Daniel Béal

Plastoquinone compartmentation in chloroplasts. I: Evidence for domains with different rates of photo-reduction

The photo-reduction of plastoquinones by Photosystem II reaction centers was investigated using fluorescence and oxygen-evolution measurements in thylakoids deprived of Photosystem I acceptors. The process appears biphasic under limiting, as well as saturating, illumination. The ‘fast’ pool fraction (about 6 PQ molecules per PS II center) represents 50–70% of the total. Its half reduction time under saturating light was found about 25–60 ms, while that of the ‘slow’ pool was 0.8–1 s. When the photo-reduction process is interrupted after reduction of the fast pool and resumed after a dark period, a redistribution of the reduced plastoquinones towards the slow pool is observed, with t12 ≈ 6 s. We interpret these results as expressing a limitation of PQ diffusion in the membrane and propose that the fast pool reflects the fraction present in the grana region where most PS II centers are located, while the slow pool corresponds to quinones from the stromal region. The relationship between the redox states of PQ and of the primary acceptor QA during photo-reduction of the fast pool expresses marked discrepancies with respect to a quasi-equilibrium relationship. This failure to achieve equilibrium on a rapid time-scale and the slow diffusion rate of quinones over long distances are accounted for by small size domains bounded by membrane proteins. In agreement with this view, we found that the amount of fast photo-reducible quinones is decreased when a fraction of PS II centers is inhibited, indicating that the domains contain, on average, about 3–4 PS II centers. We conclude that PQ cannot be responsible for the long range diffusion involved in rapid electron transfer from granal (PS II) to stromal (PS II) regions, a role that must be fulfilled by plastocyanin.

A new high-sensitivity 10-ns time-resolution spectrophotometric technique adapted to in vivo analysis of the photosynthetic apparatus

A new spectrophotometric technique has been developed in which the absorption is sampled by short flashes produced by an optical parametric oscillator (OPO) pumped by the third harmonic of a Nd:YAG laser. Continuously tunable monochromatic flashes (6 ns duration, 1 nm bandwidth) are obtained in a domain ranging from 415 to 680 nm and 730 to 2000 nm. Light-induced absorption changes can be analyzed in a time window ranging from 10 ns to 100 s, with a 10−5 noise level. This new technique opens areas of research which are not accessible to previously existing spectrophotometers. Indeed, the high energy of the detecting flashes allows the analysis of samples of large optical density such as leaves, in the blue region of the spectrum. A few examples of application of this method to the analysis of electron transfer reactions in different types of photosynthetic apparatus (leaves, unicellular algae, and bacteria) are presented. This technique is also well suited to analyze the photodissociation of ligands assoc...

Photoacoustic detection of flash-induced charge separation in photosynthetic systems. Spectral dependence of the quantum yield

Abstract A new high-sensitivity photoacoustic spectrometer is described. The volume changes occurring in a thin layer of photosynthetic suspension during the first microsecond following a monochromatic laser flash (3 ns duration) are detected by a piezoelectric ceramic. These volume changes result from two components of opposite signs: (1) thermal expansion due to the release of heat in the medium, and (2) contraction occurring in the neighbourhood of the charged photoproducts. The expansion signal measures the thermal losses in the light-conversion process. Under weak flash excitation, the difference between the amounts of heat released by an active and an inactive sample measures the energy stored by the photoreactions. As the contraction signal is also linearly related to the number of flash-induced charge separations, the overall volume change, when compared between the active and inactive states of the sample, gives a relative measure of the quantum yield. In the spectral range investigated (630–705 nm), the quantum yield is practically independent of wavelength in purified PS I and in PS II (BBY) particles. In spinach chloroplasts, a depression of the quantum yield in the absorption region of LHC II suggests that a significant fraction of the light quanta absorbed by LHC II are not transferred to open reaction centres. In intact cells of Chlamydomonas reinhardtii with closed PS II reaction centres, the quantum yield spectrum of the PS I centres was compared in state I and state II. Upon transition from state I to state II, a large proportion of the quanta absorbed by the PS II antenna was found to be transferred towards PS I reaction centres. When measured at two different temperatures, the overall volume change can be deconvoluted in its two components (expansion and contraction), and the absolute value of energy storage calculated. In purified PS I and in PS II (BBY) particles, this value was found close to 1 eV per absorbed quantum, which is approximately the energy required for the formation of the radical pairs P700 + (F A ,F B ) − and Z + Q − A .

SCREENING ALGAL MUTANT COLONIES WITH ALTERED THYLAKOID ELECTROCHEMICAL GRADIENT THROUGH FLUORESCENCE AND DELAYED LUMINESCENCE DIGITAL IMAGING

A digital imaging instrument intended to monitor fluorescence and delayed luminescence of algal colonies grown on Petri plates is described. The system includes light-emitting diodes, a cooled line transfer charge-coupled device (CCD) camera, and a personal computer. Software developments were made to capture pictures and kinetics in real time during illuminations. This instrument makes the fluorescence induction kinetics of individual colonies readily accessible with a good time resolution. It offers a great refinement for screening colonies deficient in photosynthetic electron transfer thanks to appropriate computed fluorescence images. The high sensitivity of the instrument allows it to capture fluorescence images under non-actinic illuminations, and for the first time, delayed luminescence images, opening thus the way to screening mutants that have altered thylakoid electrochemical gradients.

In vivo Measurements of Photosynthetic Activity: Methods

Functional studies of living unicellular algae such as Chlamydomonas provide the simplest way to collect information on the mechanisms of photosynthetic exciton and electron transfer. Physiological function and regulation, which can be impaired upon isolation of subcellular compartments, are best studied in intact cells. Functional analysis in vivo is particularly well suited for characterizing mutant strains obtained either by random or by site-directed mutagenesis. At variance with isolated thylakoid membrane preparations, it should be taken into account that in intact cells the thylakoid membranes are in permanent interaction with the other compartments of the cell. The most important parameter that influences the properties of the photosynthetic chain is the ATP/ADP ratio. Derived from this observation, optimal conditions for studying Photosystem II, Photosystem I and the cytochrome b6f complex are discussed.

Time-resolved electron transfer at the donor side of Rhodopseudomonas viridis photosynthetic reaction centers in whole cells

We have studied the electron transfer reactions from the tetraheme cytochrome of Rhodopseudomonas viridis to the oxidized primary donor in whole cells with a new high sensitivity spectrophotometer. In this apparatus the monochromatic detecting flashes are provided by a YAG pumped Optical Parametric Oscillator, allowing a 10 ns time resolution. When four hemes are reduced the observed electron transfer reaction sequence is the following: first the low-potential c552 heme (the number refers to the maximum absorption wavelength in the alpha-band region) is oxidized with a half time of 130 ns, in agreement with previous reports of measurements performed with purified reaction centers. Then, the electron hole is transferred to the low potential c554 heme with a half time of 2.6 µs. When only the two high potential hemes are reduced the observed electron transfer sequence is the following: oxidation of the high potential c559 heme in the hundreds of ns time range (410 ns), reduction of this heme by the high potential c556 heme in the µs time range (2.7 µs). This confirms the first steps of electron transfer observed in isolated reaction centers. However, in the microsecond time domain, the overall amount of oxidized hemes increases suggesting that, in vivo, the equilibrium constant between the P+/P and the c559ox/c559red couples is significantly lower than expected from the difference in their midpoint potentials.

New Digital Imaging Instrument For Measuring Fluorescence and Delayed Luminescence

We describe a new digital imaging instrument designed to monitor fluorescence and delayed luminescence of photosynthetic systems (algal or bacterial colonies grown on Petri dishes, leaves). The setup includes light-emitting diodes, a cooled line transfer charge-coupled device (CCD) camera, a personal computer and appropriate software which allows capture of pictures and kinetics in real time during illumination. This instrument provides fluorescence images under actinic or non-actinic illumination and makes the fluorescence induction kinetics of individual colonies readily accessible with good time resolution. It brings great refinement to procedures for screening colonies that are altered in photosynthetic electron transfer owing to appropriately computed fluorescence images. This instrument allows, for the first time, to capture delayed luminescence images, thus opening the way to screening for mutants that have an altered permanent or light-induced electrochemical gradient across thylakoid membranes.