Effect of Carbonic Anhydrase on the Activity of Ribulose Bisphosphate Carboxylase

At concentrations of CO₂ less than saturating, carbonic anhydrase(EC 4.2.1.1 [EC] ) stimulates the carboxylation of ribulose bisphosphatecatalysed by ribulose bisphosphale carboxylase (EC 4.1.1.3 [EC] .9)in vitro. This is not through any beneficial association ofthe two enzymes but is a consequence of the increased rate ofconversion of HCO₃^– ion to CO₂, the substrate for thecarboxylation. Carbonic anhydrase should always be includedin reaction mixtures used to determine the Michaelis constantof ribulose bisphosphate carboxylase for CO₂ where fixationof radioactive CO₂ into phosphoglycerate is the basis of rateestimation. The effect is to decrease the value obtained forthe Michaelis constant.     

Photosynthetic and Photorespiratory Enzymes in Widely Divergent Plant Species with Special Reference to the Moss Ceratodon purpureus: Properties of Ribulose Bisphosphate Carboxylase/ Oxygenase, Phosphoenolpyruvate Carboxylase and Glycolate Oxidase

Rintamäki, E. and Aro, E.-M. 1985. Photosynthetic and photorespiratoryenzymes in widely divergent plant species with special referenceto the moss Ceratodon purpureus: Properties of ribulose bisphosphatecarboxylase/oxygenase, phosphoenolpyruvate carboxylase and glycolateoxidase.—J. exp. Bot. 36: 1677–1684. Km(CO₂) values and maximal velocities of ribulose bisphosphatecarboxylase/oxygenase (E.C. 4.1.1.39 [EC] ) were determined for sixplant species growing in the wild, consisting of a moss, a fernand four angiosperms. The maximum velocities of the RuBP carboxylasesvaried from 0.13 to 0.;62 µmol CO₂ fixed min^–1 mg^–1soluble protein and the Km(CO₂) values from 15 to 22 mmol m^–3CO₂. The highest Km(CO₂) values found were for the moss, Ceratodonpurpureus, and the grass, Deschampsia flexuosa. These plantsalso had the highest ratios of the activities of RuBP carboxylaseto RuBP oxygenase. Glycolate oxidase (E.C. 1.1.3.1 [EC] ) activitieswere slightly lower in D.flexuosa, but not in C. purpureus,than for typical C₃ species. Phosphoenolpyruvate carboxylase(E.C. 4.1.1.31 [EC] ) was not involved in the photosynthetic carboxylationby these two plants. However, another grass, Phragmites australis,was intermediate in PEP carboxylase activity between C₃ andC₄ plants The properties of RuBP carboxylase/oxygenase are discussedin relation to the activities of PEP carboxylase and glycolateoxidase and to the internal CO₂ concentration. Key words: RuBP carboxylase, oxygenase, Km(CO₂), moss     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

Glycine-serine transformation in the photosynthetic bacterium Chromatium vinosum

The metabolic transformation of glycine into serine in the photosyntheticbacterium Chromatium vinosum was accompanied by the evolutionof CO₂ due to decarboxylation of glycine. Isonicotinylhydrazideinhibited both ¹⁴CO₂ evolution and the formation of ¹⁴C-serinefrom ¹⁴C-glycine. The results indicate that a glycine-serinetransformation reaction takes place which is analogous to thatoccurring in green leaf tissues. Glycine may be metabolisedthrough serine by this reaction. The light stimulation of ¹⁴CO₂evolution and ¹⁴C-serine formation from 14C-glycine by the Chromatiumcells are judged to be results of the light-induced enhancementof ¹⁴C-glycine uptake by the bacterial cells. ¹This is paper 53 in the series "Structure and Function of ChloroplastProteins" and paper 7 of the series "Biosynthetic Mechanismof Glycolate in Chromatium". Paper 6 of the latter series isRef. 3 by Asami and Akazawa (1978). ²This study was aided by research grants from the Ministry ofEducation, Science and Culture of Japan and the Nissan ScienceFoundation (Tokyo). ³Postdoctoral Fellow (1980) of the Japan Society for the Promotionof Science. (Received May 20, 1980; )     

Stimulatory Effect of Chloride Ions on NADP Malic Enzyme from Leaves of two C4 Species of Cyperus

NADP malic enzyme (EC 1.1.1.40 [EC] ) from leaves of two C₄ speciesof Cyperus (C. rotundus and C. brevifolius var leiolepis) exihibiteda low level of activity in an assay mixture that contained lowconcentrations of Cl^–. This low level of activity wasmarkedly enhanced by increases in the concentration of NaClup to 200 mM. Since the activity of NADP malic enzyme was inhibitedby Na₂SO₄ and stimulated by relatively high concentration ofTris-HCl (50–100 mM, pH 7–8), the activation ofthe enzyme by NaCl appears to be due to Cl^–. Variationsin the concentration of Mg²⁺ affected the K_A (the concentrationof activator giving half-maximal activation) for Cl^–,which decreased from 500 mM to 80 mM with increasing concentrationsof Mg²⁺ from 0.5 mM to 7 mM. The K_m for Mg²⁺ was decreased from7.7 mM to 1.3 mM with increases in the concentration of NaClfrom zero to 200 mM, although the increase of V_max was not remarkable.NADP malic enzyme from Cyperus, being similar to that from otherC₄ species, was able to utilize Mn²⁺. The K_m for Mn²⁺ was 5mM, a value similar to that for Mg²⁺. The addition of 91 mMNaCl markedly decreased the K_m for Mn²⁺ to 20 +M. NADP malicenzyme from Setaria glauca, which contains rather less Cl^–than other C₄ species, was inactivated by concentrations ofNaCl above 20 mM, although slight activation of the enzyme wasobserved at low concentrations of NaCl at pH7.6. (Received February 20, 1989; Accepted June 12, 1989)     

Coupling of malate decarboxylation to CO2 fixation and the reduction of 3-phosphoglyceric acid in corn bundle sheath cells,2

1. In the presence of NADP⁺ and Mg⁺⁺, the bundle sheath strandsisolated from corn (Zea mays) leaves by cellulase treatmentsdecarboxylated malate in the light at an initial rate (200 µmoles/mgchl.hr), which was sufficient to account for photosyntheticCO₂ fixation in intact leaves. This rate gradually slowed downand then stopped. The final level of the malate decarboxylatedwas approximately equal to the amount of NADP⁺ added.
2. Rapidand continued decarboxylation of malate was observed whenNADP⁺,3-phosphoglyceric acid and ATP (and Mg⁺⁺) were addedtogether.The addition of ADP instead of ATP showed a similareffect.Light did not show any effect on the malate decarboxylationin the presence of ATP or ADP.
3. When malate was added to thebundle sheath strands in the presenceof exogenous NADP⁺ NADP⁺was rapidly reduced. The reductionstopped after 2 min when,73% of the added NADP⁺ was reduced.The further addition of3-phosphoglyceric acid and ATP broughtabout a decrease in theNADPH-level, which rose again to attaina new steady level.
4. The transfer of radioactivity from (1-¹⁴C-3-phosphoglycericacid to dihydroxyacetone phosphate in the bundle sheath strandsin the presence of ATP and NADP⁺ was greatly enhanced by theaddition of malate.
5. In the presence of ribose 5-phosphateand ATP, the rate of ¹⁴C-transferfrom (4-¹⁴C)-malate to theintermediates of the reductive pentosephosphate cycle was equalto that of ¹⁴CO₂ fixation in the light.

All these results support the current view that in the bundlesheath cells of C₄ plants belonging to the NADP-malic enzyme-group,the decarboxylation of malate is coupled to the fixation ofthe released CO₂ and the reduction of 3-phosphoglyceric acidformed as a result of CO₂ fixation. ¹ Part of this research was reported at the 40th Annual Meetingof the Botanical Society of Japan Osaka, December, 1975. ³ Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, 359 Otsuka, Hachioji-City, Tokyo 173, Japan. (Received April 30, 1977; )     

ATP Synthesis in Cell-Free Extracts of Peanut (Arachis hypogaea L.) Seeds

Cell-free extracts of peanut (Arachis hypogaea L., cv. Shulamit)seeds, incubated with various substrates, synthesized ATP. Significantsynthesis occurred in the presence of AMP + PEP, NADH₂ + PEPand NAD + PEP. When the activities were examined in extractsprepared with 0.3 M mannitol, the rates were 0.6, 0.1 and 0.04nmol min^–1 mg^–1 protein, respectively. The activitiesunder such conditions were linear with time up to 90 min incubationat 30 °C. In the presence of PEP + NADH₂ there was a higherspecific activity in extracts from non-dormant seeds than fromdormant seeds. No such difference was found when PEP + AMP orNAD + PEP was used as the substrate. The temperature dependenceof the activity showed a relatively high energy of activation(Ea) for AMP + PEP and a low one if NADH₂ + PEP or NAD + PEPwas used as substrate. In buffer extracts of seeds ATP was synthesizedin the presence of the above-mentioned substrate combinationsbut the rate of activity exhibited a lag phase at the earlytime of incubation, after which higher rates of activities (ascompared with mannitol extracts) were obtained. The activitieswere Co⁺-dependent, with a K_m of about 0.7 mM. In the bufferextracts relatively high activities of adenylate kinase (EC2.7.4.3 [EC] (AK) and pyruvate kinase (EC 2.7.1.50 [EC] ) (PK) were found.AK was stimulated by ethephon (ethylene). This effect is temperature-dependentand occurs in both directions: in the presence of ADP (ATP +AMP) as well as if ATP + AMP is used as substrate to synthesizeADP. PK is Co⁺-dependent, and unaffected by ethephon. Both activitieswere stimulated by malonate. Key words: Adenylate Kinase, Arachis hypogaea, ATP synthesis, Peanut, Pyruvate kinase, Seed     

Role of carbonic anhydrase in photosynthetic CO2 fixation in Chlorella

Chlorella vulgaris 11h cells grown in air enriched with 4% CO₂(high-CO² cells) had carbonic anhydrase (CA) activity whichwas 20 to 90 times lower than that of algal cells grown in ordinaryair (containing 0.04% CO₂, low-CO₂ cells). The CO₂ concentrationduring growth did not affect either ribulose 1,5-bisphosphate(RuBP) carboxylase activity or its Km for CO₂. When high-CO₂ cells were transferred to low CO₂ conditions,CA activity increased without a lag period, and this increasewas accompanied by an increase in the rate of photosynthetic¹⁴CO₂ fixation under ¹⁴CO₂-limiting conditions. On the otherhand, CA activity as well as the rate of photosynthetic ¹⁴CO₂fixation at low ¹⁴CO₂ concentrations decreased when low-CO₂cells were transferred to high CO₂ conditions. Diamox, an inhibitor of CA, at 0.1 mM did not affect photosynthesisof low-CO₂ cells at high CO₂ concentration (0.5%). Diamox inhibitedphotosynthesis only under low CO₂ concentrations, and the lowerthe CO₂ concentration, the greater was the inhibition. Consequently,the CO₂ concentration at which the rate of photosynthesis attainedone-half its maximum rate (Km) greatly increased in the presenceof this inhibitor. When CO₂ concentration was higher than 1%, the photosyntheticrate in low-CO₂ cells decreased, while that in high-CO₂ cellsincreased. Fractionation of the low-CO₂ cells in non-aqueous medium bydensity showed that CA was fractionated in a manner similarto the distribution of chlorophyll and RuBP carboxylase. These observations indicate that CA enhances photosynthesisunder CO₂-limiting conditions, but inhibits it at CO₂ concentrationshigher than a certain level. The mechanism underlying the aboveregulatory functions of CA is discussed. ¹This work was reported at the International Symposium on PhotosyntheticCO₂-Assimilation and Photorespiration, Sofia, August, 1977 (18).Requests for reprints should be addressed to S. Miyachi, RadioisotopeCentre, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. (Received December 11, 1978; )     

Enzymic mechanism of starch breakdown in germinating rice seeds V. Sucrose phosphate synthetase in the scutellum

Some enzymic Properties of a partially purified preparationof sucrose phosphate synthetase (E.C.2.4.1.14) from germinatingrice seed scutella were studied. Examination of the reactionkinetics revealed that the rate of synthesis of sucrose phosphatefollows the Michaelis-Menten equation at an optimum PH of 7.5,having K_m of 25 mM for UDP-glucose, and of 4.9 mM for fructose6-phosphate. UDP inhibited the enzyme reaction competitively;K₁ of 3.3 mM. Fe⁺⁺ and Fe⁺⁺⁺ activated the enzyme reaction about2-fold; K_a, 0.3 mM and 2.0 mM, respectively. Co⁺⁺, Co(NH₃)₆⁺⁺⁺,Mg⁺⁺ and Mn⁺⁺ also activated the enzyme reaction. At high concentrationK⁺ activated the enzyme reaction with the maximum activationof 24% at 400 mM. The molecular weight and S_20,w value of theenzyme were determined as 4.5 ? 10⁵ and 10.4S, respectively. ¹Part IV of this series is Ref. (5). ²California Foundation for Biochemical Research Fellow (1973). (Received December 20, 1973; )     

The effects of irradiance and CO2 on the activity and activation of ribulose-1, 5-bisphosphate carboxylase/ oxygenase in the aquatic plant Spirodela polyrhiza

The activation of ribulose–1, 5-bisphosphate carb-oxylase/oxygenase(Rubisco, EC 4.1.1.39 [EC] ) from the floating angiosperm Spirodelapolyrhiza (L.) Schleid. (giant duckweed) grown at a photon irradianceof 200 or 400 mol photons m^–2 s^–1 was consistentlylow, in the range of 56–62%. Similarly low values wereobserved with four other emergent aquatic species growing underfull sun irradiance. Transference of Spirodela plants for short(minutes) or long (days) periods to the higher or lower irradianceincreased or decreased, respectively, the activation by onlyabout 15%. Activation was not greatly altered by exposure ofthe plants to full sun irradiance of >2000 mol photons m^–2s^–1 or CO₂ concentrations in air of 0 and 1170 mol mor^–1but darkness caused a slow decline to 20% activation. Transientoscillations were observed following a change in irradianceor CO₂ concentration indicating that Rubisco was responsiveto environmental perturbations. The low Rubisco activation wasnot due to the tight binding of inhibitors such as carboxyarabinitol-1-phosphate.It is concluded that a substantial proportion of the Rubiscoprotein in these naturally-occurring species may not be usedfor CO₂-fixation at any given moment. Key words: Rubisco     

Role of carbonic anhydrase in photosynthesis in Chlorella derived from kinetic analysis of 14CO2 fixation1

Time courses of photosynthetic ¹⁴CO₂ fixation and its simulationare presented for Chlorella cells grown under low CO₂ concentration(low-CO₂ cells) and subsequently exposed to 0.2 mM NaH¹⁴CO₃or 130 ppm ¹⁴CO₂ in the presence or absence of carbonic anhydrase(CA) in the suspending medium. It was shown that Chlorella cells utilized only free CO₂ whenNaHCO₃ was given in the presence or absence of CA, or when CO₂was bubbled in the absence of CA. However, the present simulationindicated that both CO₃ and HCO₃^– were utilized when CO₂was given in the presence of CA. Based on these results, weconcluded that 1) Chlorella cells absorb only free CO₂ and 2)this gas is provided to algal cells in two ways, i.e., by directand indirect CO₂ supply. Usually, the dissolved CO₂ is directlyutilized by the algal cells (direct supply of CO₂). However,when the concentration of dissolved CO₂ is extremely low andwhen there is CA, CO₂ reconverted from HCO₃^– is also utilizedby Chlorella cells (indirect supply of CO₂). The utilizationof HCO₃^– indicated by the above simulation was explainedby the indirect supply of CO₂. We further assumed that the indirectsupply of CO₂ to ribulose 1,5-bisphosphate carboxylase occursmainly in the chloroplasts of low-CO₂ cells containing highCA. Thus, under low CO₂ concentrations, low-CO₂ cells can carryout more efficient CO₂ fixation than high-CO₂ cells, resultingin the lower apparent Km(CO₂). ³Department of Biology, Faculty of Science, Niigata University,Niigata, Japan. (Received April 2, 1980; )     

An effective time for CO2 gas treatment in overcoming self-incompatibility in Brassica

An investigation was made to determine the effective time forCO₂ treatment in overcoming self-incompatibility in Brassica.CO₂ was effective when supplied to a self-pollinated flowerwhile hundreds of pollen grains were germinating on the stigma.Since the effective time coincides with the attachment of pollentubes to papilla cells, it is thought that CO₂ produces a metabolicchange in these cells during attachement. ¹Part of a thesis submitted for the Dr. of Agr. degree by thesenior author at Tohoku University. ²Present address: Faculty of Agriculture, Kobe University, Nada-ku,Kobe, Japan. (Received December 7, 1972; )     

Development of Lipid Synthesis from CO2 in Avena Leaves during Greening of Etiolated Seedlings

The development of the lipid synthesizing system in Avena leafsections was examined in connection with carbon fixation duringthe greening of etiolated seedlings under light. During theinitial 2 h illumination there was a low level of CO₂ fixationby PEP carboxylation, but its products, malate and citrate,did not serve as a carbon source for lipid synthesis, althoughlipid synthesis from acetate had already been established. Withthe initiation of Calvin cycle activity after the initial 2h illumination, lipid synthesis began, with CO₂ fixed by RuBPcarboxylation serving exclusively as the carbon source. Fattyacid synthesis in the leaves during the initial 3 h illumination,unlike the fatty acid synthesis thereafter, was insensitiveto thiolactomycin, an inhibitor of type II fatty acid synthetasecontained in the plastids, and was not dependent on light, incontrast to light-dependent activity in greened leaves. The distribution of ¹⁴C incorporated into lipid molecules fromNaH¹⁴CO₃ showed an equal ratio of ¹⁴C in fatty acid, glyceroland choline moieties of labeled phosphatidylcholine, but a denserradioactivity in the galactose moiety than in the residual moietyof mono- and di-galactosyldiacylglycerols. This suggests a regulatedsupply of glycerol, choline and fatty acid moieties for phosphatidylcholinesynthesis, and an excess supply of galactose to diacylglycerolmoiety for galactosyldiacylglycerol synthesis in Avena leaves. (Received October 31, 1984; Accepted January 25, 1985)     

Control of Glutamate Dehydrogenase from Green Tobacco Callus Mitochondria by Ca2$ and pH

Glutamate dehydrogenase (GDH) (EC 1.4.1.3 [EC] .) purified from greentobacco callus mitochondria was activated markedly by Ca^2$ inthe amination reaction. This activation was detectable evenat concentrations below 5 µM Ca^2$. Saturation curves for the three substrates of the aminationreaction showed normal Michaelis-Menten kinetics in the presenceof 1 mM of Ca^2$, but pronounced substrate inhibition occurredwithout Ca^2$. The effect of Ca^2$ was chiefly on the maximalvelocity. The saturation curve for NH₄Cl in the presence of Ca^2$ was modulatedby a change in pH. The apparent K_m value for NH₄Cl markedlydecreased whereas that for -ketoglutarate increased slightlywhen the pH was raised from 7.3 to 9.0. In contrast, the K_mfor NADH was little affected by raising the pH. The characteristicof GDH which increases its affinity for NH₄Cl when the pH israised may be compatible with the detoxification of ammonia. ¹ Present address: Mochida Pharmaceutical Co., Ltd. (Received August 24, 1981; Accepted November 28, 1981)     

Model for the Relationships betweenCO2-Concentrating Mechanism, CO2 Fixation, and Glycolate Synthesis during Photosynthesis in Chlamydomonas reinhardtii

We constructed a mathematical model for simulating the relationshipsof extracellular concentration of dissolved inorganic carbon(DIC), the rates of photosynthetic CO₂ fixation and glycolatesynthesis, and the concentrations of intrachloroplast CO₂ andO₂ in Chlamydomonas reinhardtii. When we compared the photosyntheticrates of I0W-CO₂ (air)-grown C. reinhardtii measured experimentallyand the rates simulated with the incubation conditions in themodel, the model was found to function well. The calculatedrates for glycolate synthesis also matched the measured ratesbetween 80 to 200 µM extracellular DIC, found in the presenceof 1 mM aminooxyacetate. The conformity of the calculated ratesto the measured ones of the glycolate synthesis encouraged usto estimate the O₂ concentration at the active site of ribulosebisphosphate carboxylase/oxygenase; the results were 0.36 and0.40 mM at 80 and 200 µM extracellular DIC, respectively.These high concentrations of O₂ were due to stimulation of photosyntheticCO₂ fixation and further O₂ evolution by a CO₂- concentratingmechanism in the low-CO₂-grown cells. These cells were calculatedto consume 43% of ATP formed photosynthetically for CO₂ concentrationat 200 µM extracellular DIC. The model modified to simulatethese relationships in high-CO₂ (3 to 5% CO₂)-grown C. reinhardtiipredicted O₂ concentration in chloroplasts to be 0.36 mM ina 1% CO₂ atmosphere. This high concentration of O₂ caused activeglycolate synthesis at the measured rate in the high-CO₂-growncells even in the presence of 1% CO₂. The comparisons of themeasured and simulated rates of photosynthesis in low- and high-CO₂-grownC. reinhardtii indicated that no matter how the CO₂ accumulatedin the chloroplasts, it increased the O₂ concentration in theorganelles, and consequently enhanced glycolate synthesis. ¹This paper is the twenty-first in a series on glycolate metabolismin Euglena gracilis. (Received March 11, 1987; Accepted August 17, 1987)     

Role of Carbonic Anhydrase and Identification of the Active Species of Inorganic Carbon Utilized for Photosynthesis in Chora corallina

Carbonic anhydrase (CA, EC. 4.2.1.1 [EC] ) activity in air-grown Characorallina was detected mainly in the intracellular fraction,most of which composed of chloroplasts and cytoplasmic gel,and not on the cell surface. Only minor levels of CA activity,on the basis of equivalent volumes, were detected in the cellsap and the cytoplasmic sol. The maximum rate of photosynthetic O₂ evolution by air-grownChara corallina at pH 6.0 was twice that at pH 7.6, while theapparent K_m for external inorganic carbon (C_i) at pH 7.6 wasabout three times that at pH 6.0. However, the apparent K_m(CO₂)was about three times larger at pH 6.0 than at pH 7.6. The K_m(C_i)-valueat pH 7.6 increased severalfold in the presence of acetazolamide(AZA), an inhibitor of CA, but no inhibition was observed atpH 6.0. The pH-dependence may be due to differences in the permeabilityof AZA at the given pH values. Fixation of ¹⁴CO₂ at 20 µMand of H¹⁴CO₃^– at 200 µM over the course of 5 swas very similar at pH 7.4. Addition of CA significantly suppressedthe photosynthetic ¹⁴CO₂-fixation but it stimulated the H¹⁴CO₃^–-fixation.This result indicates that free CO₂ is an active species ofC_i that is incorporated into the cell during photosynthesis. These results together suggest the following: (1) Free CO₂ isutilized for photosynthesis, (2) CA is mainly located insidethe cell and functions to increase the affinity for CO₂ in photosynthesisby facilitating the supply of CO₂ from the plasmalemma to thesite of CO₂-fixation. ³Present address: Biological Laboratory, The University of theAir, Wakaba 2-11, Chiba, 260 Japan. (Received December 9, 1988; Accepted March 22, 1989)     

The Conductance of the Plasmalemma for CO2

Permeability coefficients (P_S values) for CO₂ of the plasmamembrane (PM) of the unicellular green algae Eremosphaera viridis,Dunaliella parva, and Dunaliella acidophila, and of mesophyllprotoplasts isolated from Valerianella locusta were determinedfrom ¹⁴CO₂ uptake experiments using the rapid separation ofcells by the silicone oil layer centrifugation technique. Theexperimental P_S values were compared with calculated numbersobtained by interpolation of Collander plots, which are basedon lipid solubility and molecular size, for D. parva cells,mesophyll protoplasts isolated from Spinacia oleracea, mesophyllcells and guard cells of Valerianella, and guard cell protoplastsisolated from Vicia faba. The conductivity of algal plasma membranes for CO₂ varies between0.1 and 9 ? 10^–6 m s^–1, whereas for the plasmalemmaof cells and protoplasts isolated from leaves of higher plantsvalues between 0.3 and 11 ? 10^–6 m s^–1 were measured.By assuming that these measurements are representative for plantsand algae in general, it is concluded that the CO₂ conductivityof algal PM is of the same order of magnitude as that of thehigher plant cell PM. P_s values of plasma membranes for CO₂are lower than those for SO₂, but are in the same order of magnitudeas those measured for H₂O. On the basis of these results itis concluded that theoretical values of about 3000 ? 10^–6m s^–1 believed to be representative for higher plant cells(Nobel, 1983) and which are frequently used for computer-basedmodels of photosynthesis, lack experimental confirmation andrepresent considerable overestimations. However, with severalsystems, including higher plant cells, the conductance of thePM for CO₂ was significantly higher in light than in darkness.This suggests that in light, additional mechanisms for CO₂ uptakesuch as facilitated diffusion or active uptake may operate inparallel with diffusional uptake. Key words: Conductivity, CO₂, permeability coefficient, photosynthesis, plasmalemma     

Photosynthetic metabolism of 14CO2 in the process of the "glucose-bleaching" of Chlorella protothecoides

Changes in photosynthetic carbon metabolism during the glucosebleaching of Chlorella protothecoides cells were investigatedusing NaH¹⁴CO₃ as tracer. Several hours after incubating thegreen algal cells in the glucose medium in the dark, the ratesof ¹⁴C-incorporation into glucose polymers and sucrose decreasedand the incorporation into the lipid fraction (fatty acids)greatly increased. At this stage, the rate of photosynthetic¹⁴CO₂ fixation and the chlorophyll content were practicallythe same as in the starting green cells. Afterwards, the photosyntheticcapacity and chlorophyll content continued to decrease throughoutthe experimental period. In contrast, when photosynthetic ¹⁴CO₂fixation of green cells was carried out in the medium containingglucose, the rate of ¹⁴C-incorporation into glucose polymersincreased, though there was no change in the incorporationsinto sucrose and the lipid fraction. ¹Part of this investigation was reported at the Conference "ComparativeBiochemistry and Biophysics of Photosynthesis" (Japan-U.S. CooperativeScience Program) held at Hakone, Japan in 1967. ²Present address: Faculty of Agriculture, Tamagawa University,Machida-shi, Tokyo, Japan. (Received June 10, 1974; )     

Green Light Drives CO2 Fixation Deep within Leaves

Maximal ^l4CO₂-fixation in spinach occurs in the middle of thepalisade mesophyll [Nishio et al. (1993) Plant Cell 5: 953],however, ninety percent of the blue and red light is attenuatedin the upper twenty percent of a spinach leaf [Cui et al. (1991)Plant Cell Environ. 14: 493]. In this report, we showed thatgreen light drives ¹⁴CO₂-fixation deep within spinach leavescompared to red and blue light. Blue light caused fixation mainlyin the palisade mesophyll of the leaf, whereas red light drovefixation slightly deeper into the leaf than did blue light.¹⁴CO₂-fixation measured under green light resulted in less fixationin the upper epidermal layer (guard cells) and upper most palisademesophyll compared to red and blue light, but led to more fixationdeeper in the leaf than that caused by either red or blue light.Saturating white, red, or green light resulted in similar maximal¹⁴CO₂-fixation rates, whereas under the highest irradiance ofblue light given, carbon fixation was not saturated, but itasymptotically approached the maximal ¹⁴CO₂-fixation rates attainedunder the other types of light. The importance of green lightin photosynthesis is discussed. ¹Supported in part by grants from Competitive Research GrantsOffice, U.S. Department of Agriculture (Nos. 91-37100-6672 and93-37100-8855).     

Stomatal Responses of Variegated Leaves to CO2 Enrichment

The responses of stomatal density and stomatal index of fivespecies of ornamental plants with variegated leaves grown attwo mole fractions of atmospheric CO₂ (350 and 700 µmolmol^-1) were measured. The use of variegated leaves allowed anypotential effects of mesophyll photosynthetic capacity to beuncoupled from the responses of stomatal density to changesin atmospheric CO₂ concentration. There was a decrease in stomataldensity and stomatal index with CO₂ enrichment on both white(unpigmented) and green (pigmented) leaf areas. A similar responseof stomatal density and index was also observed on areas ofleaves with pigmentation other than green indicating that anydifferences in metabolic processes associated with colouredleaves are not influencing the responses of stomatal densityto CO₂ concentrations. Therefore the carboxylation capacityof mesophyll tissue has no direct influence on stomatal densityand index responses as suggested previously (Friend and Woodward1990 Advances in Ecological Research 20: 59-124), instead theresponses were related to leaf structure. The stomatal characteristics(density and index) of homobaric variegated leaves showed agreater sensitivity to CO₂ on green portions, whereas heterobaricleaves showed a greater sensitivity on white areas. These resultsprovide evidence that leaf structure may play an important rolein determining the magnitude of stomatal density and index responsesto CO₂ concentrations.Copyright 1995, 1999 Academic Press Leaf structure, photosynthesis, stomatal conductance, CO₂, stomatal density, stomatal index