Affinity chromatography of an ethylene-synthesizing enzyme from red alga Porphyra tenera on an immobilized inhibitor of ethylene evolution

A method was established to purify acrylate decarboxylase fromPorphyra tenera by affinity chromatography using a proteinaceousinhibitor of ethylene evolution in marine algae, isolated fromP. tenera as a ligand. The proteinaceous inhibitor was covalentlycoupled to porous glass via four different spacer arms. Theporous glass-aminopropyltriethoxysilane-succinatephenylendiamine-succinate-inhibitorappeared to be the best derivative for retaining acrylate decarboxylaseextracted from P. tenera. Acrylate decarboxylase was extracted from 10 kg of P. teneraand semi-purified by ammonium sulfate. preparation and gel filtrationon Sephadex G-100. The active fraction was applied to an affinitycolumn. Acrylate decarboxylase was eluted in the starting buffercontaining 0.2 M NaCl. Ethylene formation from acrylate wasdetected in the presence of this enzyme extract, but not inthe case of the boiled enzyme extract. Acrylate decarboxylasewas inhibited by the inhibitor isolated from P. tenera. Thesefacts indicate that the formation of ethylene in marine algaefrom acrylate proceeds enzymatically. ² Present address: Division of Environmental Biology, NationalInstitute for Environmental Studies, Yatabe, Ibaraki 300-21,Japan. (Received July 13, 1976; )     

NUTRITIONAL STUDIES OF THE EDIBLE SEAWEED PORPHYRA TENERA I. THE INFLUENCE OF DIFFERENT B12 ANALOGUES, PLANT HORMONES, PURINES AND PYRIMIDINES ON THE GROWTH OF CONCHOCELIS

The red alga Porphyra tenera has been obtained in axenic cultureby the "dip and drag" technique in an agarized medium containingantibiotics. In the axenic culture, the Conchocelis of P. teneraneeds vitamin B₁₂ for growth. The addition of other vitaminsdoes not increase growth further. The pattern of specificityis similar to that of Escherichia coli 113-3. Factor B, pseudovitaminB₁₂ and Factor Z₂ support as much growth as vitamin B₁₂, while2-methylmercaptoadenine and 5-methyl benzimidazole cobalamineincrease the growth more than B₁₂. All the analogues containingbenzimidazole can replace B₁₂. Kinetin, adenine, indoleaceticacid and especially gibberellic acid increase growth. Threepurines, (xanthine, hypoxanthine and guanine) and three pyrimidines(uracil, methylcytosine and thymine) also promote growth inthe presence of vitamine B₁₂. (Received January 18, 1965; )     

Relation between Sex Expression Types and Cotyledon Etiolation of Cucumber in vitro I. On the Role of Ethylene Evolved from Seedlings

Cucumber seedlings, when cultured in vitro, showed differencesin cotyledon etiolation rates among cultivars with differentgenetic backgrounds for sex expression. The chlorophyll contentin gynoecious cultivars (acr^F/acr^F) decreased rapidly whilethat in monoecious ones (acr⁺/acr⁺) decreased more slowly, andthat in mono-gynoecious ones (acr¹/acr¹) decreased at an intermediaterate. Etiolation was suppressed even in early-etiolating cultivarswhen the flask remained unsealed or endogenously evolved ethylenewas removed. Cotyledon etiolation was enhanced even in late-etiolatingcultivars when ethephon was added to the flask. The rate ofetiolation corresponded to the ethylene concentration in theflask; much more ethylene was detected in early-etiolating cultivarsthan in late-etiolating ones. Ethylene accumulation is one of the important factors involvedin the cotyledon etiolation observed in in vitro cultures. Thedifference in etiolation rates among seedlings with differentgenetic backgrounds for sex expression corresponds to theirability for ethylene evolution, in the order of acr^F>acr¹>acr⁺. (Received January 6, 1981; Accepted March 23, 1981)     

Ethylene Production by the Lichen Ramalina duriaei

The lichen Ramalina duriaei evolved ethylene when in a wettedstate, the rate of ethylene evolution being constant for atleast the first 20 h. Inhibitors of the ACC (I-aminocyclopropane-I-carboxylicacid) pathway did not inhibit ethylene production. Metal ionsstimulated the production, with Fe²⁺ being the most effective.This stimulation was not affected by inhibitors of the ACC pathwaybut was inhibited by free radical scavengers such as propylgallateand quercitin. Endogenous ACC content was similar whether thelichens were producing ethylene at a basal rate or during Fe²⁺-stimulatedethylene formation. Malondialdehyde and aldehyde contents werehigher in the presence of Fe²⁺. The results are discussed interms of known pathways of ethylene production by micro-organisms. ACC, ethylene, metal ions, methionine, 2-oxo-methylthiobutyric acid, Ramalina duriaei (De Not.) Bagl     

Relationships between Acetylene Reduction Activity, Hydrogen Evolution and Nitrogen Fixation in Nodules of Acacia spp.: Experimental Background to Assaying Fixation by Acetylene Reduction under Field Conditions

Hansen, A. P., Pate, J. S. and Atkins, C. A. 1987. Relationshipsbetween acetylene reduction activity, hydrogen evolution andnitrogen fixation in nodules of Acacia spp.: Experimental backgroundto assaying fixation by acetylene reduction under field conditions.—J.exp. Bot. 38: 1–12 Glasshouse grown, symbiotically-dependent seedlings of Acaciaalata R.Br., .A. extensa Lindl., and A. pulchella R.Br. wereexamined for acetylene reduction in closed assay systems usingundisturbed potted plants, excavated whole plants, nodulatedroots or detached nodules. Nitrogenase activity declined sharplyover the first hour after exposure of detached nodules to acetylene(10% v/v in air), less steeply or not at all over a 3 h periodin assays involving attached nodules. Using detached nodules,rates of acetylene reduction, nitrogen (¹⁵N₂) fixation, andhydrogen evolution in air (¹⁵N₂) and acetylene-containing atmosphereswere measured in comparable 30 min assays. Total electron flowthrough nitrogenase in air was determined from rates of nitrogen(¹⁵N₂) fixation ( ? 3) plus hydrogen evolution, that in thepresence of acetylene from rates of acetylene reduction andhydrogen evolution in air: acetylene. Values for the ratio ofelectron flow in air: acetylene to that in air ranged from 0?43to 0?83 in A. pulcheila, from 0?44 to 0?66 in A. alala and from0?37 to 0?70 in A. extensa, indicating substantial inhibitionof electron flow through nitrogenase of detached nodules byacetylene. Relative efficiencies of nitrogenase functioningbased on hydrogen evolution and acetylene reduction were from0?15 to 0?79, those based on nitrogen (¹⁵N₂) fixation and hydrogenevolution from 0?53 to 0?87. Molar ratios of acetylene reducedto nitrogen (¹⁵N₂) fixed were 2?82 ? 0?24, 201 ? 0?15, and 1?91? 0?11 (?s.e.; n = 7) for A. pulcheila,A. extensa and A. alata respectively A standard 5–10 min acetylene reduction assay, conductedon freshly detached unwashed nodules in daytime (12.00–14.00h), was calibrated for field use by comparing total N accumulationof seedlings with estimated cumulative acetylene reduction overa 7-week period of glasshouse culture. Molar ratios for acetylenereduced: nitrogen fixed using this arbitrary method were 3?58for A. alata, 4?82 for A. extensa and 1?60 for A. pulchella.The significance of the data is discussed. Key words: Acacia spp, nitrogenase functioning     

Determination of Volume of Dunaliella Cells by Lithium Dilution Measurements and Derivation of Internal Solute Concentrations

A method has been developed to measure the cell volume of theunicellular green alga Dunaliella parva 19/9 using Li⁺ measurementsonly. Concentrations of internal solutes can also be calculatedif they are assayed in the same samples as Li⁺. We found thatD. parva cells grown in 0.4 kmol m^–3 NaCl have an averageaqueous cell volume of 65.1 ?2.9 µm³, a K⁺ concentrationof 126?6 mol m^–3, a Na⁺ concentration of 11?11 mol m^–3and a glycerol concentration of 615?27 mol m–3 (n= 12).Algae grown in 1.5 kmol m^–3 NaCl have an average aqueouscell volume of 131 ?7.5 µm³, a K⁺ concentration of 109?4mol m^–3, a Na⁺ concentration of 10?39 mol m^–3 anda glycerol concentration of 1 425?59 mol m^–3 (n = 12).These results indicate that D. parva cells adapted to high salinitieshave larger cell volumes than those adapted to lower salinities.However, there is no evidence for a significant difference ininternal Na⁺ concentration, despite the almost 4-fold differencein the concentration of external NaCl. The intracellular glycerolconcentration alone accounts for 65% and 54%, respectively,of the osmotic balance in low and high salt grown cells. Key words: Dunaliella, cell volume, intracellular solutes     

Heat-stabilities of Electron Transport Related to Photosystem I in a Thermophilic Blue-green Alga, Synechococcus sp.

Heat-stabilities of photosystem I reactions in a thermophilicblue-green alga, Synechococcus sp. were studied. All the reactionsexamined were highly resistant to heat as compared with thosein ordinary higher plants and algae. Cyt c-553 photooxidation in vivo was abolished by treatmentat 75?C for 5 min. By contrast, P700 photooxidation was extremelyresistant to heat and could not be completely inactivated bytreatment of the cells or isolated thylakoids at about 100?Cfor 5 min. Photooxidation of added Cyt c-553 by isolated thylakoidmembranes was more heat-stable than was this activity in cells.This suggests that heat-treatment caused a perturbation in thestructural integrity of the membranes which is required forefficient electron transfer from Cyt c-553 to P700 in situ. At higher temperatures, the inactivation of Cyt c-553 photooxidationin the membranes parallels the decrease in the rate of P700photooxidation. Spectrophotometric studies with short flashesindicated that inactivation of the electron transport from Cytc-553 to methyl viologen is due to the denaturation of a secondaryelectron acceptor of photosystem I, A₂, and possibly anotheracceptor, P430. ¹ Present address: The Solar Energy Research Group, the Instituteof Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi,Saitama 351, Japan. (Received September 28, 1981; Accepted December 21, 1981)     

Purification and some properties of L-tyrosine carboxy-lyase from barley roots

L-Tyrosine carboxy-lyase (E. C. 4. 1. 1. 25) was extracted fromthe roots of barley seedlings and purified approximately 25fold. Optimum pH for the enzyme activity was found to be 7.3.The Km value for L-tyrosine was calulated as 4.5?10^–4M.D-Isomer did not react with the enzyme. L-DOPA, m-tyrosine ando-tyrosine were decarboxylated to some extent. Pyridoxal phosphateactivated the enzyme 4 fold. Caffeic acid and p-coumaric acidare competitive inhibitors. Ki values were 4.5?10^–5M forcaffeic acid and 1.6?10^–4M for p-coumaric acid. L-DOPAand m-tyrosine had an inhibitory effect on the decarboxylationof L-tyrosine. Hydroxylamine, semicarbazide, p-CMB, Fe⁺⁺, Cu⁺⁺,and Hg⁺⁺ inhibited the decarboxylation of tyrosine. Enzyme activitywas also found in extracts from Triticum aestivum, Zea maysand Cytisus scoparius. (Received November 30, 1973; )     

Temperature dependence of chlorophyll {alpha} fluorescence in lettuce and spinach chloroplasts at sub-zero temperatures

The temperature dependence of chlorophyll fluorescence wasmeasured in spinach and lettuce chloroplasts at sub-zero temperaturesin the presence of 50% ethylene glycol. In the presence of 5mM Mg²⁺, a fluorescence maximum appeared at –31?C in boththe spinach and lettuce chloroplasts, while in the presenceof only 5 mM Na⁺ as cations the maximum shifted to –20?Cin the spinach chloroplasts and to –11?C in the lettucechloroplasts. Since the occurrence of a maximum in the temperatureversus fluorescence curve is an indication for the transitionof the physical phase of thylakoid membrane lipids between theliquid crystalline and the phase-separation state (16, 18),these findings suggest that the (major) phase transition ofmembrane lipids occurs at these low temperatures in chloroplastsof higher plants and also that the phase transition temperatureis markedly lowered by the presence of divalent cations. Ethylene glycol at a concentration of 50% had almost no effecton the temperature dependence of chlorophyll fluorescence ina lamellar membrane preparation of Anabaena variabilis. In awater suspension of dimyristoylphosphatidylcholine, the additionof ethylene glycol to 50% did not alter the characteristic featureof the temperature dependence of fluorescence of 1-anilinonaphthalene-8-sulfonate.These findings suggest that 50% ethylene glycol does not affectthe temperature of the transition of the physical phase of membranelipids. ¹ C.I.W.-D.P.B. Publication No. 592. ² Present Address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Hongo 113, Tokyo, Japan. (Received June 22, 1977; )     

Relations Between Indoleacetic Acid, Calcium Ions and Ethylene in the Regulation of Growth and Cell Wall Composition in Picea abies

Effects of indoleacetic acid, calcium ions and ethylene on thegrowth of and deposition of different cell wall fractions inthe hypocotyl of Norway spruce (Picea abies (L.) Karst.) seedlingswere investigated. Indoleacetic acid progressively stimulated cellulose depositionas the amount of added Ca²⁺ increased. In contrast, indoleaceticacid promoted lignification and the deposition of non-cellulosicpolysaccharides only in the absence of added Ca²⁺ . When Ca²⁺was added, the indoleacetic acid effect disappeared. Similarly,indoleacetic acid promoted non-cellulosic polysaccharide depositiononly in the absence of ethylene. At increasing ethylene levelsthe effect of indoleacetic acid on non-cellulosic polysaccharidedeposition disappeared and indoleacetic acid instead promotedcellulose deposition. The response to indoleacetic acid depended on the Ca²⁺ concentrationand on the rate of ethylene production. The relationship betweenindoleacetic acid and Ca²⁺ seemed complex, but clearly indoleaceticacid could partially overcome a Ca²⁺ deficiency. The resultssuggest that ethylene may be a factor of particular importancefor the type of polysaccharide deposition during cell wall formation. Key words: Calcium, cell wall, conifers, ethylene, indoleacetic acid     

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

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

Effects of Metabolic Inhibitors on the Calcification of a Freshwater Green Alga Gloeotaenium loitlesbergarianum Hansgirg. 2. Effects of Some Inhibitors of Protein Synthesis

The effects of five inhibitors of protein synthesis, viz. streptomycin,aurin tricarboxylic acid, tetracycline, chloramphenicol, andcycloheximide, on the calcification of Gloeotaenium loitlesbergarianumHansgirg, a freshwater green alga were studied. Streptomycinhad no effect while aurin tricarboxylic acid at 50 µgml^–1 and tetracyline, chloramphenicol and cycloheximideat 20 µg ml^–1 completely inhibited calcificationin the alga. High concentrations of chloramphenicol and cycloheximidewere not completely inhibitory when added 26 h and 32 h respectivelyafter the material was incubated in the induction medium. Itis concluded that the effects by these substrates are the resultsof inhibition of protein synthesis, which is directly or indirectlylinked to calcification. calcification, Gloeotaenium loitlesbergarianum Hansgirg, green alga, chlorophyceae, protein synthesis inhibitors     

Ethylene Production by the Kudzu Strains of Pseudomonas syringae pv. phaseolicola Causing Halo Blight in Pueraria lobata (Willd) Ohwi

Significant amounts of ethylene was produced by Pseudomonassolanacearum (all strains), P. syringae pv. phaseolicola (Kudzustrains isolated from Pueraria lobata) and Erwinia rhapontici(2 strains out of 22) out of 24 species, 3 subspecies and 38pathovars of plant pathogenic bacteria tested in yeast extract-peptonebroth. The bean strains of P. syringae pv. phaseolicola causinghalo blight in kindney bean plants did not produce ethylene.The Kudzu strains produced ethylene at a rate of 7 to 100?10^–9nl cell^–1 h^–1, which was 500 to 1,000 times higherthan that of P. solanacearum and several times higher than thatof Penicillium digitatum, the most potent ethylene producerknown among microorganisms. The presence of living cells was essential for ethylene productionby the Kudzu strains. The bacterium effectively produced ethylenefrom amino acids such as glutamate, aspartate and their amides.Although glucose and succinate were also good substrates forethylene biosynthesis, the rate of ethylene production was significantlysmaller than that with glutamate. Methionine, which is knownas the precursor of ethylene in plants, had no effect on ethyleneproduction by the bacterium. 1-Aminocyclopropane-1-carboxylicacid (ACC) also had no effect on ethylene production, and therewas not enough ACC in the bacterial cells to account for thehigh rate of ethylene production. Ethylene production from glutamatewas inhibited by n-propylgallate and EDTA, but not by aminoethoxyvinylglycine.These results indicate that ACC is not involved as an intermediatein the process of ethylene biosynthesis by the bacterium, suggestingthe presence of a pathway different from that of plant tissues. (Received September 4, 1984; Accepted October 27, 1984)     

The Oxidation of 14C-labelled Glucose by Chlorella vulgaris: 1. The Time Course of 14CO2 Production

Glucose, either uniformly labelled with¹⁴C, or specificallylabelled in the I, 2, or 6 position, was added to C. vulgaris.Radio-active carbon dioxide was produced initially ten timesfaster from glucose-I-¹⁴C than from glucose-6-¹⁴C. This differencewas found with carbohydrate-starved cultures, exponentiallygrowing cultures, and cultures assimilating ammonia or nitraterapidly. A similar difference was also found with C. pyrenoidosaand Ankistrodesmus. 37 per cent. of the ¹⁴C added as glucose-¹-¹⁴Cto exponentially growing cells was recovered as carbon dioxidebut generally the recovery was less than this. Only 5 per cent.of ¹⁴C added as glucose-6-¹⁴C was recovered as carbon dioxide.The specific activity of the carbon dioxide produced was considerablylower than that of the carbon in the added glucose.     

Mechanisms of the acido- and thermophily of Cyanidium caldarium Geitler V. Acid and heat stabilities of soluble proteins

Acid and heat stabilities of water-soluble proteins extractedfrom an acidophilic and thermophilic unicellular alga, Cyanidiumcaldarium, by French pressure cell treatment were investigated.Soluble proteins from Cyanidium were not particularly acid orheat stable compared with those from mesophilic algae such asAnabaena variabilis and Anacystis nidulans. The results suggestthat the acidophilic and thermophilic mechanisms of Cyanidiumcan not be ascribed to the acid and heat stabilities of theproteins in the cells. ¹ Based on a dissertation submitted to the Tokyo MetropolitanUniversity in partial fulfillment of the requirements for thedegree of Doctor of Science. (Received October 24, 1977; )     

Energy Loss in Tissue 2Sauromatum guttatum (Schott) Analysed by Microcalorimetry

The heat evolved by 1 mm-thick tissue slices of the appendixof the Sauromatum guttatum inflorescence was measured calorimetricallyduring development. From D–5 (5 d before inflorescence-opening,designated as D-day) to D–2 about 8 µW mg^–1fresh wt. was observed, and during D–1 an increase inheat evolution to 14 µW mg^–1 fresh wt. was monitored.The heat was produced through oxidative metabolism, since replacingthe air in the microcalorimeter with nitrogen blocked heatproduction.Addition of salicylic acid to tissue slices of thermogenic organs(appendix, lowest part of the spadix and male flowers) and ofnon-thermogenic organs (female flowers, club-shaped organs andlower part of the spadix) of Sauromatum inflorescences revealedthat the acid boosted heat-production only in the thermogenicorgans. The effect of the acid manifested itself with no appreciablelag time, and it generated non-linearity in the rate of heat-productionby tissue slices of the appendix. In the appendices of the highlythermogenic Arum italicum and of the weakly thermogenic Amorphophallusrivieri, salicylic acid selectively boosted the rate of heat-productionin the appendix of A. italicum. A Q₁₀ of 24 was found between15C to 25 C for 1 mm-thick slices of D–4 appendicesof S guttatum. Addition of digitonin or deoxycholate to pre-D-daytissue slices of the appendix increased the rate of heat-production. Key words: Amorphophallus rivieri, Arum italicum, microcalorimetry, salicylic acid, Sauromatum guttatum     

HYDROGENASE ACTIVITY OF CELL-FREE PREPARATION OBTAINED FROM THE BLUE-GREEN ALGA, ANABAENA CYLINDRICA

A cell-free preparation of hydrogenase was obtained from acetone-driedcells of Anabaena cylindrica. This preparation was capable ofcatalyzing the reduction of various redox dyes by molecularhydrogen and evolution of hydrogen from reduced methyl viologen.PMS, methylene blue, DPIP and toluidine blue were effective,in this order, as hydrogen acceptors. Ferricyanide, p-quinone,phenosafranine and neutral red were not reduced by the presentpreparation. PPNR obtained from this alga was effective on neitherreduction of NAD and NADP by hydrogen nor stimulation of hydrogenuptake with methylene blue and PMS. Coupled with hydrogenasereaction, there occurred reduction of nitrate to ammonia whenmethylene blue was added to the reaction mixture. ¹Present address: Department of Zoology, University of Texas,Austin, Texas, U.S.A.     

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     

Accumulation and Utilization of Dissolved Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi

The mechanism for utilization of dissolved inorganic carbon(DIC) was investigated in the marine unicellular calcareousalga Emiliania huxleyi, grown with constant aeration. The apparentK_0.5 (DIC), the concentration of DIC which attains one-halfof the maximum velocity of apparent photosynthesis, for photosyntheticevolution of O₂, measured under saturating light, was 5.5 mM(55 µM for CO₂) at pH 8.0 and 25°C. The value of K_0.5was not affected by inhibitors of carbonic anhydrase (CA), andan electrometric assay of CA showed that the enzyme was notinvolved in photosynthesis in this alga. The rate of photosyntheticfixation of ¹⁴C-DIC into acid-stable products was about 20 timeshigher than that into CaCO₃, irrespective of the external concentrationof DIC. In short-term experiments, ¹⁴C-DIC was usually incorporatedinto the internal pool of DIC (IIC) to concentrations up to13 to 16 times higher than that of the external DIC. CO₂ addedexternally was utilized mainly for fixation of CO₂ and accumulationof IIC. By contrast, HCO^-₃ was utilized mainly for productionof CaCO₃ and accumulation of IIC. Incorporation of ¹⁴C intoIIC was partially suppressed by DCMU or in darkness but itstransfer to CaCO₃ was unaffected. These results suggest thataccumulation of IIC in this alga, even under ordinary circumstances,is only partially responsible for increasing the efficiencyof utilization of DIC by photosynthetic fixation but may bemost useful for the production of CaCO₃. (Hydroxyethylidene) bisphosphonic acid, an inhibitor of thegrowth of CaCO₃ crystals, completely suppressed production ofCaCO₃. The accumulation of IIC was also partially suppressed,but photosynthetic fixation of CO₂ was enhanced. In a pulse-chaseexperiment with ¹⁴CDIC, ¹⁴C incorporated into IIC and CaCO₃in darkness was transferred to acid-stable products of photosynthesisin the light. These results suggest that ¹⁴C-DIC in IIC andpre-formed CaCO₃ may be useful sources of carbon for fixationof CO₂. (Received July 2, 1993; Accepted January 10, 1994)     

Effect of Temperature on Nitrogenase Activity in White Clover

Single, clonal plants of white clover were grown without inorganicnitrogen in four contrasting day/night temperature regimes,with a 12 h photoperiod, in controlled environments. Root andnodule respiration and acetylene reduction activity were measuredin a flow-through system during both day and night for plantsacclimated to day/night regimes of 23/18, 15/10 and 10/5 ?C.Similar measurements were made on plants acclimated to 20/15?C and stepwise at temperatures from 4 to 33 ?C. Peak rate of ethylene production, nitrogenase-linked respirationand basal root + nodule respiration increased approximatelylinearly from 5 to 23 ?C both in temperature-acclimated plantsand in plants exposed to varying measurement temperatures. Themeasured attributes did not vary significantly between day andnight. Temperatures above 23–25 ?C did not further enhancethe rate of ethylene production, which remained essentiallythe same up to the maximum measured temperature of 33 ?C. The measurements of nitrogenase-linked respiration between 5and 23 ?C, during both day and night, demonstrated a constant‘energetic cost’ of acetylene reduction of 2.9 µmolCO₂ µmol C₂H₄^–1,. Over the same temperature range,the approximate activation energy of acetylene reduction was60 kJ mol^–1. The integrated day plus night nitrogenase-linkedrespiration accounted for 13.4–16% of the plant‘snet shoot photosynthesis in a single diurnal period: there wasno significant effect of temperature between 5 and 23 ?C. Key words: Trifolium repens, white clover, temperature, N₂ fixation, respiration