Chromatic fluorescence transitions in bean chloroplasts]

Chromatic fluorescence transients of isolated bean chloroplasts were investigated. In the presence of methylviologen we observed a decrease in the chloroplasts fluorescence under the influence of light which mainly excited the photosystem I. The effects of preliminary illumination with the white light and Mg2+ ions concentration on the fluorescence of chloroplasts were also studied.     

Chlorophyll fluorescence as a probe for the determination of the photo-induced proton gradient in isolated chloroplasts

Gramicidin D-treated chloroplasts show an acid-induced quenching of the chlorophyll fluorescence, which is composed of a reversible and irreversible part. The reversible quenching is analogous to the photo-induced quenching in coupled chloroplasts and can be taken to determine the light induced delta pH.     

Studies on the slow fluorescence decline in isolated chloroplasts

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar.At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by uncoupler. Thus the pH gradient seems to inhibit the process. The involvement of coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission.Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sig-moidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116–125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.     

The relationship between cation-induced fluorescence and membrane stacking in isolated spinach chloroplasts

In order to study the relationship between Mg⁺⁺-induced fluorescence and membrane stacking, trypsin was used as a probe. Trypsin treatment diminished to a high degree the light-induced variable fluorescence and membrane stacking. Mg⁺⁺ markedly increased the fluorescence yield near 680 nm and membrane stacking. Pretreatment of chloroplasts with Mg⁺⁺ eliminates the effect of trypsin on cation-induced fluorescence change but not on the membrane stacking. The results presented in this contribution support the evidence that the cation-induced membrane stacking and the fluorescence yield are not linked     

The effect of salt concentration on the fluorescence parameters of isolated chloroplasts

The previously reported effect of salt concentration on the fluorescence and other photochemical activities of Photosystem II is interpreted in terms of a change in the radiationless transition and the trapping probabilities. This is confirmed by quantitative comparison of the fluorescence and the photochemical activity. As a by-product of this analysis a method is devised to estimate the background fluorescence.

We did not eliminate the possibility that the radiationless transition constant may include a contribution of energy transfer from Photosystem II to Photosystem I.     

Energy-dependent uptake of phenazinemethosulfate in isolated chloroplasts]

The concentration and absorption of methylphenazinium cations (MP+) in suspensions of pea chloroplasts are simultaneously lowered during rapid (approximately 10s) illumination. The light-induced changes of absorption and concentration of MP+ reveal similar sensitivity towards some inhibitors and uncouplers and are determined by MP+ uptake by the thylakoids. The time-course of light-induced MP+ uptake was found to be modified in the presence of dithioerythritol, Mg2+ and ATP, i. e. under conditions which induce the ATPase activity and ATP hydrolysis in chloroplasts. The kinetic curve of light-induced MP+ uptake under these conditions consists of a relatively fast (approximatley 10 s) and a slow (approximately 10 min) components. The slow ATP-dependent component of MP+ uptake is enhanced by low concentrations of gramicidin and is completely inhibited by the energy transfer inhibitor--dicyclohexylcarbodiimide. The data obtained suggest that the light-induced energization of the chloroplast membrane is accompanied by the transport of MP+ into the thylakoids against the electrical potential and concentration gradients.     

Studies on the slow fluorescence decline in isolated chloroplasts.

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar. At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission. Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sigmoidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116-125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.     

Studies on chlorophyll fluorescence in chloroplasts. I. Effects of dithionite on fluorescence of chlorophyll A in isolated spinach chloroplasts

Effects of dithionite on the time-course of fluorescence emitted from chlorophyll a in isolated spinach chloroplasts were studied. Addition of dithionite markedly shortened the induction period of fluorescence and increased the steady-state level of fluorescence. However, a small but distinct induction, comparable to that observed in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea, was always observed in the presence of dithionite. When the fluorescence change was determined in the presence of DCMU, preincubation of the chloroplasts with dithionite for a prolonged period further shortened, but only slowly, the induction period. However, addition of DCMU during the incubation period abolished most of the effects of dithionite in reducing the induction period. The results obtained were interpreted in terms of the reduction by dithionite of endogenous electron carriers associated with photosystem 2.     

Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.     

Light activation of fructose bisphosphatase in isolated spinach chloroplasts and deactivation by hydrogen peroxide

Spinach chloroplast fructose bisphosphatase (EC 3.1.3.11.) exists in both oxidised and reduced forms. Only the latter has the kinetic properties that allow it to function at physiological concentrations of fructose 1,6-bisphosphate and Mg²⁺. Illumination of freshly prepared type A chloroplasts causes a conversion of oxidised to reduced enzyme. The rate of this conversion does not limit the rate of CO₂ fixation. In the dark the reduced enzyme partially reverts back to the oxidised form. If catalase is omitted from the reaction medium the rate of CO₂ fixation by chloroplasts is decreased and seems to be limited by the rate of conversion of the enzyme to the reduced form. The physiological significance of the light dependent generation of dithiol compounds (such as thioredoxin) within chloroplasts is discussed.     

The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts

Low concentrations of hydrogen peroxide strongly inhibit CO₂ fixation of isolated intact chloroplasts (50% inhibition at 10⁻⁵ M hydrogen peroxide). Addition of catalase to a suspension of intact chloroplasts stimulates CO₂ fixation 2–6 fold, indicating that this process is partially inhibited by endogenous hydrogen peroxide formed in a Mehler reaction.

The rate of CO₂ fixation is strongly increased by addition of Calvin cycle intermediates if the catalase activity of the preparation is low. However, at high catalase activity addition of Calvin cycle intermediates remains without effect. Obviously the hydrogen peroxide formed at low catalase activity leads to a loss of Calvin cycle substrates which reduces the rate of CO₂ fixation.

3-Phosphoglycerate-dependent O₂-evolution is not influenced by hydrogen peroxide at a concentration (5 · 10⁻⁴ M) which inhibits CO₂ fixation almost completely. Therefore the inhibition site of hydrogen peroxide cannot be at the step of 3-phosphoglycerate reduction. Dark CO₂ fixation of lysed chloroplasts in a hypotonic medium is not or only slightly inhibited by hydrogen peroxide (2.5 · 10⁻⁴ M), if ribulose-1,5-diphosphate, ribose 5-phosphate or xylulose 5-phosphate were added as substrates. However, there is a strong inhibition of CO₂ fixation by hydrogen peroxide, if fructose 6-phosphate together with triose phosphate are used as substrates. This indicates that hydrogen peroxide interrupts the Calvin cycle at the transketolase step, leading to a reduced supply of the CO₂-acceptor ribulose 1,5-diphosphate.