Effect of silver nanoparticles on the parameters of chlorophyll fluorescence and P700 reaction in the green alga Chlamydomonas reinhardtii

Acute toxicity of silver nanoparticle (AgNP) for photosynthesis in Chlamydomonas reinhardtii was studied using an M-PEA2 fluorimeter. Analysis of the fluorescence induction curves in the presence of AgNP at low concentrations revealed inhibited electron transport on the PS2 photosystem and increased content of Q_B-nonreducing centers. No direct effect of AgNP on the reactions of P₇₀₀ oxidation in PS1 was found, while the energization of the photosynthetic membranes was affected. Investigation of the parameters of the prompt and delayed fluorescence is proposed as a method for early detection of AgNP in the environment.     

In vivo effects of phenolic compounds on blood parameters of a marine fish (Dicentrarchus labrax)

Sea bass (Dicentrarchus labrax) were injected intraperitoneally once (single dose) or three times (fractionated dose) with phenol or OH-phenols (hydroquinone, resorcinol, and pyrocatechol). On the basis of the lethal doses, OH-phenols were more toxic than phenol, and pyrocatechol was the most powerful compound. Hematological, metabolic and antioxidant blood parameters were measured 3 days after the end of the treatment. Metabolic variations as specific effects on erythrocytes were revealed and differences between single and fractionated doses were observed. OH-phenols-treated fish showed disorders in the metabolic toxicity indicators as hypoglycemia, low blood urea nitrogen level (BUN) and decrease of alkaline phosphatase activity (ALP). In addition, quantitative structure-activity relationships were developed using the n-octanol:water partition coefficient (log K_ow). Positive correlations were found with ALP, plasma glucose and hemoglobin.     

Synthesis of L-Tyrosine or 3,4-Dihydroxyphenyl-L-alanine from DL-Serine and Phenol or Pyrocathechol

Reaction conditions for the synthesis of L-tyrosine or L-dopa from DL-serine and phenol or pyrocatechol were studied with intact cells of Erwinia herbicola (ATCC 21434) containing high tyrosine phenol lyase activity. The optimum pH for this reaction was around 8.0, and the optimum temperature range was between 37~40°C for the synthesis of L-tyrosine and between 15~25°C for that of L-dopa. Sodium sulfite and EDTA were added to protect the synthesized L-dopa from decomposition. As high concentrations of phenol or pyrocatechol denatured the enzyme, each substrate was fed to maintain the optimum concentration during incubation.

The reaction mixture (100 ml) containing 4.0 g of DL-serine, 1.0 g of phenol or 0.7 g of pyrocatechol, 0.5 g of ammonium acetate and the cells, was incubated. During incubation, phenol or pyrocatechol was fed at intervals to maintain the substrate at the initial concentration. 5.35 g of L-tyrosine or 5.10 g of L-dopa was synthesized in 100 ml of the reaction mixture.     

Theoretical fluorescence induction curves derived from coupled differential equations describing the primary photochemistry of photosystem II by an exciton-radical pair equilibrium

Fluorescence induction curves were calculated from a molecular model for the primary photophysical and photochemical processes of photosystem II that includes reversible exciton trapping by open (PHQ_A) and closed (PHQ^-_A) reaction centers (RCs), charge stabilization as well as quenching by oxidized (P⁺HQ^(-)_A) RCs. For the limiting case of perfectly connected photosynthetic units (“lake model”) and thermal equilibrium between the primary radical pair (P⁺H^-) and the excited singlet state, the primary reactions can be mathematically formulated by a set of coupled ordinary differential equations (ODE). These were numerically solved for weak flashes in a recursive way to simulate experiments with continuous illumination. Using recently published values for the molecular rate constants, this procedure yielded the time dependence of closed RCs as well as of the fluorescence yield (= fluorescence induction curves). The theoretical curves displayed the same sigmoidal shapes as experimental fluorescence induction curves. From the time development of closed RCs and the fluorescence yield, it was possible to check currently assumed proportionalities between the fraction of closed RCs and either (a) the variable fluorescence, (b) the complementary area above the fluorescence induction curve, or (c) the complementary area normalized to the variable fluorescence. By changing selected molecular rate constants, it is shown that, in contrast to current beliefs, none of these correlations obeys simple laws. The time dependence of these quantities is strongly nonexponential. In the presence of substances that quench the excited state, the model predicts straight lines in Stern-Volmer plots. We further conclude that it is impossible to estimate the degree of physical interunit energy transfer from the sigmoidicity of the fluorescence induction curve or from the curvature of the variable fluorescence plotted versus the fraction of closed RCs.     

Parallel inductive kinetics of fluorescence and photoacoustic signal in dark-adapted thalli of Bryopsis maxima

The inductive kinetics of fluorescence and photoacoustic signal were measured simultaneously in dark-adapted thalli of the green coenocytic alga Bryopsis maxima. Under illumination with weak red light modulated at 60 Hz, the fluorescence yield varied, showing three maxima P, M₁ and M₂ almost immediately, 10 s and 6 min after the onset of the illumination, respectively (Yamagishi, A., Satoh, K. and Katoh, S. (1978) Plant Cell Physiol. 19, 17–25). The photoacoustic signal also showed inductive transients which parallel well those of the fluorescence up to the M₂ stage. After M₂, the photoacoustic signal remained at a constant level, while the emission yield gradually decreased. The first peak of the fluorescence induction and a corresponding peak of the photoacoustic transients were selectively eliminated by prior illumination or methyl viologen treatment of the dark-adapted thalli. The second peaks of the two induction curves were abolished by carbonylcyanide-m-chlorophenylhydrazone, whereas dicyclohexylcarbodiimide enhanced their peak heights and suppressed the subsequent decreases. The results indicate that the fluorescence yield is mainly determined by the redox state of the Photosystem II reaction center throughout the induction period except the last phase. Mechanisms underlying inductive transients of fluorescence are discussed in the light of the present findings.     

On the function of the fluorescence quenchers in chloroplasts and their relation to the primary electron acceptor of photosystem II.

Redox titrations of the fluorescence quenching components in chloroplasts indicate the presence of two components, one with E_m7.6 = + 25 mV and the second with E_m7.6 = -270 mV. These midpoint potentials are almost the same as those of two Photosystem II components previously shown to contribute to the chloroplast electrogenic reaction measured at 518 nm (R. Malkin, 1978, FEBS Lett.87, 329–333). Comparison of light-induced fluorescence yield changes with those obtained by redox titration suggests that both fluorescence quenchers are photoreduced. A direct demonstration of the photoreduction of the low-potential fluorescence quencher was observed in experiments at defined redox potentials. Fluorescence induction curves measured at low light intensity in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) also showed a contribution from both fluorescence quenchers. An additional electron acceptor, other than the two fluorescence quenchers, was also identified in the acceptor complex. These results are discussed in terms of several electron acceptors functioning in the Photosystem II reaction center complex, and the possible function of these acceptors is considered.     

Pigment mutants of the green microalga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: Morphological properties and photosynthetic performance

Pigment mutants CC-124y-1, СС-124у-2, and СС-124р-2 of the green microalga Chlamydomonas reinhardtii Dangeard were obtained by means of UV-induced mutagenesis. Morphological properties and photosynthetic activity of these mutants were examined. The mutants displayed a lowered content of chlorophyll b and carotenoids. Analysis of fluorescence induction curves revealed the decreased rates of electron transport in photosystem II (PSII), the increase in the fraction of Q_B-nonreducing centers, and the enhancement of nonphotochemical fluorescence quenching. The mutations had no direct impact on oxidation of pigment P₇₀₀ in PSI and on the decline of delayed fluorescence. The parameters of induction curves of prompt and delayed fluorescence are proposed for use in early diagnostics of UV-induced mutagenesis. The pigment mutants obtained can be applied in biomonitoring studies.     

Light-dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5′-triphosphate

Addition of ATP to chloroplasts causes a reversible 25–30% decrease in chlorophyll fluorescence. This quenching is light-dependent, uncoupler insensitive but inhibited by DCMU and electron acceptors and has a half-time of 3 minutes. Electron donors to Photosystem I can not overcome the inhibitory effect of DCMU, suggesting that light activation depends on the reduced state of plastoquinone. Fluorescence emission spectra recorded at ?196°C indicate that ATP treatment increases the amount of excitation energy transferred to Photosystem I. Examination of fluorescence induction curves indicate that ATP treatment decreases both the initial (F_o) and variable (F_v) fluorescence such that the ratio of F_v to the maximum (F_m) yield is unchanged. The initial sigmoidal phase of induction is slowed down by ATP treatment and is quenched 3-fold more than the exponential slow phase, the rate of which is unchanged. A plot of F_v against area above the induction curve was identical plus or minus ATP. Thus ATP treatment can alter quantal distribution between Photosystems II and I without altering Photosystem II-Photosystem II interaction. The effect of ATP strongly resembles in its properties the phosphorylation of the light-harvesting complex by a light activated, ATP-dependent protein kinase found in chloroplast membranes and could be the basis of physiological mechanisms which contribute to slow fluorescence quenching in vivo and regulate excitation energy distribution between Photosystem I and II. It is suggested that the sensor for this regulation is the redox state of plastoquinone.     

lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA

Most commonly used expression systems in bacteria are based on the Escherichia coli lac promoter. Furthermore, lac operon elements are used today in systems and synthetic biology. In the majority of the cases the gratuitous inducers IPTG or TMG are used. Here we report a systematic comparison of lac promoter induction by TMG and IPTG which focuses on the aspects inducer uptake, population heterogeneity and a potential influence of the transacetylase, LacA. We provide induction curves in E. coli LJ110 and in isogenic lacY and lacA mutant strains and we show that both inducers are substrates of the lactose permease at low inducer concentrations but can also enter cells independently of lactose permease if present at higher concentrations. Using a gfp reporter strain we compared TMG and IPTG induction at single cell level and showed that bimodal induction with IPTG occurred at approximately ten-fold lower concentrations than with TMG. Furthermore, we observed that lac operon induction is influenced by the transacetylase, LacA. By comparing two Plac-gfp reporter strains with and without a lacA deletion we could show that in the lacA⁺ strain the fluorescence level decreased after few hours while the fluorescence further increased in the lacA⁻ strain. The results indicate that through the activity of LacA the IPTG concentration can be reduced below an inducing threshold concentration—an influence that should be considered if low inducer amounts are used.     

State 1-State 2 Transitions in a Unicellular Green Algae : Analysis of In Vivo Chlorophyll Fluorescence Induction Curves in the Presence of 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea (DCMU)

A study has been made on the State 1-State 2 transitions exhibited by the unicellular green algae Chlorella pyrenoidosa. Chlorophyll fluorescence induction curves from algae adapted to State 1 or State 2 have been analyzed and a comparison made with similar curves produced by decreasing the intensity of light going to the photosystem II reaction centers. In both cases, quenching of the maximum fluorescence yield (F_m) and the initial fluorescence yield (F_o) were observed so that the F_v/F_m ratio and the area above the induction curve (A_max) remained constant. The State 1-State 2 transition also produced changes in the β_max component indicative of some alteration within photosystem II organization. The implications of these experiments on the in vivo mechanism for energy redistribution between the two photosystems are discussed in terms of changes in absorption cross-section rather than being due to spillover from photosystem II to photosystem I. These changes may reflect the phosphorylation of the light-harvesting chlorophyll a/b protein complex and its subsequent migration away from the photosystem II core leading to its closer association with photosystem I.     

Effects of different mineral supplements on fertilization of phenol-contaminated soils by Corynebacterium glutamicum

This study focused on the effects of different mineral supplements on the ability of Corynebacterium glutamicum to degrade phenol in contaminated soil and convert the phenol into useful amino acids. Three types of minerals including FeSO₄, MgSO₄, and MnSO₄ were added at several concentrations to C. glutamicum culture media containing 1% yeast extract prior to treating the soil samples with 4.24 mM phenol. The reactor was incubated at 30°C and 150 rpm for 3 days, and the treated soil was sampled daily and analyzed using gas chromatography for residual phenol and the amino acids produced. Additionally, a plant toxicity assay was employed to examine the fertilization of the phenol-contaminated soil after C. glutamicum treatment supplemented with the three minerals. Our results suggested that among various tested concentrations, 72 μM of iron showed a significant effect on the utilization of phenol by C. glutamicum for conversion to amino acids, therefore enhancing fertilization of the phenol-contaminated soil.     

Simultaneous evaluation of on-line microcalorimetry and fluorometry during batch culture of Pseudomonas putida-ATCC 11172 and Saccharomyces cerevisiae ATCC 18824

Application of on-line sensors (flow calorimeter, fluorescence probe, dissolved oxygen and CO₂ probes) was assessed to monitor microbial biomass and physiological state of cells during a biological process. Two systems were studied; diauxic growth of Pseudomonas putida ATCC 11172 on glycerol and phenol, and the aerobic growth of Saccharomyces cerevisiae ATCC 18824 on glucose. The results showed that the cells produced a heat output which could be quantitatively related to the various phases of the growth cycle. The initial stage of enzymatic induction and substrate mobilization during the diauxic growth of P. putida was easily detected, and a clear oscillation phenomenon was observed during enzymatic rearrangement in shifting from phenol to glycerol metabolism. Glucose oxidation in ethanol and then in acetate was also clearly delineated from the growth of S cerevisiae. NADH (fluorescence probe) measurements gave a strong correlation with various biomass indicators such as optical density, dry weight, ATP content and cellular protein. The fluorescence signal appeared to be very sensitive to the quenching effect of the culture medium and of the cells themselves. The fluorescence emitted from the NADH molecules in a culture medium can be reduced from 30–70% depending on the chemical composition and the optical density.     

Kinetics of Phenol Biodegradation by an Immobilized Methanogenic Consortium

A phenol-degrading methanogenic enrichment was successfully immobilized in agar as shown by the stoichiometric conversion of phenol to CH₄ and CO₂. The enrichment contained members of three physiological groups necessary for the syntrophic mineralization of phenol: a phenol-oxidizing bacterium, a Methanothrix-like bacterium, and an H₂-utilizing methanogen. The immobilization technique resulted in the cells being embedded in a long, thin agar strand (1 mm in diameter by 2 to 50 cm in length) that resembled spaghetti. Immobilization had three effects as shown by a comparative kinetic analysis of phenol degradation by free versus immobilized cells. (i) The maximum rate of degradation was reduced from 14.8 to 10.0 μg of phenol per h; (ii) the apparent K_m for the overall reaction was reduced from 90 to 46 μg of phenol per ml, probably because of the retention of acetate, H₂ and CO₂ in the proximity of immobilized methanogens; and (iii) the cells were protected from substrate inhibition caused by high concentrations of phenol, which increased the apparent K_i value from 900 to 1,725 μg of phenol per ml. Estimates for the kinetic parameters K_m, K_i, and V_max were used in a modified substrate inhibition model that simulated rates of phenol degradation for given phenol concentrations. The simulated rates were in close agreement with experimentally derived rates for both stimulatory and inhibitory concentrations of phenol.     

Estimation of biophysical characteristics for <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> pigment mutants with an M-PEA-2 fluorometer

Light green pigment mutants with a reduced chlorophyll b content were constructed in the microalga Chlamydomonas reinhardtii Dangeard. A simultaneous recording of the induction curves for prompt and delayed fluorescence and the redox state of P700 in the microsecond range with a M-PEA-2 fluorometer revealed decreases in the quantum yield of electron transport in PS2 (φE₀) and the performance index (PI_ABS) and increases in the quantum efficiency of energy dissipation (φD₀) and ΔpH-dependent nonphotochemical quenching (qE and NPQ). The light-dependence curves of the fluorescence parameters confirmed a decrease in the coefficient of maximum utilization of light energy (α) for the mutants. However, the mutants showed an adequate rate of electron transport at a medium light intensity under steady-state conditions. The mutations did not directly affect the oxidation reactions of the PS1 pigment (P700) and the decrease in delayed fluorescence. Experience in using the mutants to test polluted waters of Kazakhstan confirmed that the mutants are promising for use in biomonitoring for mutagens.     

The relationship between carbon dioxide fixation and chlorophyll a fluorescence during induction of photosynthesis in maize leaves at different temperatures and carbon dioxide concentrations

The rate of CO₂ fixation (F_c) and 680 nm chlorophyll fluorescence emission (F680) were measured simultaneously during induction of photosynthesis in Zea mays L. leaves under varying experimental conditions in order to assess the validity of fluorescence as an indicator of in vivo photosynthetic carbon assimilation. Z. mays leaves showed typical Kautsky fluorescence induction curves consisting of a fast rise in emission (O to P) followed by a slow quenching via a major transient (S-M) to a steady-state (T). After an initial lag, net CO₂ assimilation commenced at a point corresponding to the onset of the S-M transient on the F680 induction curve. Subsequently, F_c and F680 always arrived at a steady-state simultaneously. Decreasing the dark-adaption period increased the rate of induction of both parameters. Alteration of leaf temperature produced anti-parallel changes in induction characteristics of F_c and F680. Reducing the CO₂ level to below that required for saturation of photosynthesis also produced anti-parallel changes during induction, however, at CO₂ concentrations tenfold greater than the atmospheric level the rate of F680 quenching from P to T was appreciably reduced without a similar change in the induction of F_c. Removal of CO₂ at steady-state produced only a small increase in F680 and a correspondingly small decrease in F680 occurred when CO₂ was re-introduced. The complex relationship between chlorophyll fluorescence and carbon assimilation in vivo is discussed and the applicability of fluorescence as an indicator of carbon assimilation is considered.Abbreviations F_c rate of CO₂ fixation - F680 fluorescence emission at 680 nm     

Abbau chlorierter Benzole,Phenole und Cyclohexan-Derivate durch Benzol und Phenol verwertende Bodenbakterien unter aeroben Bedingungen

From soil samples of different origin (field, grassland and forest soils) small numbers ofNocardin andPseudomonas spec., able to utilize benzene and phenol could be isolated. Organisms which could only utilize phenol and phenolcarboxylic acids were more numerous and consisted mainly ofArthrobacter spec. It was tested to what extent these organisms could also utilize chlorinated aromatic and cyclohexane derivatives. For the degradation studies the bacteria were precultivated on benzene or p-hydroxybenzoic acid and then the compounds used were added. These compounds were labeled by¹⁴C and their degradation rates determined by measuring the¹⁴CO₂ release.Pseudomonas andNocardia spec. precultivated on benzene could also degrade the chlorinated derivatives of benzene and phenol. The monochlorinated derivates were degraded more easily than the di- and trichlorinated derivates. The chlorinated benzenes, especially in higher concentrations, were less degraded than the chlorinated phenols, but with lower concentrations their degradation rates were about similar. This was due to a higher toxicity of the benzenes. The phenol utilizingArthrobacter spec. were only able to degrade phenol and the chlorinated phenols. Benzoic and m-chlorobenzoic acid were degraded to CO₂ by thePseudomonas andNocardia spec. only. The benzene utilizing pseudomonads released more CO₂ from γ-pentachlorocyclohexane than from γ-hexachlorocyclohexane, but none from cyclehexane. Upon precultivation of benzene utilizing pseudomonads in glucose, the aromatic compounds were also degraded, but especially the chlorinated derivatives to a lower extent. In comparison with these soil organisms in pure culture, experiments with soil samples showed a degradation of all compounds which were used by the isolated organisms after variable induction periods. Cyclohexane was degraded slowly to CO₂ by the mixed soil flora in contrast to the benzene or phenol utilizing pure cultures.     

Assessment of the effects of methylmercury and copper ions on primary processes of photosynthesis in green microalga Chlamydomonas moewusii by analysis of the kinetic curves of variable chlorophyll fluorescence

The effect of methylmercury and copper ions on the kinetics of light induction and dark relaxation of the variable fluorescence of chlorophyll a has been studied on cultures of unicellular alga Chlamydomonas moewusii. Methylmercury was effective at much lower levels. The toxicants at concentrations that did not decrease the photochemical activity of PS II (F _v/F _m) did affect the electron transport on the acceptor side of PS II, nonphotochemical quenching of excitation in the antenna, and reoxidation of the quinone pool. Our results indicate that this approach can be used for detecting the changes in plant cells at the early stages of toxicant action.     

Does long-term cultivation of saplings under elevated CO2 concentration influence their photosynthetic response to temperature?

Background and Aims Plants growing under elevated atmospheric CO₂ concentrations often have reduced stomatal conductance and subsequently increased leaf temperature. This study therefore tested the hypothesis that under long-term elevated CO₂ the temperature optima of photosynthetic processes will shift towards higher temperatures and the thermostability of the photosynthetic apparatus will increase.Methods The hypothesis was tested for saplings of broadleaved Fagus sylvatica and coniferous Picea abies exposed for 4–5 years to either ambient (AC; 385 µmol mol⁻¹) or elevated (EC; 700 µmol mol⁻¹) CO₂ concentrations. Temperature response curves of photosynthetic processes were determined by gas-exchange and chlorophyll fluorescence techniques.Key Results Initial assumptions of reduced light-saturated stomatal conductance and increased leaf temperatures for EC plants were confirmed. Temperature response curves revealed stimulation of light-saturated rates of CO₂ assimilation (A_max) and a decline in photorespiration (R_L) as a result of EC within a wide temperature range. However, these effects were negligible or reduced at low and high temperatures. Higher temperature optima (T_opt) of A_max, Rubisco carboxylation rates (V_Cmax) and R_L were found for EC saplings compared with AC saplings. However, the shifts in T_opt of A_max were instantaneous, and disappeared when measured at identical CO₂ concentrations. Higher values of T_opt at elevated CO₂ were attributed particularly to reduced photorespiration and prevailing limitation of photosynthesis by ribulose-1,5-bisphosphate (RuBP) regeneration. Temperature response curves of fluorescence parameters suggested a negligible effect of EC on enhancement of thermostability of photosystem II photochemistry.Conclusions Elevated CO₂ instantaneously increases temperature optima of A_max due to reduced photorespiration and limitation of photosynthesis by RuBP regeneration. However, this increase disappears when plants are exposed to identical CO₂ concentrations. In addition, increased heat-stress tolerance of primary photochemistry in plants grown at elevated CO₂ is unlikely. The hypothesis that long-term cultivation at elevated CO₂ leads to acclimation of photosynthesis to higher temperatures is therefore rejected. Nevertheless, incorporating acclimation mechanisms into models simulating carbon flux between the atmosphere and vegetation is necessary.     

Reactions of Egyptian landraces of Hordeum vulgare and Sorghum bicolor to drought stress, evaluated by the OJIP fluorescence transient analysis

Landraces of barley and of Sorghum bicolor from Egypt were evaluated for their tolerance to drought stress (DS) using the OJIP test of the chl fluorescence fast induction curve. Water was withheld from 4-week-old, pot-grown plants for 8–10 days, until the volumetric soil water content decreased from 30 to below 5 vol% and the leaves reached relative water contents of <60 %. The plants were rewatered and recovery measurements were taken 24 h later. Comparative studies of the most sensitive and the most tolerant lines of both cereals, as evaluated by their Performance Indices (PI_abs), revealed a similar behavior in the sensitive lines, i.e., inhibiting effects of DS on PS II connectivity (occurrence of an L band), oxygen evolving complex (occurrence of a K band) and on the J step of the induction curves, associated with an inhibition of electron transport from Q _A to Q _B . These effects persisted or were even enhanced in the rewatered plants, which resulted in similar deviations of spider plots of the OJIP parameters in the sensitive lines of both species. In the most tolerant barley accession, drought effects on “early” events (i.e., L, K bands) were much smaller or negligible, and there was no pronounced effect on the J step. However, distinct increases of the I step occurred, pointing to inhibited electron flow to the intersystem electron carriers and beyond PS I. The most tolerant Sorghum line, in contrast, revealed nearly no effects of the DS and recovery treatment on the fluorescence induction curves and OJIP parameters.