and 390 µs, respectively,that coincided with the reduction half times of P840⁺. Threedistinct iron-sulfur centers assigned to F_A, F_B and F_x, respectively,from their g-values were detected by EPR spectroscopy at cryogenictemperature. These results suggest that the present preparationcontains a minimal functional unit of the RC of this bacterium,and that this complex appears to lie on a evolutionary linebetween RC's of purple bacteria and photosystem I. (Received August 18, 1992; Accepted October 28, 1992)     

Preparation of a Photoactive Reaction Center Complex Containing Photo-reducible Fe-S Centers and Photooxidizable Cytochrome c from the Green Sulfur Bacterium Chlorobium tepidum

A photoactive reaction center (RC) complex was isolated fromthe green sulfur bacterium Chlorobium tepidum by solubilizationof membranes with Triton X-100, followed by sucrosedensity gradientcentrifugation, DEAE Bio-Gel A chromatography, and hydroxyapatitechromatography. The purified RC complex contained about 50–70bacteriochlorophyll molecules (BChl) per P840, as assayed byphotooxidafion. It showed a near-infrared BChl a absorptionpeak at 814 nm and shoulders at about 800 and 835 nm at roomtemperature. SDS-PAGE analysis revealed 6 polypeptides withapparent molecular masses of 100, 65, 41, 32, 23, and 18 kDa.The RC complex binds functional P840 and Cyt c₅₅₁, which werephotooxidized by continuous illumination at room temperature.Upon flash excitation, the bound Cyt c₅₅₁ was oxidized, andrereduced in the dark with a half-time of 16 and 400 ms in thepresence and absence of 0.1 mM 2,6-dichlorophenol indophenol,respectively, at room temperature. At 551 nm, the amount ofthe Cyt c photooxidized by continuous illumination was 60% ofthe amount determined by chemical oxidation-reduction. The functionalCyt c₅₅₁/P840 ratio was calculated to be 1.2–1.7. EPRspectroscopy at cryogenic temperatures revealed that the RCcomplex binds three photoreducible Fe-S centers designated tobe CF_A, CF_B and CF_X (C for Chlorobium). CF_A and CF_B were reducedin the dark with dithionite at pH 10. (Received May 26, 1993; Accepted October 4, 1993)     

Artificial Quinones Replace the Function of Quinone Electron Acceptor (QA) in the Isolated D1-D2-Cytochrome b559 Photosystem II Reaction Center Complex

Various benzo- and naphthoquinone derivatives were introducedinto the purified photosystem II Dl-D2-cytochrome b₅₅₉ reactioncenter complex, which lacks the intrinsic plasto-quinone electronacceptors. Effects of these quinones on the electron transferreactions in nanoseconds to milliseconds time range were studiedat room and cryogenic temperatures. 1) The addition of quinonesto the purified photosystem II reaction center complex suppressedthe nanosecond charge recombination between oxidized reactioncenter chlorophyll a (P680⁺) and reduced pheophytin a (Ph^–),and stabilized P680⁺ up to millisecond time range at 280 K andat 77 K. 2) In the reaction center complex supplemented withdibromothymoquinone (DBMIB), P68O was almost fully oxidizedand cytochrome b₅₅₉ was partially reduced by flash excitation.A semi-quinone-like signal with a peak around 320 nm was alsoinduced but the shift of pheophytin absorption band (C55O) wasnot observed. 3) Halogenated quinones, especially DBMIB, werebetter electron acceptors than unsubstituted or methylated quinones.4) The affinities of quinones to the reaction center complexwere weakly dependent on their molecular structure. (Received July 9, 1991; Accepted August 15, 1991)     

Dibromothymoquinone (DBMIB) Replaces the Function of QA at 77 K in the Isolated Photosystem II Reaction Center (D1-D2-Cytochrome b559) Complex: Difference Spectrum of the P680+(DBMIB-) State

Transient absorbance changes of the primary electron donor chlorophylla (P680) and acceptor pheophytin a (H) were measured at 77 Kby nanosecond laser spectroscopy in the D1-D2-cytochrome b₅₅₉photosystem II reaction center complex containing dibromomethylisopropylbenzoquinone (DBMIB). After the laser excitation of the reactioncenter in the presence of DBMIB, only the P680⁺-(DBMIB^-) statewas detected. P680⁺ mainly decayed with a t_1/e of 11 ms. Inthe absence of DBMIB, the excitation produced the P680⁺H^- radicalpair. The radical pair produced the triplet state (P680^T) witha t_1/e of 50 ns, and P680^T then decayed with a t_1/e of 2.1 ms.It was concluded that H_- was oxidized by DBMIB in a time rangefaster than the detecting time resolution (3.5 ns) even at 77K. The rapid oxidation of H^- by DBMIB was also confirmed bythe suppression of delayed fluorescence with a decay t_1/e of50 ns. The P680⁺(DBMIB^-)/P680(DBMIB) difference spectrum exhibiteda Q_y, band with a peak at 682 nm with a shoulder at 673 nm.The spectral shape was almost temperature insensitive between77 and 265 K. The feature of this spectrum in the wavelengthrange between 330 and 720 nm was compared with that of P680_T/P680or H^-/H at 77 K. (Received May 8, 1996; Accepted June 24, 1996)     

Reaction of Reconstituted Acceptor Quinone and Dynamic Equilibration of Electron Transfer in the Photosystem I Reaction Center

Electron transfer mechanism in the spinach photosystem I reactioncenter that contains artificial quinones in place of phylloquinone(2-methyl-3-phytyl-1,4-naphthoquinone, vitamin K₁) as the secondaryelectron acceptor, Q_ø (or A₁) was discussed. (1) Mostof the reconstituted quinones oxidized the primary acceptorchlorophyll a, A⁰, at a rate rapid enough to compete againstthe charge recombination between A₀^– and the oxidizeddonor chlorophyll P700⁺. (2) The pathway of electron transferfrom the semiquinone^– varied depending on the redox potentialvalue of each semiquinone^– /quinone couple. Low potentialquinones reduced the tertiary acceptor iron-sulfur center, F_x,while the high potential ones reduced P700⁺ directly with a200-µs halftime. (3) The E_m value of each semiquinone^–/quinone couple in situ in the reaction center was estimatedto be shifted by about 0.3 volt to the negative side from theirhalf wave redox potential values that were measured polarographicallyin dimethylformamide. The shift seems to represent the acceptorproperty of the protein environment at the Q_ø site. (4)The E_m of reconstituted phylloquinone was estimated to be 50–80mV more negative than that of F_x. (5) The mechanism of efficientelectron transfer in the reaction center was discussed basedon the dynamic equilibria between the electron transfer componentsand on the estimated E_m values. (Received April 9, 1994; Accepted July 7, 1994)     

Luminal L-alanine stimulates exocytosis at the K+-conductive apical membrane of Aplysia enterocytes

In Aplysia intestine,stimulation of Na⁺ absorption withluminal alanine increases apical membraneK⁺ conductance(G_K,a), whichpresumably regulates enterocyte volume during stimulatedNa⁺ absorption. However, themechanism responsible for the sustained increase in plasma membraneK⁺ conductance is not known forany nutrient-absorbing epithelium. In the present study, we have begunto test the hypothesis that the alanine-induced increase inG_K,a inAplysia enterocytes results fromexocytic insertion of K⁺ channelsinto the apical membrane. We used the fluid-phase marker horseradishperoxidase to assess the effect of alanine on apical membraneexocytosis and conventional microelectrode techniques to assess theeffect of alanine on fractional capacitance of the apical membrane(fC_a). Luminalalanine significantly increased apical membrane exocytosis from 1.04 ± 0.30 to 1.39 ± 0.38 ng · min¹ · cm².To measure fC_a,we modeled the Aplysia enterocyte as adouble resistance-capacitance (RC) electric circuit arranged in series. Several criteria were tested to confirm application of the model to theenterocytes, and all satisfied the model. When added to the luminalsurface, alanine significantly increasedfC_a from 0.27 ± 0.02 to 0.33 ± 0.04 (n = 10)after 4 min. There are two possible explanations for our findings:1) the increase in exocytosis, whichadds membrane to the apical plasma membrane, prevents plasma membranefracture, and 2) the increase inexocytosis delivers K⁺ channels tothe apical membrane by exocytic insertion. After the alanine-induceddepolarization of apical membrane potential (V_a), there isa strong correlation (r = 0.96)between repolarization ofV_a, whichreflects the increase inG_K,a, andincrease in fC_a. This correlation supports the exocytic insertion hypothesis for activation ofG_K,a.

     

Studies on the absorption spectra of chlorophyll a in aqueous dispersions of lipids from the photosynthetic membranes

The absorption spectra of chlorophyll a were studied in aqueousdispersions of four major lipid components present in the thylakoidmembranes. Chlorophyll a in aqueous dispersions of uncharged galactolipidsrevealed two absorption bands, at 670 and 745 nm, when the molecularratio of chlorophyll to lipid was higher than 0.2. The latterband may be due to the formation of microcrystals of chlorophylla. Chlorophyll a in aqueous dispersions of negatively chargedlipids revealed a single absorption band at 670 nm. However,chlorophyll a was decomposed during measurement in these lipiddispersions. The absorption spectra of chlorophyll a in aqueous dispersionsof mixture of galactolipid and charged lipid were apparentlysimilar to those of chlorophyll a in the charged lipid dispersion.Chlorophyll a, however, was not decomposed in these aqueousdispersions of lipid mixtures. It is concluded that the presence of both galactolipid and chargedlipid are necessary to reconstruct the state of chlorophylla dissolved in the lipid phase in the thylakoid membranes. The red absorption band of chlorophyll a in the reconstructedsystem composed of chlorophyll a, charged and uncharged lipids,appeared at 670 nm with a half bandwidth of 22 nm. Analysisof the absorption spectrum in the fourth derivative and thecurve-fitting methods indicated that the red band was composedmainly of a single band with a peak at 670–671 nm. ¹ Present address: Department of Biology, College of GeneralEducation, University of Tokyo, Komaba, Meguro-ku, Tokyo 153,Japan. (Received October 13, 1977; )     

Temperature-Induced Change of Chlorophyll a Forms in Phosphatidylcholine Liposomes

Absorption spectra of chlorophyll a in phosphatidylcholine liposomesat different temperatures were analyzed by a curve fitting method.The absorption spectrum was found to be composed of one majorband with a peak at 670–671 nm and minor bands with peaksat 650–652, 662–663 and 684–686 nm. Upon coolingbelow the phase transition temperature of the lipid, the componentabsorbing at 670–671 nm increased significantly at theexpense of the component absorbing at 662–663 nm. No changein the extents of other bands was observed. ¹ CIW-DPB Publication No. 795. ²On leave from the Department of Biology, Faculty of Science,Kanazawa University, Marunouchi, Kanazawa 920, Japan. (Received December 20, 1982; Accepted April 27, 1983)     

The optical properties of a turbid reservoir and its phytoplankton in relation to photosynthesis and growth (Mount Bold Reservoir, South Australia)

In situ light measurements were used to obtain information oninherent and apparent optical properties. The average verticalattenuation coefficient K_d(ave) varied from 1.1 to 4.6 In unitsm^–1 During three periods the variation in K_d(ave) correlatedwith changes in chlorophyll a concentration and specific attenuationcoefficients K_s, of 0.013, 0.014 and 0.022 m² mg Chl a^–1were calculated. Chlorophyll-specific diffuse absorption coefficients(A,) for these periods were 0.012. 0.013 and 0.017 m² mg Chla^–1 and only varied significantly from estimates of K_sin the period when scattering was intense. Absorption coefficientsa(z^mid) and scattering coefficients b(z^mid) calculated for themid-point of the euphotic zone ranged between 0.45 and 2.9 mand 3.5–52.0 m respectively. Chlorophyll-specific absorptioncoefficients K_a, of 0.005, 0.006 and 0.007 m² mg Chl a^–1and scattering coefficients K_b of 0.05. 0.09 and 0.191 m² mgChl a^–1 were measured during the three periods. The highK_b value occurred when gas-vacuolate cyanobactena were dominant.Algal photosynthesis and light absorption were related throughthe maximum quantum yield _m which varied between 0.019 and 0.11mol C Einstein^–1 while average quantum yields _a, variedbetween 0.006 and 0.024 with a mean of 0.013 mol C Einstein^–1A comparison of changes in the mean irradiance of the mixedzone and chlorophyll concentration indicated that growth waslight limited below 0.04–0.05 Einsteins absorbed mg Chla^–1 day^–1.     

Photoconversion of a Water-Soluble Chlorophyll Protein from Chenopodium Album: Resonance Raman and Fourier Transform Infrared Study of Protein and Pigment Structures

A water-soluble Chl a/b-protein complex, CP668, from Chenopodiumalbum converts to another form of protein complex, CP743, uponlight illumination. Structural changes of pigments and proteinsupon photoconversion were studied using resonance Raman (RR)and Fourier transform infrared (FTIR) spectroscopies. RR spectraof CP668 and CP743 and a light-induced FTIR difference spectrumshowed that the macrocyle C=C bands of Chl a in CP668 considerablychanged upon conversion to the pigment (not chemically identifiedyet) in CP743. The C=C band pattern of the RR spectrum of CP743was similar to that of bacteriochlorophyll a, suggesting thatthe conjugated system of the CP743 pigment resembles a bacteriochlorinring. Judging from the C=O frequencies, the 13¹-keto C=O groupsof Chl a and b in CP668 are free from hydrogen bonding, whereasthe 13²-ester C=O groups of both Chl a and b and the 7-formylC=O of Chl b in CP668 are hydrogen bonded. Upon conversion toCP743, interactions of the 13¹-keto and 13²-ester C=O groupswere basically unaffected, demonstrating no drastic changesaround these C=O groups. FTIR spectra in the amide I' regionof CP668 and CP743 in D₂O buffer showed a peak at 1,633 cm^–1,which represents a major component of ß-sheet conformation.Second-derivative spectra of the amide I' bands as well as alight-induced FTIR difference spectrum suggested that drasticchange in the protein conformation does not occur upon photoconversion. (Received November 1, 1998; Accepted December 24, 1998)     

Estimation of the sensitivity to photoinhibition in Striga hermonthica-infected sorghum

Sensitivity to photoinhibition was assessed in sorghum infectedwith the angiosperm root parasite Striga her-monthica and inuninfected sorghum plants, at four times during the developmentof the host-parasite association. Photoinhibition was inducedby exposing either leaf discs or intact leaves to a photosyntheticphoton flux density of 2000 µmol m^–2 s^–1 for4 h. The inhibition of apparent quantum yield (_a) and photosynthesisin high light (A₁₅₀₀) were assessed in leaf discs using an oxygenelectrode and the recovery of these from photoinhibition wasfollowed in intact leaves using an infra-red gas analyser. Fromsoon after attachment of the parasite, infected sorghum plantshad a lower A₁₅₀₀. During the period when Striga induced a loweringof A₁₅₀₀, _a was more sensitive to photoinhibition in Striga-infectedplants. However, at the same time, the high-light-induced inhibitionof A₁₅₀₀ was similar in Striga-infected and uninfected plants.Recovery of both _a and A₁₅₀₀ was incomplete after 6 h and thetime-course of recovery was similar in Striga-infected and uninfectedplants. The results indicate that Striga-infected plants weremore sensitive to photoinhibition and that photoinhibition wasprimarily due to damage to electron transport/photo-phosphorylationand not disablement of the recovery processes. Key words: Photoinhibition, quantum yield, recovery from photoinhibition, parasitic plants     

Photosynthetic Properties of Guard Cell Protoplasts from Vicia faba L.

Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O₂ uptake (175 µmol mg^–1 Chl hr^–1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN₃ by about 60%. On illumination,this O₂ uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg^–1 Chl hr^–1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982)     

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     

Factors controlling primary production in a hypertrophic lake (Hartbeespoort Dam, South Africa)

The physical factors controlling algal primary production weredemonstrated from data collected for a hypertrophic lake. A_maxranged between 12.4 and 5916 mg C m^–3 h^–1. Arealrates (A) varied between 46.9 and 3381 mg C m^–2 h^–1.The factors permitting and controlling production were subjectivelyseparated into two categories. In category 1, nutrients (N +P), which were in overabundance, permitted large standing cropsof Microcystis aeruginosa to develop (>1000 µg chla 1^–1). Wind patterns determined the dramatic spatialand temporal changes in algal standing crop which could dropto 2.7 µg chl a 1^–1. In category 2 were the factorswhich affected the rate processes. The buoyancy mechanism ofMicrocystis usually kept the alga in the euphotic zone. A powerrelationship (r = 0.92, n = 54) between A and A_max/_min showedthat with increasing phytoplankton vertical stratification,A_max was increasingly important in the integral. The saturationparameter I_K and photosynthetic capacity were temperature dependent.Variations of A were significantly related to changes in watercolumn stability (g cm cm^–2) because both axes of thephotosynthesis depth-profile were affected by stability changes.     

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     

The Effect of Root Temperature on Growth and Uptake of Ammonium and Nitrate by Brassica napus L. cv. Bien venu in Flowing Solution Culture: II. UPTAKE FROM SOLUTIONS CONTAINING NH4NO3

Macduff, J. H., Hopper, M. J. and Wild, A. 1987. The effectof root temperature on growth and uptake of ammonium and nitrateby Brassica napus L. CV. Bien venu in flowing solution culture.II. Uptake from solutions containing NH₄NO₃.—J. exp. Bot.38: 53–66 The effects of root temperature on uptake and assimilation ofNH₄⁺ and NO₃^– by oilseed rape (Brassica napus L. CV. Bienvenu) were examined. Plants were grown for 49 d in flowing nutrientsolution at pH 6?0 with root temperature decrementally reducedfrom 20?C to 5?C; and then exposed to different root temperatures(3, 5, 7, 9, 11, 13, 17 or 25?C) held constant for 14 d. Theair temperature was 20/15?C day/night and nitrogen was suppliedautomatically to maintain 10 mmol m^–3 NH₄NO₃ in solution.Total uptake of nitrogen over 14 d increased threefold between3–13?C but was constant above 13?C. Net uptake of NH₄⁺exceeded that of NO₃^– at all temperatures except 17?C,and represented 47–65% of the total uptake of nitrogen.Unit absorption rates of NH₄⁺ and of 1?5–2?7 for NO₃^–suggested that NO₃^– absorption was more sensitive thanNH₄⁺ absorption to temperature. Rates of absorption were relativelystable at 3?C and 5?C compared with those at 17?C and 25?C whichincreased sharply after 10 d. Tissue concentration of N in theshoot, expressed on a fresh weight basis, was independent ofroot temperature throughout, but doubled between 3–25?Cwhen expressed on a dry weight basis. The apparent proportionof net uptake of NO₃^– that was assimilated was inverselyrelated to root temperature. The results are used to examinethe relation between unit absorption rate adn shoot:root ratioin the context of short and long term responses to change ofroot temperature Key words: Brassica napus, oilseed rape, root temperature, nitrogen uptake     

Uptake of Imidazole and its Effects on the Intracellular pH and Ionic Relations of Chara corallina

Ammonia (pK_a 9.25) and methylamine (pK_a, 10.65) increase cytoplasmicpH and stimulate Cl^– influx in Chara corallina, theseeffects being associated with influx of the amine cations ona specific porter. The weak base imidazole (pK_a 6.96) has similareffects but diffuses passively into the cell both as an unionizedbase and as a cation. When the external pH is greater than 6.0influx of the unionized species predominates. Imidazole accumulates to high concentrations in the vacuole,where it is protonated. Cytoplasmic pH and vacuolar pH riseby only 0.2–0.3 units, suggesting a large balancing protoninflux across the plasma membrane. Balance of electric chargeis partially maintained by net efflux of K⁺ and net influx ofCl^–. Calculation of vacuolar concentrations of imidazole(from (¹⁴C] imidazole uptake, assuming that there is no metabolism)plus K⁺ and Na⁺ indicates an excess of cations over inorganicanions (Cl^–). However, although the osmotic potentialof the cells increases, also indicating increased solute concentrations,the increase is less than that predicted by the calculated ionicconcentrations. This discrepancy remains to be resolved. Becausethe osmotic potential also increases when imidazole is absorbedfrom Cl^–-free solutions it is likely that maintenanceof charge-balance can also involve synthesis and vacuolar storageof organic or amino acids. Key words: Imidazole, potassium, intracellular pH, membrane transport, Chara     

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     

Properties of Chlamydomonas Photosystem II Core Complex with a His-Tag at the C-Terminus of the D2 Protein

A His-tagged PSII core complex was purified from recombinantChlamydomonas reinhardtii D2-H thylakoids by single-step Ni²⁺-affinitycolumn chromatography and its properties were partially characterizedin terms of their PSII functions and chemical compositions.The PSII core complex that has a His-tag extension at the C-terminusof the D2 protein evolved oxygen at a high rate of 2,400 µmol(mg Chl)^–1h^–1 at the optimum pH of 6.5 with ferricyanideand 2,6-dichlorobenzoquinone as electron acceptors in the presenceof Ca²⁺ as an essential cofactor, and approximately 90% of theactivity was blocked by 10 µM DCMU. The core complex exhibitedthe thermoluminescence Q-band but not the B-band regardlessof the presence or absence of DCMU, although both bands wereobserved in the His-tagged thylakoids. The core complex wasfree from PSI and contained one Y_D, Tyr 160 of the D2 protein,four Mn atoms, two cytochrome b-559, about 46 Chl a molecules,and probably one Q_A, the primary acceptor quinone of PSII. Itwas inferred from these results that His-tagging at the C-terminusof the D2 protein does not affect the functional and structuralintegrity of the PSII core complex, and that the ‘His-tagstrategy’ is highly useful for biochemical, physicochemical,and structural studies of Chlamydomonas PSII. (Received October 22, 1998; Accepted December 25, 1998)     

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