Johann Lavaud
email: lavaud@biologie.ens.fr

Hans J Van Gorkom

Anne-Lise Etienne

Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms

Keywords: diatoms, electron transport cycle, oxygen evolution, Phaeodactylum tricornutum, photoprotection, photosystem II

) 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.

Introduction

Although marine diatoms contribute substantially to global primary production [START_REF] Van Den Hoek | Algae: An Introduction to Phycology[END_REF], their bioenergetics has received relatively little attention. Diatoms often dominate the phytoplankton in turbulent waters [START_REF] Harris | Phytoplankton Ecology: Structure, Function, and Fluctuation[END_REF], where vertical mixing continuously exposes them to large fluctuations in light intensity on time scales of minutes to days [START_REF] Lewis | Turbulent motions may control phytoplankton photosynthesis in the upper ocean[END_REF]. They must be able to survive long periods of darkness when carried down by deep mixing as well as short periods of full sunlight when they hit the surface. They have indeed been shown to survive many days of darkness without carbon assimilation [START_REF] Griffiths | Factors affecting the photosynthetic capacity of laboratory cultures of the diatom Phaeodactylum tricornutum[END_REF]) and to cope with a wide variation in light intensity [START_REF] Richardson | Adaptation of unicellular algae to irradiance: an analysis of strategies[END_REF]. Their main protective mechanism against over-excitation appears to be non-photochemical quenching of the chlorophyll excited state (NPQ) [START_REF] Ting | Photochemical and non-photochemical fluorescence quenching processes in the diatom Phaeodactylum tricornutum[END_REF]) associated with xanthophyll de-epoxidation [START_REF] Arsalane | Influence of the pool size of the xanthophyll cycle on the effects of light stress in a diatom: Competition between photoprotection and photoinhibition[END_REF][START_REF] Olaizola | Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom[END_REF]. By a quantitative study of the quenching by NPQ, both with open (Q A oxidized) and with closed (Q A reduced) photosystem II (PS II) reaction centers, we recently demonstrated that the xanthophyll cycle provides the planktonic diatom Phaeodactylum tricornutum with a unusually effective and adaptable mechanism to down-regulate PS II activity at high light intensity [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF].

Illumination for an hour at a light intensity corresponding to full sunlight (50 times the intensity at which the cells were grown) was required to inactivate a large fraction of PS II, as measured by the yield of O 2 production upon illumination by repetitive singleturnover flashes. Surprisingly, the chlorophyll fluorescence yield increase associated with photoreduction of Q A was not affected. When the oxygen evolving complex is not functional, charge separations in PS II would be expected to lead to rapid photooxidative destruction, either directly by the strong oxidants resulting from charge separation or indirectly by charge recombination to the triplet state, leading to singlet oxygen production [START_REF] Aro | Photoinhibition of Photosystem II. Inactivation, protein damage and turnover[END_REF]. The only likely way to avoid that would seem to be an electron transfer cycle around PS II.

Cyclic electron flow around PSII, presumably via cytochrome b 559 (for review see [START_REF] Whitmarsh | Form and function of cytochrome b-559[END_REF], has been suggested earlier as a photoprotection mechanism that could retard both acceptor and donor side photoinhibition [START_REF] Nebdal | Redox state of a one electron component controls the rate of photoinhibition of Photosystem II[END_REF][START_REF] Barber | A functional model for the role of cytochrome b559 in the protection against donor and acceptor side photoinhibition[END_REF]. Such a cycle was shown to occur in vivo in the green alga Chlorella pyrenoidosa at high light intensities [START_REF] Falkowski | Evidence for cyclic electron flow around Photosystem II in Chlorella pyrenoidosa[END_REF]. [START_REF] Prasil | Cyclic electron flow around Photosystem II in vivo[END_REF] studied its expression in a temporary decrease of O 2 production by saturating flashes during the first tens of seconds after a light to dark transition, and concluded that the cycle occurs as long as the plastoquinone pool is reduced.

In order to verify the possible induction of an electron transfer cycle in

Phaeodactylum tricornutum at high light intensity, we have also investigated the flash number dependence of the O 2 yields in a series of single turnover flashes. For darkadapted cells, these yields initially show a period 4 oscillation with flash number due to the 4-state oxidation cycle of the O 2 -evolving complex [START_REF] Joliot | Un nouveau modèle des centres photochimiques du système II[END_REF][START_REF] Kok | Cooperation of charges in photosynthetic O2 evolution-I. A linear four step mechanism[END_REF]). To produce an O 2 molecule, 4 consecutive charge separations in the same PS II reaction center are required, each raising the oxidation state ('S-state') by one equivalent. S 0 and S 1 are stable, but the higher S-states decay to S 1 in a few minutes darkness, resulting in an oscillating O 2 yield with maxima on flash numbers 3, 7, etc.

The oscillation is damped in a few cycles to a constant steady state yield, Y SS , due to loss of synchrony caused mainly by a non-zero probability of 'misses', usually near 10 %. The oscillation pattern is extremely sensitive to the miss probability and would change significantly already if only a few percent of the electrons would come from a cyclic electron transfer pathway rather than from water oxidation.

After exposure to high light intensity a very high miss probability that disappeared in approximately 15 flashes, quite similar to that reported by [START_REF] Prasil | Cyclic electron flow around Photosystem II in vivo[END_REF] for Chlorella, was indeed observed. In Phaeodactylum and other planktonic diatoms, it could still be measured after very long dark times and its properties suggest a somewhat different interpretation, but nevertheless provide evidence for a photoprotective electron transfer cycle around PS II at high light intensity.

Materials and methods

Cultures

Phaeodactylum tricornutum Böhlin and other marine diatoms: Cylindrotheca fusiformis, Cylindrotheca cryptica and Navicula sp. were grown photoautotrophically at 18 °C in sterile natural seawater F/2 medium [START_REF] Guillard | Studies of marin planktonic diatoms. 1. C. nana (Hustedt) and D. confervacea (Cleve)[END_REF] in 300 mL batches, continuously flushed with sterile air (except for Navicula sp. which is a benthic species)and illuminated at 40 µE.m -2 .s -1 by white fluorescent tubes in a 16 h light -8 h dark cycle. The cells were harvested in exponential phase, resuspended in their culture medium at 10 µg Chl a.mL -1 , and kept continuously stirred under low light at 18 °C until use. All measurements were done at 18°C. Before measurement 4 mM sodium bicarbonate was added.

Measurement of oxygen evolution

O 2 production on illumination by a series of short (5 µs) saturating flashes (Strobotac, General Radio) fired at 2 Hz was measured polarographically by a rate electrode [START_REF] Joliot | A polarographic method for detection of oxygen production and reduction of hill reagent by isolated chloroplasts[END_REF]) as described [START_REF] Lemasson | Photo-inactivation of system II centers by carbonyl cyanide m-chlorophenylhydrazone in Chlorella pyrenoidosa[END_REF], after 7 min settling of the cells in the dark. For the experiments shown by the diamonds in Figure 3A, the setup was modified to allow saturating illumination via an optic guide that could be removed within a few seconds before the flash series. Illumination was provided by a 150 W quartz-iodine lamp through a heat filter. The O 2 deficit was quantified as 20 x Y SS , the steady-state flash yield, minus the sum of signals in a 20-flash series, expressed in units Y SS . The contribution to the deficit resulting from normal S-state deactivation during dark-adaptation was corrected for by subtraction of the deficit in a non-illuminated sample.

Fluorescence yield measurements

Chlorophyll fluorescence yield changes were recorded by a modified [START_REF] Ritz | pH-dependent fluorescence quenching and its photoprotective role in the unicellular red alga Rhodella violacea[END_REF] PAM 101 module (Walz). The cells were dark-adapted for 20 min before actinic illumination of adjustable intensity, as measured by a PAR sensor (Licor), and the development of NPQ was probed by 600 ms pulses of 4 mE.m -2 .s -1 white light from a quartz-iodine lamp admitted by an electronic shutter (Vincent Uniblitz). NPQ was calculated as (F m -F m ')/F m ' [START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF].

Results

Typical O 2 flash yield measurements are shown in Figure 1a. Phaeodactylum tricornutum cells were allowed to settle in darkness on a rate electrode and the polarographic current changes induced by a series of single-turnover flashes were recorded. The dashed line shows the classical period four oscillation of the O 2 yield with flash number due to the 4-step redox cycle of the O 2 -evolving complex [START_REF] Joliot | Un nouveau modèle des centres photochimiques du système II[END_REF][START_REF] Kok | Cooperation of charges in photosynthetic O2 evolution-I. A linear four step mechanism[END_REF]. The damping to a constant steady-state yield, Y SS , results from a normal probability of about 10 % that a photoreaction fails to contribute to H 2 O Fig. 1A First mention oxidation. Early in the sequence this 'miss probability' was much higher in cells that had been exposed to saturating light for 5 min, but it returned to 10 % during the flash series so that Y SS was the same (solid line). This phenomenon is better visualized by plotting the sums of 4 consecutive flash yields in order to remove most of the oscillatory pattern (Figure 1b). The solid symbols show that it took about 15 flashes before the normal O 2 yield was fully recovered in the illuminated sample.

We quantify the oxygen deficit as 20 Y SS minus the total oxygen production of the 20 flashes and subtract the O 2 deficit of the non-illuminated sample, which is 0.8-1.0 Y SS . This is expected because the higher S-states are reduced to S 1 in the dark, so the average number of stored oxidizing equivalents is expected to decrease from approximately (0+1+2+3)/4 = 1.5 in the steady state to (0+1+1+1)/4 = 0.75 after darkadaptation. The difference of 0.75 equivalents should cause a deficit of 0.83 Y SS , taking the 10 % miss probability into account. It will be somewhat larger because the miss probability and hence the steady state S-state concentration is higher for high S-states than for low S-states (de Wijn and van Gorkom 2002). The deficit in the illuminated sample in Figure 1 was higher than that in the non-illuminated sample by 2.8 Y SS , or 2.5 electrons per PS II. The shape of the oscillation pattern does not suggest a phase delay by 3 flashes but rather an increased miss probability that gradually disappears in 15 flashes. From our previous study [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF] we know that the large extra miss probability during the first S-state cycle in these conditions cannot be attributed to a non-saturating flash energy or to the presence of closed PS II centers. It appears, therefore, that the O 2 deficit is due to the presence of reductants that can compete with water as an electron donor to PS II. Decreasing the flash frequency did not change the deficit, however. Similar data were obtained with other planktonic marine diatoms from Cyclotella cryptica), but not or much less with a benthic diatom (Navicula sp.), a green alga (Chlorella fusca) and a cyanobacterium (Synechococcus sp.). The duration and intensity of illumination producing the maximum O 2 deficit were rather different for the different species but it was for the planktonic diatoms that the O 2 deficit was the highest.

The O 2 deficit was induced by conditions similar to those that elicit NPQ, suggesting a connection to photoprotective mechanisms, but there are clear differences in detail. Figure 2a shows that it appeared earlier and continued to rise longer. At much longer times, while NPQ continues to increase slowly [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF], Y SS and the O 2 deficit decreased in parallel (Table 1), i.e. the deficit per active PS II remained unchanged. This observation indicates that the electron donors responsible for the O 2 deficit are intrinsic components of PS II and not a common pool accessible to all remaining active PS II centers. Figure 2b Presumably both conditions affect NPQ primarily via the extent of the steady-state pH gradient maintained during illumination. When the pH gradient is lowered by decreasing the electron transport rate (using a lower light intensity) or by uncoupling (which probably increases the electron transport), a similar decrease of O 2 deficit with decreasing NPQ is seen. Therefore the deficit appears to depend on the pH gradient, but not in the same way as NPQ: a deficit is detectable already at a pH gradient too small to induce NPQ. Low concentrations of N,N'-dicyclohexyl-carbodiimide (DCCD) enhanced NPQ but decreased the deficit (Table 1). DCCD can have multiple effects but After illumination, the size of the O 2 deficit changed in complicated ways (Fig. 3a). The open symbols in panel A show that recovery from NPQ was largely complete in 5 min, independent of illumination time. Decay of the O 2 deficit (solid symbols) was much slower and depended strongly on illumination time. Two components can be distinguished: a rapid decay starting after a lag time that also depended on illumination time, followed by a much slower component. Addition of DCCD after illumination led to a slower decay of the O 2 deficit and to an increase of NPQ after its initial postillumination decrease (not shown). The first point of these O 2 data is at 7 min after illumination, the time required for settling of the cells on the electrode. The diamonds represent a light treatment equivalent to that for the circles but which was applied directly on the rate electrode. These data show that the deficit was fully developed immediately after illumination and did not decrease during a half-hour in the semianaerobic conditions on the electrode. Figure 4 shows the kinetics of re-appearance of the O 2 deficit in a second flash series as a function of dark time, on the electrode, after it was removed by a first series of 20 flashes. Depletion of the electron source by (chloro)respiration is slow in the semi-anaerobic conditions on the polarized electrode and should not become limiting on this time scale (see Fig. 3a,diamonds). The deficit increased with a half time 2 min to the same value as in the first flash series. This is not shorter than the lifetime of the high S-states, which limits the minimum light intensity where PS II can function, and will not decrease the efficiency of photosynthesis in low light. The appearance of the deficit after strong illumination could not be resolved (diamonds in Figure 3a) and must be much faster than its measurement (a few seconds). This suggests that immediately after, and presumably during, high intensity illumination the time required for reduction of the PS II components responsible for the O 2 deficit must be at least several orders of magnitude shorter than 2 min and might well be short enough to play a photoprotective role.

Discussion

The large deficit that appears after saturating illumination in the O 2 yields of a series of single turnover flashes could not be attributed to a decreased flash yield of stable charge separation and shows that a substantial fraction of the charge separations did not contribute to water oxidation. Therefore competition by an alternative electron donor had to be assumed. The O 2 deficit of up to 3 Y SS is much larger than can be accounted for by possible changes in steady state S-state distribution or deactivation kinetics induced by high intensity illumination.

Cofactors involved

The proportional decrease of Y SS and the deficit by long exposure to high light intensity shows that the alternative electron donors causing the deficit in O 2 production are probably endogenous PS II components that are not exchangeable between different PS II centers. Perhaps reduction of S 1 to S 0 might account for a fraction of the deficit, but it is not possible to attribute the deficit to an over-reduced 'S -2 ' state of the oxygen evolving complex itself, because the oscillation pattern shows a decreased efficiency rather than a delay in O 2 production. The PS II reaction center contains several cofactors known to play a role as alternative electron donors in specific experimental circumstances, especially at cryogenic temperatures: -carotene, Chl Z , and cytochrome b 559 [START_REF] Butler | The kinetics of light-induced changes of C550, Cyt b559 and fluorescence yield in chloroplasts at low temperature[END_REF][START_REF] Visser | Photooxidation of chlorophyll in spinach chloroplasts between 10 and 180 K[END_REF][START_REF] Schenk | Flash-induced carotenoid radical cation formation in photosystem II[END_REF][START_REF] Thompson | Cyt b559 may function to protect photosystem II from photoinhibition[END_REF]. Tyrosine D (Y D ) is the only PS II component that normally competes with, but does not completely out-compete, the electron transfer reactions leading to water oxidation. The high S-states can oxidize it in seconds [START_REF] Babcock | The photosynthetic oxygen evolving process[END_REF], presumably via their redox equilibria with the secondary electron donor Y Z and the primary electron 1993). Normally Y D remains in its oxidized tyrosyl radical form during photosynthesis, but we found no literature data to confirm that for diatoms. If high light intensity would enable its rapid reduction, e.g. via Cyt b 559 and one other component, a cyclic electron transfer path would result that could explain both the O 2 deficit reported here and the lack of photo-oxidative damage when the oxygen evolving complex is inactivated (Lavaud et al., submitted).

Reduction of the alternative electron donors during illumination

In the conditions of Figure 4, the reduction rate of the alternative electron donors is approximately 1 equivalent per PS II per minute. With an oxidation rate near 1 equivalent per S-state cycle and a reasonable light-saturated rate of O 2 evolution of the order of 10 S-state cycles per PS II per second, the alternative electron donors should be in their oxidized state during saturating illumination. The O 2 deficit measured in seconds after illumination, however, shows that the alternative electron donors were largely in the reduced state. At high light intensity their oxidation is unlikely to be slower, so their reduction must be very much faster than in the dark.

Electrons reducing the endogenous PS II components that act as alternative electron donors would be expected to come from Q A , Q B , or the plastoquinone pool.

The steady state reduction level of the plastoquinone pool remains unusually low on illumination of P. tricornutum, in spite of normal antenna sizes and relative amounts of PS I and PS II [START_REF] Owens | Light-harvesting function in the diatom Phaeodactylum tricornutum. II. Distribution of excitation energy between the photosystems[END_REF][START_REF] Caron | Chlorophyll fluorescence transients from the diatom Phaeodactylum tricornutum : relative rates of cyclic phosphorylation and chlororespiration[END_REF]) and the absence of a 'state 1 -state 2 transition' [START_REF] Owens | Light-harvesting function in the diatom Phaeodactylum tricornutum. II. Distribution of excitation energy between the photosystems[END_REF]). This would be consistent with an electron transfer cycle around PS II that, at saturating light intensity, is not slow compared to linear electron flow to PS I. A time constant of the order of 10 ms would be indicated, long enough not to interfere with commonly used methods to determine the extent of photochemical quenching, but short enough to prevent acceptor side photoinhibition [START_REF] Nebdal | Redox state of a one electron component controls the rate of photoinhibition of Photosystem II[END_REF]) and keep some of the alternative electron donors in the reduced state. Those would decrease the quantum yield of O 2 evolution substantially and would also account the non-linearity between O 2 evolution and photochemical quenching at high light intensity that [START_REF] Geel | Estimation of oxygen evolution by marine phytoplankton from measurement of the efficiency of photosystem II electron flow[END_REF] attribute to a redox state dependent Mehler-type O 2 reduction.

Reduction in the dark [START_REF] Prasil | Cyclic electron flow around Photosystem II in vivo[END_REF] studied a similar post-illumination decrease of O 2 flash yields seen in the green alga Chlorella pyrenoidosa on a time scale of seconds, which they attributed to a Cyt b 559 -switched cyclic electron transport around PS II. The main difference with our data seems to be the absence of period 4 oscillations due to the absence of a dark-adaptation time between illumination and flash series. The recovery to normal values after illumination was attributed to a decreasing number of centers in which the cycle was switched on and was related to the oxidation of the plastoquinone pool. Indeed a faster recovery of the oxygen yield per flash during the flash series was observed with background illumination by far-red light that selectively excited PS I, and a slower recovery was obtained in anaerobic conditions. Measurements at a different flash spacing than 2.5 s were not reported, but would not be expected to show a much different oxidation time of the pool in this case.

The O 2 deficit in P. tricornutum did not depend on flash frequency, so the time required to recover Y SS yields was proportional to the spacing of the flashes. Apparently it is the flashes that cause the recovery, not the time spent between them. If the deficit in O 2 production is attributed to cyclic electron transport during the flash series, as proposed by [START_REF] Prasil | Cyclic electron flow around Photosystem II in vivo[END_REF], it implies a temporary shortage also in reduction of the plastoquinone pool by PS II compared to its oxidation by PS I. So the flash series itself might indeed cause some oxidation of the pool if it was in the reduced state. Since the deficit accounts for at most 3 electrons/PS II, however, an unlikely small pool size or PS II/PS I ratio would have to be assumed to explain the recovery of normal Y SS yields by PS I-induced oxidation of the total plastoquinone pool. If only an arbitrary fraction of the pool was involved an invariable maximum O 2 deficit of 3 Y SS would not be expected. We conclude that cyclic electron transfer during the flash series is unlikely to explain our results.

After a few minutes darkness, the reduction of the alternative electron donors appeared to depend on the total pool of reducing equivalents accumulated by water oxidation during the preceding illumination. After saturating illumination an accelerated O 2 uptake was observed that consumed approximately the same amount of O 2 as had been produced in the light (Fig. 3b). The diatoms are evidently unable to use the temporary >10-fold excess of light, compared to the light intensity at which they were grown, for net carbon fixation on this time scale. Instead, they seem to use the accumulated reducing equivalents as fuel for subsequent (chloro)respiratory ATP synthesis in the dark. Such a process could extend the useful dynamic range of photosynthetic electron transport. This could be an important advantage at fluctuating light intensity, e.g. in allowing carotenoid biosynthesis to proceed [START_REF] Bennoun | Chlororespiration and the process of carotenoid biosynthesis[END_REF][START_REF] Carol | A plastid terminal oxidase comes to light: implications for carotenoid biosynthesis and chlororespiration[END_REF]. We have shown earlier that the capacity of photoprotection by NPQ in P. tricornutum is proportional to the diadinoxanthin content of the cells.

A temporary storage of excess reductant for later use was recently proposed to be the function of the bi-directional hydrogenase in a cyanobacterium [START_REF] Appel | The bidirectional hydrogenase of Synechocystis sp. PCC 6803 works as an electron valve during photosynthesis[END_REF].

H 2 seems in fact the most, if not the only, likely reductant to have direct access to the donor side of PS II and cause an O 2 deficit. The electron source involved here cannot be H 2 , however, since it was not washed from the sample on the rate electrode. Instead, the reducing equivalents are presumably accumulated in the form of reduced carbon compounds produced by the Calvin cycle or C 4 metabolism [START_REF] Reinfelder | Unicellular C 4 photosynthesis in a marine diatom[END_REF] and should have no access to the donor side of PS II. The electron transfer pathway reducing the alternative electron donors in PS II is probably very indirect in this case.

When added after illumination, DCCD caused not only to a slower decay of the O 2 deficit but also an increase of NPQ after its initial post-illumination decrease. This indicates that the oxidation of the accumulated photosynthetic products takes place at least in part in the chloroplast via the chlororespiration coupled to ATP synthesis reported earlier [START_REF] Caron | Chlorophyll fluorescence transients from the diatom Phaeodactylum tricornutum : relative rates of cyclic phosphorylation and chlororespiration[END_REF][START_REF] Ting | Photochemical and non-photochemical fluorescence quenching processes in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Jakob | Activation of diadinoxanthin de-epoxidase due to a chlororespiratory proton gradient in the dark in the diatom Phaeodactylum tricornutum[END_REF].

Chlororespiration is thought to proceed via the plastoquinone pool [START_REF] Bennoun | Chlororespiration and the process of carotenoid biosynthesis[END_REF], so in principle the same electron transfer pathway from plastoquinol to the PS II donor side could be involved in the dark as well as in the light, only at a 10 4 times slower rate. This rate difference is much too large to be attributed merely to a lower steady state plastoquinol concentration if chlororespiration can build up a sufficiently large pH gradient to induce substantial NPQ in the dark. An effective switching mechanism must exist to ensure that the cycle operates only at high light intensity. Cyt b 559 has often been postulated to play a role in such a switching mechanism [START_REF] Thompson | Cyt b559 may function to protect photosystem II from photoinhibition[END_REF][START_REF] Nebdal | Redox state of a one electron component controls the rate of photoinhibition of Photosystem II[END_REF][START_REF] Allakhverdiev | Evidence for the involvement of cyclic electron transport in the protection of photosystem II against photoinhibition: influence of a new phenolic compound[END_REF].

Concluding remarks

We have presented evidence for the induction of an electron transfer cycle around PS II in planktonic diatoms at high light intensity. With an estimated rate of 10 2 s -1 at saturating light intensity acceptor-side photoinhibition due to over-reduction of Q A is prevented and the cofactors involved in the cycle are kept largely in the reduced state that competes with water as an electron donor. This is tentatively attributed to electron transfer from Y D to Y Z ox that competes with the 2 ms reduction of Y Z ox S 3 to Y Z S 0 by water and that will also prevent photo-oxidative damage when the O 2 -evolving complex is inactivated. The fact that we can still see the competition long after the cycle has been shut down is attributed to a very slow, physiologically irrelevant leak of electrons from the chlororespiratory pathway in the dark to the cofactors responsible for the PS II electron transport cycle at high light intensity. T im e a f te r fir s t f la s h s e q u e n c e ( m in )

  Fig.1B First mention

  shows the relation between O 2 deficit and NPQ when their amplitude was varied using either different light intensities (solid symbols) or different concentrations of an uncoupler (NH 4 Cl) (open symbols).

  Fig.2A First mention

Figure

  Figure3bshows the O 2 deficit as a function of dark time after a fixed

  Fig.3A First mention

  donor P 680 . Y Z ox can oxidize Y D in a ms (Boussac and Etienne 1982) and this reaction competes with the 2 ms reduction of Y Z ox S 3 to Y Z S 0 by water (van Leeuwen et al.

Figure 1 .

 1 Figure 1. (a) Oxygen production in a series of single-turnover flashes by dark-adapted cells (dashed line) or after 5 min saturating illumination (450 µE.m -2 .s -1 ) in the PAM

Figure 2 .

 2 Figure 2. (a) Induction of the O 2 deficit (circles) during saturating illumination in the

Figure 3 .

 3 Figure 3. O 2 deficit (solid symbols) as a function of time after illumination. Panel (a):

Figure 4 .

 4 Figure 4. O 2 deficit as a function of dark time on the electrode after its removal by a

F

  Figure 2 Lavaud J.

Table 1

 1 Effects of saturating light on NPQ and O 2 flash yield Effects of illumination (in the PAM vial) at 450 µE.m -2 .s -1 on 20 min dark-adapted P. tricornutum. NPQ: non-photochemical quenching expressed as (F m -F m ')/F m '. Y SS : steady state O 2 flash yield measured after 7 min sedimentation on the electrode, normalized to non-illuminated control. DCCD was added at 10 µM at the start of the 20 min dark-adaptation. *DCCD decreased the dark-adapted Y SS substantially. The O 2 deficit is given in units Y SS of the non-illuminated control.

		NPQ	Y SS	O 2 deficit
	5 min Light	1.6 ± 0.1	0.98 ± 0.2	2.8 ± 0.2
	60 min Light	2.5	0.69	1.7
	DCCD + 5 min L	2.1	0.95*	1.5

Acknowledgements

We thank J.-C. Duval for experimental assistance and helpful discussions and B. Rousseau for culture maintenance. JL thanks the French Ministry of National Education, Research and Technology for its financial support. HJvG thanks the Ecole Normale Supérieure for an invited professorship.


Johann Lavaud
email: lavaud@biologie.ens.fr

Bernard Rousseau

Anne-Lise Etienne

† J L Thanks

Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms †

published or not. The documents may come

Enrichment of the light-harvesting complex in diadinoxanthin and implications for the non-photochemical fluorescence quenching in diatoms

The pigment composition of diatoms differs from that of green algae and plants. Diatoms contain chlorophyll (Chl 1 ) c, fucoxanthin and diadinoxanthin (DD). An intermittent light regime during growth induced a large increase in the DD content in the marine planktonic diatom Phaeodactylum tricornutum.

Light-harvesting complex (LHCF) subunits were purified on a sucrose gradient after treatment of thylakoid membranes with a mild detergent. DD was found all the LHCF fractions: a "major" composite LHCF fraction and the two fractions where some LHCF was associated with photosystem centers. For cells enriched in DD, most of the additional DD molecules were bound to the major LHCF fraction. The DD enrichment of the major LHCF fraction was accompanied by a decrease in the fucoxanthin to Chl a ratio. Either some fucoxanthin molecules were replaced by DD or there could be a relative enrichment of subunits rich in DD at the expense of fucoxanthin/Chl c rich subunits. Under high light illumination, a higher degree of de-epoxidation of DD into DT was observed for the major LHCF of cells enriched in DD. This fraction has the higher DD content and the higher degree of de-epoxidation. These results show that the distal antennae, probably mostly isolated as the major LHCF fraction, plays a crucial role in the formation of NPQ, its amplitude depending on the amount of DD bound and on the degree of deepoxidation [Lavaud et al. (2002) Plant Physiology 129, 1398-1406].

Diatoms are unicellular photosynthetic eukariots which play a key role in the ocean carbon, silica and azote cycles (1,2). They are ubiquitous and the major group of phytoplanktonic marine algae. They experience large fluctuations in light intensity due to unpredictable water motions that can vary over several orders of magnitudes [START_REF] Harris | Phytoplankton Ecology: Structure, Function, and Fluctuation[END_REF][START_REF] Fogg | [END_REF][START_REF] Falkowski | Aquatic Photosynthesis[END_REF]. As other photosynthetic organisms [START_REF] Falkowski | [END_REF]7), they have developed efficient photoprotective mechanisms (8)(9)(10)(11)(12)(13) in order to minimize photoinhibition that could result from their periodic exposure to excess light intensities when transported to the water column surface (14).

Among the short term "safety valves" which are activated by a sudden increase in light intensity, the photoprotective non-radiative dissipation of the light energy absorbed in excess is an important mechanism (15). This phenomenon is attributed to rapid modifications within the light-harvesting complexes (LHC) of photosystem II (PS II) leading to a decrease in the excitation pressure on PS II (16,17). It is designated as the non-photochemical fluorescence quenching (NPQ) process and measured by a decrease in chlorophyll (Chl) fluorescence intensity under high light. In higher plants and green algae, NPQ is controlled by the formation of a proton gradient across the thylakoid membrane and by reversible conversion of epoxidized (violaxanthin) to de-epoxidized (antheraxanthin and zeaxanthin) forms of xanthophylls (the so-called xanthophyll cycle, for reviews see refs (16)(17)(18)(19)(20)). In diatoms, the xanthophyll cycle consists in the conversion of the mono-epoxide diadinoxanthin (DD) into the deepoxide diatoxanthin (DT) under excess light, and vice-versa under limiting light or darkness (21). The formation of DT is associated with the onset of NPQ within the light-harvesting complexes (LHCF) (9,11,12,22). In higher plants, the location and cycling of xanthophylls within LHC subunits have been the subject of many investigations (see refs (23)(24)(25) and citations within). It has been shown recently that in higher plants, a specific LHC subunit, the CP22 (PsbS), binds zeaxanthin in vitro [START_REF] Aspinall-O'dea | Proc. Natl. Acad. Sci. USA[END_REF]. It plays a crucial role in NPQ: the Arabidopsis psbS deletion mutant is not capable of NPQ [START_REF] Li | [END_REF] and there is a stoichiometric dependence of NPQ capacity on the amount of PsbS (28,[START_REF] Li | Proc. Natl. Acad. Sci[END_REF].

There is a lack of knowledge concerning the exact nature and organization of LHCF subunits in diatoms, and especially the location of DD and DT in the antenna complex. The whole organization of the photosynthetic apparatus in diatoms differs in many aspects from that of green plants: i) their pigment composition is different, with Chl c and fucoxanthin as the main xanthophyll, ii) the xanthophyll concentration relative to Chl can be two to four times higher than in higher plants [START_REF] Wilhelm | [END_REF]31), and iii) there is no grana stacking and no segregation of PS II and PS I (32). LHCF subunits are made of several highly homologous proteins encoded by a multigene family (Fucoxanthin Chlorophyll Proteins, FCP, (33)). No obvious orthologs of some of the important components of LHC of higher plants PS II (e. g. the minor antennae CP 26, CP 29 and PsbS) have been found in Phaeodactylum tricornutum Böhlin or within the genome sequence of Thalassiosira pseudonana Hustedt (Hasle and Heimdal) (17), C. Bowler, personal com.).

It is known that the DD content of diatoms can be modulated by the light regime during culturing (34)(35)(36). We used an intermittent light regime to grow the marine planktonic diatom P. tricornutum. As a result there was a specific increase in the DD content of cells. The additional DD was convertible to DT under excess light exposure of whole cells and concomitantly photoprotective energy dissipation was increased (12).

In the present study, our goal was to locate the DD molecules and determine their degree of conversion into DT within the different pigment-protein complexes. We present the pigment content and spectroscopic properties of LHCF fractions isolated from P. tricornutum cells grown under an intermittent light regime, and compare them with those of cells grown under a "classical" light regime which have a smaller DD content. The data obtained provide a possible explanation for the larger NPQ found in cells enriched in DD (12).

MATERIALS AND METHODS

Cultures.

Phaeodactylum tricornutum Böhlin (from the algal culture collection of Laboratoire Arago, Banyuls, France) cells were grown photoautotrophically in sterile natural seawater F/2 medium (37). Cultures of 300 mL were incubated at 18 C in airlifts continuously flushed with sterile air. They were illuminated at a light intensity of 40 µmol photons.m -2 .s -1 with white fluorescent tubes (Claude, Blanc Industrie, France) with a 16 h light/8 h dark cycle for continuous light (CL) cells, or with a 5 min light/55 min dark cycle for intermittent light (IL) cells. The growth rate under IL illumination was reduced from 0.65 d -1 to 0.065 d -1 , partly because of the 8-fold lower total amount of light supplied. CL cells were collected after 3-4 days and IL cells between 30-40 days after dilution. Part of the culture was then diluted in fresh medium to maintain cells in exponential growth phase.

Pigment content.

Pigment analyses were performed by HPLC as described previously (11). Cells were filtered onto a filter paper (Millipore AP-20 prefilter) rapidly frozen into liquid nitrogen. After thawing, the filter was placed in a methanol:acetone solution (70:30, v/v) to extract the pigments. For the light-harvesting complexes isolated on the sucrose gradient, pigments were extracted by a phase separation procedure: samples were first mixed with a methanol:acetone (50:50, 1v) solution completed with ether (1 v) and water-NaCl 10 % (2 v). Published extinction coefficients for chlorophylls (38) and for diadinoxanthin (DD) and diatoxanthin (DT) (39) were used. Cell counts were performed with a Thoma hematocymeter, using the public domain NIH Image program (US National Institute of Health).

Spectroscopy.

77 K absorption spectra were performed with a DW-2 Aminco spectrophotometer equipped with a home-made Dewar holder. 77 K fluorescence excitation spectra were measured with a F-4500 Hitachi spectrophotometer. Half-bandwidth for emission and excitation was 2.5 nm. Samples were dark-adapted and then concentrated on a Millipore AP-20 prefilter that was immediately frozen in liquid nitrogen before measurement.

Isolation of light-harvesting complexes.

The light-harvesting complexes subunits were isolated as described (31). Chloroplasts were extracted and suspended in a 50 mM Hepes-KOH buffer (pH 7.6) with 5 mM MgCl 2 , 2 mM MnCl 2 , 10 mM KCl, 6 mM Na 2 -EDTA, 5 mM amino caproic acid, 0.6 M sorbitol and protease inhibitors (1 mM phenylmethanesulfonylfluoride and 1 mM benzamidine). They were disrupted in a French pressure cell, then resuspended in the same buffer, without sorbitol, in the presence of digitonin at a detergent/Chl a ratio of 80:1 and incubated for 1 h at 4 °C. The homogenate was loaded on top of a sucrose gradient (10-55 %) and centrifuged at 140 000 x g for 15 h at 4 °C. The three bands obtained corresponded, from the bottom to the top of the gradient, to PS I and PS II enriched fractions with some LHCF remaining bound, and the major free LHCF band (see Results section for a detailed description). For preilluminated cells, epoxidation of DT into DD was prevented by keeping all fractions on ice and by a faster handling of the samples.

RESULTS

Ultrastructure, photosynthetic parameters and pigment content of the CL and IL cells.

Phaeodactylum tricornutum Böhlin cells were grown at low light intensity (40 µmol photons.m -2 .s -1 ) under two different light regimes: a 16 h light/8 h dark cycle for "continuous light" (CL) cultures and a 5 min light/55 min dark cycle for "intermittent light" (IL) cultures. Cell size and chloroplast ultrastructure, as illustrated by the number of thylakoids and their organization, were similar under the two light regimes. The photosynthetic parameters of both types of cells have been previously described (12). They were all similar, especially photosystem stoichiometries, PS II antenna size and oxygen emission versus light intensity curve. The larger change in the pigment content of whole cells was that of DD which was more than twice in IL cells (Table 1). Fucoxanthin and Chl c were decreased to a much lesser extent. At the low light intensity used for culturing, no DT was present before exposure of the cells to excess light (Table 1). DD absorbed in the blue region of the visible spectrum with a peak at 495 nm clearly visible in both the absorption spectra of cells enriched in DD (Fig. 1A, continuous line) and in the difference spectrum of the absorption spectra of cells with different DD content (Fig. 1A, bottom part). In the 77 K fluorescence excitation spectra of the fluorescence emission at 687 nm (characteristic of PS II), the contribution of Chl a, Chl c and fucoxanthin to the transfer of excitation energy were visible (Fig. 1B). The contribution of DD was less marked showing that all DD molecules, and specifically the additional DD molecules in IL cells (Fig. 1B, arrow), would thus not be able to transmit their excitation energy to Chl a.

Pigment content of the light-harvesting complexes of dark-adapted CL and IL cells.

After disruption of the chloroplasts of dark-adapted cells in presence of mild detergent, the homogenate was loaded onto a sucrose gradient and centrifuged. Three LHCF bands were obtained (Fig. 2A). The band on the top of the gradient (a brown band, at 10-15 % sucrose) contained 50 % of the loaded Chl a (Fig. 2B) and the majority of DD (Fig. 2C). The two other heavier bands contained 15-20 % Chl a each. This distribution is similar to what was observed when the same purification procedure was used with brown algae which belong to the same phylum (Heterokontophyts) (40,41).

The three bands obtained in P. tricornutum can be identified as follows. The top band corresponds to a band enriched in LHCF designated as the "major" LHCF (Fig. 2A). In brown algae, it is quasi devoid of photosystems reaction centers (31,42). According to (38) and ( 43), the two other bands are enriched in photosystems with their respective LHCF. In the present study, the intermediate band at 20 % sucrose is enriched in PS II and the bottom band in PS I (Fig. 2A). Pellets represented 15-20 % of the total loaded Chl a (Fig. 2B) and their pigment content was similar for both types of cells as shown in Table 1. This similarity in the pigment content of the pellets is important for valuable comparison of LHCF subunits pigment content of CL and IL cells.

It is known that the core complexes of the two photosystems contain only Chl a and -carotene.

Therefore, proportionally to Chl a, Chl c and fucoxanthin were more abundant in the major LHCF fraction than in the fractions enriched in PS I and PS I core complexes (Table 1). The situation was reversed for -carotene which is more abundant in the fractions enriched in photosystems. The traces of -carotene in the major LHCF were indicative of a small contamination as already reported for isolation of LCH-II in higher plants (24). When comparing the DD/Chl a in whole cells and in different fractions for IL and CL cells, it is obvious that the main enrichment in DD, when the DD pool was increased (IL cells), was in the major LHCF fraction (Table 1, Fig. 2C).

Spectroscopic properties of the light-harvesting complexes of dark-adapted CL and IL cells.

The 77K absorption spectra of the major LHCF and the two photosystems fractions in the two types of cells are shown Figure 3. The spectra of the major LHCF fractions differed. The enrichment in DD of the major LHCF fraction of IL cells was clearly visible by the increased 495 nm peak (Fig. 3B,arrow).

The spectra of PS II and PS I fractions of both types of cells were quite similar. They showed a smaller absorption in the blue region, as compared to the major LHCF fraction, indicative of a decrease in Chl c, fucoxanthin and DD content. The two photosystem fractions have also a larger concentration in long wavelength Chl a than the major LHCF fractions.

The 77 K fluorescence emission of the major LHCF of CL and IL cells was maximal at 678 nm with a secondary peak at 735 nm (Fig. 4A). The small fluorescence at 635 nm showed that the majority of Chl c molecules still transferred excitation energy to Chl a and were not fluorescing. In this context, it is noticeable that the difference already observed in the Chl c content in the major LHCF of the two types of cells was not due to an increase in free pigments in IL cells but reflected the difference observed between whole cells (Table 1). When comparing the 77 K excitation spectra of the emission at 678 nm (maximum emission of the major LHCF fractions) of whole cells and major LHCF of the two types of cells, one can see a large band in the 500-550 nm range in the LHCF fractions, which showed that most of the fucoxanthin molecules remained correctly bound and transferred their excitation energy to Chl a (Fig. 4B). It was not the case for the DD molecules which contribution around 495 nm was no longer detectable especially for IL cells (compare Fig. 1B and Fig. 4B). However, the fact that the DD absorption peak was not blue-shifted after purification of LHCF (Fig. 3) is a possible indication that DD remained connected to complexes, as already reported for the analogue violaxanthin (44), but probably not in a configuration that still allowed energy transfer to Chl a as observed for whole cells (Fig. 1B).

Hence, considering that free pigments are a minority (no free pigments were visible on the top of the sucrose gradient, Fig. 2A) one can further analyze the pigment content of LHCF fractions (Table 2). The pigment content of the two major LHCF fractions was different. There was significantly less fucoxanthin and Chl c but more DD in the IL fraction. However, the xanthophyll (fucoxanthin+DD) to Chl a ratio as well as the ratio Chl c/fucoxanthin remained constant.

Pigment content and spectroscopic properties of the light-harvesting complexes of illuminated IL cells.

With the low light intensity used for culturing, no DT was present in whole cells (Table 1). When cells were shifted to excess light, the de-epoxidation of DD into DT took place (22). We have previously shown that under excess light, the degree of de-epoxidation was higher in IL cells, resulting in a larger accumulation of DT (22). For a light intensity of 2000 µmol photons.m -2 .s -1 during 1 h, the amount of DT formed was 5 and 12.5 mol DT/100 mol Chl a in CL and IL cells, respectively. It corresponded to a de-epoxidation degree of 50% and 65% respectively (12).

In order to determine the degree of de-epoxidation in the three LHCF fractions in IL cells, they were strongly illuminated during 20 min prior to purification of the fractions. When comparing the pigment content of dark-adapted and illuminated cells, one can observed that during illumination, the amount of all pigments remained constant with a small increase in the sum DD+DT due to a slight de novo synthesis of DT (9). While the major LHCF fraction showed a degree of de-epoxidation similar to whole cells, the degree was clearly less in the two photosystems fractions (Table 2): the amount of DT reached in the major LHCF fraction was three times that reached in the PS+LHCF fractions. The conversion of DD into DT was illustrated by peaks at 438, 474 and 508 nm in the difference absorption spectrum of the major LHCF of illuminated cells minus dark-adapted cells (Fig. 6). The 508 nm peak has already been assigned to DT (45). The three bands are very similar to those observed in the zeaxanthin-minusviolaxanthin difference spectrum in higher plants (46).

DISCUSSION

Additional DD binds to the 'major' light-harvesting complex in DD enriched cells.

In P. tricornutum, the size of the DD pool increases under intermittent light and a larger fraction of the pool is susceptible to de-epoxidation (12,22). It was important to determine the exact location of the additional DD molecules in the IL cells. Indeed, the corresponding DT molecules produced under high light were shown to enhance the dissipation of excess energy and were therefore likely to be bound to the antenna subunits responsible for the NPQ process (12).

In brown algae which belong to the same phylum than diatoms, it has been shown that a LHCF monomer can contain seven Chl a and as much as height fucoxanthin molecules (41,42). Nothing is known about the number of violaxanthin (the analogue of DD in brown algae) bound per subunit. After solubilization of the P. tricornutum thylakoids membranes by a mild detergent and fractionation of the pigment-protein complexes, all the fractions containing LHCF antenna are found to contain DD as reported for its violaxanthin analog in higher plants (24,47). In IL cells, compared to CL cells, nearly all of the additional DD molecules are bound to the major composite LHCF fraction with only a minor enrichment for the LHCF fractions which remain associated with the photosystem centers. Two hypotheses can be proposed to explain the quasi specific DD enrichment of the major composite LHCF fraction in IL cells. The first could be a pigment-pigment "replacement". If we assume that the Chl a stoichiometry of LHCF subunit in diatoms is the same than in brown algae, we can estimate that in IL cells one subunit would bind two DD molecules. In CL cells, only one DD would be bound per LHCF subunit. Such a doubling of the DD amount bound to one LHCF subunit would be explained by a replacement of some fucoxanthin molecules by DD. This is supported by the observation that the enrichment in DD in IL cells is correlated with a stoichiometric parallel decrease in fucoxanthin (Table 3). Such a "competition" of two xanthophylls of related structures, as it is the case for DD and fucoxanthin [START_REF] Lohr | Proc. Natl. Acad. USA[END_REF][START_REF] Lohr | [END_REF], for a common binding site is conceivable. In higher plants, the possibility that a given site of a LHC protein can bind different xanthophylls has been demonstrated both in vitro (20) and in vivo (25,50,51). However, the existence of LHCF subunits with different pigment content is very likely. Since, in parallel to the increase in DD content, Chl c decreases in the same extent as fucoxanthin, some subunits could be specifically rich in DD while others could mainly bound fucoxanthin and Chl c. The relative abundance of the different subunits would be modulated by light. It has already been shown that the expression of the different lhcf genes is light-dependent (52,53). The regulation of the LHCF protein synthesis has been studied as a function of light intensity (54)(55)(56)(57) but not as a function of the light period. In IL cells, the amount of subunits rich in DD would be increased and that of subunits rich in fucoxanthin and Chl c decreased compared to the situation in CL cells.

Fucoxanthin, Chl c and Chl a are the main contributors of the energy harvesting (Fig. 1A) and transfer to the photosystem reaction centers (Fig. 1B). Therefore, the modulation in DD content allows the cells to regulate their capacity for dissipating energy under excess light without changing significantly the light-harvesting under limiting conditions.

The xanthophyll cycle within light-harvesting complexes in DD enriched cells.

Under excess light, a higher degree of de-epoxidation occurs in DD enriched cells (12,22). To be deepoxidized, xanthophylls have to be accessible to the de-epoxidase which is localized in the lipid matrix (19). The fraction of DD which can be transformed to DT is thus likely located at the periphery of the pigment-protein complexes (25). The fact that DD is more abundant and de-epoxidized to a larger extent within the major LHCF of IL cells leads to propose a peripheral location for the additional DD molecules. Comparison of absorption and excitation spectra (Fig. 1) shows that part of additional DD molecules interact with Chl a with a low efficiency. This could also suggests a peripheral location of these molecules. In brown algae, among the eight fucoxanthin molecules that one LHCF monomer can bind, two are proposed to have a role in the stabilization of the LHCF (41,58) by analogy with the two central lutein molecules in the LCHII of higher plants (59). The binding sites of the six other molecules of fucoxanthin remain unknown. In the framework of our "DD-fucoxanthin replacement hypothesis" and if we assume the same fucoxanthin stoichiometry per monomer in P. tricornutum, at least two fucoxanthin molecules could be bound at the periphery of the LHCF. Under intermittent light, these molecules would be likely to be the ones that could possibly be replaced by DD, then easily accessible to the de-epoxidase.

In a previous report, we have shown that NPQ is enhanced in IL grown cells and linearly related to the amount of DT formed upon light exposure (12). Our present results show that the majority of the additional DD molecules are bound to the major LHCF and that the largest degree of de-epoxidation is observed in this fraction. It indicates that NPQ mostly takes place in the distal antennae, essentially isolated as the major LHCF fraction. Similar conclusions were recently reached for higher plants (44,60). Considering the most recent results on the understanding of the NPQ process in higher plants about the essential role of PsbS and the fact it can bind zeaxanthin (the analogue of DT) [START_REF] Aspinall-O'dea | Proc. Natl. Acad. Sci. USA[END_REF][START_REF] Li | Proc. Natl. Acad. Sci[END_REF], one can conclude that the orthologue of PsbS in diatoms (if it exists) is likely to be found in the composite major LHCF fraction.

Concluding remarks

By regulation of the light-harvesting through a fine-tuning of their xanthophyll content, diatoms are well suited to avoid the serious damages that may result from light stresses without any significant decrease in their capacity for light collection under light limiting conditions. As planktonic diatoms can modulate their DD pool size in the field (61)(62)(63), this ability likely explain their successful adaptation to the fluctuating light environment [START_REF] Harris | Phytoplankton Ecology: Structure, Function, and Fluctuation[END_REF][START_REF] Fogg | [END_REF].

Because DD de-epoxidation into DT can be readily observed by absorption changes (see Fig. 6), these changes will be used for further studies on the regulation of energy dissipation mechanism within the light-harvesting complexes. Such measurements have previously been performed with higher plants (64,65). Intermittent light-grown diatoms being enriched in DD provide a unique model for further studies on photoprotective energy dissipation process. a DED: de-epoxidation degree (in %): DT/(DD+DT) x 100 for 20 min induced a 67% de-epoxidation of DD into DT (see Table 3). Three absorption bands (438,

FIGURE 1 :

 1 FIGURE 1: A-77 K absorption spectra of CL (dotted line) and IL (continuous line) cells. The main

FIGURE 2 :

 2 FIGURE 2: A-Distribution of the three LHCF bands on the sucrose gradient after solubilization of the

FIGURE 3 :

 3 FIGURE 3: 77 K absorption spectra of the three LHCF fractions isolated on sucrose gradient for the two

FIGURE 4 :

 4 FIGURE 4: A-77 K emission fluorescence spectrum of Chl a excited at 440 nm of the major LHCF

FIGURE 5 :

 5 FIGURE 5: 77 K absorption of the "major" LHCF fraction of dark-adapted (dotted line) and illuminated (continuous line) IL cells. Spectra are normalized to 667 nm. Illumination at 5300 µmol photons.m -2 .s -1

Figure 1

 1 Figure 1 -Lavaud, Rousseau and Etienne

Table 1 :

 1 Pigment composition of dark-adapted CL and IL cells grown under an intensity of 40 µmol photons.m -2 .s -1 , and of corresponding sucrose gradient fractions containing lightharvesting complexes (LHCF) and photosystems (PS). Total Chl a. cell -1 was 0.38  0.05 pg for CL cells and 0.35  0.05 pg for IL cells; Data ( SD) are the average of three to five independent measurements.

	Pigments	CL cells	Major LHCF PS II+LHCF PS I+LHCF	Pellets
	(mol/100 mol Chl a) a
	Chl c	15.9 ± 0.3	24.8 ± 1.0	8.7 ± 0.06	5.8 ± 1.0	8.4 ± 1.8
	Fucoxanthin	68.4 ± 3.0	110.0 ± 5.9	47.3 ± 3.2	31.0 ± 1.5 52.0 ± 3.4
	DD	9.5 ± 1.5	9.6 ± 2.3	8.9 ± 0.4	10.5 ± 0.7	6.7 ± 1.0
	DT	0	0	0	0	0
	ß-carotene	8.0 ± 1.1	1.8 ± 0.2	12.3 ± 2.7	15.1 ± 1.8	8.6 ± 1.9
	Pigments	IL cells	Major LHCF PS II+LHCF PS I+LHCF	Pellets
	(mol/100 mol Chl a) a
	Chl c	13.4 ± 0.4	19.9 ± 1.2	8.0 ± 2.4	5.1 ± 0.4	8.7 ± 2.4
	Fucoxanthin	65.5 ± 3.5	95.6 ± 3.3	46.0 ± 3.3	30.0 ± 3.6 51.4 ± 0.2
	DD	21.6 ± 1.8	27.6 ± 3.1	13.4 ± 1.2	13.2 ± 1.7	8.6 ± 0.2
	DT	0	0	0	0	0
	ß-carotene	8.4 ± 0.2	2.3 ± 0.7	12.0 ± 1.4	14.3 ± 2.2	8.3 ± 1.6

a

Table 2 :

 2 Pigment composition of the major LHCF fractions of IL and CL cells. Data ( SD) are the average of three to five independent measurements; a, b Student test (statistical test for mean values comparison): means are significantly different (p<0.05); c Student test: means are not significantly different (p<0.05).

	Pigments	IL cells	CL cells
	(mol/100 mol Chl a)
	Fucoxanthin	110.0 ± 5.9 a	95.6 ± 3.3 a
	DD	27.6 ± 3.1	9.6 ± 2.3
	Chl c	24.8 ± 1.0 b	19.9 ± 1.2 b
	DD + Fucoxanthin	119.6 ± 6.0 c	123.2 ± 7.3 c
	Chl c / Fucoxanthin	0.225	0.208

Table 3 :

 3 Pigment composition of illuminated IL cells (20 min, 5300 µE.m -2 .s -1 ) and of corresponding sucrose gradient fractions containing light-harvesting complexes (LHCF) and photosystems (PS).

	Pigments	IL cells	IL cells	Major	PS II	PS I
	(mol/100 mol Chl a)	dark-adapted	illuminated	LHCF	+LHCF	+LHCF
	Chl c	13.0	13.2	18.2	8.9	4.4
	Fucoxanthin	68.0	66.7	93.2	55.6	27.2
	DD	20.7	9.1	9.3	7.7	10
	DT	0	13.9	19.1	6.0	6.3
	ß-carotene	8.5	8.4	2.1	10.6	15.4
	DD+DT	20.7	23.0	28.4	13.7	16.3
	DED a	0	60	67	44	39

Abbreviations: Chl, chlorophyll; CL, continuous light regime (16 h light/8 h dark per day); DD, diadinoxanthin; DT, diatoxanthin; IL, intermittent light regime (5 min light per hour); LHC, Lightharvesting complex; LHCF, Light-harvesting complex containing Fucoxanthin; NPQ, nonphotochemical chlorophyll fluorescence quenching; PS II and I, photosystem II and I.

and 508 nm) due to the conversion of DD in DT during light exposure are visible in the difference spectrum (illuminated cells minus dark-adapted cells, bottom dashed line).

AKNOWLEDGMENTS

We thank C. Lichtlé for electron microscopic photographs of sections of diatom cells, J.-C. Thomas for his assistance with spectroscopic measurements, A. Korber for artwork and Drs A. Ruban, A. Pascal and J. Houmard for critical reading and improvement of the manuscript. The paper is dedicated to J.-C.

Duval, who initiated this work, for his sixtieth anniversary.