Loss of photosystem II induced by a nitrate deficiency in photoorganotrophically grown Anabaena variabilis

When photoorganotrophically trained cells of Anabaena variabiliswere grown in nitrate-free medium, they lost the activity ofphotosynthetic oxygen evolution and became devoid of phycobilinpigments. These cells (H cells) lacked the fluorescence emissioncharacteristic of photosystem II chlorophyll, and their lamellarfragments failed to photoreduce DPIP even in the presence ofdiphenylcarbazide as the electron donor, suggesting that theloss of photosynthetic oxygen evolution in H cells is primarilydue to degeneration of an integral part of photosystem II. These characteristics of H cells closely resembled those ofheterocysts differentiated from normal, vegetative cells in[i] the deficiency of phycobilin pigments, [ii] the loss ofphotosystem II activity, [iii] the photoorganotrophic mode ofcell growth, depending upon the organic substances furnishedexternally or provided from neighboring vegetative cells, and[iv] the manner of transformation from normal cells, both typesof cells being induced in the absence of nitrate. In spite ofsuch similarity, light and electron microscopic observationsrevealed that H cells differed significantly from heterocysts.Furthermore, the readiness with which H cells resumed photosystemII activity and the independence of this resumption from cellgrowth exclude the possibility that the nutritional enrichmentof heterocysts was responsible for the loss of photosyntheticactivity. ¹Present address: Ocean Research Institute, University of Tokyo,Nakano, Tokyo 164. (Received May 14, 1975; )     

Incompetence of dark-synthesized phycocyanin in excitation transfer to photosystem II chlorophyll

Summary When cells of Anabaena variabilis, all the phycobilin pigments of which had been newly synthesized in the dark, were excited by light absorbed in phycocyanin, the fluorescence emission spectrum showed a peak corresponding to the emission from allophycocyanin, but no emission from chlorophyll. These cells were active in photosynthesis and, when excited by light absorbed by chlorophyll, the emitted fluorescence was characteristic of photosystem II chlorophyll. This indicates that dark synthesized phycocyanin is capable of excitation transfer to allophycocyanin but not to photosystem II chlorophyll.Abbreviation CMU 3-(p-chlorophenyl)-1,1-dimethylurea     

Mechanism of the light state transition in photosynthesis. IV. Picosecond fluorescence spectroscopy of Anacystis nidulans and Porphyridium cruentum in state 1 and state 2 at 77 K

The sequential energy-transfer pathway through the phycobilin pigments to chlorophyll a was investigated as a function of the state transition in the cyanobacterium Anacystis nidulans and the red alga Porphyridium cruentum. The fluorescence decay kinetics of the phycobilin pigments and chlorophyll a were determined for cells frozen at 77 K in state 1 and state 2 using a single-photon timing fluorescence spectroscopy apparatus with picosecond resolution. Time-resolved 77 K fluorescence emission spectra were also obtained for both species in state 1 and state 2. In both A. nidulans and P. cruentum the transition to state 1 was accompanied by a large increase in the apparent fluorescent lifetime of chlorophyll a associated with PS II (emission peak at 695 nm). There were smaller increases in the lifetime of the terminal phycobilin emitter (685 nm) in both species and no change in phycocyanin (645 nm) or allophycocyanin (660 nm). Time-resolved spectra showed sequential emission from phycocyanin, allophycocyanin, the terminal phycobilin emitter and chlorophyll a. Spectral red shifts were observed with time for all emission peaks with the exception of the terminal phycobilin emitter. In A. nidulans this peak showed a small blue shift with time. The results are interpreted as evidence for an effective uncoupling of PS II chlorophyll a from subsequent energy transfer to PS I chlorophyll a upon transition to state 1. Our recently proposed model for the mechanism of the state transition in phycobilisome-containing organisms is discussed in terms of a decrease in the energy transfer overlap between PS II chlorophyll a and PS I chlorophyll a in state 1.     

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; )     

Spectral effectiveness in chlorophyll and 5-aminolevulinic acid formation during regreening of glucose-bleached cells of Chlorella protothecoides

Regreening of glucose-bleached cells of Chlorella protothecoidesis stimulated by light. Spectral effectiveness in the processshowed maxima around 370, 440 and 480 nm, suggesting a flavoproteinas primary photoreceptor. Action spectra of ALA synthesis provedto be similar to those of chlorophyll formation, indicatingthat light stimulation of greening in this alga is regulatedat the first step of chlorophyll biosynthesis. ¹ Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo 113, Japan. (Received March 27, 1978; )     

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)     

EFFECT OF CHROMATIC LIGHTS ON PHYCOBILIN FORMATION IN A BLUE-GREEN ALGA, TOLYPOTHRIX TENUIS

1. The formation of phycobilin pigments in a blue-green alga Tolypothrixtenuis was investigated with special reference to the effectsof preillumination with colored lights.
2. It was discoveredthat the algal cells are capable of formingphycobilin pigmentsin the dark, if they have been previouslyilluminated for severalhours in the presence of CO₂.
3. The color of light applied inthe later period of preillumination(chromatic illumination)was found to affect the ratio of phycoerythrinto phycocyaninformed in the subsequent dark period. A greenlight acceleratesthe dark-formation of phycoerythrin, a redlight that of phycocyanin,and the two lights counteractingwith each other in their effects.
4. These directive effects of the "chromatic illumination" canbe accomplished within a very short period, for instance, in3 minutes if it is preceded by sufficient "preillumination"with an incandescent or day light fluorescent light. The reactionsoccurring during the period of chromatic illumination does notrequire the presence of CO₂ and the aerobic condition.
5. Thealga can be grown heterotrophically when supplied with casaminoacids and glucose. Under such a condition the alga forms phycocyanintogether with chlorophyll and carotenoids, but not phycoerythrin.
6. On the basis of the results obtained, a tentative scheme forthe biosynthesis of phycobilin pigments in the alga was proposed,assuming the light-induced formation of unknown precursors whichare converted into phycocyanin and phycoerythrin in the subsequentdark period.

(Received July 4, 1960; )     

Acclimation of Tomato Leaves to Changes in Light Intensity; Effects on the Function of the Thylakoid Membrane

When young tomato plants grown in high light (400 µmolquanta m^–2s^–1 PAR) were transferred to low light(100 µmol quanta m^–2s^–1 PAR), non-cyclic electrontransport capacity was decreased and the rate of dark re-oxidationof Q^–, the first quinone electron acceptor of photosystemII, was decreased within 1–2 d. In contrast, the amountof coupling factor CF₁, assayed by its ATPase activity, decreasedmore gradually over several days. The total chlorophyll contentper unit leaf area remained relatively constant, although thechlorophyll a/chlorophyll b ratio declined. When young tomato plants grown in low light were transferredto high light, the ATPase activity of isolated thylakoids increasedmarkedly within 1 d of transfer. This increase occurred morerapidly than changes in chlorophyll content per leaf area. Inaddition, in vivo chlorophyll fluorescence induction curvesindicate that forward electron transfer from Q^– occurredmore readily. The functional implications of these changes arediscussed. Key words: Tomato, leaves, light intensity, thylakoid membrane     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Formation of protochlorophyll(ide) in wild type and mutant C-2A' cells of Scenedesmus obliquus

Protochlorophyll(ide) was isolated from dark-grown wild typeand mutant C-2A' cells of Scenedesmus obliquus after dark incubationwith 5-aminolevulinate. Proto-chlorophyll(ide) was detectedin mutant cells grown heterotrophically at 29°C or at 21°C.At the latter temperature chlorophyll synthesis was significant.Regulation of chlorophyll synthesis in algae is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otsuka, Hachioji, Tokyo 192-03, Japan. (Received July 14, 1980; )     

Post-transcriptional Control of Nitrate Reductase Formation in Green Algae

Cycloheximide (2·0 µg ml^–1) inhibits theincorporation of [¹⁴C]phenylalanine and [¹⁴C]adenine into insolublecompounds in Ankistrodesmus braunii. 6-Methylpurine (1·0mM) inhibits only the incorporation of [14C]adenine and it isconcluded that it inhibits RNA synthesis. When ammonium-growncells of Ankistrodesmus or Chlorella are nitrogen-starved orwhen ammonium-grown cells of Dunalitlla are resuspended in nitratemedium, the appearance of nitrate reductase in these organismsis not inhibited by 6-methylpurine. The appearance of nitratereductase activity in Ankistrodesmus or Chlorella is inhibitedby 6-methylpurine when ammonium-grown organisms are preincubatedwith this substance for 1-2 h before nitrogen starvation. Itis concluded that cells growing with ammonium and lacking nitratereductase activity nevertheless contain preformed mRNA for nitratereductase synthesis.     

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.     

Studies on chlorophyll formation in Chlorella protothecoides III. Effects of chloramphenicol, cycloheximide, and ethionine on chlorophyll formation

Effects of chloramphenicol, cycloheximide, puromycin and ethionineon the light-independent and subsequent light-dependent processesof chlorophyll formation in "glucose-bleached" cells of Chlorellaprotothecoides were studied. These substances, except puromycin,strongly suppressed different phases of chlorophyll formation.Ethionine most strongly suppressed the light-independent phaseand chloramphenicol an early, relatively short process in thelight-dependent phase of chlorophyll formation. Cycloheximideseverely suppressed all phases of chlorophyll formation. Possibleimplications of these results for the biosynthesis of chlorophyllin algal cells are discussed. ¹ Present address: National Food Research Institute, Ministryof Agriculture and Forestry, Koto-ku, Tokyo 135, Japan. ² Laboratory of Entomology, Faculty of Agriculture, TamagawaUniversity, Machida-shi, Tokyo, Japan (Received October 5, 1972; )     

Studies on the Formation of Phycocyanin, Porphyrins, and a Blue Phycobilin by Wild-Type and Mutant Strains of Cyanidium caldarium

The synthesis of chlorophyll a and the bile-pigment and protein moieties of phycocyanin were arrested in illuminated cells of Cyanidium caldarium, strain III-D-2, incubated with chloramphenicol, ethionine, p-fluorophenylalanine, and p-chloromercuribenzoate. Pigment synthesis was similarly retarded in illuminated cells provided with nutrient medium lacking nitrogen.

Porphobilinogen, porphyrins, and a blue phycobilin were excreted into the nutrient medium by illuminated and unilluminated cells of wild-type and mutant C. caldarium strains incubated with δ-aminolevulinic acid in darkness. Pigment production from δ-aminolevulinic acid was sensitive to treatment with chloramphenicol and ethionine.

Cells of C. caldarium excreted 7 red-fluorescing porphyrins into the suspending medium during incubation with δ-aminolevulinic acid. Three of these porphyrins were identified as uroporphyrin III, coproporphyrin III, and protoporphyrin on the basis of their spectral properties and by paper chromatogaphy with standards.

The blue phycobilin was characterized spectrally and compared with biliverdin. The algal phycobilin displayed properties of a pigment with a violin-type structure. The phycobilin may be an immediate precursor of phycocyanobilin, the phycocyanin chromophore, or identical to it.

     

Phytochrome Control of Its Own Synthesis in Pisum sativum

An analysis of phytochrome synthesis in Pisum seedlings by measuringthe activity of polysomal polyadenylated RNA (poly-A⁺-RNA) codingfor phytochrome apoprotein showed phytochrome control of itsown synthesis; brief red-light irradiation of pea seedlingsinhibited the activity of the RNA, and the red-light effectwas red/far-red reversible. ⁴ Permanent address: Biology Department, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. (Received August 13, 1984; Accepted September 17, 1984)     

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; )     

Enhancing effects of CO2 on chloroplast regeneration in glucose-bleached cells of Chlorella protothecoides II. Role of carbamylphosphate in chloroplast regeneration

Levels of the activities of glutamine-dependent carbamylphosphatesynthetase, ornithine-and aspartate-transcabamylase and phosphoenolpyruvatecarboxylase were followed in greening cells of Chlorella prolothecoides.Among the enzymes examined the activity of carbamylphosphatesynthetase was extremely low, especially at the early phaseof greening. Arginine (but not ornithine or aspartate), when administeredto algal cells at the 24th hour of greening, stimulated thesyntheses of RNA, protein and chlorophyll in the subsequentperiod. It also affected the metabolic pathway of the ¹⁴CO₂supplied simultaneously with arginine in the presence of CMU.Arginine produced a decreased incorporation of ¹⁴C into proteinand an increased incorporation into nucleic acid. The mechanismof the action of CO₂ on chloroplast regeneration is discussed.We concluded that chloroplast regeneration in glucose-bleachedcells is limited by the synthesis of carbamylphosphate, especiallyin the early phase of greening. (Received August 19, 1975; )     

ABA Increases the Desiccation Tolerance of Photosynthesis in the Afromontane Understorey Moss Atrichum androgynum

The effect of pretreatment with abscisic acid (ABA) on the physiologyof the moss Atrichum androgynum during a desiccation–rehydrationcycle was examined. During rehydration following desiccationfor 16 h, net CO₂fixation recovered much more slowly than photosystemII (PSII) activity, conditions conducive to the formation ofreactive oxygen species (ROS) in the photosynthetic apparatus.Pretreatment with ABA increased the rate of recovery of photosynthesisand PSII activity, and also doubled non-photochemical quenching(NPQ). Increased NPQ activity will reduce ROS formation, andmay explain in part how ABA hardens the moss to desiccation.In ABA-pretreated, but not untreated mosses, desiccation significantlyincreased the concentration of soluble sugars. Sugar accumulationmay promote vitrification of the cytoplasm and protect membranesduring desiccation. Starch concentrations in freshly collectedA. androgynum were only approx. 40 mg g^-1dry mass; they roseslightly during desiccation but were only slightly affectedby ABA pretreatment. ABA did not reduce chlorophyll breakdownduring desiccation. Copyright 2001 Annals of Botany Company Moss, desiccation, abscisic acid, photosynthesis, chlorophyll fluorescence     

{beta}-1,4-Glucan occurring in homogenate of Phaseolus aureus seedlings. Possible nascent stage of cellulose biosynthesis in vivo

A small amount of cytoplasmic ß-1,4-glucan, whichmight be involved in the synthesis of cellulose in the cellwall, was found in the homogenate prepared from the hypocotylsof seedlings of Phaseolus aureus. Upon hydrolysis by cellulaseof the 20,000?g pellet from the cytoplasmic fraction of segmentsincubated in a [¹⁴C]-glucose solution, [¹⁴C]-cellobiose wasproduced, with specific radioactivities 3 to 10 times greaterthan those of the cellobiose from cellulose in the cell wallat various incubation periods. The incoporation of radioactivityfrom [¹⁴C]-glucose into this cytoplasmic ß-1,4-glucanwas therefore faster than that into cellulose constituting thecell wall. Hence, it seemed that the former ß-1,4-glucancould be turned over. To examine whether the- cytoplasmic ß-1,4-glucanis carried by some subcellular components, cytoplasmic ß-1,4-glucanin the cell was fractionated by differential centrifugation,two enzyme activities being measured as the markers of subcellularcomponents. The distribution of ß-1,4-glucan was similarto that of UDPG-glucosyltransferase activity but not to thatof IDP-ase activity. The result suggests that the cytoplasmicß-1,4-glucan has some relation to plasma membranes. Coumarin, known as a specific inhibitor for the biosynthesisof cellulose in plant cells, was shown to inhibit the incorporationof radiocarbon from [¹⁴C]-glucose into cytoplasmic ß-1,4-glucanto the same extent as that into cellulose in the cell wall ofthe hypocotyls. ¹ Present address: Department of Biological Science, TohokuUniversity, Kawauchi, Sendai 980, Japan. (Received May 31, 1976; )     

The development of structure and function in chloroplasts of greening mutants of Scenedesmus III. Biosynthesis of {delta}-aminolevulinic acid

Cells of the mutant C-2A' of Scenedesmus obliquus which requirelight for chlorophyll formation were assayed for in vivo activityof ALA synthesis. In general, ALA and chlorophyll syntheseswere coupled during the greening process. The action spectrafor ALA and chlorophyll syntheses both show the highest activitiesin the blue region, but were different in details. Under certainconditions, ALA synthesis occurred without a corresponding synthesisof chlorophyll. Reasons for these variances were discussed. The controlling action of light on ALA synthesis may occur throughthree different, but related, mechanisms. The principle mechanismappeared to be linked to lightenhanced respiration since itsinhibition by cycloheximide blocks ALA synthesis. The Hill coefficientof this inhibition is 2. After the light-induced enhancementof respiration had ceased, the Hill-coefficient of inhibitionof ALA synthesis became 1. Thus, in addition to enhanced respiration,ALA formation depends on its sensitizing enzyme having a half-lifetime of less than 1 hr. Finally, the dependence of the synthesisof ALA precursors on light was evident. ¹ On leave from the Institute of Applied Microbiology, Universityof Tokyo, Tokyo, Japan. (Received November 11, 1974; )