Gérard Guglielmi
email: ggugliel@biologie.ens.fr

Johann Lavaud

Bernard Rousseau

Anne-Lise Etienne

Jean Houmard

Alexander V Ruban

The light-harvesting antenna of the diatom Phaeodactylum tricornutum: Evidence for a diadinoxanthin binding sub-complex

Keywords: Diatom, light-harvesting complex, photoprotection, xanthophyll cycle, fucoxanthin

Diatoms differ from higher plants by their antenna system, in terms of both polypeptide and pigment contents. A rapid isolation procedure was designed for the membrane-intrinsic light harvesting complexes (LHC) of the diatom Phaeodactylum tricornutum to show whether different LHC subcomplexes may exist, as well as an uneven distribution between them of pigments and polypeptides. Two distinct fractions were separated that contain functional oligomeric complexes. A major and more stable complex (about 75% of total polypeptides) carries most of the Chla, and almost only one type of carotenoid, fucoxanthin. The minor component, carrying approximately 10-15% of the total antenna chlorophyll and only a little chlorophyll c, is highly enriched in diadinoxanthin, the main xanthophyll cycle carotenoid. The two complexes also differ in their polypeptide composition suggesting specialized functions within the antenna. The diadinoxanthin-enriched complex could be where de-epoxidation of diadinoxanthin into diatoxanthin mostly occur.

Introduction

Diatoms constitute a dominant group of phytoplanktonic algae, playing an important role in the carbon, silica and nitrogen biogeochemical cycles [START_REF] Field | Primary production of the biosphere: integrating terrestrial and oceanic components[END_REF][START_REF] Tréguer | The silica balance in the word ocean: a reestimate[END_REF][START_REF] Smetacek | Diatoms and the ocean carbon cycle[END_REF]. Their photosynthetic efficiency and subsequent productivity depend upon the light environment which can vary greatly due to water motions [START_REF] Falkowski | Aquatic Photosynthesis[END_REF][START_REF] Macintyre | The effect of water motion on short-term rates of photosynthesis by marine phytoplankton[END_REF]. Fluctuating irradiances and especially excess light exposure can be harmful for photosynthesis, in particular photosystem II (PS II), causing a decrease in productivity and fitness [START_REF] Long | Photoinhibition of photosynthesis in nature[END_REF][START_REF] Külheilm | Rapid regulation of light harvesting and plant fitness in the field[END_REF]. One of the photoprotective mechanisms used by diatoms is the dissipation of excess energy in the light-harvesting complex (LHC) of PS II to prevent over excitation of the photosystems (so-called NPQ for non-photochemical chlorophyll fluorescence quenching). NPQ is triggered by a transthylakoidal proton gradient (ΔpH) and results from a modulation in the xanthophyll content [START_REF] Ting | Photochemical and non-photochemical fluorescence quenching processes in the diatom Phaeodactylum tricornutum[END_REF][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][START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF][START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF]. In diatoms, the xanthophyll cycle (XC) is made up of diadinoxanthin (DD), which is converted under an excess of light into its de-epoxidised form, diathoxanthin (DT) [START_REF] Hager | Das Carotinoidmuster und die Verbreitung des lichtinduzierten Xanthophyllcyclus in verschieden Algenklassen. V. Einzelne Vertreter der Cryptophyceae, Euglenophyceae, Bacillariophyceae, Chrysophyceae und Phaeophyceae[END_REF]. The presence of DT is mandatory for NPQ [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF][START_REF] Lavaud | In diatoms, a transthylakoid proton gradient alone is not sufficient to induce a non-photochemical fluorescence quenching[END_REF][START_REF] Gilmore | Mechanistics aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves[END_REF][START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]. Additionally, a reverse deepoxidation of violaxanthin into zeaxanthin via antheraxanthin, similar to that which occurs in plants, has been demonstrated in diatoms submitted to a prolonged exposure to excess light [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF].

The diatom photosynthetic apparatus differs in many aspects from that of green plants and algae.

There are no grana stacking and no segregation of the photosystems [START_REF] Pyszniak | Immunocytochemical localization of photosystem I and the fucoxanthin-chlorophyll a/c light-harvesting complex in the diatom Phaeodactylum tricornutum[END_REF]. The main components of the antenna are the Fucoxanthin Chlorophyll (Chl) Proteins (FCP) encoded by a multigene family [START_REF] Bhaya | Characterization of gene clusters encoding the fucoxanthin chlorophyll proteins of the diatom Phaeodactylum tricornutum[END_REF]. FCP share common features with plants CAB (Chl a binding) proteins [START_REF] Dunford | The fucoxanthin-chlorophyll proteins from a chromophyte alga are part of a large multigene family: Structural and evolutionary relationships to other light harvesting antennae[END_REF], and in the diatom Cyclotella meneghiniana, two 18 and 19 kDa subunits were recently shown to form trimers and higher oligomers [START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF]. However, no obvious orthologues of some of the plant LHC minor components (e. g. PsbS, CP26 and 29) have been found in the fully sequenced diatom genome [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF].

In diatoms, the accessory pigments are also different. Chl c is the secondary chlorophyll, fucoxanthin is the main xanthophyll, and the XC pigments are DD and DT. The xanthophylls / Chl ratio can be 2-4 times higher than in plants [START_REF] Wilhelm | The biochemistry and physiology of light-harvesting processes in chlorophyll b-and chlorophyll c-containing algae[END_REF]. In higher plants, the XC pigments are bound to both major (LHC II trimers) and minor (PsbS, CP 24, 26 and 29) components of the LHC [START_REF] Ruban | Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes[END_REF][START_REF] Aspinall-O'dea | In vitro reconstitution of the activated zeaxanthin state associated with energy dissipation in plants[END_REF]. In diatoms, DD and DT are mainly associated with the FCP antenna [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF] but their exact localization in the different sub-fractions of the antenna has not yet been determined.

The goal of the present report was to address this question. Different isolation procedures were applied to obtain purified LHC fractions. The apparent molecular size, polypeptide and pigment compositions, as well as the spectroscopic properties of the various fractions were compared.

The data show that oligomeric FCP subcomplexes exist which have different polypeptides and pigment contents.

Results

Sucrose gradient preparations of pigment-protein complexes from Phaeodactylum

tricornutum. The mild detergent -DM was used for the solubilization of the pigmentprotein complexes from the thylakoid membrane [START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF]. Freshly solubilized P. tricornutum pigment-protein complexes were loaded onto a sucrose density gradient under either low salt (LS) or high salt (HS) conditions. The two lower bands at densities of approximately 0.6 and 0.75 M sucrose (Fig. 1) correspond to PSII and PSI, respectively, as deduced from comparison with the sucrose gradient separation of the PSII-enriched particles from spinach chloroplasts (Fig. 1B), and already published data [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF][START_REF] Burke | Involvement of light-harvesting complex in cation regulation of excitation-energy distribution in chloroplasts[END_REF]. The upper bands correspond to the fucoxanthin containing light-harvesting protein complexes (FCP or LHCF) fraction. The major brown colored band designated F ran where FCP's were reported to be localized [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF][START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF] and at a density similar to that of the spinach LHCIIb monomers (compare Fig. 1A and1B). This fraction contained approximately 80-85% of the total LHC chlorophyll (Chl) a. A lighter yellow band (termed D) ran at ~ 0.3 M sucrose and contained about 10-15% of the total LHC Chl a. With the high salt buffer, conditions known to better keep the integrity of the oligomeric states of protein complexes, two F bands were resolved: F1, similar to F, and F2. F2 ran at a higher sucrose concentration (0.45 M, Fig. 1C), i.e. in between the position for the spinach LHCII monomers and trimers.

Further purification of the F and D fractions by gel filtration.

The LHC fractions D and F from the sucrose gradients were buffer-exchanged and immediately applied onto a FPLC column.

Figure 2 shows typical elution profiles recorded by absorption at 280 nm, except for the free pigment fraction (fp) which did not contain any polypeptide and was recorded at 436 nm.

Isolated LHCII trimers, monomers and the free pigment fraction obtained from spinach chloroplasts by the sucrose gradient procedure (Fig. 1B) were used for size calibration. Fraction F eluted like the LHCII monomers (at 47 min) with a shoulder around 49-50 min (Fig. 2, trace 1), and fraction D at 50 min, with a minor shoulder at 47-48 min (Fig. 2, trace 3). The shoulders are likely to be due to a cross-contamination of F by D, and vice versa. All the elution times were reproducible with at most a 8% variation, and co-chromatographies of P. tricornutum F and D with monomeric spinach LHC fractions were performed to validate the comparison of the elution times (data not shown). Regardless of the salt conditions used for the sucrose gradients, the apparent molecular size of the F and D complexes was always the same. For F, it corresponded to that observed for the spinach LHCII monomer while D eluted at about 50 min well ahead of the spinach free pigment fraction. Absorption at 280 nm showed that both fractions do contain polypeptides. No significant differences in terms of pigment composition, spectroscopic properties or chromatographic behaviour on gel filtration columns could be detected between the D fractions, whether they were isolated from LS or HS conditions, nor between the F, F1 and F2 fractions. A higher salt concentration allowed fractioning of the F fraction into F1 and F2, the latter likely representing a higher aggregation state (dimers?) of the same subcomplexes which is not stable enough to be kept during the gel chromatography.

In another set of experiments, fractions F and D were additionally treated with -DM before gel filtration. These stronger detergent conditions led to more loosening of the subcomplex interactions. Following this treatment, the retention time increased for the D fraction (Fig. 2, trace 4), and fraction F (Fig. 2, trace 2) appeared as two peaks, the second one corresponding to that obtained with the -DM-treated D fraction. Table 1 shows the pigment composition of the different fractions. Fucoxanthin is the major pigment in all the fractions. Its concentration is higher than that of Chl a, in contrast to what is observed for lutein, the main xanthophyll of the higher plant LHCs [START_REF] Peter | Biochemical composition and organization of higher plant photosystem II light-harvesting pigment-proteins[END_REF]. DD, as well as Chl c, are unevenly distributed. Compared to F, D fractions are highly enriched in diadinoxanthin (DD) and contain less Chl c. Absorption (Fig. 3) and 77K fluorescence spectra (Fig. 4) were recorded for fractions F and D.

Fraction F exhibited an absorption spectrum (Fig. 3B) which reflected its pigment content: Chl c peaks at 463 nm and 636 nm, and a large fucoxanthin 500-550 nm absorbance band was visible with two distinct peaks at 505 and 536 nm. This is characteristic of the absorption properties of the LHC bound fucoxanthin observed with whole cells [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF][START_REF] Werner | The diatoms[END_REF]. In agreement with the low DD content of the F fractions, no peak corresponding to the DD absorption (around 490 nm) is observed. The 77 K emission (Fig. 4A) and excitation (Fig. 4B) fluorescence spectra confirmed that energy couplings between Chl c, as well as fucoxanthin and Chl a, are preserved in the F fraction. The lack of a 635 nm peak in the emission spectrum and the peak at 463 nm in the excitation spectrum were indicative of a coupled Chl c. The two shoulders at 505 and 536 nm in the excitation spectrum were indicative of a coupled fucoxanthin. We therefore concluded that fraction F was obtained in a form very close to the in vivo state. The fraction D showed different absorption and fluorescence spectra. According to its low Chl c content, no peak corresponding to the Chl c absorption (at 463 and 636 nm) was visible in the absorption (Fig. 3A) and the excitation (at 463 nm) spectra (Fig. 4B). Although the amount of fucoxanthin was higher in fraction D, no 500-550 nm LHC bound fucoxanthin band could be observed on the absorption spectrum (Fig. 3A). The uncoupling of fucoxanthin was confirmed by the excitation spectrum (Fig. 4B). It resulted in an increased absorption in the Soret region (Fig. 3A) with a specific 486 nm peak characteristic of the blue shifted absorbance of decoupled fucoxanthin [START_REF] Werner | The diatoms[END_REF]. Since the ratio DD/fucoxanthin was small, the absorption peak corresponding to DD was not detectable (Fig. 3A). Hence, in fraction D, only the Chl a molecules (Fig. 4) appear to be still energetically coupled. Finally, the D fraction showed a broader Chl a band in the red absorption region than did the F fraction (compare the respective 670 nm peaks in Fig. 3A andB). This indicated a somewhat different environment for the chlorophyll molecules in the two fractions, which was confirmed by a 2 nm shift of the Chl a fluorescence peak in the F fraction (Fig. 4A).

The polypeptide composition of the two fractions was analyzed by SDS-PAGE electrophoresis (Fig. 5). Diatom FCPs have molecular sizes ranging from 17 to 23 kDa [START_REF] Bhaya | Characterization of gene clusters encoding the fucoxanthin chlorophyll proteins of the diatom Phaeodactylum tricornutum[END_REF][START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF][START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF]. The D and F fractions share a common band at ~18.5 kDa that is a doublet at least in D. A second polypeptide of ~18 kDa is present only in F. Additional polypeptides in the 10-17 and 20-66 kDa range are present in the D fraction. The F fraction is particularly rich in FCP polypeptides.

Direct gel filtration of the solubilized pigment-protein complexes.

To obtain LHC fractions that have kept their in vivo oligomeric state as far as possible, a new procedure was devised.

Following plastid isolation, the detergent treatment was reduced to a minimum and the sucrose gradient step avoided. Plastids were solubilized by a 5 min -DM treatment in 600 mM NaKPO 4 , and immediately loaded on a gel filtration column. Three fractions were obtained, with the first two that elute corresponding to the photosystems, and the third one to a large FCP oligomer termed LHCo (Fig. 6). This LHCo started to elute at 40 min, with a peak at 43 min, and presented a tail which extended up to about 50 min. Spinach LHCII trimers used for size calibration eluted between 41 and 45 min, peaking at 43 min (see Fig. 2, dashed line). The majority of the soluble proteins were not embedded into micelles and eluted as a very broad peak cantered at around 80 min (not shown). These new conditions thus allow the isolation in a stable form of a LHC of higher apparent molecular size, suggesting that it corresponds to an oligomeric complex.

The LHCo elution peak was asymmetric, indicative of heterogeneity. This fraction further treated with -DM and rechromatographed gave a two-peak profile (Fig. 6, dashed line). From the OD 280 profile, the first peak (F) would contain about 75% of the LHCo polypeptides. Pigment composition and absorption spectra showed that these peaks corresponded to the above described F and D fractions (data not shown). We thus decided to separately collect and analyze, from this LHCo, the fractions that eluted between 40 and 44 min (LHCo-1, first fraction) and between 45 and 50 min (LHCo-2, second fraction). Pigment compositions are presented in Table 2.

Compared to the whole LHCo, LHCo-1 contained two times less DD, whereas LHCo-2 was enriched in DD (3 fold) and fucoxanthin (1.7 fold), and contained about two times less Chl c.

After treatment with -DM and a new gel filtration, the LHCo-1 gave a major peak with an elution time corresponding to that of the F fraction (47 min), and a minor one eluting at 52 min that resembled D fraction (retention times similar to traces 2 and 4 of Fig. 2). The opposite was observed for LHCo-2 which gave a major peak corresponding to a D fraction. The pigment composition of each of the major peaks is close to that of the F and D fractions obtained from sucrose gradients, once treated with -DM (Table 1). The oligomeric LHCo isolated with the new procedure thus corresponds to the association of F and D sub-complexes which likely reflects the in vivo spatial state of the LHC. This statement is well supported by the spectral properties of the LHCo-1 and -2 fractions which both showed energy coupling between Chl c, fucoxanthin and Chl a (Fig. 7). The lowest band of the 15 kDa doublet and the 17 kDa polypeptide are clearly specific to LHCo-2, and the 22 kDa polypeptide specific of the LHCo-1. Compared to the F and D fractions obtained after sucrose gradients (Fig. 5), the polypeptide patterns of the latter two -DM treated fractions show that they almost only contain polypeptides in the 12-20 kDa range. The lower contaminations by high molecular mass polypeptides suggest that with the new isolation procedure, all the macromolecular complexes, including PSI and PSII, have kept a more "native" aggregation state.

Discussion

In contrast to the plant light-harvesting complexes, LHCI and LHCII, the diatom LHC is presently poorly characterized even in terms of polypeptide and pigment composition.

Concerning the FCPs, six genes have been described for P. tricornutum whose products share 86-99% similarity [START_REF] Bhaya | Characterization of gene clusters encoding the fucoxanthin chlorophyll proteins of the diatom Phaeodactylum tricornutum[END_REF], but up to 20 or even more would exist in Cyclotella cryptica and Thalassiosira pseudonana [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF][START_REF] Eppard | Investigations on gene copy number, introns and chromosomal arrangement of genes encoding the fucoxanthin chlorophyll a/c-binding proteins of the centric diatom Cyclotella cryptica[END_REF]. On the other hand, the diatom xanthophyll cycle required for the establishment of the photoprotective non-photochemical fluorescence quenching (NPQ) also differs. This cycle mainly occurs between two forms, diadinoxanthin (DD) and its de-epoxidized form, diatoxanthin (DT), while three different ones are required in higher plants [START_REF] Hager | Das Carotinoidmuster und die Verbreitung des lichtinduzierten Xanthophyllcyclus in verschieden Algenklassen. V. Einzelne Vertreter der Cryptophyceae, Euglenophyceae, Bacillariophyceae, Chrysophyceae und Phaeophyceae[END_REF]. Our goal was to better characterize the P. tricornutum LHC, looking for the existence of putative subcomplexes that would contain the xanthophyll pigments. Because it is known from studies on higher plant LHC antennae that pigment-protein complexes can have different stabilities and that their binding affinity for pigment can vary greatly [START_REF] Sandona | Higher plants light harvesting proteins. Structure and function as revealed by mutation analysis of either protein or chromophore moieties[END_REF][START_REF] Bassi | Lhc proteins and the regulation of photosynthetic light harvesting function by xanthophylls[END_REF], we designed a new fractionation procedure, and compared it with previously used isolation techniques.

The organization of the light-harvesting antenna in diatoms.

Omitting the sucrose gradient step and using a mild and short detergent treatment under high salt conditions, immediately followed by gel filtration chromatography, we could separate from PSI and PSII a diatom LHC as an oligomer, LHCo, whose molecular size resembles that of the spinach trimers. We further showed that this LHCo is made up of two different subcomplexes. The first part of the LHCo peak essentially corresponds to the F fraction that was previously isolated from sucrose gradient preparations [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF], and the second to the D fraction obtained by the same procedure. The isolation of the LHCo as an asymmetric peak (see Fig. 6) strongly suggests that interactions between the two subcomplexes do exist in vivo. Compared to the total LHCo, the LHCo-1 (F analogue) is depleted in diadinoxanthin while LHCo-2 (D analogue) is depleted in Chl c and highly enriched in diadinoxanthin. Our analyses also demonstrated that the two oligomeric subcomplexes that were isolated have a different polypeptide composition (Fig. 8). Moreover, both fractions can efficiently transfer energy from fucoxanthin to Chl a. Thus none of them correspond to free pigments. This means that two subcomplexes do exist and that, by using the newly designed procedure, they have kept a more "native" state than the F and D fractions obtained from the sucrose gradients. A recent study was conducted on the Cyclotella meneghiniana LHC, in which two FCP fractions were separated from sucrose gradients, A and B, the latter having a higher apparent molecular size than A [START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF]. Fraction A is mainly composed of 18 kDa polypeptides and exhibits a 486 nm absorption shoulder; fraction B does not have this shoulder and is made up of 18 and 19 kDa polypeptides. The pigment content of each of these fractions was however not provided. In the present study it is shown that only the D fraction and its analogue (LHCo-2) from the LHCo have a 486 nm absorption peak, and they contain polypeptides of lower molecular masses than the F and LHCo-1 fractions. Fractions D and LHCo-2 thus resemble the fraction A, whereas F and LHCo-1 correspond to the fraction B of C. meneghiniana. Büchel [START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF] also reported that the B fraction is more stable than the A, and our results show that the F fraction (LHCo-1) is similarly more stable than the D (LHCo-2) one. Indeed, a -DM treatment modifies the molecular size of D (Fig. 2) and LHCo-2 but not that of F, and a loss of energy coupling between fucoxanthin and Chl a was observed for the D but not for the F fractions. All of the presently available data confirm that, though sharing a common ancestor, diatoms exhibit an organization and pigment composition for their LHC that clearly differ from that of extant higher plants, in terms of both polypeptide and pigment content.

Consequences of the LHC organization on the mechanism of the excess energy dissipation (NPQ). The spatial organization of the LHC in diatoms is very likely at the origin of the huge

NPQs that diatoms can exhibit [START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]. Different minor LHC polypeptides (in particular CP26, CP29), as well as PSII small subunits (PsbS = CP22 and PsbZ = Ycf9) have been implicated in the NPQ formation in plant and green algae, underlying that it is a rather complex phenomenon not yet totally understood [START_REF] Muller | Non-photochemical quenching. A response to excess light energy[END_REF][START_REF] Holt | Toward an understanding of the mechanism of nonphotochemical quenching in green plants[END_REF][START_REF] Swiatek | The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture[END_REF]. In plants and green algae, the PsbS protein binds zeaxanthin (the DT analogue) and is required for the NPQ to develop [START_REF] Holt | Toward an understanding of the mechanism of nonphotochemical quenching in green plants[END_REF]. No CP26, CP29 and PsbS orthologues have been recognized in the fully sequenced diatom genome [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism[END_REF]. In the green microalga Chlamydomonas reinhardtii a CAB polypeptide, PsbZ, involved in the oligomeric organization of the LHC was found to affect: i) the deepoxidation of xanthophylls; and ii) the kinetics and amplitude of nonphotochemical quenching [START_REF] Swiatek | The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture[END_REF]. PsbZ (Ycf9) genes are also present in red algae, diatoms and cyanobacteria. Our working hypothesis is that the functional diatom orthologues of such polypeptides are present in the D and LHCo-2 minor sub-fractions that we purified from P. tricornutum. One of the two polypeptides (15 or 17 kDa) specifically found in this LHC subcomplex might play a functional role in DD binding and NPQ formation. When grown under an intermittent light regime, P. tricornutum cells show a very high NPQ that was correlated with a specific up to 3-fold enrichment of the LHC in DD and DT [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF][START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF][START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]. In this context, the intermittent-light grown P. tricornutum cells could constitute a unique model to elucidate the exact role played by the organization of the LHC in the photoprotective energy dissipation. Compared to plants and green algae, the different organization of the diatom LHC, as well as the distribution of the xanthophyll pigments between the two subcomplexes, might ensure more flexibility and thus quicker responses to the important light intensity fluctuations that diatoms encounter in their natural habitat.

Materials and Methods

Culture conditions -Phaeodactylum tricornutum Böhlin cells (Laboratoire Arago algal collection, Banyuls-sur Mer, France) were grown photoautotrophically in sterile seawater as described in [START_REF] Guillard | Studies of marin planktonic diatoms. 1. C. nana (Hustedt) and D. confervacea (Cleve)[END_REF]. Briefly, cultures were incubated at 18 °C in airlifts continuously bubbled with air to maintain the cells in suspension, and under a 16 h light/8 h dark cycle. They were grown under a white light of 40 µmol photons.m -2 .s -1 provided by fluorescent tubes (Claude, Blanc Industry, France). Under this light intensity there is no DT formed during the light periods and therefore after purification, the antenna only contains DD. When needed, DT is formed by exposure of the cells to a strong illumination [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF][START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF].

Plastid preparation and membrane solubilization -Diatoms were collected after 4 to 5 days in their exponential growth phase by centrifugation at 3,000 g for 10 min and resuspended in medium containing 600 mM NaKPO 4 buffer, pH7.5, 5 mM EDTA, 1/100 (v/v) of the Sigma protease inhibitor cocktail. Cells were broken by two 15 s cycles of sonication and centrifuged for 5 min at 400 g. The pellet was sonicated for a second time and centrifuged as above.

Chloroplasts from the two supernatants were pelleted by centrifugation at 12,000 g for 10 min and resuspended in the same high salt buffer, at a chlorophyll concentration of 2 mg.ml -1 .

P. tricornutum chloroplasts were solubilized with -DM at a 1/15 chlorophyll detergent ratio for 15-30 min and centrifuged in Eppendorf tubes at 12,000 g for 10 min to remove insoluble material. All these steps were performed at 4°C. Sucrose gradients -Exponential 7-step sucrose gradients were prepared in either a low-salt buffer containing 50 mM HEPES, pH 7.5, 5 mM EDTA, 0.03 % of -DM and 1mM PMSF or a high-salt buffer containing 100 mM NaKPO 4 , pH 7.5, 5 mM EDTA, 0.03% of -DM and 1 mM PMSF. Detergent-solubilized membrane fractions in 600 mM NaKPO 4 were buffer exchanged on PD-10 column (Amersham Pharmacia) against low-salt or high-salt buffers before loading on appropriate gradients. Centrifugation was performed using a SW41 rotor in a Beckman XL-90 ultracentrifuge at 250,000 g for 17-20 hrs at 4°C. Monomeric and trimeric forms of LHCII from market spinach were used as standards for the sucrose gradient and gel filtration procedures. The preparation of PSII membranes from spinach thylakoids was performed as described by Burke et al. [START_REF] Burke | Involvement of light-harvesting complex in cation regulation of excitation-energy distribution in chloroplasts[END_REF].

Gel filtration -Gel filtration chromatographies were performed using the Biologic Duo flow system (Biorad). Fractions from the gradients were collected and further purified by gel filtration on a Superdex TM 200 10/300GL Tricorn column (Amersham) with a flow rate of 0.3 ml.min -1 .

The running buffer was 20 mM NaKPO 4 , pH 7.5 supplemented with 10 mM EDTA, 1mM PMSF and 0.03% -DM. Elution profiles were recorded at 280 nm to detect proteins or 436 nm to detect chlorophylls, and 0.3 ml fractions were collected. A further purification using -DM was sometimes used as indicated in the text and figure legends.

A direct gel filtration, without any previous sucrose gradient step, was performed following a shorter detergent treatment (5 min -DM solubilization of thylakoids on ice, using a 1/15 chlorophyll/detergent ratio). This new procedure was devised in an attempt to get betterpreserved fractions. Solubilized membrane fractions were centrifuged at 12,000 g in Eppendorf tubes to remove insoluble material before applying the samples onto the column. Spectroscopic analyses -Absorption measurements were performed using a DW-2 Aminco spectrophotometer. 77K fluorescence emission and excitation spectra were measured on a Hitachi F-4500 spectrophotometer with 2.5 nm spectral resolution for both types of measurements.

Pigment analysis -Pigment contents of cells, plastids and isolated fractions were determined by the HPLC method as described earlier [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical fluorescence quenching in diatoms[END_REF]. Extraction was performed using the phase separation procedure first with one volume of a methanol/acetone (50:50, v/v) solution followed by one volume of ether and two volumes of a 10% NaCl solution.

Gel electrophoresis -Polyacrylamide gel electrophoresis was performed using 10-15 % gels according to Laemmli and stained with silver nitrate (Amersham Biosciences kit).

Table 1. Pigment composition of P. tricornutum plastids and antenna fractions obtained

after solubilization of the plastids by n-dodecyl-,D-maltoside followed by separation on the sucrose gradient (see Fig. 1). Whenever indicated (+) the fractions have been treated with ndodecyl-,D-maltoside before gel filtration. Pigment composition is given in mol per 100 mol of Chla. Absorption was monitored at 280 nm, except for the free pigment (fp) monitored at 436 nm.

Table 2. Pigment composition of

Plastids

Figure 8

 8 Figure 8 presents the SDS-PAGE polypeptide profiles of the LHCo fractions. Subfractions

  LHCo fractions prepared from isolated P. tricornutum plastids solubilized with n-dodecyl-,D-maltoside and separated on the gel filtration column. Whenever indicated (+) the fractions have been treated with n-dodecyl-,D-maltoside before gel filtration. Pigment composition is given in mol per 100 mol of Chla.

Figure 1 .

 1 Figure 1. Schematic representation of the pigment-protein complexes separated by sucrose

Figure 2 .

 2 Figure 2. Elution profiles after gel filtration of LHC fractions obtained from sucrose gradients.

Figure 3 .

 3 Figure 3. Absorption spectra of purified D and F fractions obtained by the gel filtration

Figure 4 .

 4 Figure 4. 77K chlorophyll fluorescence spectra of purified D (solid line) and F (dashed line)

Figure 5 .

 5 Figure 5. SDS-PAGE analysis of LHC fractions prepared by gel filtration: D and F originate

Figure 6 .

 6 Figure 6. Gel filtration profiles of P. tricornutum plastids solubilized with -D-maltoside (solid

Figure 7 .

 7 Figure 7. 77K excitation spectra of chlorophyll fluorescence emission at 672 nm for the two

Figure 8 .

 8 Figure 8. SDS-PAGE analysis of the fractions obtained after direct gel filtration of -D-

Figure 1 .Figure 2 .Figure 3 .Figure 4 . 27 Figure 5 . 29 Figure 6 . 30 Figure 7 .Figure 8 . 32 Figure 9 .

 12342752963078329 Figure 1. Guglielmi et al.

Acknowledgements

Authors thank Gérard Paresys and Jean-Pierre Roux for their help in electronic and informatic maintenance. This work was supported by grants from the Centre National de la Recherche Scientifique to the FRE 2433. AVR thanks administration of the Ecole Normale Supérieure for invited Professorship and CNRS for a visiting Fellowship and UK BBSRC for financial support.


Johann Lavaud
email: johann.lavaud@uni-konstanz.de

Peter G Kroth

photochemical fluorescence quenching beyond its control on the xanthophyll cycle

Keywords: fluorescence quenching, PSII, photosystem II, XC, xanthophyll cycle, pH, transthylakoid proton gradient diatom, diatoxanthin, Phaeodactylum tricornutum, NPQ, xanthophyll cycle, transthylakoid delta pH

).

In order to maintain an optimal photosynthetic activity in a permanently changing light environment, plants and algae have evolved a number of mechanisms aimed at the feed-back regulation of the light-dependent reaction of photosynthesis [START_REF] Niyogi | Safety valves for photosynthesis[END_REF].

The thermal dissipation of light energy absorbed in excess (so-called NPQ for nonphotochemical chlorophyll (Chl) fluorescence quenching) by the light-harvesting antenna complex (LHC) of photosystem II (PSII) is one of the most important of the rapidly activated regulatory mechanisms [START_REF] Külheim | Rapide regulation of light harvesting and plant fitness in the field[END_REF][START_REF] Holt | Toward an understanding of the mechanism of the nonphotochemical quenching in green plants[END_REF][START_REF] Horton | Control of the light-harvesting function of chloroplast membranes: The LHCII-aggregation model for non-photochemical quenching[END_REF]. It avoids energy imbalance within the photosynthetic apparatus and protects the PSII activity from high light stress. In higher plants, NPQ is triggered by the light-driven build-up of a transthylakoid proton gradient (ΔpH). The acidification of the lumen results in a protonation of specific sites of the PSII LHC antenna proteins (PsbS,.

Protonation induces conformational changes within the antenna leading to the dissipation of excess energy [START_REF] Horton | Control of the light-harvesting function of chloroplast membranes: The LHCII-aggregation model for non-photochemical quenching[END_REF]. The switch of the antenna to the dissipative quenching state is additionally regulated by the binding of de-epoxidised forms of xanthophylls [START_REF] Holt | Toward an understanding of the mechanism of the nonphotochemical quenching in green plants[END_REF][START_REF] Horton | Control of the light-harvesting function of chloroplast membranes: The LHCII-aggregation model for non-photochemical quenching[END_REF]) produced via a so-called xanthophyll cycle (XC). The XC results in the ΔpH-dependent conversion of epoxidised to deepoxidised forms of xanthophylls. Hence, in higher plants, both the protonation of the LHC antenna and the production of de-epoxidised xanthophylls are involved in NPQ.

Diatoms are a key taxon of the phytoplankton microalgae which dominate in deeply mixed waters [START_REF] Falkowski | The evolution of modern phytoplankton[END_REF] where they may experience large changes in irradiance. NPQ has been shown to allow the diatoms to keep photosynthesis effective in the fluctuating underwater light climate of the Northern marine waters [START_REF] Kashino | Strategies of phytoplankton to perform effective photosynthesis in the North Water[END_REF]. The diatom NPQ process shows specific properties when compared to the NPQ of plants/green algae [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. The polypeptide organisation and composition of the LHC antenna is different with two major points regarding NPQ: 1) the plant minor components PsbS, CP26-29 are absent in diatoms [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: Ecology, evolution and metabolism[END_REF], 2) the spatial arrangement of the polypeptides [START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF] as well as the nature and location of the xanthophylls are specific for diatoms [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF][START_REF] Guglielmi | The light-harvesting antenna of the diatom Phaeodactylum tricornutum: Evidence for a diadinoxanthin binding sub-complex[END_REF]. The XC is also different from the one in plants. It is simpler and involves different pigments: the diadinoxanthin (DD) is converted into diatoxanthin (DT) by a deepoxidase and vice versa (thanks to an epoxidase) depending on the ΔpH. The XC enzymes show specific features: the de-epoxidase is activated at a higher lumenal pH [START_REF] Jakob | Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum[END_REF], Grouneva et al. 2005) than in higher plants and its activity is strongly depending on the acidic form of the co-factor ascorbate (Grouneva et al. 2005). As a consequence, the DD de-epoxidase is characterised by a very rapid activation even when only small changes in the ΔpH occur. The reactivity of the enzyme can be reduced by DCCD (N, N'-dicyclohexyl-carbodiimide) which binds to the domain which is responsible for the sensitivity of the enzyme to the pH [START_REF] Jahns | Dicyclohexylcarbodiimide alters the pH dependence of violaxanthin de-epoxidation[END_REF]. The DT epoxidase is also very sensitive to a change in the ΔpH, being inactivated by high ΔpH conditions [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF].

The light-dependent accumulation of DT is mandatory for NPQ, regulating its kinetics and extent in a quantity dependent way (Lavaud et al. 2002a[START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. As compared to land plants, the ratio xanthophylls over chlorophyll is much more larger [START_REF] Wilhelm | The biochemistry and physiology of light-harvesting processes in chlorophyll b-and c-containing algae[END_REF][START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF]. Consequently, NPQ in diatoms can reach higher values (Lavaud et al. 2002a). It has been recently shown that, once the antenna dissipative state is formed, the extent and kinetics of NPQ solely depend on amount of DT and are independent from the ΔpH [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF], which explains the prolonged stability of NPQ after the illumination [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]). Still, it cannot be excluded that both the ΔpH and DT are required to generate the antenna dissipative state (Lavaud et al. 2002b[START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. This has been difficult to show because the kinetics of the build-up of ΔpH and the accumulation of the DT are tightly related (Lavaud et al. 2002b[START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]. Even though, in some cases DT can be present with no development of NPQ depending on the extent of the ΔpH (Lavaud et al. 2002b[START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. This observation and the specific properties of the XC regulation in diatoms were used to design a special experimental procedure aimed at uncoupling the relationship between ΔpH and the XC and studying the effect on NPQ. For our experimental approach, we used five protocols (Prot.) and measured NPQ and DD deepoxidation in Phaeodactylum tricornutum (Table 1). Table 1 here.

It has been previously shown in higher plants that pre-illumination of leaves changes the relationship between the ΔpH, the XC and NPQ [START_REF] Noctor | Modulation of delta-pH-dependent nonphotochemical quenching of chlorophyll fluorescence in spinach chloroplast[END_REF]) and accelerates the NPQ kinetics [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]), during the following high light exposure. We thus decided to apply this technique to the diatoms. In the two first protocols (Prot. n°1 and 2), the cells were pre-illuminated before inducing NPQ and DD de-epoxidation by high light (HL) exposure (2000 µmol photons m -2 s -1 ). The measurements during HL were compared with cells dark-adapted for the same time than the pre-illumination. In Prot. n°1, the cells were incubated at the growth light (GL) intensity (50 µmol photons m -2 s -1 ) for a time long enough (45 min) to re-acclimate the cells to the growing conditions after the sampling and the preparation (see Lavaud et al. 2002a). We designed this experiment as a control to check the feasibility in diatoms of accelerating the NPQ kinetics by pre-illuminating the cells. It is a preparatory procedure to Prot. n°2. After incubation of the cells to 45 min darkness and exposure to HL, NPQ 1min was 3.7 and its development paralleled the accumulation of DT formed by the de-epoxidation of DD (Table 2) according the previous observations (Lavaud et al. 2002a, Lavaud et al. 2004). When adapted to GL before the same HL exposure, NPQ was multiplied by a factor 1.6 (6 vs 3.7) similar to the increase of DT (Table 2). The irradiance used during the GL pre-incubation was not high enough to accumulate any DT (see Table 2). The cause of the acceleration is then likely to be a small difference in the ΔpH, compared to the dark-adapted cells, due to the basal linear electron transport driven by the pre-illumination [START_REF] Cruz | Plasticity in light reactions of photosynthesis for energy production and photoprotection[END_REF]. This small ΔpH change might be sufficient to stimulate the DD de-epoxidase [START_REF] Jakob | Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum[END_REF], Grouneva et al. 2005). As the backward DT epoxidation reaction has the same [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF] or even higher [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]) velocity in low light, no DT accumulated and consequently no NPQ developed (Lavaud et al. 2002b) during the GL pre-illumination. Nevertheless, in the conditions of Prot. n°1, the initial ΔpH and the stimulated de-epoxidase lead to the concomitant acceleration of both DD de-epoxidation and NPQ kinetics during the following HL illumination as reported before in higher plants [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]. Table 2 here. A special pre-illumination procedure (Prot. n°2) consisting in 3 successive 5 min steps of progressively increasing irradiance (PL, from 50 to 150 via 100 µmol photons m - 2 s -1 ) allowed breaking the proportionality between DT and NPQ. This pre-illumination procedure was designed regarding the results of Prot. n°1. Its first purpose has been to accelerate to a maximum the kinetics of both NPQ and DD de-epoxidation during HL by driving the highest linear electron transport and consequently the highest ΔpH without generating any NPQ and DT during the pre-illumination. The PL pre-illumination allowed reaching this situation (see Table 2). It has been tested that for a longer illumination at intermediate irradiance (like 100 µmol photons m -2 s -1 ) and that from 200 µmol photons m -2 s -1 on, whatever the illumination time, NPQ developed (not shown).

Figure 1 shows a recording of the Chl a fluorescence emission changes induced by a 5 min HL illumination after pre-incubation of the cells with PL or darkness. The spikes resulted from short pulses of saturating intensity (down arrows). The maximum fluorescence yield, F m was reached when such a pulse was applied to dark-adapted cells that have a low fluorescence yield (F 0 ). During the illumination, NPQ (= (F m -F m ')/F m ') rapidly developed, as shown by the lowering of the maximum yields reached during the pulses (F m '). Figure 1 here. When the cells were adapted to PL before HL exposure, the NPQ kinetics was accelerated during the first 30-45 s HL (see Inset Fig. 1A). It was already visible after few seconds of illumination (Fig. 1B, note the decrease after the G peak for pre-illuminated cells [START_REF] Lazar | The polyphasic chlorophyll a fluorescence rise measured under high intensity of exciting light[END_REF]). Nevertheless, in these conditions NPQ development and DD de-epoxidation were not accelerated to the same extent. The DT accumulated exceeded the amount which is effectively used for NPQ (Table 2, compare bold lines) (see also Lavaud et al. 2002b). Still, after 5 min HL (the time to reach steadystate conditions for Chl a fluorescence emission) the increase in NPQ and DT accumulation was similar (Table 2). In higher plants, de-epoxidised xanthophylls need to enter a so-called 'activated state' to drive NPQ [START_REF] Holt | Toward an understanding of the mechanism of the nonphotochemical quenching in green plants[END_REF][START_REF] Horton | Control of the light-harvesting function of chloroplast membranes: The LHCII-aggregation model for non-photochemical quenching[END_REF]) by binding to protonated LHC antenna proteins (such as PsbS). In diatoms, there is no PsbS [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: Ecology, evolution and metabolism[END_REF]), but a DT 'activated' state does exist [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]) arguing for a LHC antenna protonation event as in higher plants. A possible explanation for the data of Prot. n°2 might be that the PL pre-illumination drove a linear electron transport (as seen by the increased steady-state F S level (Fig. 1A) and the increased I shoulder (Fig. 1B) resulting from Q A reduction) which generated a change in the ΔpH which was high enough to ensure conditions for which the proportionality between the accumulation of DT and the protonation of LHC antenna polypeptides was lost: more DT molecules accumulated than LHC antenna sites have been protonated because of the high reactivity of the XC enzymes towards the ΔpH [START_REF] Jakob | Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum[END_REF], Grouneva et al. 2005[START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. Indeed, the DD de-epoxidase and the DT epoxidase are, respectively, rapidly activated and inactivated by high ΔpH conditions driven by a sudden increase in light intensity.

Addition of exogenous ascorbate to the cells (Prot. n°3) confirmed this proposal.

Ascorbate is one of the co-factors of the DD de-epoxidase. It shifts the activity of the enzyme at higher pH and consequently a lower ΔpH is required for DD de-epoxidation (Grouneva et al. 2005). The presence of ascorbate lead to an increase of DD deepoxidation without virtually any effect on NPQ at moderate light conditions (ML, 350 µmol photons m -2 s -1 , Table 3). For higher intensities (above the light for saturation of photosynthesis, 450 µmol photons m -2 s -1 ), the effect of ascorbate on the relationship DT/NPQ was not visible anymore (data not shown), surely due to the fact that the change in the ΔpH was too high and fast. Table 3 here.

Hence, as shown by Prot. n°2 and 3, increasing only the amount of DT molecules is not sufficient for enhancing NPQ, it needs a simultaneous acidification of the lumen and subsequent capacity for switching to DT 'activated' molecules, probably via protonation of LHC antenna sites as recently proposed [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]).

The relationship between ΔpH and NPQ was further investigated by using DCCD to vary the extent of the light-dependent lumen acidification. DCCD inhibits the activity of the ATP synthase, by blocking the proton conductive channel, and thus the reflux of lumenal protons [START_REF] Mccarty | The inhibition and stimulation of photophosphorylation by N,N'-dicyclohexylcarbodiimide[END_REF]. DCCD increases NPQ in diatoms in an incubation time-dependent way (Lavaud et al. 2002b). The amount of DCCD which penetrates the chloroplast is likely to be dependent on the incubation time. Prot. n°4 was designed to further test the influence of the DCCD incubation time on the kinetics of NPQ. It is a preparatory procedure to Prot. n°5. DCCD greatly enhanced the extent of NPQ at a low light irradiance (LL, 100 µmol photons m -2 s -1 ) which generated a small NPQ (0.09) in the absence of DCCD (Fig. 2A). At the low concentration of 10 µM, the effect of DCCD on NPQ kinetics was dependent on the incubation time before the illumination even if after 5 min light the extent of both NPQ (1.7 ± 0.1) and DT accumulation (2 ± 0.3 mol/100 mol Chl a) were similar for 5 and 15 min incubation. In higher plants, at higher concentrations, DCCD binds to the proton binding sites of the CP26-29 and PsbS antenna polypeptides thus inhibiting NPQ [START_REF] Bassi | Lhc proteins and the regulation of photosynthetic light harvesting function by xanthophylls[END_REF]Caffarri 2000, Horton et al. 2005). These polypeptides do not exist in diatoms [START_REF] Armbrust | The genome of the diatom Thalassiosira pseudonana: Ecology, evolution and metabolism[END_REF] and the addition of DCCD at low concentrations enhances NPQ, as previously reported (Lavaud et al. 2002b), by enhancing the amount of protons which accumulate within the lumen. Figure 2 here.

Regarding the results of Prot. n°4, a dynamic study was then performed (Prot. n°5) where the effect of DCCD incubation time on NPQ and the DD de-epoxidation were observed over 30 min to reach a steady-state of the penetration of DCCD in the chloroplast. As for Prot. n°3, ML irradiance was chosen to avoid a too high and fast change in the ΔpH upon illumination, a better way to uncouple the NPQ and DD deepoxidation kinetics. Without DCCD in the light conditions used, an NPQ of 1.3 formed concomitantly with DD de-epoxidation (15 %). For short DCCD incubation times, NPQ was enhanced with no parallel increase in DT; then the DD de-epoxidation suddenly increased (to above 25 %) (Fig. 2B). It is proposed that the uncoupling between NPQ and the DD de-epoxidation is due to the DCCD effect on the XC enzymes. DCCD reduces the de-epoxidase reactivity to changes in pH [START_REF] Jahns | Dicyclohexylcarbodiimide alters the pH dependence of violaxanthin de-epoxidation[END_REF], and simultaneously induces high ΔpH which decreases the epoxidase activity [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. This situation generated a clear deviation from the linear NPQ-DT relationship usually observed (Lavaud et al. 2002a[START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]). Hence, it was possible to enhance the NPQ extent with a constant amount of already present DT molecules. In analogy to higher plants, we hypothesise that this was made possible through additional protonation of LHC antenna polypeptides and subsequent conformational changes [START_REF] Horton | Control of the light-harvesting function of chloroplast membranes: The LHCII-aggregation model for non-photochemical quenching[END_REF][START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF].

Altogether, these observations strongly support the hypothesis that in diatoms, the ΔpH plays a central role in the NPQ process not only in controlling the activity of the XC enzymes. As protons accumulate in the lumen during illumination, protonation of LHC antenna polypeptides is likely to occur, as in higher plants, enabling the switch of DT molecules to an 'activated' quenching state previously reported [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]; which is an essential step in the development of NPQ. In diatoms, the mechanism of NPQ might be similar to that in higher plants [START_REF] Holt | Toward an understanding of the mechanism of the nonphotochemical quenching in green plants[END_REF][START_REF] Horton | Control of the light-harvesting function of chloroplast membranes: The LHCII-aggregation model for non-photochemical quenching[END_REF]), confirming the model recently proposed [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]). Nevertheless, one has to keep in mind that the organisation and xanthophyll/protein composition of the diatom LHC [START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF][START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF][START_REF] Guglielmi | The light-harvesting antenna of the diatom Phaeodactylum tricornutum: Evidence for a diadinoxanthin binding sub-complex[END_REF] as well as the regulation of the XC [START_REF] Jakob | Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum[END_REF], Grouneva et al. 2005) are very different from higher plants. These specific characteristics probably explain why the extent and kinetics of NPQ in diatoms show striking features when compared to higher plants [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]). An important question which still remains to be solved is the capacity for protonation of LHC polypeptides to drive alone (i. e. without the presence of DT) a basal NPQ via LHC conformational changes such as in plants [START_REF] Holt | Toward an understanding of the mechanism of the nonphotochemical quenching in green plants[END_REF][START_REF] Horton | Control of the light-harvesting function of chloroplast membranes: The LHCII-aggregation model for non-photochemical quenching[END_REF]. So far there is no clear evidence for such a mechanism in diatoms (Lavaud et al. 2002b[START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. Because of the characteristics of the XC enzymes, only an in vitro approach would allow to clarify this point. The recent improvement in the isolation of native LHC complexes from diatoms [START_REF] Büchel | Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states[END_REF][START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF][START_REF] Guglielmi | The light-harvesting antenna of the diatom Phaeodactylum tricornutum: Evidence for a diadinoxanthin binding sub-complex[END_REF]) now provides the possibility to follow the same successful approach as in higher plants [START_REF] Ruban | Modulation of chlorophyll fluorescence quenching in isolated light-harvesting complex of Photosystem II[END_REF].

Hence, in diatoms, there is a very tight and concerted activation of the XC and of the NPQ process via the ΔpH build-up. This is surely due to the fact that the de-epoxidase is very rapidly activated by the acidification of the lumen [START_REF] Jakob | Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum[END_REF], Grouneva et al. 2005) while the epoxidase is simultaneously inhibited by the increase of ΔpH [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]) ensuring a fast supply of NPQ with DT molecules (Lavaud et al. 2002b). This feature is important for a fast switch of the LHC function to a photoprotective state of energy dissipation in a highly variable underwater light climate. In this context, it is noteworthy that the first-order reaction time constant of the XC in diatoms is faster than the time needed for the irradiance to double in the coastal/estuarine turbulent waters where the diatoms flourish [START_REF] Macintyre | The effect of water motion on short-term rates of photosynthesis by marine phytoplankton[END_REF].

Methods

We used five experimental protocols (Prot.) and measured NPQ and DD deepoxidation. All the details for the experimental approach are given in Table 1 and the corresponding parts of the text. The photosynthetic properties as well as the growing and preparation for experiments of the low and high NPQ cells of Phaeodactylum tricornutum Böhlin were described previously (Lavaud et al. 2002a[START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF]. Because high NPQ cells exhibit faster NPQ kinetics (Lavaud et al. 2002a), they were preferred for Prot. n°1 and 2. Xanthophyll pigment analyses were performed by HPLC as described [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF]). The DD de-epoxidation (DEP) extent was calculated as (DT/DD+DT) x 100 where diadinoxanthin (DD) is the epoxidised form and diatoxanthin (DT) is the deepoxidised form. NPQ (= (F m -F m ')/F m ') was calculated from standard modulated Chl a fluorescence measurements (see the text for details) performed with a PAM-101 fluorometer (Walz, Effeltrich, Germany) as described [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]). Fluorescence induction kinetics (FIK) of P. tricornutum cells were obtained with a home-made fluorometer as described [START_REF] Parésys | Quantitative and qualitative evaluation of phytoplankton populations by trichromatic chlorophyll fluorescence excitation with special focus on cyanobacteria[END_REF]. Ascorbate (Sigma) was obtained from a 2 M fresh stock solution in distilled water and DCCD (Sigma) from a fresh 1 M stock solution in absolute ethanol. For all the measurements, sodium bicarbonate was added to the algal suspension (4 mM final, from a freshly prepared 0.2 M stock solution) to prevent any limitation of the photosynthetic rate by carbon supply. Incubation was performed in the dark prior to the illumination (5 min, 350 µE m -2 s -1 ).

Values are mean of three measurements  SD. Fig. 2A and 2B stand for Prot. n°4 and 5, respectively.

Figure legends Figure 1

 legends1 Figure legends

Figure 2 .

 2 Figure 2. (A) Effect of DCCD (N,N'-dicyclohexyl-carbodiimide ) (10 µM final) on the

  photosynthesis (450 µmol photons m -2 s -1 ,Lavaud et al. 2002a); HL, high light (2000 µmol photons m -2 s -1 ) allowing to rapidly (in few seconds) obtain a large NPQ(Lavaud et

Table 1

 1 Descriptions of the five experimental protocols (Prot.) used.

	Prot.	Cells	Pre-incubation	Light treatment
	1	High NPQ	45 min Darkness or GL	HL, 5 min
	2	High NPQ	15 min Darkness or PL	HL, 5 min
	3	Low NPQ	15 min darkness, +/-100 mM ascorbate	ML, 5 min

Acknowledgments

We thank B. Rousseau for his assistance with the pigment analyses and Dr. Anne-Lise Etienne for critical reading and improvement of the manuscript.


domain: stat math info

text:
Sebastian Hitziger

Maureen Clerc

Alexandre Gramfort

Sandrine Saillet

Christian G Bénar

Théodore Papadopoulo

Jitter-Adaptive Dictionary Learning -Application to Multi-Trial Neuroelectric Signals

come

Real data

Idea: as is finite, we can first apply all possible shifts to , yielding the "unrolled" dictionary .

In an animal model of epilepsy, local field potentials were recorded during one hour with an intra-cranial electrode in a Winster-Han rat. Biccuculine (a blocker of inhibition) was injected in the cortex to elicit epileptic-like discharges. 169 of these spikes were then selected visually and segmented into epochs of 10 seconds.

Epoch 41 Epoch 111 Epoch 161 Average

Learned dictionaries

Only JADL clearly separates spikes from oscillations.

DL smoothes the periodic wave, as its atoms do not adapt to phase shifts.

Learned coefficients and shifts provide insight into data

Goal: detect similar waveforms and describe how they change across trials.

Averaging

Matrix factorization (PCA, ICA, dictionary learning [START_REF] Mairal | Online Learning for Matrix Factorization and Sparse Coding[END_REF]) Linear approach, does not account for temporal shifts [START_REF] Hanselmayr | Alpha phase reset contributes to the generation of ERP's[END_REF]. Variants of matching pursuit [START_REF] Benar | Consensus matching pursuit for multi-trial EEG signals[END_REF] Do not learn waveforms but require predefined dictionary.

Loses the information present in individual trials.

finite set of allowed shifts, Sparse coding can now be performed over , the non-zero coefficients show which shifts are used.

A uniqueness constraint on the coefficients ensures, that at most one shifted version of each atom is used; the LARS algorithm [START_REF] Efron | Least angle regression[END_REF] can be modified to guarantee this constraint. where the inverse shift functions as a realignment operator. This is followed by normalization.

Relations to other DL approaches

Dictionary learning (DL) Minimization (1) becomes DL for .

Shift-invariant sparse coding (SISC) [START_REF] Blumensath | Sparse and shift-invariant representations of music[END_REF] Atoms can shift arbitrarily.

Atoms typically shorter than signal. Structure of algorithms of JADL and DL are similar.

No uniqueness constraint as in JADL: multiple shifts per atom allowed in each signal.

Performance

While both (the number of allowed shifts) and influence the size of the "unrolled" dictionary, the increase in computation time due to is only linear. This comes from the uniqueness constraint which bounds the complexity as well as the use of fft-based convolution for large .

On the 169 epochs, dictionaries were learned using DL and JADL, each algorithm performing 200 iterations. The spikes (first four atoms) are well aligned across trials. The periodic atom (five) occurs with uniformly distributed phase.

*For each , we selected the parameter that gave the smallest error. This was for DL and and for JADL and .

Existing approaches L 1 -regularized optimization

Conclusion

We presented a new method (JADL) which is an extension to dictionary learning and designed to analyze multi-trial neuroelectric datasets. We evaluated JADL on synthetic and real data and showed its superiority to common dictionary learning. In particular, JADL showed the following qualities:

Ability to learn main waveforms in trials and to separate them.

Learned shifts and coefficients give insight into the changes of waveforms (phase, latency, amplitude).

Robustness and denoising qualities.

Computational efficiency, even for high shift-tolerance.

  t r i a l s time Assumption: trials can be generated from a dictionary. Trials: recordings of neuronal electromagnetic activity under similar conditions.

The simultaneous analysis of

  multiple recordings of neuronal electromagnetic activity is an important task requiring sophisticated and extremely noise robust techniques. A general goal is to find a representation of the similarities (e.g. repeating Jitter-Adaptive Dictionary Learning -Application to Multi-Trial Neuroelectric Signals Sebastian Hitziger 1 , Maureen Clerc 1 , Alexandre Gramfort 2 , Sandrine Saillet 3 , Christian Bénar 3 , Théodore Papadopoulo 1 1 Project-Team Athena, INRIA Sophia Antipolis, France 2 Institut Mines-Telecom, Telecom ParisTech CNRS LTCI, France 3 Institut de Neurosciences des Systèmes, UMR 1106 INSERM, Aix-Marseille Université, Faculté de Médecine La Timone, Marseille, @inria.fr https://team.inria.fr/athena/ waveforms) as well as the differences (e.g. varying shape, latency, phase, or amplitude of waveforms) across the signals. Here, we present an extension to dictionary learning that explicitly accounts for small variations in latency and phase of atoms. This work was supported by the ANR grants CoAdapt (09-EMER-002-01) and MultiModel (2010-BLAN-0309-04).