Anne-Lise Etienne

Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum.

The pool size of the xanthophyll cycle pigment diadinoxanthin (DD) in the diatom Phaeodactylum tricornutum depends on illumination conditions during culture. Intermittent light caused a doubling of the DD pool without significant change in other pigment contents and photosynthetic parameters, including the photosystem II (PSII) antenna size. On exposure to high-light intensity, extensive de-epoxidation of DD to diatoxanthin (DT) rapidly caused a very strong quenching of the maximum chlorophyll fluorescence yield (F m, PSII reaction centers closed), which was fully reversed in the dark. The non-photochemical quenching of the minimum fluorescence yield (F o, PSII centers open) decreased the quantum efficiency of PSII proportionally. For bothF m and F o, the non-photochemical quenching expressed asF/F′ − 1 (with F′ the quenched level) was proportional to the DT concentration. However, the quenching of F o relative to that ofF m was much stronger than random quenching in a homogeneous antenna could explain, showing that the rate of photochemical excitation trapping was limited by energy transfer to the reaction center rather than by charge separation. The cells can increase not only the amount of DT they can produce, but also its efficiency in competing with the PSII reaction center for excitation. The combined effect allowed intermittent light grown cells to down-regulate PSII by 90% and virtually eliminated photoinhibition by saturating light. The unusually rapid and effective photoprotection by the xanthophyll cycle in diatoms may help to explain their dominance in turbulent waters.

Quenching of the System II chlorophyll fluorescence by the plastoquinone pool

Abstract If 3-(3,4-dichlorophenyl)-1,1-dimethylurea is added to dark adapted chloroplasts, the maximum fluorescence F stat when Q is completely reduced, is lower than the maximum fluorescence reached with no 3-(3,4-dichloropheny)-1,1-dimethylurea present during a continuous illumination F p . If 3-(3,4-dichlorophenyl)-1,1-dimethylurea is added during illumination a quenching develops and the fluorescence drops from F p to F stat . A study was made of that quenching and we show that it corresponds to a non-photochemical quenching by the oxidized pool of plastoquinones A: 1. 1. When 3-(3,4-dichlorophenyl)-1,1-dimethylurea is added during illumination, A initially reduced is reoxidized by System I. The rate of the fluorescence quenching which develops upon addition of 3-(3,4-dichlorophyl)-1,1-dimethylurea is also dependent on System I activity. 2. 2. If A is reduced by a strong illumination, it is slowly reoxidized in the dark by oxygen. The maximum fluorescence level F t reached during an illumination following 3-(3,4-dichlorophenyl)-1,1-dimethylurea addition is related to the oxidation level of A. 3. 3. In low light intensity, the amount of reduced plastoquinone is increasing with increasing MgCl 2 . The quenching observed also depends on MgCl 2 concentration. 4. 4. If A is maintained reduced by dithionite, the quenching is abolished. This quenching exerted by the oxidized plastoquinones is 20% of the maximum fluorescence. It is weak compared to the very different photochemical quenching of open Photosystem II centers.

GENERAL FEATURES OF PHOTOPROTECTION BY ENERGY DISSIPATION IN PLANKTONIC DIATOMS (BACILLARIOPHYCEAE)

Planktonic diatoms (Bacillariophyceae) have to cope with large fluctuations of light intensity and periodic exposure to high light. After a shift to high light, photoprotective dissipation of excess energy characterized by the nonphotochemical quenching of fluorescence (NPQ) and the concomitant deepoxidation of diadinoxanthin to diatoxanthin (DT) were measured in four different planktonic marine diatoms (Bacillariophyceae): Skeletonema costatum (Greville) Cleve, Cylindrotheca fusiformis Reimann et Lewin, Thalassiosira weissflogii (Grunow) Fryxell et Hasle, and Ditylum brightwellii (West) Grunow in comparison to the model organism Phaeodactylum tricornutum Böhlin. Upon a sudden increase of light intensity, deepoxidation was rapid and de novo synthesis of DT also occurred. In all species, NPQ was linearly related to the amount of DT formed during high light. In this report, we focused on the role of DT in the dissipation of energy that takes place in the light‐harvesting complex. In S. costatum for the same amount of DT, less NPQ was formed than in P. tricornutum and as a consequence the photoprotection of PSII was less efficient. The general features of photoprotection by harmless dissipation of excess energy in planktonic diatoms described here partly explain why diatoms are well adapted to light intensity fluctuations.

Photosystem II fluorescence quenching in the cyanobacterium Synechocystis PCC 6803: involvement of two different mechanisms.

The structural changes associated to non-photochemical quenching in cyanobacteria is still a matter of discussion. The role of phycobilisome and/or photosystem mobility in this mechanism is a point of interest to be elucidated. Changes in photosystem II fluorescence induced by different quality of illumination (state transitions) or by strong light were characterized at different temperatures in wild-type and mutant cells, that lacked polyunsaturated fatty acids, of the cyanobacterium Synechocystis PCC 6803. The amplitude and the rate of state transitions decreased by lowering temperature in both strains. Our results support the hypothesis that a movement of membrane complexes and/or changes in the oligomerization state of these complexes are involved in the mechanism of state transitions. The quenching induced by strong blue light which was not associated to D1 damage and photoinhibition, did not depend on temperature or on the membrane state. Thus, the mechanism involved in the formation of this type of quenching seems to be unrelated to the movement of membrane complexes. Our results strongly support the idea that the mechanism involved in the fluorescence quenching induced by light 2 is different from that involved in strong blue light induced quenching.

In diatoms, a transthylakoid proton gradient alone is not sufficient to induce a non‐photochemical fluorescence quenching

Non‐photochemical fluorescence quenching (NPQ) in diatoms is associated with a xanthophyll cycle involving diadinoxanthin (DD) and its de‐epoxidized form, diatoxanthin (DT). In higher plants, an obligatory role of de‐epoxidized xanthophylls in NPQ remains controversial and the presence of a transthylakoid proton gradient (ΔpH) alone may induce NPQ. We used inhibitors to alter the amplitude of ΔpH and/or DD de‐epoxidation, and coupled NPQ. No ΔpH‐dependent quenching was detected in the absence of DT. In diatoms, both ΔpH and DT are required for NPQ. The binding of DT to protonated antenna sites could be obligatory for energy dissipation.

Effect of the 33-kDa protein on the S-state transitions in photosynthetic oxygen evolution

The effect of the extrinsic 33-kDa protein on the photosynthetic oxygen evolution was studied by comparing spinach Photosystem II particles depleted of the 33-kDa protein with those reconstituted with the protein. The light-intensity dependence of the oxygen-evolution activity under continuous illumination suggests that a dark step, but not a light step, in the oxygen-evolving reaction is accelerated by the 33-kDa protein. Consistently, the pattern of oxygen yield with a series of short saturating flashes, which showed a maximum on the third flash and a damped oscillation with a period of 4, was not much affected by the removal and rebinding of the 33-kDa protein, when the dark interval between the flashes was long enough, i.e., longer than 0.5 s. The millisecond kinetics of oxygen release after the third flash was retarded by the removal of the 33-kDa protein and stimulated by its rebinding, suggesting that the transition from S3 to S0 is accelerated by the 33-kDa protein. The stability of the S2 and S3 states in darkness was higher in the absence of the 33-kDa protein than its presence.

Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms.

The dominance of diatoms in turbulent waters suggests special adaptations to the wide fluctuations in light intensity that phytoplankton must cope with in such an environment. Our recent demonstration of the unusually effective photoprotection by the xanthophyll cycle in diatoms [Lavaud et al. (2002) Plant Physiol 129 (3) (in press)] also revealed that failure of this protection led to inactivation of oxygen evolution, but not to the expected photoinhibition. Photo-oxidative damage might be prevented by an electron transfer cycle around Photosystem II (PS II). The induction of such a cycle at high light intensity was verified by measurements of the flash number dependence of oxygen production in a series of single-turnover flashes. After a few minutes of saturating illumination, the oxygen flash yields are temporarily decreased. The deficit in oxygen production amounts to at most 3 electrons per PS II, but continues to reappear with a half time of 2 min in the dark until the total pool of reducing equivalents accumulated during the illumination has been consumed by (chloro)respiration. This is attributed to an electron transfer pathway from the plastoquinone pool or the acceptor side of PS II to the donor side of PS II that is insignificant at limiting light intensity but is accelerated to milliseconds at excess light intensity. Partial filling of the 3-equivalents capacity of the cyclic electron transfer path in PS II may prevent both acceptor-side photoinhibition in oxygen-evolving PS II and donor-side photoinhibition when the oxygen-evolving complex is temporarily inactivated.

Reactivation of oxygen evolution of NaCl-washed Photosystem-II particles by Ca2+ and/or the 24 kDa protein

We have studied the conditions required to reactivate oxygen evolution in NaCl-washed Photosystem-II particles. Restoration of oxygen evolution by Ca2+ revealed an heterogeneity in these Photosystem-II particles: 30% possess a low affinity site for Ca2+ (1–2 mM), 70% a high affinity site for Ca2+ (50–100 μM), even in the absence of the 24 kDa protein. The sole effect of the 24 kDa protein added back to Photosystem-II particles shortly before illumination was to stabilize oxygen evolution. Added back more than half an hour before, to Photosystem II particles at a high chlorophyll concentration, it increased oxygen evolution from approx. 40% of the control to 60–70% of the control. After reconstitution, an appreciable fraction of the low-affinity site for Ca2+ was still present.

The charge accumulation mechanism in NaCl-washed and in Ca2+-reactivated Photosystem-II particles

Abstract In this report we show that, under flashing light, NaCl-washed, Photosystem-II particles which do not evolve oxygen, are unable to go beyond the S 3 Z + state. Addition of Ca 2+ alone restores the S 3 Z + → S 0 transition and oxygen evolution. These conclusions were reached by analysis of the oxygen and luminescence oscillations.

Prompt and delayed fluorescence of chloroplasts upon mixing with dichlorophenyldimethylurea.

Abstract 1. 1. The kinetics of prompt and delayed fluorescence of isolated chloroplasts or algae have been monitored after flash preillumination (in the time-range extending from 0.4 s after the flash). A rapid mixing with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) may take place after the last flash. 2. 2. 1 s after the mixing with DCMU, the prompt fluorescence displays binary oscillations with the number of preilluminating flashes, similarly to the observation of Velthuys, B.R. and Amesz, J. ((1974) Biochim. Biophys. Acta 333, 85–94), with chloroplasts to which an artificial Photosystem II donor was added. These oscillations are due to a back-transfer of electrons from the secondary acceptor, B, to the primary acceptor, Q, caused by DCMU. At longer times after the mixing, charge recombination takes place to a variable extent according to the charge storage state Si on the donor side, yielding the oscillatory pattern observed by Wollman, F.A. ((1978) Biochim. Biophys. Acta 503, 263–273). 3. 3. Shifting the pH from 6 to 8 causes an acceleration of the DCMU-induced back-transfer to Q and an about 2-fold increase in the amplitude of the fluorescence oscillations. The rate of the DCMU-induced rise of fluorescence is sensitive to the pH during the mixing, whereas the amplitude of the oscillations depends on the pH during the preillumination. Even under optimal conditions, the oscillations account only for a fraction of the total variable fluorescence. 4. 4. The delayed light emitted by isolated chloroplasts in the 100 ms—seconds range oscillates weakly (periodicity 4) with the number of preilluminating flashes. Mixing with DCMU after the preillumination causes a delayed light stimulation which varies with the flash number. The enhancement factor oscillates with periodicities of both 2 and 4. The amplitude of the period-2 contribution varies with the amount of B oxidized in the dark, while that of the period-4 contribution depends on the extent of this type of oscillation in the control experiment. 5. 5. The period-2 oscillations of the DCMU-stimulation of delayed light behave similarly to the fluorescence oscillations. It is shown that they are not due to a modulation of luminescence by the fluorescence yield, but rather to the variations of the amount of Q− as a substrate. 6. 6. It is concluded that in the absence of DCMU, the reduced secondary acceptor B− is not the main source of electrons involved in radiative recombination of functional centers in the time-range we have studied. Possible models are discussed.