Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

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.     

Characterisation of Non-Uniform Photosynthesis Induced by Abscisic Acid in Leaves Having Different Mesophyll Anatomies

The effects of abscisic acid (ABA) on photosynthesis in leavesof Helianthus annuus L. were compared with those in leaves ofVicia faba L. After the ABA treatment, the response of photosyntheticCO₂ assimilation rate, A, to calculated intercellular partialpressure of CO₂, P_i, (A(p_i) relationship) was markedly depressedin H. annuus. A less marked depression was also observed inV.faba. However, when the abaxial epidermes were removed fromthese leaves, neither the maximum rate nor the CO₂ responseof photosynthetic oxygen evolution was affected by the applicationof ABA. Starch-iodine tests revealed that photosynthesis was not uniformover the leaves of H. annuus treated with ABA. The starch contentwas diffferent in each bundle sheath extension compartment (thesmallest subdivision of mesophyll by veins with bundle sheathextensions, having an area of ca. 0.25 mm² and ca. 50 stomata).In some compartments, no starch was detected. The distributionof open stomata, examined using the silicone rubber impressiontechniques, was similar to the pattern of starch accumulation.In V.faba leaves, which lack bundle sheath extensions, distributionof starch was more homogeneous. These results indicate that the apparent non-stomatal inhibitionof photosynthesis by ABA deduced from the depression of A(pⁱ)relationship is an artifact which can be attributed to the non-uniformdistribution of transpiration and photosynthesis over the leaf.Intercellular gaseous environment in the ABA-treated leavesis discussed in relation to mesophyll anatomy. ¹ Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received September 30, 1987; Accepted January 13, 1988)     

Purification and Characterization of Sucrose Synthase from Peach (Prunus persica) Fruit

Sucrose synthase (EC 2.4.1.13 [EC] ) was purified from peach fruit(Prunus persica) to a single band of protein on SDS-PAGE byammonium sulfate fractionation, DEAE-cellulose (DE-52) chromatography,Sepharose CL-6B gel filtration, PBA-60 affinity chromatographyand Sephadex G-200 gel filtration. The molecular weight wasestimated to be 360,000 by gel filtration. The enzyme was foundto be a tetramer of identical 87-kDa subunits. The maximum activityfor the synthesis and cleavage of sucrose was observed at pH8.5 and pH 7.0, respectively. The enzymatic reaction followedtypical Michaelis-Menten kinetics in both directions, with thefollowing parameters: K_m(fructose), 4.8 mmM; K_m(UDPglucose),0.033 mM; K_m(sucrose), 62.5 mM; K_m(UDP), 0.080 mM. Other properties,such as substrate specificity and the effects of divalent cations,were also investigated. The relationship between the enzymeand the accumulation of sucrose in peach fruit is discussed. Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Chikusa, Nagoya 464, Japan. (Received May 2, 1988; Accepted September 14, 1988)     

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)     

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     

Isolation and Characterization of Temperature-Sensitive, High-CO2 Requiring Mutant of Anacystis nidulans R2

Temperature-sensitive (ts), high-CO₂ requiring mutants of Anacystisnidulans R2 were isolated by N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) mutagenesis and ampicillin enrichment. One of these mutantswas able to grow under ordinary air enriched with 5% CO₂, butnot under ordinary air at 40?C. At 40?C, the concentration ofCO₂ at which the rate of oxygen evolution reached half the maximumvelocity (apparent Km(CO₂) in photosynthesis) was 1,000 timeshigher in mutant cells than in wild type cells, whereas therewas no significant difference in the maximum rate of photosynthesis. When wild type and mutant cells were incubated with 55 µmNaHC0₃ under illumination at 40?C, the initial rate of inorganiccarbon (IC) transport from the medium into the cells and themaximum internal IC accumulation were significantly higher inwild type cells than in mutant cells. These results indicate that the isolated high-CO₂ requiringmutant lacks the ability in transporting IC into the cells at40?C. Furthermore, the finding that the mutant cells which aredefective in IC transport cannot grow under ordinary air suggeststhe importance of IC concentrating system(s) in photosynthesisof cyanobacteria. Present address: Department of Molecular Biology, The Universityof Wyoming, Laramie, Wyoming 82071, U.S.A. To whom reprints should be requested. (Received April 28, 1988; Accepted September 14, 1988)     

Studies on the change of the respiratory system in roots in relation to the growth of plants II. Activity of iron-containing oxidases in roots of cereal crops with the aging of plants

Distribution of iron-containing oxidases in aging nodal rootsof rice and wheat was studied. Activities of cytochrome c oxidase(1.9.3.1 [EC] , cytochrome c : O₂ oxidoreductase), catalase (1.11.1.6 [EC] ,H₂O₂: H₂O₂ oxidoreductase) and peroxidase (1.11.1.7 [EC] , donor:H₂O₂ oxidoreductase) in wheat roots were comparatively higherthan were those in rice roots at corresponding stages. Cytochromec oxidase in roots remained active throughout the lives of therice and wheat crops. In rice roots, catalase seemed to playa distinct role around the panicle formation stage. Decay ofcatalase activity took place earlier than did that of peroxidaseand cytochrome c oxidase activities. In wheat roots similarenzyme activity changes were not observed. Data may suggestthat the high activity of iron containing oxidases at the panicleformation stage (I) may be chiefly due to catalase activityin rice roots. ¹Paper presented at the 14th Annual Meeting of the Society ofthe Science of Soil and Manure, Japan (1968). (Received November 21, 1968; )     

Ultrastructure of Dunaliella tertiolecta Cells Grown under Low and High CO2 Concentrations1

The cells of Dunaliella tertiolecta grown under ordinary air(low-CO₂ cells) had a well developed pyrenoid with many morestarch granules than those grown under air enriched with CO₂(high-CO₂ cells). The chloroplast was located close to the plasmamembranein low-CO₂ cells, while that in high-CO₂ cells was located inthe inner area of the cells. Chloroplast envelope was electronicallydenser in low-CO₂ cells than in high-CO₂ cells, while the oppositeeffect of CO₂ was observed for the plasmamembrane. ²On leave from Institute of Biology, University of Novi Sad,Novi Sad, Yugoslavia. (Received November 7, 1985; Accepted March 5, 1986)     

Nutrient Dilution by Starch in CO2-enriched Chrysanthemum

Increasing growth irradiance and CO₂ generally decreases foliarnutrient concentration on a dry weight basis and increases foliarstarch concentration. However, the extent to which starch concentrations‘dilute’ foliar nutrient concentrations when thelatter are expressed on a dry weight basis is not known. Todetermine the importance of differential starch accumulationin calculating nutrient concentrations on a dry weight basis,leaf nutrient and starch concentrations were measured in Chrysanthemum? morifolium ‘Fiesta’ (Ramat.) cuttings grown atthree irradiance levels and two CO₂ levels for eight weeks inboth winter and spring. On a dry weight basis, foliar concentrationsof most nutrients were lower in both seasons as a result ofthe elevated CO₂ and irradiance levels, and total dry weightswere higher. Per cent starch was greater at the high CO₂ levelin both seasons but was only greater at higher irradiances inthe winter experiment. When starch was subtracted from the leafdry weights, the differences between CO₂ and irradiance treatmentsdisappeared with respect to N, P, K, Ca, Mg, S, and B but notfor Fe, Mn, Zn, and Cu. Key words: CO₂ enrichment, starch, nutrients, irradiance     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

Participation of Hydrogen Peroxide in the Inactivation of Calvin-Cycle SH Enzymes in SO2-Fumigated Spinach Leaves

In SO₂-fumigated spinach leaves under light, chloroplast SHenzymes, glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD)(EC 1.2.1.13 [EC] ), ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19 [EC] )and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11 [EC] ) weremore remarkably inactivated than other chloroplast enzymes.Their activities recovered after removal of SO₂. The inactivationparalleled light-dependent CO₂-fixation in spinach leaves. Inilluminated chloroplasts isolated from SO₂-fumigated spinachleaves, NADP-GAPD and Ru5PK were more specifically in activatedthan other chloroplast enzymes. These two enzymes could be protectedfrom the inactivation by adding catalase. The NADP-GAPD inactivationwas suppressed by DCMU, cytochrome c or anaerobic conditions.By adding thiol compounds, the NADP-GAPD inactivation was dischargedand the activity increased. In chloroplasts or crude extractsfrom non-fumigated spinach leaves, NADP-GAPD and Ru5PK weremore strongly inhibited by externally added H₂O₂ than otherchloroplast enzymes. All results supported the idea that thesuppression of photosynthesis at the beginning of SO₂ fumigationwas caused by the reversible inhibition of chloroplast SH enzymewith H₂O₂. (Received October 7, 1981; Accepted June 16, 1982)     

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO₂ concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO₂-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO₂ conditions. These growth conditions also partly inhibit the induction of the CO₂-concentrating mechanism. When cells are grown with acetate in the dark, the CO₂-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO₂-concentrating mechanism under low-CO₂ conditions. We suggest that this ultrastructural reorganization under lowCO₂ conditions plays a role in the CO₂-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.     

Carbonic Anhydrase of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1 [EC] ; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

The enzyme system for the entrance of 14CO2 in the dark CO2-fixation of brown algae

High activity of phosphoenolpyruvate (PEP)-carboxykinase, orADP: oxalacetate (OAA) carboxy-lyase activity (a kind of EC4. 1. 1. 32) was discovered in enzyme extracts or partiallypurified preparations obtained from the brown algae, Eiseniabicyclis, Dictyota dichotoma, Spatoglossum pacificum; and Hizikiafusiformis. Enzyme activities were determined by measuring theradioactivity incorporated in the products of dark ¹⁴CO₂-fixationand by spectrophotometric determinations. Except for the lowactivity of "malic enzyme" (EC 1. 1. 1.40), no activities ofother carboxylases, i.e. PEP-carboxylase, PEP-carboxytransphosphorylase,and pyruvate carboxylase could be detected in algal extractsprepared under various conditions. Malate dehydrogenase (EC1. 1. 1. 37), fumarase (EC 4. 2. 1. 2), and glutamic: oxalacetictransaminase (EC 2. 6. 1. 1) were also detected. The algal PEP-carboxykinase required ADP and Mn²⁺ for maximumactivity in the carboxylation reaction; and ATP and Mn²⁺, butnot GTP, for maximum activity in both the decarboxylation andOAA-¹⁴CO₂-exchange reactions. The optimum pH of purified PEP-carboxykinase was in the regionof 7.0 to 7.3 in both the carboxylation and decarboxylationreactions, and its Km values for HCO₃^–, PEP, and ADP were10 mM, 0.3 mM, and 0.07 mM, respectively, in the carboxylationreaction, and values for OAA and ATP were 0.05 mM and 0.4 mM,respectively, in the decarboxylation reaction. Furthermore,the decarboxylation reaction was markedly inhibited by 20 mMHCO₃^–. The physiological role of PEP-carboxykinase as the enzyme responsiblefor the entrance reaction of the dark CO₂-fixation is discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 236. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and Matsunaga Science Foundation (to T.Ikawa). ² Present address: Department of Antibiotics, the National Instituteof Health, Shinagawa, Tokyo, Japan. (Received February 22, 1972; )     

Ammonia Induces Starch Degradation in Chlorella Cells

When ammonia was added to cells of Chlorella which had fixed¹⁴CO₂ photo synthetically, ¹⁴C which had been incorporated intostarch was greatly decreased. A similar effect was observedwhen potassium nitrate and sodium nitrite were added. The ammonia-induceddecrease in ¹⁴C-starch was observed in all species of Chlorellatested. With cells of C. vulgaris 11h, most of the radioactivityin starch was recovered in sucrose, indicating that ammoniainduces the conversion of starch into sucrose. The percent of¹⁴C recovered in sucrose differed from species to species andpractically no recovery in sucrose was observed in C. pyrenoidosa.In most species tested, the enhancing effects of blue lightand ammonia on O₂ uptake as well as the ammonia effect on starchdegradation were greater in cells which had been starved inphosphate medium in the dark than in non-starved cells. In contrast,the enhancing effect of ammonia on dark CO₂ fixation was muchgreater in non-starved cells. C. pyrenoidosa was unique in thatblue light did not show any effect on its O₂ uptake. (Received August 15, 1984; Accepted November 16, 1984)     

Changes in Photosynthetic Characteristics Induced by Transferring Air-Grown Cells of Chlorococcum littorale to High-CO2 Conditions

When air-grown cells of Chlorococcum littorale was enrichedwith CO₂, growth was enhanced after a lag period of one to twodays at 20% CO₂, and 3 to 6 days at 40% CO₂. Changes in therate of photosynthesis measured as oxygen evolution and CO₂fixation, were similar to those observed for growth. Duringthe initial inhibition of photosynthesis in 40% CO₂, the activityof PSII was suppressed. In contrast, PSI activity was greatlyenhanced. Air-grown cells of C. littorale possessed comparatively highcarbonic anhydrase (CA) activity which was localized insidethe cells and on the cell surface. Under high CO₂ concentrationsextracellular CA activity was greatly suppressed and intracellularactivity almost completely abolished. Phosphoenol pyruvate carboxylaseactivity was also suppressed in high CO₂-grown cells. Ribulose-l,5-bisphosphatecarboxylase activity was higher in high-CO₂ grown cells thanin air-grown cells. The above results indicated that the lagphase induced by 40% CO₂ was due to suppression of PSII activity. ¹Part of this work was reported in the International PhotosynthesisCongress, Nagoya, 1992.     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Influence of Drought on Mitochondrial Activity, Photosynthesis, Nocturnal Acid Accumulation and Water Relations in the CAM Plants Prenia sladeniana(ME-type) and Crassula lycopodioides(PEPCK-type)

The activity of photosynthesis and mitochondrial respiration,nocturnal organic acid accumulation and water relations wereinvestigated in Prenia sladeniana L. Bol. [malic enzyme (ME)-type]andCrassula lycopodioides Lam. [phosphoenolpyruvate carboxykinase(PEPCK)-type] to compare the physiological responses to waterdeficit in crassulacean acid metabolism (CAM) plants differingin their decarboxylating enzyme systems. Withholding water inhibiteddaytime gas exchange within 2 d while night time CO₂gain andmalic acid accumulation remained relatively unchanged in bothspecies. In P. sladeniana, maximum photochemical efficiency(F_v/F_m) and photosynthetic electron transport declined to nearlythe same degree as CO₂supply was restricted during drought.Despite limited CO₂availability, photosynthetic activity waslargely unaffected in C. lycopodioides, as were mitochondrialproperties. There is no indication of a drought-induced increasein the capability to totally oxidize malate, yielding 4 CO₂, in either species. Nevertheless, the enhanced ratio of malateto glycine oxidation may have increased the in vivo capabilityfor malate oxidation in P. sladeniana. Although pressure potentialwas maintained throughout the experiment in both species, activeosmotic adaptation occurred only inP. sladeniana. The observeddecrease in photosynthetic and mitochondrial activity may haveresulted from the large increase in osmotic concentration inthis species. Copyright 2000 Annals of Botany Company Chlorophyll fluorescence analysis, Crassula lycopodioides Lam., crassulacean acid metabolism, citric acid, gas exchange, malic acid, mitochondria, photosynthetic electron transport, Prenia sladeniana L. Bol., water relations     

Activation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase by Inorganic Phosphate under Nocturnal Conditions

In vivo activation states of ribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCO; EC 4.1.1.39 [EC] ) in the dark and light phases were measuredin intact leaves of Phaseolus and radish. The activation statewas high in the dark and comparable to the activation stateunder illumination at saturating light intensity. Then, we examined,using RuBisCO purified from spinach leaves, a mechanism forthe activation of RuBisCO in the dark when the stroma is neutralizedand lossess Mg²⁺ partly. Activation was not obserevd when theenzyme was incubated at air-level CO₂ and 10 mM Mg²⁺ at pH rangingfrom 6.2 to 7.5. However, the activation was highly promotedin this pH range when the activation mixture contained 10 mMinorganic phosphate. The activation state was 50 to 60% betweenpH 7.0 and 7.8 and maximum over pH 8.2 in the presence of 10mM inorganic phosphate. Studies of the initial rate of activationshow that the promotion of activation was through stabilizationof the active form of the enzyme by inorganic phosphate, notby altering the pKa of the activator -amino group of Lys-201.The physiological significance of the activation of RuBisCOby inorganic phosphate in the dark is discussed. ³ Present address: Department of Biochemistry, University ofNebraska, Lincoln, NE 68588-0664, U.S.A.