Studies on the Hill reaction of membrane fragments of blue-green algae II. The reaction steps inactivated at high water concentration

DPIP-photoreduction by membrane fragments of Anabaena cylindricaand A. variabilis was studied to determine which step(s) ofthe Hill reaction system is inactivated on incubation of themembrane fragments in a medium with a high water concentration(cf. 1). Supplementary experiments were done with Anacystisnidulans and Plectonema boryanum. After inactivation of the Hill system at a high water concentration,DPIP-photo-reducing activity was strongly enhanced in the A.variabilis system but less so in the A. cylindrica system byadding DPC, NH₂OH, Mn⁺⁺ or H₂0₂. The activity supported by theadded electron donor was inhibited by DCMU. The steady statelevel of chlorophyll fluorescence was lowered by the inactivationtreatment. In the A. variabilis system, the fluorescence yieldincreased to the original level on the addition of an electrondonor. In the A. cylindrica system, the yield was not so stronglyenhanced as in the A. variabilis system. We inferred that, in A. variabilis, inactivation occurs in thereaction system before the site which receives electrons fromartificial donors, probably including the water oxidation system.In A. cylindrica, besides this site, a site at or near the photochemicalsystem is also blocked. Similar types of inactivation were observed in DPIP-Hill reactionsusing Anacystis nidulans and Plectonema boryanum preparations.The characteristic stability of the Hill reaction system observedin two Anabaena preparations is probably common to the blue-greenalgae. (Received December 10, 1971; )     

Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Studies on the Hill reaction of membrane fragments of blue-green algae V. A correlation between the solubilization of a photochemically active chromoprotein (ACP) and the inactivation of photosystem II activity in membrane fragments of Anabaena cylindrica

The relationship between a photochemically active chromoprotein(ACP) (cf. ref. 1) and photosystem II was investigated withmembrane fragments of Anabaena cylindrica, A. variabilis andP. boryaman. ACP was solubilized from membrane fragments of A. cylindricabut not from those of A. variabilis or P. boryanum, when themembrane fragments had been incubated in a dilute buffer andhad lost their Hill or photosystem II activity. In A. cylindrica,ACP-solubilization always occurred, independent of photosystemII inactivation, on incubation of the membrane fragments inmedia without PEG. However, the amount of ACP solubilizationaccompanying photosystem II inactivation was twice that withoutphotosystem II inactivation. The increase in ACP solubilizationaccompanying photosystem II inactivation. The kinetics resembledthose for the decrease in 695 nm fluorescence emitted by membranefragments at — 196?C (cf. 2). The ACP solubilized independent of photosystem II inactivationwas assumed to have been released during disruption of intactcells in the preparation of membrane fragments. The slow ACPsolubilization upon the inactivation of photosystem II was attributedto the pigment being bound to membranes. We assume that thephoto-reactive component of ACP, P690 (cf. 3, 4), is releasedfrom the membranes during photosystem II inactivation, and thatP690 is a component of photosystem II which emits the 695 nmfluorescence at — 196?C. (Received March 22, 1974; )     

Further investigation of the light-induced cytochrome b-559 reaction in membrane fragments of the blue-green alga Anabaena variabilis

Oxidation-reduction reactions of the low redox potential cytochromeb-559 were studied for membrane fragments of the blue-greenalga Anabaena variabilis. Cytochrome photooxidation was observableat room temperature when the membrane fragments had been preincubatedat room temperature in the dark. A CCCP addition (10^–4M) strongly enhanced the reaction. Oxidation consisted of a DCMU-sensitive and an insensitive reaction.The former depended on actinic illumination of short wavelength.The latter showed a dependency on longer wavelength light. Theformer was assumed to be induced by the action of photosystemII and the latter by that of photosystem I. The photosystem II oxidation was small before preincubation,and was enhanced by added DPIP or Ferro. This was interpretedas photosystem II action inducing oxidation as well as reduction;reduction being inactivated during dark incubation or beingsuppressed by added redox reagents which compete for electronacceptance from photosystem II so that oxidation was apparentlyenhanced. The oxidationreduction reactions of this cytochromewith low redox potential were assumed to be almost identicalwith those of the high redox potential form, at least in themembrane fragments of Anabaena variabilis. (Received June 8, 1975; )     

Electron donor-specificity observed in photosystem I reactions of membrane fragments of the blue-green alga Anabaena variabilis and the higher plant Spinacea oleracea

Electron donating activities of plastocyanins and c-type cytochromesof various organisms for photosystem I reactions were studiedwith membrane fragments of the blue-green alga Anabaena variabilisand the higher plant Spinacea oleracea. In the Anabaena photosystem I reaction, basic but not acidicplastocyanin and c-type cytochromes acted as efficient electrondonors, while only acidic redox proteins were active in thespinach photosystem I reaction. The selective reactivity ofredox proteins in the two photosystem I reactions was observedwith both plastocyanin (or cytochrome) limited and saturatedconditions. These data support our previous observation that photosystemI of blue-green algae differs from those of other green plantswith respect to specificity to the proteinous electron donor(1). (Received August 17, 1971; )     

Studies on the Hill reaction of membrane fragments of blue-green algae IV. Garotenoid photobleaching induced by photosystem II action

Carotenoid photobleaching induced by the action of photosystemII was investigated with membrane fragments of Anabaena cylindricaand A. variabilis. Carotenoid photobleaching occurred only when ferricyanide and/orCCCP was present in the reaction mixture. Maximum velocity ofthe reaction was obtained in the presence of ferricyanide andCCCP. Difference spectra (light minus dark) indicated that afast carotenoid photobleaching was accompanied by a slow chlorophyllbleaching. The pattern of the difference spectra was identicalto that reported by Yamashita et al. (1) with spinach chloroplasts.The reaction was DCMU-sensitive, though a portion of the activitywas insensitive to DCMU when ferricyanide was present in thereaction mixture. The effect of CCCP on stimulation of carotenoidphotobleaching showed the same function against CCCP concentrationas that on inhibition of DPIP-photoreduction with DPC. Carotenoidphotobleaching was stimulated by ferricyanide and suppressedby ferrocyanide; thereaction rate was reduced fifty percentwith a ferri- and ferrocyanide mixture giving 510 mV redox potential(pH 7.5). Benzoquinone was stimulatory, but DPIP had no effect. Incubation of membrane fragments in a dilute buffer inactivatedthe Hill reaction but neither the DPC-supported DPIP-photoreductionnor carotenoid photobleaching in A. variabilis. In A. cylindrica,incubation inactivated all three reactions. Inactivation ofthe latter two reactions followed the same kinetics. (Received August 31, 1972; )     

Toxicity of Selenium to the Blue-green Algae, Anacystis nidulans and Anabaena variabilis

Selenate, selenite, selenomethionine, and selenopurine are toxicto Anacystis nidulans and Anabaena variabilis. These compoundsare less toxic in culture medium containing sulphate than insulphur-free medium, thus suggesting a protective role of sulphuragainst selenium toxicity Selenite is more toxic in agar plate cultures and less toxicin liquid cultures than selenate. Cells grown in the highestgrowth-permitting concentration of selenite in liquid mediumform a variable number of red granules No such granulation occursin cells treated with other seleno-compounds, indicating therebythat the mode of selenite action in blue-green algae differsfrom that of other selenium compounds     

Effect of nitrate on nitrogen-fixation by the blue-green alga Anabaena cylindrica

The effect of nitrate on nitrogen-fixation in the blue-greenalga Anabaena cylindrica Lemm (Fogg strain) was investigated.At concentrations up to 2x10^–2 M, nitrate neither inhibitedthe activity of nitrogenase nor repressed its formation. Atthe late logarithmic phase, more than 50% of cell nitrogen wasprovided by nitrogen-fixation when the cells were grown in thepresence of nitrate. Ammonia at a concentration of 1x10^–3M completely repressed the formation of nitrogenase, but hadno effect on its activity. Nitrogen-fixing activity in the algavaried to a considerable extent during growth on N₂ and themaximum activity was attained at the middle logarithmic phase.However, atmospheric nitrogen did not directly affect the inductionof nitrogenase. The formation of nitrogenase in A. cylindricaappears to be controlled by the intracellular level of a certainnitrogenous metabolite. ¹ This work was supported by grant No. 38814 from the Ministryof Education. (Received January 26, 1972; )     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

The Effects of Modifying Sucrose Concentration on the Development of Maize Kernels Grown in vitro

Intact Zea mays L. kernels attached to cob tissue develop tomaturity when grown in vitro. This experiment was designed todetermine if it is possible to prolong kernel growth by refreshingthe culture medium. Blocks of maize kernels were grown in vitroon media containing several concentrations of sucrose. Kernels,at all concentrations of sucrose, developed to maturity at 30–35d post-pollination, indicating that it is not possible to extendthe kernel growth phase by supplying a carbohydrate source.Kernels grown on media containing 80 g 1^–1 or higher sucroseconcentration had a significantly greater percentage of kernelsthat developed to maturity, and had greater weight and starchcontent per seed. Zea mays, kernel culture, seed development, starch     

Reversible and Irreversible Inactivation of Photosynthesis in Relation to the Lipid Phases of Membranes in the Blue-Green Algae (Cyanobacteria) Anacystis nidulans and Anabaena variabilis

The Arrhenius plots of photosynthetic oxygen evolution in theblue-green algae Anacystis nidulans and Anabaena variabiliswere composed of two straight lines with break points whichwere very close to temperatures for the onset of phase separationof the thylakoid membranes. Irreversible inactivation of photosynthesisbegan to appear at the same temperature as the onset of phaseseparation of the cytoplasmic membranes in A. nidulans. Electrolytesbegan to leak from the cytoplasm into the outer medium, indicatingthat the permeability of the cytoplasmic membranes increasedwhen they entered the phase separation state. In A. variabilis,in which the cytoplasmic membranes had remained in the liquidcrystalline state above 0?C, no irreversible damage to photosynthesisnor leakage of electrolytes was observed between 0 and 20?C.These findings suggest that photosynthesis of the blue-greenalgae is reversibly suppressed when only the thylakoid membranesare in the phase separation state, and irreversibly inactivatedwhen the cytoplasmic membranes are in the phase separation state. (Received April 9, 1984; Accepted June 19, 1984)     

Effect of NaCl and polyethylene glycol on solute content and glycosidase activities during germination of lentil seeds

Abstract Lentil seeds were sown in water and with different concentrations of polyethylene glycol (PEG) 4000 or NaCl. Radicle emergence and growth were delayed by these substances. In cotyledons under stress no variations in solutes occurred, whereas in embryonic axes an accumulation of soluble sugars was observed. The major constituents of the soluble carbohydrates were sucrose, galactose and mannose. Glycosidase activities were not significantly affected in PEG- or NaCl-germinatcd seeds, except axis α-galactosidase, whose activity during axis growth was higher under stress. Water and salt stress did not have a marked effect on carbohydrate metabolism in intact seedlings. The rate of release of ¹⁴C from [6-¹⁴C]-glucose was similar in the stressed seeds and in the control seeds.     

Studies on the effect of certain enzymic poisons on the metabolism of storage organs III. Uptake and utilization of sucrose by radish root slices in presence of iodoacetate

Iodoacetate greatly retarded the uptake of sucrose and slightlyaffected its inversion by radish root slices. Carbohydrate content of the samples decreased substantiallyboth in water and in iodoacetate. Feeding with sucrose led tomarked accumulation of carbohydrates and supplemental additionof iodoacetate induced less accumulation of carbohydrates. Iodoacetate caused exudation of nitrogen fractions into theculture media. Protein synthesis via amino acids seems to beoperative in iodoacetate treated slices. It is also suggestedthat nitrate-N, in presence of sucrose, is converted into peptidesand proteins. Addition of iodoacetate to sucrose media inhibitedthis pathway of protein synthesis. Both sucrose and iodoacetate (4 x 10^–4M) stimulated theCO₂ output whereas 25 x 10^–4M iodoacetate did not changethe CO₂ output when compared with that of controls. Sucroseand iodoacetate (4 x 10^–4 M) when joined together maskedthe accelerating effect of each other. ¹Present address: Department of Botany, Faculty of Science,University of A'in Shams, Abbassia, Cairo, Egypt, U. A. R. (Received November 6, 1968; )     

Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties

The effects were studied of various carbohydrates and osmoticstress, created by high agarose or carbohydrate concentrations,on the regeneration of fertile plants from protoplast-denvedcolonies of several indica (IR43, Jaya, Pusa Basmati 1) andjaponica (Taipei 309) rice varieties. Observations of the culturesdeveloped on media containing one of these carbohydrates (cellobiose,fructose, glucose, lactose, maltose, mannitol, sorbitol or sucrose),each at 88 mM, indicated that maltose was the preferential carbonsource for the proliferation of embryogenic callus and shootregeneration. Maltose-containing medium induced shoot formationin 24–66% of the protoplast-derived tissues, dependingupon the rice variety, compared to shoot regeneration from 4–32%of the tissues in sucrose-supplemented medium. Media containing288 mM maltose or an equimolar combination of 88 mM maltoseand 200 mM mannitol, caused water loss from calli and promotedthe growth of embryogenic calli. These calli formed shoots withgreater frequencies when subsequently transferred to shoot regenerationmedium with 88 mM maltose. A medium containing 88 mM maltoseand semi-solidified with 1.0% (w/v) instead of 0.5% (w/v) agarosehad a similar beneficial effect on the growth of embryogeniccalli and simultaneously supported high-frequency (48–55%)shoot formation. The optimum shoot regeneration frequencies(60–78%) were obtained when protoplast-derived colonieswere serially cultured on to shoot regeneration medium containing1.0% (w/v) agarose for 4 weeks, followed by a 2-week cultureperiod on the same medium with 0.5% (w/v) agarose. Plants regeneratedon medium containing maltose and/or 1.0% (w/v) agarose werephenotypically normal and fertile. Key words: Carbohydrates, Oryza sativa L, indica and japonica rice, osmotic stress, plant regeneration, protoplast-derived colonies     

The light-induced oxidation-reduction reaction of cytochrome b-559 in membrane fragments of the blue-green alga Anabaena variabilis

Light-induced oxidation-reduction reactions of cytochrome b-559were investigated with membrane fragments of Anabaena variabilisand supplementarily with Plectonema boryanum. The oxidation-reduction reactions of cytochrome b-559 observedwith membrane fragments were similar in their kinetics to thoseof the cytochrome in aged chloroplasts. The reactions were annihilatedby the addition of Ferro, indicating that the cytochrome ofhigh redox potential (E'_o=+373 mV, pH 6.5) was photoreducedand oxidized. Titration with reducing agents indicated that cytochrome b-559is contained in Anabaena membrane fragments in an amount 1.5times as much as the content of P700 on a molar basis; the contentof the species of high redox potential was estimated to be around70%. Kinetic treatment of the photoreduction indicated that the cytochromewas reduced at some site of the electron transport system betweenthe two photosystems. The photo-oxidation depended on the actionof either photosystem II or I even in the presence of DCMU,indicating that the photooxidation was induced by both photosystems.The oxidation by photosystem I action was inhibited by HgCl₂-treatment,indicating that this reaction is mediated by plastocyanin. EDTA (5?10^-3 M) suppressed the cytochrome photoreduction andenhanced the rapid phase of the photooxidation. The latter effectappeared only when an appropriate dark time (3 min) was insertedafter the cytochrome photoreduction. The phenomenon was interpretedas showing that EDTA modifies the reactivity of the electroncarrier which directly donates electrons to cytochrome b-559.The oxidation, and probably also the reduction of cytochromeb-559, was assumed to be regulated by the oxidation-reductionstate of this carrier. (Received April 26, 1974; )     

Dependence of wheat callus growth,differentiation and mineral content on carbohydrate supply

The effects of different carbon sources on the growth, differentiation and mineral content of wheat callus were investigated. Callus originating from immature embryos showed optimal growth produced the highest ratio of shoots when it was cultured on the medium containing 0.058 M sucrose. Higher carbohydrate concentrations reduced both shoot formation and growth. On the other hand, when sucrose concentration was less than 0.029 M neither differentiation nor greening was observed. Mannitol had a stimulating effect on shoot formation when the medium containing 0.029 M sucrose was supplemented by mannitol to get the final concentration of 0.058 M. The respiration rate increased along with increasing concentration of sucrose and glucose, and reached a maximum in the case of sucrose at the concentration of 0.263 M. On the addition of different concentrations of mannitol to a 0.058 M sucrose medium the respiration remained essentially unchanged. The mineral content of the tissue cultures also depended on the carbohydrate concentration. The water content decreased with increasing carbohydrate concentrations and among carbohydrates examined, sucrose was the most effective. The nitrogen and potassium contents of the calli reached their maximum values at 0.117 M–0.175 M carbohydrate content. The highest phosphorus contents were detected at 0.350 M–0.468 M carbohydrates. Phosphorus proved to be the most sensitive to osmotic changes.     

The C550 photoresponse at room temperature observed in membrane fragments of the blue-green alga Anabaena variabilis

The C550 photoresponse at room temperature was studied withmembrane fragments of the blue-green alga Anabaena variabilis.The kinetics, light minus dark difference spectrum and DCMUeffect were the same as those reported for spinach chloroplasts.The photoresponse size suggested that the number of the photosystemII center is half that for the photosystem I center in thisorganism grown under our autotrophic culture conditions. (Received September 16, 1975; )     

Carbohydrate requirements for the development of black spruce (Picea mariana (Mill.) B.S.P.) and red spruce (P. rubens Sarg.) somatic embryos

Different carbohydrates were investigated for somatic embryo development of black spruce and red spruce. They were tested in a basal maturation medium consisting of Litvay's salts at half-strength containing 1 g l^-1 glutamine, 1 g l^-1 casein hydrolysate, 7.5 M abscisic acid, and 0.9% Difco Bacto-agar. A comparison of different sucrose concentrations showed that 6% was optimal for embryo development. Among the nine carbohydrates tested, sucrose, fructose, glucose, maltose, and cellobiose supported embryo development while arabinose, mannitol, myo-inositol, and sorbitol did not. A comparison of sucrose, glucose, and fructose at three concentrations showed that the general pattern of response for both species followed concentration expressed as a percentage, independent of the molarity of carbohydrate in the medium. Interspecific differences were observed concerning carbohydrate requirements. For red spruce, 6% fructose was found best for embryo development, while no such preference was observed for black spruce. No significant difference was observed in the number of embryos produced with 6% sucrose or 3% sucrose plus an equimolar concentration of either mannitol, sorbitol, or myo-inositol in the maturation medium, suggesting that the effect of the carbohydrate on the maturation was partly osmotic.     

Investigations into the Role of Sucrose in Potato cv. Estima Microtuber Production in vitro

Sucrose has been the carbohydrate traditionally used for potatomicrotuber production. Added to nutrient media, sucrose canact solely as a carbon source, or as an osmoticum, or both.Preliminary tests showed that the osmolarity of sucrose solutionswas increased by autoclaving, indicating some breakdown of thesugar. This was taken into consideration in experiments whichinvolved supplementing 4% sucrose media with sucrose, maltose,glucose or fructose, while keeping the osmotic potential ofthe media constant. A medium concentration of about 400 mM withonly sucrose was more suitable for microtuber production thanmedia supplemented with maltose, glucose or fructose. However,a better microtuber yield was obtained when hexoses were addedthan with unsupplemented 4% sucrose media. When glucose wassupplied at concentrations which had the same number of carbonatoms as 8% sucrose, the high osmolarity inhibited microtuberisation.Sugar movement in the tubering plantlet was followed using radio-labelledsucrose, glucose and fructose. The sucrose was translocatedand used at a faster rate than the other sugars, which tendedto remain in the roots of the plantlets. Furthermore, therewas no difference in microtuber production on media to whichthe sucrose was added before or after autoclaving, indicatingthat levels of breakdown were not severe enough to affect theprocess. Therefore, it is concluded that sucrose acts primarilyas a suitable carbon source for uptake and utilization by theplantlets, but, at 8%, it also provides a favourable osmolarityfor the development of microtubers.Copyright 1995, 1999 AcademicPress Solanum tuberosum (L.), potato, microtuber, media, sugar, sucrose, osmolarity, pH     

Effects of amino acids on methionine-sulfoximine-induced heterocyst formation in Anabaena

Heterocyst development in ammonia-grown cultures of Anabaena variabilis and Anabaena 7120 was fully induced by the amino-acid analog methionine sulfoximine (MSO) at concentrations of 0.5–1.0 M. Glutamine, glutamate, aspartate, and alanine at 0.5 mM blocked the induction of heterocysts by MSO in A. variabilis. With Anabaena 7120, glutamine and glutamate were fully effective and alanine partially effective in preventing MSO-induced heterocyst formation. In MSO-treated algae, glutamine synthetase activity was reduced to less than 15% of control values within 4–6 h. Inactivation of the enzyme was prevented by all four amino acids tested.