Photoinhibition and Recovery of Photosynthesis in psbA Gene-Inactivated Strains of Cyanobacterium Anacystis nidulans

The susceptibility of photosynthesis to photoinhibition and the rate of its recovery were studied in cyanobacterium Anacystis nidulans strain R2 and its two psbA gene-inactivated mutants R2S2C3 and R2K1. Changes in the fluorescence kinetics at 77K as well as the rate of O₂ evolution were measured when cells were exposed to high photosynthetic photon flux densities in the range of 0 to 2,000 micromoles per square meter per second. The R2S2C3 mutant has an active psbAI gene highly expressed under low and normal light intensities, whereas R2K1 possesses psbAII and psbAIII genes highly expressed under very high light intensities. The level of overall susceptibility of photosynthesis to photoinhibition was more pronounced in the wild type and the mutant R2S2C3 than in the mutant R2K1, especially at higher light intensities. In constrast, all three strains showed an increased but similar sensitivity to photoinhibition after addition of the translational inhibitor streptomycin; mutant R2K1 being slightly less sensitive at lower light intensities. The result is interpreted as demonstrating similar intrinsic susceptibility to photoinhibition of the two different forms of the D1 protein, form I and form II, encoded by the psbAI and psbAII/psbAIII genes, respectively. The increased resistance to photoinhibition of the R2K1 mutant was ascribed to an approximately 3 times higher rate of recovery than the wild type and the mutant R2S2C3. On the basis of our experiments we conclude that the susceptibilities to photoinhibition of the Anacystis nidulans psbA genes mutants studied are regulated mainly by modifying the rate of repair, i.e. the rate of turnover of the D1 protein.     

Ammonia Uptake in the Alkalophilic Cyanobacterium Spirulina platensis

Ammonia uptake was studied in the alkalophilic cyanobacteriumSpirulina platensis. In continuous cultures under optimal growthconditions ammonia supported optimal growth (doubling time of9.3 h), causing a reduction of glutamine synthetase activityto 25% of that found in cultures grown on NO₃. Long term (20min) ammonia uptake assays were performed to study the dependencyon metabolism: 1) Ammonia uptake proceeded at the same ratesin the light and in the dark, the pH dependency pattern correlatingwith light-dependent O₂ evolution and dark O₂ consumption. 2)The uptake of ammonia was pH dependent with an optimum at pH9.3. 3) The uptake was totally dependent upon the activity ofglutamine synthetase and was completely inhibited by methoininesulfoximine. To study the mechanism by which NH₄⁺/NH₃ enters the cells, shortterm experiments (up to 1 min) were performed at pH 7.0 andpH 10.0: At pH 7.0 the uptake was slow and at a constant rate.At pH 10.0, the uptake did not saturate even at 1 mM ammoniaand the kinetics were biphasic, consisting of a fast componentlasting less than 5 seconds and of a subsequent slower component.The fast phase was insensitive to methionine sulfoximine, whereasthe slower phase was completely inhibited by this compound.We suggest that under optimal (alkaline) pH the entry of ammoniainto Spirulina cells is likely to be a pH driven diffusion process,continuously supported by its intracellular assimilation. ¹Contribution number 35 of the Microalgal Biotechnology Laboratory. (Received September 19, 1988; Accepted January 16, 1989)     

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO₂ or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP⁺ is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O₂ to H₂O₂. Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.     

钝顶螺旋藻突变株FBL细胞超微结构

利用透射电镜技术观察钝顶螺旋藻出发株和突变株FBL的细胞超微结构。观察结果表明L出发株和突变株均为多细胞丝状体,细胞间横隔膜清晰,细胞壁均由四层结构组成,细胞质膜内陷形成类囊体,类囊体由双层膜堆积而成,膜上附着藻胆体,类囊体与细胞壁呈垂直方向排列,细胞质内包含有充气液泡等细胞器。与出发株相比,突变株细胞壁表面较光滑,四层结构电子密度较深;类囊体膜增多、变发达;羧化体数量增多;横隔膜收缢明显。     

Function of cAMP as a Mat-Forming Factor in the Cyanobacterium Spirulina platensis

A dense suspension of Spirulina platensis trichomes aggregatedrapidly and formed a diskshaped algal mat when cAMP was added.Cyclic AMP significantly stimulated algal mat formation at concentrationsas low as 10^–7 M. Stimulation of the mat formation wasmost rapid at about 10^–5M cAMP, but higher concentrationswere not increasingly effective. Other nucleotides such as cGMP,ATP and AMP showed no stimulatory effect on algal mat formation.CCCP, an inhibitor of ATP synthesis, was found to suppress thecAMP-stimulated algal mat formation. Cyclic AMP also stimulatedrespiration and gliding movement of this cyanobacterium. (Received September 2, 1991; Accepted October 15, 1991)     

Effect of Light Quality on Phycobilisome Components of the Cyanobacterium Spirulina platensis

Phycobilisomes from the nonchromatic adapting cyanobacterium Spirulina platensis are composed of a central core containing allophycocyanin and rods with phycocyanin and linker polypeptides in a regular array. Room temperature absorption spectra of phycobilisomes from this organism indicated the presence of phycocyanin and allophycocyanin. However, low temperature absorption spectra showed the association of a phycobiliviolin type of chromophore within phycobilisomes. This chromophore had an absorption maximum at 590 nanometers when phycobilisomes were suspended in 0.75 molar K-phosphate buffer (pH 7.0). Purified phycocyanin from this cyanobacterium was found to consist of three subparticles and the phycobiliviolin type of chromophore was associated with the lowest density subparticle. Circular dichroism spectra of phycocyanin subparticles also indicated the association of this chromophore with the lowest density subparticle. Absorption spectral analysis of α and β subunits of phycocyanin showed that phycobiliviolin type of chromophore was attached to the α subunit, but not the β subunit. Effect of light quality showed that green light enhanced the synthesis of this chromophore as analyzed from the room temperature absorption spectra of phycocyanin subparticles and subunits, while red or white light did not have any effect. Low temperature absorption spectra of phycobilisomes isolated from green, red, and white light conditions also indicated the enhancement of phycobiliviolin type of chromophore under green light.     

Photoinhibition in outdoor Spirulina platensis cultures assessed by polyphasic chlorophyll fluorescence transients

Photoinhibition in outdoor cultures of Spirulina platensis was studied by measuring the polyphasic rise of chlorophyll fluorescence transients, which provide information on the primary photochemistry of PSII. The maximum efficiency of PSII photochemustry (F_v/F_m) declined in response to daily increasing irradiance and recovered as daily irradiance decreased. The greatest inhibition (15%) in F_v/F_m was observed at 12:00 hr which responded to the highest irradiance. The absorption flux, the trapping flux, and the electron transport flux per PSII reaction center increased in response to daily increasing irradiance and decreased as irradiance decreased. The daily change in the concentration of PSII reaction centers followed the same pattern as F_v/F_m. However, no significant changes in the probability of electron transport beyond Q_A (Ψ_o) were observed during the day. The results suggest that the decrease in F_v/F_m induced by photoinhibition in outdoor Spirulina cultures was a result of the inactivation of PSII reaction centers. The results also suggest that the measurement of polyphasic fluorescence transients is a powerful tool to study the mechanism of photoinhibition in outdoor Spirulina cultures and to screen strains for photoinhibition tolerance. This revised version was published online in June 2006 with corrections to the Cover Date.     

A New Type of Adaptation of the Cyanobacterium Spirulina platensis to Illumination Conditions

Incubation of cells of the cyanobacterium Spirulina platensis under conditions of exposure to low-intensity (2–3 E m^–2 s^–1) red light, which was predominantly absorbed by photosystem I (PS I), caused atypical adaptation changes. Invariable pigment composition and stoichiometry of the photosystems was observed in the cells incubated under these conditions against the background of a decrease in the rate of photosynthetic fixation of ₂ (by one-half) and a 1.5-fold increase in the rate of dark respiration relative to cells incubated under conditions of exposure to green light. Comparison of these data with a high rate of dark relaxation of P700⁺ in the presence of diuron suggests that deficiency of reduced equivalents on the donor side of PS I in Spirulina cells exposed to red light is compensated by electron supply from the respiratory chain NAD(P)H dehydrogenase complex.     

Two tuf genes in the cyanobacterium Spirulina platensis.

Probes derived from the tufA gene of Escherichia coli have been utilized to detect homologous sequences on Spirulina platensis DNA. A 6-kilobase-pair fragment of S. platensis DNA appears to contain two sequences homologous to the E. coli gene. Thus, as reported for gram-negative bacteria, the cyanobacterium presumably contains two tuf genes.     

Isolation and Characterization of the Plasma Membrane by Two-Phase Partitioning from the Alkalophilic Cyanobacterium Spirulina platensis

Partition in aqueous dextran-polyethylene glycol two-phase systemwas used to isolate the plasma membranes from the alkalophiliccyanobacterium Spirulina platensis. The upper phase containeda colorless membranes obtained in relatively short time, 3–4h. This fraction had a different protein profile than that ofthe thylakoid fraction obtained in the lower phase. It did notcontain cytochrome c-oxidase activity, but retained characteristicMg²⁺-ATPase activity that is sensitive to vanadate, stimulatedby K⁺, and has a pH optimum near 8.5. These data support ourassumption that the upper phase of the gradient consist of theplasma membrane of S. platensis. (Received November 25, 1993; Accepted April 12, 1994)     

Calcium and Phosphate Regulation of Nitrogen Metabolism in the Cyanobacterium Spirulina platensis Under the High Light Stress

High light stress (40 W/m²)-induced alterations in the nitrogen assimilatory enzymes in Spirulina platensis were studied under the Ca²⁺ and phosphate (Pi)-supplemented as well as starved conditions. Results revealed that activities of nitrate reductase (NR), amino acid transferases (AST/GOT and ALT/GPT), and protease enzymes in the high-light-incubated cells were relatively higher under the Ca²⁺- and Pi-starved conditions. On the contrary, relative rates of glutamine synthetase (GS) and ATPase activities were lower in the Ca²⁺- and Pi-starved cells. But the Spirulina cells under the Ca²⁺- and Pi-added conditions showed enhanced activity of both GS and ATPase enzymes. During the high-light stress, a decline in the GS activity, particularly under the Ca²⁺- and Pi-starved conditions, was indicative of a nitrogen starvation-like condition. This could be one of the reasons for induction of the NR and protease enzymes. A higher rate of GS activity was recorded under both the Ca²⁺- and Pi-supplemented conditions, perhaps owing to the enhanced rate of ATPase activity in such conditions. But a declining pattern of both NR and protease activities in the presence of Ca²⁺ and Pi, despite the higher rate of ATPase activity, might involve some other mechanism like the protein-kinase system. Received: 11 May 2000 / Accepted: 13 June 2000     

Photoinhibition induced alterations in energy transfer process in phycobilisomes of PS II in the cyanobacterium, Spirulina platensis

Exposure of algae or plants to irradiance from above the light saturation point of photosynthesis is known as high light stress. This high light stress induces various responses including photoinhibition of the photosynthetic apparatus. The degree of photoinhibition could be clearly determined by measuring the parameters such as absorption and fluorescence of chromoproteins. In cyanobacteria and red algae, most of the photosystem (PS) II associated light harvesting is performed by a membrane attached complex called the phycobilisome (PBS). The effects of high intensity light (1000-4000 micromol photons m(-2) s(-1)) on excitation energy transfer from PBSs to PS II in a cyanobacterium Spirulina platensis were studied by measuring room temperature PC fluorescence emission spectra. High light (3000 micromol photons m(-2) s(-1)) stress had a significant effect on PC fluorescence emission spectra. On the other hand, light stress induced an increase in the ratio of PC fluorescence intensity of PBS indicating that light stress inhibits excitation energy transfer from PBS to PS II. The high light treatment to 3000 micromol photons m(-2) s(-1) caused disappearance of 31.5 kDa linker polypeptide which is known to link PC discs together. In addition we observed the similar decrease in the other polypeptide contents. Our data concludes that the Spirulina cells upon light treatment causes alterations in the phycobiliproteins (PBPs) and affects the energy transfer process within the PBSs.     

An Increase in the Intracellular Concentration of cAMP Triggers Formation of an Algal Mat by the Cyanobacterium Spirulina platensis

The intracellular concentration of cAMP increased rapidly when3-isobutyl-l-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterase,was added to a suspension of Spirulina platensis cells. Additionof IBMX also enhanced respiratory activity and cell motilityand, as a consequence, a floating algal mat was induced. (Received July 31, 1992; Accepted November 9, 1992)     

Photoinhibition in Spirulina platensis: Response of Photosynthesis and HCO3- Uptake Capability to CO2 Depleted Conditions

The apparent photosynthetic affinity-for inorganic carbon inSpirulina is severely reduced by exposing the cells to CO₂-depletedconditions in the light. The extent of reduction depends onthe length of exposure, the light intensity, and the O₂ concentration.The photosynthetic dependence on inorganic carbon (C_Inorg) concentration,however, is similar at 3% O₂ and at 25% O₂ but in the presenceof catalase. The amount of O₂ released following the additionof catalase to the cell suspension also depends on the O₂ andCO₂ concentrations. The addition of H₂O₂ either to the cellsuspension or by a treatment with methyl viologen caused a severeinhibition of the capability of the cells to accumulate inorganiccarbon internally. It is suggested that the reduction of thephotosynthetic apparent affinity to C_Inorg upon subjecting thecells to photoinhibitory conditions is caused by an alterationof their ability to accumulate C_Inorg due to the accumulationof H₂O₂.     

Osmotic adjustment in Spirulina platensis

Unilateral irradiation of maize (Zea mays L.) seedlings results in a fluence-rate gradient, and hence below saturation, a gradient of the far-red-absorbing form of phytochrome (Pfr). The Pfr-gradients established by blue, red and far-red light were spectrophotometrically measured in the mesocotyl. Based on these Pfr-gradients and the fluence-response curves of phytochrome photoconversion the fluence-rate gradients were calculated. The fluence-rate gradient in the blue (460 nm) was steeper than that in the red (665 nm), which in turn was steeper than that in the far-red light (725 nm). The fluence-rate ratios front to rear were 1:0.06 (460 nm), 1:0.2 (665 nm), and 1:0.33 (725 nm). The assumption that phytochrome-mediated phototropism of maize mesocotyls is caused by local phytochrome-mediated growth inhibition was tested in the following manner. Firstly, the Pfr response curve for growth inhibition was calculated; these calculations were based on measurements of Pfr-gradients and data from red-light-induced phototropism. Secondly, the Pfr response curve for growth inhibition was used as a basis for calculating fluence-response curves for blue-and far-red-light-induced phototropism. Finally, these calculated results were compared with experimental data. It was concluded that the threshold for phytochrome-mediated phototropism of maize mesocotyls reflects the apparent photoconversion cross section of phytochrome whereas the maximal inducable curvature depends on the steepness of the light (Pfr) gradient across the mesocotyl.Abbreviations Pfr far-red-absorbing form of phytochrome - Ptot total phytochrome - Fr far-red light     

News & Notes: Efficient Library Construction with a TA Vector and Its Application to Cloning of the Phytoene Synthase Gene from the Cyanobacterium Spirulina platensis

An efficient and simple method for constructing a genomic DNA library is presented by use of a TA cloning vector. It is based on sonicative cleavage of genomic DNA and modification of the fragment ends with Taq DNA polymerase, followed by ligation with a TA vector. This method was successfully applied to cloning of the phytoene synthase gene crtB from Spirulina platensis. The method is useful when the genomic DNA is not well digested with restriction enzymes owing to methylation or other reasons. Received: 25 February 1988/Accepted: 12 May 1998