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Wild-type Nostoc calcicola carried out oxygenic photosynthesis extremely sensitive to copper. A Cu(2+)-resistant mutant (Cu-R1) of the cyanobacterium grew normally at high concentrations of Cu2+. Its ability to grow under such conditions was found to be due to mutational acquisition of an energy-dependent efficient system of Cu(2+)-efflux, which rendered Cu(2+)-inhibited oxygenic photosynthesis fully reversible.
The ureas and phenolics are two major classes of herbicides that act on Photosystem II (PSII) and are normally inactive in the photosynthetic reaction centers of purple bacteria. However, the triazine-resistant mutant T4 from Rhodopseudomonas (Rps.) viridis, which has the tyrosine residue at position 222 on the L subunit substituted for phenylalanine (TyrL222Phe), is sensitive to both ureas and phenolics. Since for the first time structural data on urea binding are available, T4 is a particularly interesting model for the herbicide-binding site of PSII.
The rate of cyclic electron flow measured in dark-adapted leaves under aerobic conditions submitted to a saturating illumination has been performed by the analysis of the transmembrane potential changes induced by a light to dark transfer. Using a new highly sensitive spectrophotometric technique, a rate of the cyclic flow of approximately 130 s(-1) has been measured in the presence or absence of 3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU). This value is approximately 1.5 times larger than that previously reported [Proc. Natl. Acad. Sci. U. S. A. 99 (2001) 10209]. We have characterized in the presence or absence of DCMU charge recombination process (t(1/2) approximately 60 micros) that involves P(700)(+) and very likely the reduced form of the iron sulfur acceptor F(X). This led to conclude that, under saturating illumination, the PSI centers involved in the cyclic pathway have most of the iron sulfur acceptors F(A) and F(B) reduced. In the proposed mechanism, electrons are transferred from a ferredoxin bound to a site localized on the stromal side of the cytochrome b(6)f complex to the Q(i) site. Two possible models of the organization of the membrane complexes are discussed, in which the cyclic and linear electron transfer chains are isolated one from the other.
The effects of DCMU (3-(3',4'-dichlorophenyl)-1,1-dimethylurea) on the fluorescence induction transient (OJIP) in higher plants were re-investigated. We found that the initial (F(0)) and maximum (F(M)) fluorescence levels of DCMU-treated leaves do not change relative to controls when the treatment is done in complete darkness and DCMU is allowed to diffuse slowly into the leaves either by submersion or by application via the stem. Simultaneous 820 nm transmission measurements (a measure of electron flow through Photosystem I) showed that in the DCMU-treated samples, the plastoquinone pool remained oxidized during the light pulses whereas in uninhibited leaves, the F(M) level coincided with a fully reduced electron transport chain. The identical F(M) values with and without DCMU indicate that in intact leaves, the F(M) value is independent of the redox state of the plastoquinone pool. We also show that (i) the generally observed F(0) increase is probably due to the presence of (even very weak) light during the DCMU treatment, (ii) vacuum infiltration of leaf discs leads to a drastic decrease of the fluorescence yield, and in DCMU-treated samples, the F(M) decreases to the I-level of their control (leaves vacuum infiltrated with 1% ethanol), (iii) and in thylakoid membranes, the addition of DCMU lowers the F(M) relative to that of a control sample.
Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells (Synechocystis sp. PCC 6803) and spores (Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image ('phase thicknesses' Deltah) and cell energization. It was demonstrated that the phase thickness Deltah decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Deltah and cell diameter d demonstrated that a decrease in the phase thickness Deltah could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.
Under biotic/abiotic stresses, the red alga Kappaphycus alvarezii reportedly releases massive amounts of H(2)O(2) into the surrounding seawater. As an essential redox signal, the role of chloroplast-originated H(2)O(2) in the orchestration of overall antioxidant responses in algal species has thus been questioned. This work purported to study the kinetic decay profiles of the redox-sensitive plastoquinone pool correlated to H(2)O(2) release in seawater, parameters of oxidative lesions and antioxidant enzyme activities in the red alga Kappaphycus alvarezii under the single or combined effects of high light, low temperature, and sub-lethal doses of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which are inhibitors of the thylakoid electron transport system. Within 24 h, high light and chilling stresses distinctly affected the availability of the PQ pool for photosynthesis, following Gaussian and exponential kinetic profiles, respectively, whereas combined stimuli were mostly reflected in exponential decays. No significant correlation was found in a comparison of the PQ pool levels after 24 h with either catalase (CAT) or ascorbate peroxidase (APX) activities, although the H(2)O(2) concentration in seawater (R=0.673), total superoxide dismutase activity (R=0.689), and particularly indexes of protein (R=0.869) and lipid oxidation (R=0.864), were moderately correlated. These data suggest that the release of H(2)O(2) from plastids into seawater possibly impaired efficient and immediate responses of pivotal H(2)O(2)-scavenging activities of CAT and APX in the red alga K. alvarezii, culminating in short-term exacerbated levels of protein and lipid oxidation. These facts provided a molecular basis for the recognized limited resistance of the red alga K. alvarezii under unfavorable conditions, especially under chilling stress.
The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by "mobile phycobilisome (PBS)" or "energy spillover" has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the "mobile PBS" but also an "energy spillover". Secondly, the "energy spillover" is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H(+) concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the "energy spillover". Thus, it can be concluded that the "mobile PBS" is a common rule for light-induced state transition while the "energy spillover" is only a special case when dark condition is involved.
Prochlorococcus is one of the most important primary producers on Earth; its unusual features and ecological importance have made it a model organism, but nutrient assimilation has received little attention. Glutamine synthetase (GS) plays a key role in nitrogen metabolism and its central position justifies the fine regulation of this enzyme. The aim of this work is to demonstrate the involvement of metal-catalyzed oxidation (MCO) in the control of the biological activity and turnover of GS from Prochlorococcus. In order to study the physiological role of MCO, we have first characterized the in vitro biosynthetic inactivation and degradation of GS in the axenic PCC 9511 strain, testing then the effect of several stress conditions, such as the presence of electron transport inhibitors, darkness and aging, on the inactivation and degradation of GS. It is noteworthy that the physiological substrates of GS could protect the enzyme from the oxidative inactivation and ATP partially reverted this inactivation once the enzyme had been oxidized, being this effect higher in the presence of glutamate. We have also found that the GS from aged cultures is degraded to the same smaller size fragments obtained in the in vitro degradation of GS by an oxidative model system (Fe3+/NADH/NADH oxidase/O2). These results suggest the implication of MCO in the age- and oxidative state-dependent degradation of GS from Prochlorococcus.
Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.
Phospholipase D (PLD) activity was found to be higher in etiolated oat seedlings than in green seedlings. White and red (R) light exposure inhibited PLD activity in etiolated seedlings. Far-red light eliminated R-light-induced decrease in PLD activity, indicating phytochrome participation in observed photomodulation. Inhibitor of electron transport in chloroplast 3-(3,4-dichlorophenyl)-1,1-dimethylurea stimulated and glucose suppressed PLD activity in green and etiolated oat seedlings, respectively. These results suggest that PLD activity in oat seedling is regulated by light with involvement of phytochrome photoreceptor, and associated with photosynthesis process.
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Works correlating fluctuating asymmetry with environmental stress or genetic damages have been largely reported in multicellular organisms but not in single-celled ones. We hypothesize that asymmetry analysis could also be applied to single-celled organisms, because the asymmetry between two sister cells originated from a cellular division event (same genotype in similar environment) must tend to zero in the absence of environmental or genetic perturbations. Laboratory experiments with copper sulphate and DCMU-herbicide treatments as well as experiments in a water reservoir after treatment with copper sulphate algaecide show that environmental stress increases asymmetry between sister cells of Microcystis aeruginosa (Cyanobacteria). Even low Cu(2+) or DCMU doses, which were unable to reduce growth rate, considerably enlarge asymmetry with respect to untreated controls. Asymmetry between sister cells of cyanobacteria seems to be a reliable indicator of environmental perturbation. Analysis of asymmetry in single-celled organisms could become as important as fluctuating asymmetry of multicellular organisms is today.
Capsaicin, a natural quinone analog, was found to block electron transport, in both plant photosystem II (PSII) and bacterial reaction center (RC) from Rhodobacter sphaeroides, at the QB site. The mode of action of capsaicin was investigated by O2 evolution measurements and fluoresence induction studies in the case of PSII, and flash-induced absorbance spectroscopy in the case of the bacterial RC. Structural details of capsaicin binding to the bacterial RC complex were determined by X-ray crystallographic analysis.
We investigate the role of plastoquinone (PQ) diffusion in the control of the photosynthetic electron transport. A control analysis reveals an unexpected flux control of the whole chain electron transport by photosystem (PS) II. The contribution of PSII to the flux control of whole chain electron transport was high in stacked thylakoids (control coefficient, CJ(PSII) =0.85), but decreased after destacking (CJ(PSII)=0.25). From an 'electron storage' experiment, we conclude that in stacked thylakoids only about 50 to 60% of photoreducable PQ is involved in the light-saturated linear electron transport. No redox equilibration throughout the membrane between fixed redox groups at PSII and cytochrome (cyt) bf complexes, and the diffusable carrier PQ is achieved. The data support the PQ diffusion microdomain concept by Lavergne et al. [J. Lavergne, J.-P. Bouchaud, P. Joliot, Biochim. Biophys. Acta 1101 (1992) 13-22], but we come to different conclusions about size, structure and size distribution of domains. From an analysis of cyt b6 reduction, as a function of PSII inhibition, we conclude that in stacked thylakoids about 70% of PSII is located in small domains, where only 1 to 2 PSII share a local pool of a few PQ molecules. Thirty percent of PSII is located in larger domains. No small domains were found in destacked thylakoids. We present a structural model assuming a hierarchy of specific, strong and weak interactions between PSII core, light harvesting complexes (LHC) II and cyt bf. Peripheral LHCII's may serve to connect PSII-LHCII supercomplexes to a flexible protein network, by which small closed lipid diffusion compartments are formed. Within each domain, PQ moves rapidly and shuttles electrons between PSII and cyt bf complexes in the close vicinity. At the same time, long range diffusion is slow. We conclude, that in high light, cyt bfcomplexes located in distant stromal lamellae (20 to 30%) are not involved in the linear electron transport.
Complementary chromatic adaptation was studied in two unicellular diazotrophic Synechocystis-type cyanobacteria, strains BO 8402 and BO 9201. Strain BO 8402 was isolated from Lake Constance as a mutant lacking phycobilisomes due to an insertion sequence element in the gene apcA, encoding alpha-allophycocyanin. Strain BO 9201 recovered the ability to assemble functional phycobilisomes after a spontaneous excision of the insertion sequence element in apcA. Simultaneously, the strain became able to perform group II complementary chromatic adaptation by regulating the synthesis of phycoerythrin. The two strains had identical phycoerythrin operons, cpeBA, and similar-sized transcripts were formed upon induction by green light. However, in strain BO 8402 the cpeBA transcript level was approx. 20-fold lower than in strain BO 9201. Because strain BO 8402 cannot synthesize allophycocyanin and phycocyanin is sequestered in paracrystalline inclusion bodies, non-assembled phycoerythrin may accumulate inside the cells. It was examined whether non-assembled phycoerythrin or other effects caused by the absence of phycobilisomes, such as a permanently oxidized redox status of the photosynthetic electron transport chain or a distorted ratio of C and N assimilation mediated the repression of cpeBA transcription in strain BO 8402. No such links could be established. We therefore concluded that in these diazotrophic Synechocystis-type cyanobacteria the green light-induced transcription of the cpe operon directly required a functional apc operon.
Loss by recombination of the charge separated state P(680+)Q(A-) limits the performance of Photosystem II (PS II) as a photochemical energy converter. Time constants reported in literature for this process are mostly either near 0.17 ms or near 1.4 ms. The shorter time is found in plant PS II when reduction of P(680+) by the secondary electron donor Tyrosine Z cannot occur because Y(Z) is already oxidized. The 1.4 ms recombination is seen in Y(Z)-less mutants of the cyanobacterium Synechocystis. However, the rate of P(680+)Q(A-) recombination that actually competes with the stabilization of the charge separation has not been previously reported. We have measured the kinetics of the flash-induced fluorescence yield changes in the microsecond time domain in Tris-washed spinach chloroplasts. In this way the kinetics and yield of P(680+) reduction by Y(Z) were obtained, and the rate of the competing P(680+)Q(A-) recombination could be evaluated. The recombination time was less than 0.5 ms; the best-fitting time constant was 0.1 ms. The presence of Y(Z)(ox) slightly decreased the efficiency of excitation trapping but did not seem to accelerate P(680+)Q(A-) recombination. The two P(680+)Q(A-) lifetimes in the literature probably reflect a significant difference between plant and cyanobacterial PS II.
Bicarbonate (HCO3-) causes a significant and reversible stimulation of anion-inhibited electron flow in photosystem II of higher plants and cyanobacteria. To test if selected arginine (Arg) residues are involved in the binding of HCO3-, we utilized oligonucleotide-directed mutagenesis to construct Synechocystis sp. PCC 6803 mutants carrying mutations in Arg residues in the D2 protein. Measurements of oxygen evolution showed that the D2 mutants R233Q (arginine-233----glutamine) and R251S (arginine-251----serine) were 10-fold more sensitive to formate than the wild type. The formate concentration giving half-maximal inhibition of the steady-state oxygen evolution rate was 48 mM, 4.5 mM and 4 mM for the wild type, R233Q and R251S, respectively. Measurements of oxygen evolution in single-turnover flashes confirm that the mutants are more sensitive to formate than the wild type. Measurements of chlorophyll a fluorescence decay kinetics after the second saturating actinic flash indicated that, after formate treatment, the halftime of QA- oxidation was decreased by approximately a factor of 2, 4 and 6 in the wild type, R251S and R233Q, respectively. The recombination rate between QA- and S2 was approx. 2-fold slower in R251S and R233Q than in the wild type. In the presence of 100 mM sodium formate, reactivation of the Hill reaction by bicarbonate showed that the wild type had an apparent Km for bicarbonate of 0.5 mM, while the Km values for R233Q and R251S were 1.4 and 1.5 mM, respectively. We suggest that Arg-233 and Arg-251 in the D2 polypeptide contribute to stabilization of HCO3- binding in Photosystem II.