Nitrate and nitrite as ‘in vivo’ quenchers of chlorophyll fluorescence in blue-green algae

The effect of nitrate and nitrite on long-term chlorophyll fluorescence has been studied in filamentous blue-green algae. Cells grown autotrophically with nitrate as nitrogen source show, under argon atmosphere, a high level of fluorescence. The addition of either nitrete or nitrite induces a significant fluorescence quenching, but, whereas in the case of nitrite no previous treatment is required, in the case of nitrate the cells have to be sonicated or treated with Triton X-100 in advance without destroying their cellular integrity. DCMU strongly inhibits the quenching of fluorescence caused by nitrate or nitrite. Using cells grown with ammonia, a nutritional repressor of the two enzymes of the nitrate-reducing system, the fluorescence quenching observed in either case becomes negligible. These results clearly indicate that both nitrate and nitrite can physiologically act as primary Hill reagents in photosynthesis in blue-green algae.

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Résumé L'effect du nitrate et du nitrite sur la fluorescence de la chlorophylle a été étudié chez quelques algues bleues filameneuses. Les cellules alimentées avec du nitrate montrent, sous argon, un haut niveau de fluorescence. L'addition de quantités égales de nitrate et de nitrite induit une diminution significative de la fluorescence, mais si, dans le cas du nitrite, un traitement préalable n'est pas nécessaire, dans le cas du nitrace, les cellules doivent être traitées légèrement aux ultra sons ou avec du Triton X-100, sans détruire leur intégrité cellulaire. Le DCMU inhibe sévèrement la diminution de fluorescence causée par le nitrate ou le nitrite. Dans les cellules alimentées avec l'ammoniaque, un répresseur des deux enzymes du système de réduction du nitrate, la diminution de fluorescence devient negligeable quel que soit le composé employé. Enfin, ces résultats montrent clairement que tant le nitrate que le nitrite peuvent être des réactifs primaires et physiologiques de la réaction de Hill dans la photosynthèse de nitrate en algues bleues.

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The effect of iron supply on the activity of nitrate and nitrite reduction in green algae

Summary An improved supply of iron, produced by an addition of EDTA to the culture medium, specifically increases the reduction of nitrite by green algae, whereas it has no influence on the reduction of nitrate to nitrite. Correspondingly, growth of the algae under iron-deficient conditions results in an inhibition of only nitrite reduction.     

Comparison of the long-wave chlorophyll fluorescence in various green and blue-green algae and diatoms

Two types of long-wave fluorescence bands with similar band shape occur at room temperature in various algae: F_II700 and F_I715. F_II700 occurs in a limited number of algae, follows PS II transients, increases with culture age and is moderately increased by cooling to 83 K. F_I715 occurs in most algae, especially Anabaena, but much less in most diatoms and Tribonema. It does not follow PS II transients, does not increase with culture age and is much increased by cooling to 83 K.An interpretation for the characteristics of F_II700 and F_I715 is given.     

Immunocytochemical localization of nitrite reductase in green algae

The distribution of nitrite reductase (EC 1.7.7.1) in the green algae Chlamydomonas reinhardtii, Monoraphidium braunii, Chlorella fusca, and Scenedesmus obliquus was studied by immunoelectron microscopy. The labeling of ultrathin cryosections was performed with anti-nitrite reductase antibodies followed by gold-labeled goat anti-rabbit antibodies. In C. reinhardtii sections, gold label was mainly associated with the pyrenoid, tonoplast, and plasmalemma. Significant labeling was also detected in the thylakoid region. In all other organisms, label density was lower but distributed in the same locations, except that the plasmalemma of S. obliquus was not significantly labeled. From estimates of the relative volume of different cell regions, we found that approximately 80% of the total enzyme is located in the chloroplastic region (thylakoids plus pyrenoid) of C. reinhardtii, M. braunii, and C. fusca, and 97% in the case of S. obliquus.     

Action spectra for nitrate and nitrite assimilation in blue-green algae

Action spectra for the assimilation of nitrate and nitrite have been obtained for several blue-green algae (cyanobacteria) with different accessory pigment composition. The action spectra for both nitrate and nitrite utilization by nitrate-grown Anacystis nidulans L-1402-1 cells exhibited a clear peak at about 620 nanometers, corresponding to photosystem II (PSII) C-phycocyanin absorption, the contribution of chlorophyll a (Chl a) being barely detectable. The action spectrum for nitrate reduction by a nitrite reductase mutant of A. nidulans R2 was very similar. All these action spectra resemble the fluorescence excitation spectrum of cell suspensions of the microalgae monitored at 685 nanometers—the fluorescence band of Chl a in PSII. In contrast, the action spectrum for nitrite utilization by nitrogen-starved A. nidulans cells, which are depleted of C-phycocyanin, showed a maximum near 680 nanometers, attributable to Chl a absorption. The action spectrum for nitrite utilization by Calothrix sp. PCC 7601 cells, which contain both C-phycoerythrin and C-phycocyanin as PSII accessory pigments, presented a plateau in the region from 550 to 630 nanometers. In this case, there was also a clear parallelism between the action spectrum and the fluorescence excitation spectrum, which showed two overlapped peaks with maxima at 562 and 633 nanometers. The correlation observed between the action spectra for both nitrate and nitrite assimilation and the light-harvesting pigment content of the blue-green algae studied strongly suggests that phycobiliproteins perform a direct and active role in these photosynthetic processes.     

The effect of phenols on the parameters of chlorophyll fluorescence and reactions of P700 in green algae Scenedesmus quadricauda

The effect of phenolics, present in wastes of pulp and paper industry, on photosynthesis in microalgae Scenedesmus quadricauda has been studied, analyzing the induction curves of prompt and delayed chlorophyll fluorescence and light curves of nonphotochemical quenching of fluorescence. Energization of photosynthetic membranes was impaired at low concentrations (0.1 mM) of phenol and pyrocatechol. At higher concentrations, phenol and pyrocatechol inhibited electron transport in PS II and increased the share of Q_B-nonreducing centers. As a result, the rate of P₇₀₀ reduction declined. These results indicate that the parameters of fluorescence induction curves can be used for detecting phenol and pyrocatechol in the environment at early stages of toxic effects.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

Temperature dependence on the delayed fluorescence of chlorophyll a in blue-green algae.

1. The delayed fluorescence of chlorophyll a was measured with a phosphoroscope by changing the temperature in a range of room temperatures in intact cells of blue-green algae, Anacystis nidulans, two strains of Anabaena variabilis and Plectonema boryanum, and other kinds of algae, Cyanidium caldarium and Chlorella pyrenoidosa. The induction of delayed fluorescence remarkably depended on the temperature of measurment. Nevertheless, the induction pattern was characterized by three levels of intensity; the initial rise level at the onset of excitation light, the maximum level after a period of excitation and the steady-state level after 10 min of excitation. 2. In A. nidulans and a strain of A. variabilis grown at various temperatures, close relationship was found between the phase transition of membrane lipids and the initial rise and the steady-state levels of delayed fluorescence. The initial rise level showed the maximum at the temperature of phase transition between the liquid crystalline and the mixed solid-liquid crystalline states, The steady-state levels showed a remarkable change from a high in the liquid crystalline state to a low level in the mixed solid-liquid crystalline state. 3. The millisecond decay kinetics of the delayed fluorescence measured at the steady-state level in A. nidulans grown at 38 degrees C consisted of two components with different decay rates. The half-decay time of the fast component was about 0.17 ms and was constant throughout the temperature range of measurement. The half decay time of slow component ranged from 0.6 to 1.5 ms, depending on the temperature of measurment.     

Non-photochemical quenching of chlorophyll fluorescence in the green alga Dunaliella

The relaxation of the non-photochemical quenching of chlorophyll fluorescence has been investigated in cells of the green alga Dunaliella following illumination. The relaxation after the addition of DCMU or darkening was strongly biphasic. The uncoupler NH₄Cl induced rapid relaxation of both phases, which were therefore both energy-dependent quenching, qE. The proportion of the slow phase of qE increased at increasing light intensity. In the presence of the inhibitors rotenone and antimycin the slow phase of qE was stabilised for in excess of 15 min. NaN₃ inhibited the relaxation of almost all the qE. The implications of these results are discussed in terms of the interpretation of the non-photochemical quenching of chlorophyll fluorescence in vivo and the mechanism of qE.Abbreviations PS II Photosystem II - qQ photochemical quenching of chlorophyll fluorescence - qNP non-photochemical quenching of chlorophyll fluorescence - qE energy-dependent quenching of chlorophyll fluorescence - F _m maximum level of chlorophyll fluorescence for dark adapted cells - F _m level of fluorescence at any time when qQ is zero     

Examination of chlorophyll fluorescence decay kinetics in sulfur deprived algae Chlamydomonas reinhardtii

Chlorophyll fluorescence decay kinetics was measured in sulfur deprived cells of green alga Chlamydomonas reinhardtii with a home made picosecond fluorescence laser spectrometer. The measurements were carried out on samples either shortly adapted to the dark ('Fo conditions') or treated to reduce Qa ('Fm conditions'). Bi-exponential fitting of decay kinetics was applied to distinguish two components one of them related to energy trapping (fast component) and the other to charge stabilization and recombination in PS 2 reaction centers (slow component). It was found that the slow component yield increased by 2.0 and 1.2 times when measured under 'Fo' and 'Fm conditions', respectively, in sulfur deprived cells as compared to control ones. An additional rapid rise of the slow component yield was observed when incubation was carried out in a sealed bioreactor and cell culture turned to anaerobic conditions. The obtained results strongly indicate the existence of the redox control of PS 2 activity during multiphase adaptation of C. reinhardtii to sulfur deficiency stress. Probable mechanisms responsible for the observed increased recombinant fluorescence yield in starved cells are discussed.     

Blue light photoreactivation of nitrate reductase from green algae and higher plants

The inactive form of NADH-nitrate reductase from spinach and $\text{Chlorella fusca}$ is fully reactivated in short periods of time when the enzyme-complex is illuminated with white or blue light but not with red light. Flavin nucleotides greatly accelerate the photoreactivation process. The results suggest that blue light might act as a modulating agent in the assimilation of nitrate in green algae and higher plants.     

Effect of two nutrient solution temperatures on nitrate uptake,nitrate reductase activity,NH4+ concentration and chlorophyll a fluorescence in rose plants

The effect of two nutrient solution temperatures (cold (10 °C) and warm (22 °C)) during two flowering events of rose plants (Rosa × hybrida cv. Grand Gala) were examined by measuring chlorophyll (Chl) a fluorescence, ammonium (NH₄⁺) content and nitrate reductase (NR) activity in four different leaf types, that is, external and internal leaves of bent shoots and lower and upper leaves of flowering stems. Besides, nitrate (NO₃^?) uptake and water absorption, total nitrogen (N) concentration in the plant, dry biomass, and the ratios of shoot/root and thin-white roots/suberized-brown roots were determined. Generally, cold solution increased NO₃^? uptake and thin-white roots production but decreased water uptake, so plants grown at cold solution had to improve their NO₃^? uptake mechanisms to obtain a higher amount of nutrient with less water absorption than plants grown at warm solution. The higher NO₃^? uptake can be related to an increase in NR activity, NH₄⁺ content and total N concentration at cold solution. Nutrient solution temperature also had an effect on the photosynthetic apparatus. In general terms, the effective quantum yield (?_PSII) and the fraction of open PSII reaction centres (q_L) were higher in rose plants grown at cold solution. These effects can be associated to a higher NO₃^? uptake and total N concentration in the plants and were modulated by irradiance throughout all the experiment. Plants could adapt to cold solution by enhancing their metabolism without a decrease in total dry biomass. Nevertheless, the effect of nutrient solution temperature is not simple and also affected by climatic factors.     

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.