Tetrazolium reduction and nitrogenase activity in heterocystous blue-green algae

Summary Heterocysts reduce triphenyl tetrazolium chloride (TTC) faster than vegetative cells apparently because the absence of the O₂-evolving photosystem II and the high electron transport activity in these cells. Although the rate of TTC reduction in vegetative cells is increased by the continuous removal of O₂ evolved in photosynthesis, it has not been possible to obtain rates of TTC reduction comparable with those in heterocysts probably because of the continued competition for electrons between TTC and O₂. The use of nitro-blue tetrazolium chloride (NBT) as a redox indicator has revealed the presence in filaments under aerobic conditions of a gradient of electron transport activity with strongest reducing power in the heterocysts, proheterocysts and vegetative cells next to heterocysts, and with gradually diminishing activity midway between two heterocysts. This pattern is indistinct in filaments grown under micro-aerophilic conditions. The strong electron transport activity in vegetative cells adjacent to heterocysts appears to promote reducing conditions in the heterocysts. Both, red-formazan formation in the heterocysts and blue-formazan deposition in vegetative cells greatly inhibit nitrogenase activity, and this was adversely affected also by the detachment of heterocysts from vegetative cells. The findings are consistent with the idea that the association of heterocysts with vegetative cells in essential for nitrogen fixation to occur in heterocystous blue-green algae.     

Regeneration of photosystem II and phycobilin pigments in photoorganotrophically grown Anabaena variabilis

Regeneration of photosynthetic activity and phycobilin pigmentswas studied with cells of Anabaena variabilis lacking photosystemII activity and phycobilin pigments. Regeneration was achievedonly when the cells were incubated in the presence of nitrateor nitrite. The addition of ammonium salts or urea was far lesseffective. Nitrate-directed regeneration was independent oflight and inhibited by chlorate. Dark-regenerated cells, however,differed from light-regenerated ones in that the former wereincapable of excitation transfer from phycocyanin to pigmentsystemII chlorophyll a, although they emitted fluorescence of pigmentsystem II chlorophyll a origin, if illuminated by the lightabsorbed by chlorophyll. The regeneration process inAnabaenacells is assumed to consist of two steps: [1] light-independent,nitratesupported synthesis of phycobilin pigments and photosystemII integrity, followed by [2] light-directed formation of excitationtransfer from phycocyanin to pigment system II chlorophyll a.An antibiotic study revealed that the former is associated withprotein synthesis, while the latter isnot. ¹ Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164, Japan. (Received November 19, 1975; )     

Preparation and Characterization of Heat-Stable and Very Active Oxygen-Evolving Photosystem II Particles from the Thermophilic Cyanobacterium, Synechococcus elongatu

Very active and heat-stable oxygen-evolving photosystem II particleswere isolated from the thermophilic cyanobacterium Synechococcuselongatus by treatment of thylakoid membranes with a non-ionicdetergent, sucrose monolaurate (SML). The particles were analyzedin a comparison with photosystem II particles prepared withß-octylglucoside (OG). The two preparations had similarpolypeptide compositions, which were caracterized by high levelsof polypeptides from phycobilisomes. The ratio of chlorophylla to QA was 45 and there were four Mn atoms and one tightlybound Ca²⁺ ion per QA in the particles prepared with SML. Thepreparations were thermophilic, showing substantial rates ofoxygen evolution at temperatures up to 60°C. The maximumrates attained at 45°C were as high as 6.0 mmoles O₂ mg^–1Chl h^–1. PS II particles prepared with OG were similarlythermostable but were less active in oxygen evolution at alltemperatures examined. Kinetic analysis of flash-induced absorptiontransients revealed that about 22% and 28% of photosystem IIreaction centers were not associated with the functional QBsite in the SML- and OG-particles, respectively. When correctedfor the inactive reaction centers, the maximum rates of oxygenevolution by SML- and OG-particles were 7.7 and 7.0 mmoles O₂mg^–1 Chl h^–1, which correspond to half times of1.9 and 2.1 ms for the first-order electron transfer, respectively.Comparison of these half times with those of the S-state transitionand the release of oxygen indicates that the overall photosystemII electron transport is limited by the reduction of added electronacceptors and not by release of oxygen. ³On leave from National Chemical Laboratory for Industry, Higashi1-1, Tsukuba, Ibaraki 305     

Inhibition of the Chloroplast Photochemical Reactions by Treatment of Wheat Seedlings with Low Concentrations of Cadmium: Analysis of Electron Transport Activities and Changes in Fluorescence Yield

The effect of treatment of wheat plants with Cd²⁺ ions on thephotochemical activity of the primary leaves was examined. Threeday-old etiolated seedlings were treated with Cd²⁺ ions for24 h in dark, and after this treatment the plants were grownin the light until the primary leaves were fully developed.Cd²⁺ ions (30–120 µM) induced a significant decreasein activities of both photosystem II and photosystem I. Theextent of the decrease in PS II activity was much greater thanthat in the PS I activity. Analysis of changes in the fluorescenceyield of chlorophyll also indicated that Cd²⁺ ions drasticallyaffect the photochemistry of photosystem II. Cd²⁺ ions induceddecrease in the rates of photoreduction of 2,6-dichlorophenolindophenol even in the presence of the exogenous electron donor,hydroxylamine, both in Tris-treated and untreated chloroplasts.This result suggests that the site of inhibition is near thesite of donation of electrons by hydroxylamine. Treatment withCd²⁺ ions impairs the electron transport system on the reducingside of PS II. The decrease in the fluorescence yield of Chi is less than that in the evolution of O₂ mediated by oxidizedphenylenediamine. This difference may be a result of inhibitionon the reducing side of PS II. In addition to inhibition onthe reducing side, Cd²⁺ ions may affect the oxidizing side ofPS II. A comparative study of the rates of evolution of O₂ withp-benzoquinone and dichloro-p-benzoquinone as electron acceptorswas performed since the halogenated benzoquinones have beenshown to accept electrons from both active and inactive centersof photosystem II while some of the benzoquinones accept electronsonly from active centers. The results suggest that Cd²⁺ ionsinduced a marginal increase in the number of inactive reactioncenters in PS II. Analysis of light-saturation-kinetics of theevolution of O₂ catalysed by PS II indicates a reduction inthe size of the antennae as well as in the concentration ofthe active (-type) reaction centers of PS II. Thus, the Cd²⁺-inducedeffects on the photochemistry of PS II involve changes on thereducing side of PS II as well as possible changes in the sizesof the populations of active and inactive centers. Thus, short-termexposure to Cd²⁺ ions during establishment of seedlings hasa severely detrimental effect on photochemical activities inchloroplasts. (Received October 17, 1990; Accepted July 3, 1991)     

Effects of Leaf Chilling on Thylakoid Functions, Measured at Room Temperature, in Cucumis sativus L. and Oryza sativa L.

Photosynthetic functions in leaves of cucumber (Cucumis sativusL.) and rice (Oryza sativa L.) were examined before and aftervarious chilling treatments. Cucumber leaves lost the capacityfor the photosynthetic oxygen evolution after chilling at 0°Cin the dark for 48 h. Thyla-koids isolated from such leaveswere not able to reduce dichloroindophenol (DCIP), but the additionof diphenylcarbazide (DPC), an electron donor to PS II, restoredthe ability to reduce DCIP, indicating that the site of damageis in the water-splitting machinery of PS II. In moderate light (500 jumol quanta m^–2s^–1), chillingof cucumber leaves at 5°C for 5 h was sufficient to inducethe complete loss of the capacity for photosynthetic oxygenevolution. Electron transport rates measured in thylakoids wereunaltered, but thylakoids were totally permeable to protons.Since the addition of dicyclohexylcarbodiimide (DCCD) restoredcoupling and the capacity for proton uptake, the primary siteof damage was deduced to be in the ATPase. In rice, both chilling treatments had barely any effect on thylakoidfunctions, although some negative effects was apparent in photosynthesisin leaves. ¹Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113 Japan. ²Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received January 11, 1989; Accepted June 12, 1989)     

Lipid-Linked Desaturation of Palmitic Acid in Monogalactosyl Diacylglycerol in the Blue-Green Alga (Cyanobacterium) Anabaena variabilis Studied in Vivo

The mechanism of desaturation of palmitic acid in monogalactosyldiacylglycerol in Anabaena variabilis was studied by labelingin vivo with ¹³C and mass spectrometry. When the cells werefed with [¹³C]Na₂CO₃ for 2.5 h, 19% of the palmitic, but virtuallynone of the palmitoleic, acid at the C-2 position of the lipidwas enriched with ¹³C. During subsequent incubation for 7.5h, the [¹³C]palmitic acid was desaturated to [¹³C]palmitoleicacid. Mass spectrometric analysis of the 2-acylglycerol moietyof the lipid indicated that [¹³C]palmitoyl-[¹³C]glycerol and[¹²C]palmitoyl-[¹²C]glycerol were converted to [¹³C]palmitoleoyl-[¹³C]glyceroland [¹²C]palmitoleoyl-[¹²C]glycerol, respectively. These resultssuggest that the palmitic acid was converted to palmitoleicacid in vivo by lipid-linked desaturation but not via a pathwayconsisting of deacylation, desaturation and reacylation. ⁴Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan ⁵Present address: Department of Physiological Chemistry andNutrition, Faculty of Medicine, University of Tokyo, Hongo,Tokyo 113, Japan (Received December 7, 1985; Accepted April 16, 1986)     

Formation of singlet molecular oxygen in illuminated chloroplasts. Effects on photoinactivation and lipid peroxidation

The formation of singlet molecular oxygen (¹O₂) in illuminatedchloroplasts and the effects of ¹O₂ on oxidation or destructionof components and functional integrity of chloroplasts werestudied. The rate of photoreduction of 2,6-dichloroindophenol(DCIP) and the extent of the 515-nm absorbance change were decreasedby light irradiation and by xanthine oxidase treatment. Malondialdehyde(MDA) formation, an indicator of lipid peroxidation, was observedin the light-irradiated chloroplast fragments, but not in thexanthine-xanthine oxidase-treated chloroplast fragments. MDAformation was absent under anaerobic conditions. MDA formation was stimulated when electron transfer on the oxidizingside of photosystem II (or I) was inhibited or inactivated bycarbonylcyanide m-chlorophenylhydrazone (CCCP), Tris-treatment,prolonged illumination, etc. MDA formation was also stimulatedby 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) when electrontransfer between water and the reaction center of photosystemII was intact. CCCPor DCMU-stimulated MDA formation was inhibitedby 1,4-diazabicyclo[2.2.2]octane, a quencher of singlet molecularoxygen (¹O₂). DCMU and electron donors for photosystem II, suchas ascorbate, hydroquinone and semicarbazide, inhibited MDAformation by illumination of the Tris-washed or CCCP-poisonedchloroplast fragments. Reduced DCIP, an electron donor for photosystemI, also inhibited MDA formation in the presence of DCMU. These results lead to the conclusion that MDA formation wasinitiated by ¹O₂ formed in illuminated chloroplasts. Of thethree mechanisms discussed for ¹O₂ generation in illuminatedchloroplasts, the formation by the electron transfer reactionbetween superoxide anion radical and the oxidant formed on theoxidizing side of photosystem II (or I) is mostimportant. (Received March 31, 1975; )     

Site of Nitrogenase Activity in the Blue-Green Alga <Emphasis Type="Italic">Anabaena</Emphasis> sp. L-31

MANY blue-green algae fix nitrogen, assimilate carbon dioxide and evolve oxygen and as algal nitrogenase is inhibited^1–3 by high oxygen pressure, enhanced nitrogen fixation accompanying photosynthesis is surprising. Heterocysts do not contain⁴ or have comparatively less amounts^4–7 of photosystem II (PS II) pigments, which are responsible for the evolution of oxygen. This tends to favour the suggestion of Fay et al.⁸ that these cells are the sites of nitrogenase activity. Until now, however, attempts at obtaining unequivocal evidence for heterocysts as principal loci for nitrogenase activity have yielded conflicting results. Stewart et al.⁷ first demonstrated nitrogenase activity in heterocysts incubated aerobically, a finding confirmed by Wolk and Wojciuch⁹ and Van Gorkom and Donze¹⁰. By contrast, Smith and Evans^3,11 and Kurz and La Rue¹² reported results favouring vegetative cells as the major site of nitrogenase activity. Other evidence^2,13 showed high nitrogenase activity in cell-free preparations of Anabaena cylindrica and the non-heterocystous alga Plectonema boryanum strain 594.     

Excitation-energy transfer in heterocysts isolated from the cyanobacterium Anabaena sp. PCC 7120 as studied by time-resolved fluorescence spectroscopy

Heterocysts are formed in filamentous heterocystous cyanobacteria under nitrogen-starvation conditions, and possess a very low amount of photosystem II (PSII) complexes than vegetative cells. Molecular, morphological, and biochemical characterizations of heterocysts have been investigated; however, excitation-energy dynamics in heterocysts are still unknown. In this study, we examined excitation-energy-relaxation processes of pigment-protein complexes in heterocysts isolated from the cyanobacterium Anabaena sp. PCC 7120. Thylakoid membranes from the heterocysts showed no oxygen-evolving activity under our experimental conditions and no thermoluminescence-glow curve originating from charge recombination of S₂Q_A^?. Two dimensional blue-native/SDS-PAGE analysis exhibits tetrameric, dimeric, and monomeric photosystem I (PSI) complexes but almost no dimeric and monomeric PSII complexes in the heterocyst thylakoids. The steady-state fluorescence spectrum of the heterocyst thylakoids at 77 K displays both characteristic PSI fluorescence and unusual PSII fluorescence different from the fluorescence of PSII dimer and monomer complexes. Time-resolved fluorescence spectra at 77 K, followed by fluorescence decay-associated spectra, showed different PSII and PSI fluorescence bands between heterocysts and vegetative thylakoids. Based on these findings, we discuss excitation-energy-transfer mechanisms in the heterocysts.     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

O-Linked fucose in glycoproteins from Chinese hamster ovary cells

We report our discovery that many glycoproteins synthesizedby Chinese hamster ovary (CHO) cells contain fucose in O-glycosidiclinkage to polypeptide. To enrich for the possible presenceof O-linked fucose, we studied the lectin-resistant mutant ofCHO cells known as Lec1. Lec1 cells lack N-acetylglucosaminyltransferaseI and are therefore unable to synthesize complex-type N-linkedoligosaccharides. Lec1 cells were metabolically radiolabelledwith [6-³H]fucose and total glycoproteins were isolated. Glycopeptideswere prepared by proteolysis and fractionated by chromatographyon a column of concanavalin A (Con A)— Sepharose. Thesets of fractionated glycopeptides were treated with mild base/borohydrideto effect the ß-elimination reaction and release potentialO-linked fucosyl residues. The ß-elimination produced[³H]fucitol quantitatively from [³H]fucose-labelled glycopeptidesnot bound by Con A-Sepharose, whereas none was generated bytreatment of glycopeptides bound by the lectin. The total [³H]fucose-labelledglycoproteins from Lec1 cells were separated by SDS—PAGEand detected by fluorography. Treatment of selected bands ofdetectable glycoproteins with mild base/borohydride quantitativelygenerated [³H]fucitol. Pretreatment of the glycoproteins withN-glycanase prior to the SDS—PAGE method of analysis causedan enrichment in the percentage of radioactivity recovered as[³H]fucitol. Trypsin treatment of [³H]fucose-labelled intactCHO cells released glycopeptides that contained O-linked fucose,indicating that it is present in surface glycoproteins. Thesefindings demonstrate that many glycoproteins from CHO cellscontain O-linked fucosyl residues and raise new questions aboutits biosynthesis and possible function. fucose glycoproteins monosaccharide O-linked     

Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets

Human and rabbit platelets fully aggregated byplatelet-activating factor (PAF) underwent slow disaggregation but wererapidly disaggregated by the PAF receptor antagonists WEB-2086,Y-24180, SM-12502, and CV-3988. Whereas the1-alkyl-2-[³H]acetyl-sn-glycero-3-phosphocholine([³H]acetyl-PAF)specifically bound to platelet receptors underwent slow and spontaneousdissociation, it dissociated promptly from its receptor when WEB-2086was added, in parallel with platelet disaggregation and disappearanceof P-selectin on the cell surface. Extracellular[³H]acetyl-PAF wasrapidly deacetylated by normal rabbit platelets; some of the[³H]acetyl-PAF wasbound to the cells and a very small amount of [³H]acetate wasdetected in the cells. In contrast, when1-[³H]alkyl-2-acetyl-sn-glycero-3-phosphocholinewas added to the platelets, the radioactivity was rapidly incorporatedinto the 1-alkyl-2-acyl-sn-glycero-3-phosphocholinefraction. These results indicate that1) continuous binding of PAF to itsreceptor is necessary for prolonged platelet aggregation, which may bemediated through an unknown signaling system for a long-term cellresponse rather than a transient signaling system, and2) most of the[³H]acetyl-PAF boundto platelets is metabolized extracellularly by ecto-type PAFacetylhydrolase, with the lyso-PAF generated being incorporated rapidlyinto the cells and converted to1-alkyl-2-acyl-sn-glycero-3-phosphocholine.

     

Nitrogen fixation and heterocysts in the blue-green alga Anabaena cylindrica

The site of nitrogen fixation in the blue-green alga Anabaenacylindrica Lemra (Fogg strain) was investigated. Less than 4%of the total nitrogen fixed during a relatively short period(5-15 min) was recovered in heterocysts. When estimated on thecellular nitrogen basis, vegetative cells can fix molecularnitrogen at the same rate as do heterocysts. There was no positivecorrelation between nitrogen fixation and heterocyst formation.Results do not support the hypothesis that the heterocyst isthe main site for nitrogen fixation in blue-green algae. ¹ This work was supported by grant (No. 38814) from the Ministryof Education. (Received July 23, 1971; )     

Quantum yield and conformational changes during greening and bleaching of Chlorella protothecoides

Measurements of quantum requirement of oxygen evolution in greeningand bleaching cultures of Chlorella proiothecoides reveal aconstant low-quantum requirement during greening and the firsthours of bleaching. Thereafter the values increase drastically. The light-induced "conformational change," measured as straylight-dependent absorbance change, is biphasic; the second partof die signal is due to the absorbance changes caused by theshrinking of the chloroplast. Its value was used as a measureof photophosphorylation, which follows, after a certain delay,the photosynthetic oxygen evolution during greening and bleachingofthe cells. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, 113 Tokyo, Japan. (Received January 27, 1976; )     

Biosynthetic mechanism of glycolate in Chromatium IV. Glycolate-glycine transformation

The metabolic transformation of glycolate to glycine occurringin photosynthesizing cells of Chromatium was investigated bythe radioisotopic technique and by amino acid analysis. By analyzingthe distribution of radiocarbon upon feeding [1-¹⁴C] glycolate,[2-¹⁴C] glyoxylate and [1-¹⁴C] glycine to bacterial cells, itwas demonstrated that glycolate is converted to glycinc viaglyoxylate, and both glycolate and glycine are excreted extracellularly.Although the formation of serine was barely detected by theabove two techniques in both N₂ and O₂ atmospheres, it was foundthat ¹⁴CO₂ is evolved quite markedly from both [1-¹⁴C] glycolateand [1-¹⁴C] glycine fed to the Chromatium cells. Analyticalresults of transient changes in amino acid compositions underatmospheric changes of N₂O₂ and by the addition of exogenousglycolate in N₂ confirm the notion that glycolate is convertedto glycine. Acidic amino acids (glutamic acid and aspartic acid)appear to take part in glycine formation as amino donors. Theformation of glycine from glycolate in a N₂ atmosphere suggeststhat an unknown glycolate dehydrogenation reaction may operatein the overall process. ¹ This is paper XXXVII in the series ‘Structure and Functionof Chloroplast Proteins’. Paper XXXVI is ref. (5). Theresearch was supported in part by grants from the Ministry ofEducation of Japan (No. 111912), the Toray Science Foundation(Tokyo) and the Naito Science Foundation (Tokyo). (Received July 14, 1976; )     

Regulation of photosynthesis during heterocyst differentiation in Anabaena sp. strain PCC 7120 investigated in vivo at single-cell level by chlorophyll fluorescence kinetic microscopy

Changes of photosynthetic activity in vivo of individual heterocysts and vegetative cells in the diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 during the course of diazotrophic acclimation were determined using fluorescence kinetic microscopy (FKM). Distinct phases of stress and acclimation following nitrogen step-down were observed. The first was a period of perception, in which the cells used their internally stored nitrogen without detectable loss of PS II activity or pigments. In the second, the stress phase of nitrogen limitation, the cell differentiation occurred and an abrupt decline of fluorescence yield was observed. This decline in fluorescence was not paralleled by a corresponding decline in photosynthetic pigment content and PS II activity. Both maximal quantum yield and sustained electron flow were not altered in vegetative cells, only in the forming heterocysts. The third, acclimation phase started first in the differentiating heterocysts with a recovery of PS II photochemical yields $F_{\text{v}} /F_{\text{m}} ,\;F^{\prime}_{\text{v}} /F^{\prime}_{\text{m}}.$ F v / F m , F v ′ / F m ′ . Afterwards, the onset of nitrogenase activity was observed, followed by the restoration of antenna pigments in the vegetative cells, but not in the heterocysts. Surprisingly, mature heterocysts were found to have an intact PS II as judged by photochemical yields, but a strongly reduced PS II-associated antenna as judged by decreased F ₀. The possible importance of the functional PS II in heterocysts is discussed. Also, the FKM approach allowed to follow in vivo and evaluate the heterogeneity in photosynthetic performance among individual vegetative cells as well as heterocysts in the course of diazotrophic acclimation. Some cells along the filament (so-called “superbright cells”) were observed to display transiently increased fluorescence yield, which apparently proceeded by apoptosis.     

Carbon-specific phytoplankton growth rates: a comparison of methods

Measurements of biomass and growth rate of two axenic algalcultures were carried out using three different methodologicalapproaches: the specific ¹⁴C-labelling of chlorophyll a, [³H]adenineincorporation into DNA and net organic carbon assimilation.Time-course experiments revealed that the specific activitiesof chlorophyll a were significantly higher than the specificactivity of total algal carbon in six of seven experiments.When the specific activity of chlorophyll a is used to calculatethe carbon biomass and growth rate, the carbon biomass of thealgae will thus be underestimated and the specific growth ratewill be too high. Determination of growth rates from incorporationof [³H]adenine gave lower values than those obtained from netorganic carbon assimilation and from ¹⁴C incorporation intochlorophyll a. Problems with adenine saturation are suggested.When [³H]adenine is used to measure growth rates in dense algalcultures, additions of >1 µM [³H]adenine are oftenrequired to maximally label the extracellular and intracellularadenine pools and hence DNA.     

Comparative Network Biology Discovers Protein Complexes That Underline Cellular Differentiation in Anabaena sp.

The filamentous cyanobacterium Anabaena sp. PCC 7120 can differentiate into heterocysts to fix atmospheric nitrogen. During cell differentiation, cellular morphology and gene expression undergo a series of significant changes. To uncover the mechanisms responsible for these alterations, we built protein–protein interaction (PPI) networks for these two cell types by cofractionation coupled with mass spectrometry. We predicted 280 and 215 protein complexes, with 6322 and 2791 high-confidence PPIs in vegetative cells and heterocysts, respectively. Most of the proteins in both types of cells presented similar elution profiles, whereas the elution peaks of 438 proteins showed significant changes. We observed that some well-known complexes recruited new members in heterocysts, such as ribosomes, diflavin flavoprotein, and cytochrome c oxidase. Photosynthetic complexes, including photosystem I, photosystem II, and phycobilisome, remained in both vegetative cells and heterocysts for electron transfer and energy generation. Besides that, PPI data also reveal new functions of proteins. For example, the hypothetical protein Alr4359 was found to interact with FraH and Alr4119 in heterocysts and was located on heterocyst poles, thereby influencing the diazotrophic growth of filaments. The overexpression of Alr4359 suspended heterocyst formation and altered the pigment composition and filament length. This work demonstrates the differences in protein assemblies and provides insight into physiological regulation during cell differentiation.     

COMPARATIVE STUDIES OF PHOTOCHEMICAL OXIDATION-REDUCTION REACTIONS IN LAMELLAR FRAGMENTS OF VARIOUS ALGAE AND SPINACH

The activity of various electron carriers, including DPIP, spinachplastocyanin, mammalian cytochrome c, and Anabaena cytochrome553, as donor in the reaction induced by the photochemical systemI was examined with lamellar fragments of various algae andspinach. Reduced DPIP was an effective electron donor irrespective ofthe organisms, when it was supplied at a high concentration(10^–3 M). Spinach plastocyanin was effective in the reactionswith the lamellae of green algae, Euglena, diatom Phaeodactyrumand red algae Porphyra yezoensis and Porphyra sp. Yamamoto II,whereas it was inactive in the lamellae of blue-green algae.Horse-heart cytochrome c and Anabaena cytochrome 553 were activein the reaction with the lamellae of bluegreen algae. The formercytochrome was also active in the reactions in Porphyridiumand Cyanidium. The cytochromes were less active in the reactionsin which spinach plastocyanin acted as effective electron donor. The data were interpreted as that the photochemical system Iin bluegreen algae differs from that of other photosyntheticorganisms with respect to the properties of the site of theelectron-input. ¹ Present address: Nomura Research Institute for Technologyand Economics, Kamakura, Kanagawa. ² Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo.     

Formation of a series of myo-inositol phosphates during growth of rice plant cells in suspension culture

A series of myo-inositol phosphates including myo-inositol mono-to hexa-phosphates was observed during growth of cultured riceplant cells. We also found that ³²Pi and myo-[2-³H] inositolwere incorporated into all these myo-inositol phosphates. myo-Inositolphosphorylating activity, which depended on ATP and Mg²⁺, wasdetected in the soluble fraction from the cells, and the reactionproduct was identified as myo-inositol-2-phosphate. (Received January 21, 1980; )