The role of transmembrane electrochemical potential and phosphorylation of PS II proteins in temperature induced light emission from ATP-treated lettuce thylakoids.

Changes in characteristics of flash-induced thermoluminescence (TL) glow curves in thylakoids of lettuce following incubation of the organelles with ATP, under illumination or in the dark, were investigated. TL bands were induced by 1 or 2 flashes fired at -10°C or 1°C in thylakoids: TL curves in control thylakoids which were dark-adapted or submitted to an illumination without ATP, can be deconvoluted as the sum of one single B band and minor contributions. In thylakoids incubated for 90 s with 0.5 mM ATP, either under light or in the dark (after a 90 s preillumination), bands presented complex shapes; after deconvolution, they appeared composed of a B band with a low Ea (activation energy): 0.6 e.v. as compared to 0.75 in control, and a supplementary band peaking at about 10°C. The band at low temperature was suppressed by low concentrations (10-20 nM) of valinomycin, nigericin or FCCP as well as by 10 mM ammonium chloride, leaving B bands with the same characteristics as in control material. Finally with higher nigericin concentrations, the bands became single B bands with high Ea (0.9 e.v.). These characteristics would define 3 different energized states (in the form of a transmembrane electrochemical potential) for thylakoids based upon the presence of the 10°C band and the value of the activation energy for the B band component. The presence of a large 10°C band was also correlated to the existence of a larger transmembrane pH gradient, in the dark, after an ATP-treatment, than in controls. The 10°C band was specifically suppressed by the action of low concentrations of alkaline phosphatase with minor changes in characteristics of the remaining B band suggesting that phosphorylation of PS II proteins is also involved in the appearance of this low temperature band. The main mechanism at the origin of the low temperature band would be a destabilization of S2/3QB (-) charge pairs in energized membranes.

Compared thermoluminescence characteristics of pea thylakoids studied in vitro and in situ (in leaves). The effect of photoinhibitory treatments.

Characteristics of thermoluminescence (TL) glow curves were studied in thylakoids (isolated from pea leaves) or in intact pea leaves after an exposure to very high light for 2 min in the TL device. The inhibition of photosynthesis was detected as decreases of oxygen evolution rates and/or of variable fluorescence.In thylakoids exposed to high light, then dark adapted for 5 min, a flash regime induced TL glow curves which can be interpreted as corresponding to special B bands since: 1) they can be fitted by a single B band (leaving a residual band at -5°C) with a lower activation energy and a shift of the peak maximum by -5 to -6°C and, 2) the pattern of oscillation of their amplitudes was normal with a period of 4 and maxima on flashes 2 and 6. During a 1 h dark adaptation, no recovery of PS II activity occurred but the shift of the peak maximum was decreased to -1 to -2°C, while the activation energy of B bands increased. It is supposed that centers which remained active after the photoinhibitory treatment were subjected to reversible and probably conformational changes.Conversely, in intact leaves exposed to high light and kept only some minutes in the dark, TL bands induced by a flash regime were composite and could be deconvoluted into a special B band peaking near 30°C and a complex band with maximum at 2-5°C. In the case of charging bands by one flash, this low temperature band was largely decreased in size after a 10 min dark adaptation period; parallely, an increase of the B band type component appeared. Whatever was the flash number, bands at 2-5°C were suppressed by a short far red illumination given during the dark adaptation period and only remained a main band a 20°C; therefore, the origin of the low temperature band was tentatively ascribed to recombinations in centers blocked in state S2QA (-)QB (2-). In vivo, the recovery of a moderately reduced state in the PQ pool, after an illumination, would be slow and under the dependence of a poising mechanism, probably involving an electron transfer between cytosol and chloroplasts or the so-called 'chlororespiration' process.

Oxygen-evolving system and secondary quinonic acceptors are highly reduced in dark adapted Euglena cells: A thermoluminescence study.

Characteristics of thermoluminescence glow curves were compared in three types of Euglena cells: (i) strictly autotrophic, Cramer and Myers cells; (ii) photoheterotrophic cells sampled from an exponentially growing culture containing lactate as substrate repressing the photosynthetic activity; (iii) semiautotrophic cells, sampled when the lactate being totally exhausted, the photosynthesis was enhanced.In autotrophic and semiautotrophic cells, composite curves were observed after series of two or more actinic flashes fired at -10°C, which can be deconvoluted into a large band peaking in the range 12-22°C and a smaller one near 40°C, This second band presents the characteristics of a typical B band (due to S2/3QB (-) recombination), whereas the first one resembled the band, shifted by -15-20°C, which is observed in herbicide resistant plants. The amplitude of this major band, which was in all cases very low after one flash, exhibited oscillations of period four but rapidly damping, with maxima after two and six flashes. In contrast, photoheterotrophic Euglena displayed single, non-oscillating curves with maxima in the range 5-10°C.In autotrophic and semiautotrophic cells, oxidizing pretreatments by either a preillumination with one or more (up to twenty-five) flashes, or a far-red preillumination in the presence of methylviologen, followed by a short dark period, induced thermoluminescence bands almost single and shifted by +3-5°C, or +12°C, respectively. In autotrophic cells, far-red light plus methyl viologen treatment induced a band peaking at 31°C, as in isolated thylakoids from Euglena or higher plants, while it had barely any effect in photoheterotrophic cells.Due to metabolic activities in dark-adapted cells, a reduction of redox groups at the donor and acceptor sides of PS II dark-adapted cells is supposed to occur. Two different explanations can be proposed to explain such a shift in the position of the main band in dark-adapted autotrophic control. The first explanation would be that in these reducing conditions a decreasing value of the equilibrium constant for the reaction: SnQA (-)QB⇌SnQAQB (-), would determine the shift of the main TL band towards low temperatures, as observed in herbicide resistant material. The second explanation would be that the main band would correspond to 'peak III' already observed in vivo and assigned to S2/3QB (2-) recombinations.

Dependence of energization of thylakoids on frequency of exciting flashes in intact chloroplasts.

We investigated the frequency-dependence of the flash-induced electrochromic absorbance change, ΔA515, and of the pH-indicating absorbance change of neutral red in isolated intact chloroplasts. The energization pattern of thylakoids depended strongly on the frequency (f) of the exciting flashes, tested between 0.05 and 2 s(-1). When the frequency was increased from 0.1 to 1 s(-1) the total initial change and the slow rise of ΔA515 decreased by about 30% and 70%, respectively, and both the slow rise and decay were considerably accelerated. These changes were fully reversible, even after prolonged excitation at 1 s(-1), if the frequency was decreased again to 0.1 s(-1). Accumulation of an appreciable transmembrane electric field strength could not be detected in any of our experiments, at high frequency, since the decay of ΔA515 was considerably accelerated when the frequency was increased. In contrast, ΔpH significantly increased at higher frequencies of the exciting flashes. In the steady-state (after about 100 flashes) ΔpH was about 0.5-0.8 pH unit higher than in the dark or at low frequencies. In the presence of nigericin or dithionite, both of which prevented accumulation of protons in the lumen, the total initial change in ΔA515 at f=1 s(-1) relative to that at f=0.1 s(-1) decreased to a similar extent as in the control. The proportion of the slow rise relative to the initial amplitude, however, did not decrease. Our data support the suggestion that ΔpH controls the amplitude of the slow rise of ΔA515. However, contrary to a previous statement (B. Bouges-Bouquet (1981) Biochim. Biophys. Acta 535, 327-340), we show that the ΔpH effect cannot be accounted for by variation of the rate of this kinetic component of ΔA515.

Effect of dibromothymoquinone (DBMIB) on reduction rates of Photosystem I donors in intact chloroplasts.

Dual effect of dibromothymoquinone ( DBMIB ), inhibitor and reducing agent at the donor side of Photosystem I, was investigated in isolated intact chloroplasts by flash-induced absorbance changes at 820 and 515 nm. We show that in the absence of other electron donors, rereduction of P700+ by DBMIB proceeds at a very low rate (half-time of approximately 10 s) Dual effect of DBMIB explains that the initial rise of electrochromic absorbance change induced by repetitive flashes is usually not diminished while the slow rise is fully inhibited by this compound.

Is antimycin A a specific inhibitor of the slow rise of the electrochromic absorbance change in intact chloroplasts?

The inhibitory effect of antimycin A on the slow rise of the flash-induced electrochromic absorbance change was reinvestigated in intact chloroplasts isolated from pea leaves. It is shown that in the absence of nigericin and +K at low repetition rates (less than 0.5 s-1) of the excitation flashes not only the slow (approximately 10 ms) rise but also the initial (much less than 1 ms) rise generated by photosystem 1 is inhibited by antimycin A.

Paramylon synthesis by Euglena gracilis photoheterotrophically grown under low O2 pressure : Description of a mitochloroplast complex.

Special culture conditions for Euglena gracilis Z and ZR are described. They induce interactions between the chloroplast and mitochondrial metabolisms leading to paramylon synthesis. When grown in continuous light under pure nitrogen and in the presence of lactate as the sole carbon source, sugar synthesis occurs during the first 24 h of culture with the participation of both mitochondria (using lactate) and of chloroplasts (fixing CO2 from lactate decarboxylation). The activities of ribulose bisphosphate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase are very high and mitochondria and chloroplasts develop then a common network of vesicles in which paramylon grains can be seen. Electron micrographs demonstrate membrane continuity between the two types of organelles. Occasionally the mitochondrial matrix and the chloroplast stroma are separated by only a unit membrane.

Evolution of enzymes involved in carbon metabolism (phosphoenolpyruvate and ribulose-bisphosphate carboxylases, phosphoenolpyruvate carboxykinase) during the light-induced greening of Euglena gracilis strains Z and ZR.

Phosphoenolpyruvate carboxykinase activity decreases when Euglena gracilis Z and ZR undergo light-induced chloroplast development in batch "resting" medium lacking utilizable organic carbon and CO2. This enzyme is present in heterotrophically grown cells (Briand et al. 1981) and assures gluconeogenesis. It was consistently more active in strain ZR. Decreased carboxykinase activities were accompanied by parallel increases in the activities of ribulose bisphosphate carboxylase and phosphoenolpyruvate carboxylase. The rates of O2 evolution in light were much lower than those of CO2 fixed simultaneously. The incorporation of (14)CO2 into early C-4 dicarboxylic acids was higher in green cells than in etiolated cells, and it was even higher in green cells assayed in light in the presence of (DCMU). A hypothesis has been proposed, according to which there is a possible cooperation of phosphoenolpyruvate carboxylase in photosynthetic CO2 fixation, especially under conditions of limiting CO2.High temperatures (34° C) depress carboxylation enzyme activities to a greater extent than that of the carboxykinase without a great effect on cellular chlorophyll content. In the presence of 25 µm DCMU, however, chlorophyll accumulation is reduced without any detectable changes in enzyme activities in the Z strain. The ZR strain displayed its characteristic resistance to DCMU.

Evolution of carboxylating enzymes involved in paramylon synthesis (phosphoenolpyruvate carboxylase and carboxykinase) in heterotrophically grown Euglena gracilis.

Heterotrophically grown Euglena synthesize grains of paramylon, its reserve carbohydrate, in a vesicular complex of mitochondrial origin. A CO2 fixation activity in dark grown Euglena was demonstrated in the mitochondria via paramylon. At the beginning of the exponential phase of growth, the activity of phosphoenolpyruvate carboxykinase increases before the augmentation of paramylon.At the end of the exponential phase, the activity of this enzyme decreases, and low residual levels persist in the transition and stationary phases of growth. The activity of phosphoenolpyruvate carboxylase evolves inversely during the heterotrophic growth of the algae in succinate- or a lactate-containing medium. A compartmentalized scheme of carbon metabolism in mitochondria is presented.

Light versus Dark Carbon Metabolism in Cherry Tomato Fruits: II. Relationship Between Malate Metabolism and Photosynthetic Activity.

The possible relationship between malate metabolism and photosynthetic activity in green tomato fruit tissues (Lycopersicum esculentum var. cerasiforme Dun A. Gray) was investigated. Initial experiments consisted of vacuum-infiltrating (14)C-3 or (14)C-4-malate into isolated tissues in darkness and then incubating the tissues under photosynthetic conditions. Other experiments involved a short pulse with (14)C-bicarbonate in darkness to label the malate pool(s), followed by a chase in the light in the presence of nonradioactive bicarbonate. Both series of experiments were followed by the separation and identification of labeled metabolic intermediates.Label initially in carbon atoms 3 and 4 of malate, corresponding also to C-3 of pyruvate and CO(2) after malate decarboxylation, was recovered as citrate + isocitrate, sugars and starch following incubations of tissues in the light. These data demonstrate that the reductive pentose phosphate cycle utilizes CO(2) furnished by malate metabolism due to the operation of the citric acid cycle and perhaps also to malic enzyme activity. Some synthesis of sugars and starch from C-3 of malate was observed in darkness or in the light 3-(3,4-dichlorophenyl)-1,1-dimethyl which could be due to gluconeogenesis. Pulse-chase experiments indicated a rapidly turning over malate pool.

[Metabolism of some phosphorylated compounds and photophosphorylation in Maize leaves]. / Métabolisme de quelques composés phosphorylés et photophosphorylation "in vivo" chez les feuilles de Maïs.

Following a period of steady state photosynthesis, in air, maize leaves were illuminated in a CO2-free atmosphere consisting of N2 or N2-O2 (80-20, v/v).Isotopic techniques have been used for the measurement in cells of pool sizes of some phosphorylated intermediates and for the study of the turn-over of these compounds.In N2 atmosphere and white light, a rapid decrease of the PGA level and an accumulation of Ru 1-5 P2 are observed, whereas DHAP and Fru 1-6 P2 levels remain high. With (32)P feeding in short-time experiments, Ru 1-5 P2, ATP and ADP are highly labelled, and there is a low but significant labelling of PGA and DHAP. Our interpretation is that basic reactions of the CALVIN cycle are occurring: phosphorylation of Ru 5 P to Ru 1-5 P2 (phosphorylating step), carboxylation of Ru 1-5 P2 to PGA (the CO2 belongs to an intracellular pool of unknown nature), reduction of PGA to trioses phosphates (reductive step), regeneration of the CO2 acceptor via the synthesis of Ru 5P (regenerative step). In N2 atmosphere the second step is the limiting one because of the low intracellular CO2 level; the consequence is an increase in the amount of the CO2 acceptor and of the compounds belonging to the regenerative step.When leaves have been treated with CMU before an illumination (white light) in N2 atmosphere, increased levels of PGA are observed as compared with the preceding experiments, whereas those of DHAP and Fru 1-6 P2 are lower; in short-time experiments, the PGA is labelled to a much greater extent than Ru 1-5 P2 and DHAP. In this case the limiting steps of the CALVIN cycle are: a) the reductive step, affecting the levels of the intermediates of the regenerative step: DHAP, Fru 1-6 P2 and Ru 5P. b) the phosphorylative step, because of the inhibition of the non-cyclic photophosphorylations: there is a decrease of the ATP cellular level. In N2 atmosphere a far-red illumination has the same effect on pool-sizes and labelling of compounds as CMU in white light, owing to the inhibition of the reducing power.In N2-O2 atmosphere (white light), the PGA level is higher than in N2 atmosphere; the effect of O2 is discussed (stimulation of the oxydative pentose phosphate cycle?).A physiological part played by the important reservoir of PGA accumulating in vivo, especially in far-red light, is suggested in "Discussion".