Betty Kientz
email: betty.kientz@gmail.com

Hélène Agogué

Céline Lavergne

Pauline Marié

Eric Rosenfeld
email: eric.rosenfeld@univ-lr.fr

Kientz Betty

Isolation and distribution of iridescent Cellulophaga and further iridescent marine bacteria in the Charente Maritime coast, French Atlantic coast

Keywords: Iridescence, iridescent bacteria, marine bacteria, Bacteroidetes, Flavobacteriaceae, Cellulophaga, Tenacibaculum, Aquimarina, culturable bacteria, epibiont, seaweed, coastal shore, rocky shore

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Different color processes are present in the natural environment. Pigmentation, a biochemical coloration, can be generated by molecular absorption of specific wavelengths.

The exhibited color is uniform. A very different coloration process corresponding to structural colorations is referred as iridescence. An intense coloration with angle-dependent color changes is then produced due to micron-and sub-micron-sized periodic structures interfering with light [START_REF] Fox | Animal biochromes and structural colors: physical, chemical, distributional & physiological features of colored bodies in the animal world[END_REF][START_REF] Vukusic | Structural color: now you see it-now you don't[END_REF]. This appearance has been well studied in higher organisms particularly in insects [START_REF] Ghiradella | Light and color on the wing: structural colors in butterflies and moths[END_REF][START_REF] Seago | Gold bugs and beyond: a review of iridescence and structural color mechanisms in beetles (Coleoptera)[END_REF] and birds [START_REF] Prum | Structural coloration of avian skin: convergent evolution of coherently scattering dermal collagen arrays[END_REF]. Iridescence is also encountered in marine organisms such as fishes [START_REF] Lythgoe | The structural basis for iridescent color changes in dermal and corneal iridophores in fish[END_REF], ctenophores [START_REF] Welch | Optical properties of the iridescent organ of the comb-jellyfish Beroë cucumis (Ctenophora)[END_REF], macroalgae [START_REF] Craigie | Cuticules from Chondrus crispus (Rhodophyta)[END_REF][START_REF] Gerwick | Structural, chemical and ecological studies on iridescence in Iridaea (Rhodophyta)[END_REF] and diatoms [START_REF] Noyes | Light manipulation in a marine diatom[END_REF].

Bacterial iridescence has been a vague and poorly documented concept. However, a marine bacterium -Cellulophaga lytica CECT 8139-has been recently described to exhibit, on solid media, a unique intense iridescence in reflection [START_REF] Kientz | a) Iridescence of a marine bacterium and classification of prokaryotic structural colors[END_REF]. Colonies have colored centers of varying brightness distributed across the biofilm giving a glitter-like character. Iridescent green is the dominant color, but red and blue-violet are also observed at the colonies' peripheral edges. Iridescence of C. lytica CECT 8139's colonies was proved with physical methods and the phenomenon was named glitter-like iridescence. Since this type of iridescence occurred under direct natural light exposure it was described as a more natural coloration effect compared to the other bacterial iridescences.

We recently demonstrated that the iridescent "Cellulophaga lytica" CECT 8139 belongs to the species Cellulophaga lytica by a DNA-DNA hybridization against C. lytica LIM-21 T (88.9% similarity, unpublished data obtained from DSMZ company). The C. lytica species is adapted to a wide range of marine biotopes in free-living, biofilm and associated forms [START_REF] Bernardet | An introduction to the family Flavobacteriaceae[END_REF][START_REF] Lewin | Isolation, cultivation and characterization of Flexibacteria[END_REF].

Diverse intensities of iridescence were found within the C. lytica strains [START_REF] Kientz | a) Iridescence of a marine bacterium and classification of prokaryotic structural colors[END_REF]. Non-iridescent or less iridescent C. lytica strains Cy I2 (DSM 2039), LIM-21 T (CIP 103822 and DSM 7489) were all isolated from muddy sediment of Costa Rica beaches [START_REF] Johansen | Description of Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola gen. nov., sp. nov. and reclassification of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb[END_REF][START_REF] Lewin | Isolation, cultivation and characterization of Flexibacteria[END_REF]. In contrast, iridescent strains C. lytica WFB 21 (DSM 2040) and ACEM 21 were isolated from seawater in an aquarium outflow and an estuary, respectively [START_REF] Lewin | Isolation, cultivation and characterization of Flexibacteria[END_REF][START_REF] Skerratt | Algicidal bacteria associated with blooms of a toxic dinoflagellate in a temperate Australian estuary[END_REF]. The iridescent C. lytica CECT 8139 was isolated from a red anemone Actinia equine surface at Oléron island rocky shore on the Charente maritime coast in France [START_REF] Kientz | a) Iridescence of a marine bacterium and classification of prokaryotic structural colors[END_REF]. Therefore, different ecotypes exist and unlike the benthic origin, the epibiontic and planktonic origins seemed favorable for the iridescent trait.

However, an extended study on origins and biotopes favoring the iridescence trait has not yet been performed. In addition, even if bacterial iridescence has not been yet directly observed in the marine environment, investigating the specific distribution of iridescent strains could be of major importance to determine the possible roles of marine bacterial iridescence.

In this work, we studied the occurrence and distribution of iridescent bacterial strains in the Charente maritime coast in France, with special attention paid to the C. lytica species. A specific observation device was employed to detect iridescent colonies. Isolated strains were affiliated to a genus by sequence analysis of the 16S rRNA gene.

Materials and methods

Sample collection and bacterial isolation

The sampling was performed between October and December 2011. The seven sites were located on the Charente maritime coast in France (Fig. 1). Diverse biotopes as rocky shore, mudflat and rocky-sandy shores were included (Table 1). A total of 139 samples were collected at low tide for bacterial isolation: seawater [START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF], sediments [START_REF] Ludwig | ARB: a software environment for sequence data[END_REF], red macroalgae (Rhodophyta) [START_REF] Kientz | Effect of abiotic factors on the unique glitterlike iridescence of Cellulophaga lytica[END_REF], green macroalgae (Chlorophyta) [START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF], brown macroalgae (Phaeophyta) [START_REF] Ludwig | ARB: a software environment for sequence data[END_REF], decomposed macroalgae [START_REF] Felsentein | Inferring Phylogenies[END_REF], marine plants (4), sponges [START_REF] Craigie | Cuticules from Chondrus crispus (Rhodophyta)[END_REF], cnidarians [START_REF] Fox | Animal biochromes and structural colors: physical, chemical, distributional & physiological features of colored bodies in the animal world[END_REF], worms (3), crustaceans [START_REF] Felsentein | Inferring Phylogenies[END_REF], mollusks [START_REF] Lewin | Isolation, cultivation and characterization of Flexibacteria[END_REF], echinoderms [START_REF] Doucet | Iridescence: a functional perspective[END_REF] and detritus [START_REF] Bernardet | An introduction to the family Flavobacteriaceae[END_REF].

A "fresh" inoculation protocol was defined in order to isolate environmental strains and to avoid population changes during transport. For seawater samples, a side of the agar plate was immerged directly in the water and the Petri dish was rapidly closed. For solid samples, a fragment was rinsed in artificial sterile seawater (ASW) Instant Ocean © (30 g L -1 in pure water) in order to remove loosely attached epibionts and then scraped onto the agar plate [START_REF] Kientz | a) Iridescence of a marine bacterium and classification of prokaryotic structural colors[END_REF]. For sediment samples, about 1 ml was directly added onto the agar. Three days of incubation at 20°C were needed to observe iridescent colonies. Picked colonies were then subcultured, purified and stored at -80°C in Cryovials© (Dutscher).

Culture conditions of iridescent marine bacteria

The solid medium Cytophaga (CYT) was used to isolate iridescent marine bacteria [START_REF] Johansen | Description of Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola gen. nov., sp. nov. and reclassification of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb[END_REF]. CYT medium contained 1 g tryptone, 0.5 g yeast extract, 0.5 g CaCl 2 .2H 2 O, 0.5 g MgSO 4 .7H 2 O, and 15 g agar in 1 L of artificial seawater (ASW) Instant Ocean© (30 g L -1 in pure water).

Incubation temperature was fixed at 20°C.

Iridescence observation

Plates from the collection were observed using a direct illumination set up (see below) to elicit iridescent colonies. Additionally, iridescence of the pure strains was observed using a Fig. 1 streaking procedure. One colony from a 24 h-old plate was subcultured in triplicate plates drawing thin 5 cm-linear streaks. Cultures were photographed after 24 h and 72 h of growth in a dark room using an experimental arrangement of oblique epi-illumination at a fixed illumination angle of 60 °C [START_REF] Kientz | a) Iridescence of a marine bacterium and classification of prokaryotic structural colors[END_REF]. The light source was a lamp (Kaiser RB 218N HF copy lighting unit) of 18 watts, 5400 Kelvin, the operating voltage correspond to AC 220-240 V, 50

Hz with an operating frequency of 40 kHz. The camera was a Nikon D1500 18-55VR on Av program with f22, the lens was a macro, large size (12.1 Mega pixels) used in superfin mode.

Molecular characterization of the strains

Identification of each iridescent isolate was determined by 16S rRNA gene sequencing. The DNA suspension for the PCR consisted of colonies picked on agar plates and resuspended in 500 µl of sterile water. Before the PCR, 3 heat shocks (incubation at 60°C during 2 min) and cold shocks (incubation in liquid nitrogen during 2 min) were applied on the bacterial suspension in order to lyse the cells and liberate the DNA. Primers used for 16S RNA full length gene sequencing were 27F (5'-AGA GTT TGA TCC TGG CTC AG-3'), and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3') [START_REF] Lane | 16S/23S rRNA sequencing[END_REF][START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[END_REF]. Reaction mixtures of 50 µl contained 20 µl of PCR master mix (DyNAzyme II, Finnzymes), 100 pmol of each primer, 2 µl of washed cells and MilliQ water to the 50 µl volume. PCR reaction was carried out in a Labcycler SensoQuest. The thermal PCR profile was as follows: initial denaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, primer annealing at 60°C for 1 min, and elongation at 72°C for 1 min. The final elongation step was 9 min at 72°C. The 16S rDNA products were analysed by electrophoresis in 1% agarose gels. The 16S rDNA products were then sequenced with an automatic DNA analysis system (Genoscreen, Lille, France and GATC, Konstanz, Germany). Sequences were compared with sequences available in the GenBank database by using the BLAST (Basic Local Alignment Search Tool) service to determine their approximate phylogenetic affiliations [START_REF] Altschul | Basic local alignment search tool[END_REF]. Sequences of representative strains were deposited in the GenBank database under accession numbers

Phylogeny

Sequences have been analyzed using the ARB software package (November 2011 version) [START_REF] Ludwig | ARB: a software environment for sequence data[END_REF] and the corresponding SILVA 16S RNA-based LTP released 106 SSU database [START_REF] Yarza | The All-Species Living Tree project: A 16S rRNAbased phylogenetic tree of all sequenced type strains[END_REF].

After importing, all sequences were automatically aligned according to the SILVA SSU [START_REF] Felsentein | Inferring Phylogenies[END_REF] reference alignment thanks to the online tool SINA. Manual refinement of the alignment was carried out taking into account the secondary structure information of the rRNA in ARB. Tree reconstruction was using the Neighbour Joining (ARB), Maximum Parsimony (PHYLIP DNA-Parsimony v 3.6a3 [START_REF] Felsentein | Inferring Phylogenies[END_REF]) and Maximum Likelihood (PHYML v2.4.5 [START_REF] Guindon | A Simple, Fast, and Accurate Algorithm to Estimate Large Phylogenies by Maximum Likelihood[END_REF]) methods. Tree topology was further tested by the application bacterial variables positional filters and by the application of 30%, 40% and 50% positional conservatory filters. The final tree was calculated with 43 sequences based on 1274 valid columns (50 % filtering) with PHYML (model: GTRGAMMA). Partial sequences have been added to the tree using the ARB parsimony tool.

Results

Collection and isolation of iridescent marine bacteria

For isolation of iridescent marine bacteria, the CYT medium was preferred to rich media such as marine agar to avoid too rapid growth. Using our collection and isolation procedures, we could easily observe several iridescent marine strains from diverse origins from the Charente maritime coast. Diverse bacterial colonies grew on CYT plates but iridescent colonies were only visible after three days of incubation. Interestingly, various visual effects were observed under direct illumination (Fig. 2, arrows). Mostly large green or red iridescent colonies were present. Small red pigmented colonies with red iridescence were also observed.

Iridescence and distribution of the novel strains of Cellulophaga

Iridescence in the Cellulophaga strains is characterized by a green bright effect with red/violet edges. This type of iridescence was observed on several plates of diverse origins (Fig. 2) and colonies resembling to C. lytica were isolated and then identified by molecular analysis. The 34 strains belonging to the genus Cellulophaga were grouped in OTU 1 (Table 2). Different iridescent intensities and color variations were visible. Some colonies exhibited yellow or blue colors (Fig. 3). Most of the strains were positive for agarolysis and gliding motility. The rocky shore biotope was found to contain a lot of iridescent Cellulophaga species (Fig. 4). These strains were isolated from a wide range of samples but mostly from macroalgae, and in particular from Rhodophyta and Phaeophyta, and from mollusks. Most intense iridescent strains were isolated from the surface of Rhodophyta and mollusks (Fig. 4).

Genotypic diversity of other iridescent bacteria

While searching for Cellulophaga iridescent strains, we observed other colonies with a bright effect but with different types of iridescence (Fig. 2). A total of 97 strains (including strains of Cellulophaga) were isolated and affiliated to genus by sequencing of the of 16S rRNA gene (Table 2). Similarity values for the 16S rRNA gene sequences with previously reported isolated species in databases are presented in Table 2. Overall, 11 different OTUs were found. For 3 OTUs (5, 7 and 9), the isolated strains showed sequence similarities lower than 97% to previously reported species, suggesting the possible presence of new species. All isolates belonged to the phylum of Bacteroidetes and formed two clusters (Fig. 5). The Tenacibaculum isolates named OTU 3, 4, 5, 6, 7, 8, 9, 10 and 11 clustered with the genus Tenacibaculum exhibiting between 96 and 99% 16S rRNA sequence similarity (Table 2). In the other cluster, isolates belonging to the OTU 1 had 99% similarities with Cellulophaga lytica and isolates belonging to the OTU 2 had 98% similarities with Aquimarina latercula (Table 2).

The irisdescent strains belonging to the genus Cellulophaga (OTU 1) were the most representative strains with 35.1% (Table 2). Next, 59.8% of isolated strains (OTUs 3, 4, 5, 6, 7, 8, 9, 10 and 11) belong to the genus Tenacibaculum. Finally, 5.2% of isolated strains belong to the genus Aquimarina.

Phenotypic diversity of the others iridescent strains

Different iridescent colonies were visible on the original plates (Fig. 2). Identified strains showed diversity in iridescence coloration and intensity when cultivated by the streaking method. Green or red iridescence was often observed (Fig. 6). The majority of Tenacibaculum strains expressed bright angle-dependent green iridescence, except for OTU 5, 7 and a few strains of OTU 8, 9 expressing a red iridescence. The red iridescence of OTU 5 could be observed on colony edges only after three days of incubation. OTU 3 and 4 strains expressed a particular iridescence resembling to holograms with multicolor effects observed by eyes but difficult to photograph (Fig. 6 c,d). Strains affiliated to Aquimarina sp.

(OTU 2) were red-pigmented with red iridescence. Most isolated strains were motile by gliding, especially for the bright iridescent isolates.

Source of isolation of iridescent bacteria

Iridescent bacteria were isolated from all types of marine biotope and samples except from the mudflat environment where no iridescent strain was isolated. As for Cellulophaga species, the rocky shore ecosystem contained most iridescent bacteria (Fig. 4). Efficient sources for the isolation of iridescent bacteria were mollusks, Rhodophyta, Phaeophyta, seawater and cnidarians. Rhodophyta species possessing at their surface iridescent bacteria were, for example, Palmaria palmata, Chondrus crispus or Corallina officinalis. Mollusk samples and, in particular, gastropods could contain a wide diversity of iridescent species within the same sample. For example, six different OTUs (OTU 1, 3, 4, 6, 10, 11) within three genera were isolated from the same decomposed oyster shell. The original source of the isolated species is informed in the phylogenic tree (Fig. 5). All isolates related to the genus Aquimarina (OTU 2) were from the surface of red macroalgae. The strains belonging to OTU 5 were isolated from the same brown macroalgae Fucus vesiculosus. The 2 isolates of OTU 6 were isolated from the surface of different gastropods (winkle).

Discussion

Bacterial iridescence has been ambiguously described or ignored in the literature. We have recently isolated a marine bacterium intensively iridescent, C. lytica CECT 8139, in a rocky shore of the Charente-Maritime [START_REF] Kientz | a) Iridescence of a marine bacterium and classification of prokaryotic structural colors[END_REF]. This discovery raised the question whether iridescent marine bacteria are widely distributed in coastal biotopes.

In this study, iridescent strains belonging to the genus Cellulophaga were isolated for a second time in the Charente Maritime coast. These strains were the most representative iridescent isolates in the collection but diverse bacterial strains belonging to others genera were also isolated. In total, iridescent strains were affiliated to 4 different genera. All isolated strains were belonging to the Bacteroidetes phylum. Three groups of isolates (OTU 5, 7 and 9) had 16S rRNA sequence similarity equal or lower to 97%. Therefore, we found putative new species in the genus Tenacibaculum that were never detected before through both cultivation and culture independent studies and that could have interest for future research. However, resolution of 16S rRNA sequence analysis is insufficient to distinguish closely related organisms [START_REF] Clayton | Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa[END_REF][START_REF] Stackebrandt | Molecular identification, systematics, and population structures of prokaryotes[END_REF]. More fine phylogenetic analyses on the DNA gyrase B subunit gene or the ITS region and the determination of the DNA-DNA hybridization values should confirm the novelty [START_REF] Stackebrandt | Molecular identification, systematics, and population structures of prokaryotes[END_REF][START_REF] Suzuki | Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria: proposal for Tenacibaculum gen. nov. with Tenacibaculum maritimum comb. nov. and Tenacibaculum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum amylolyticum sp[END_REF]. These iridescent bacterial strains must be further characterized by a polyphasic approach including genetic, phenotypic and biochemical studies.

This study permitted to illustrate and describe precisely colonies' coloration effects on agar plates. Different iridescence effects were observed among the new isolates of Cellulophaga.

This data confirms that these strains can be differentiated phenotypically regarding iridescence. Different iridescence appearances were also identified in the other isolates belonging to the Bacteroidetes phylum. For few species, vague or ambiguous descriptions of the phenomenon were found in the literature but the occurrence of a true iridescent trait remained unclear. The red pigmentation was used to differentiate the Aquimarina spp. but any information described iridescence [START_REF] Nedashkovskaya | Aquimarina intermedia sp. nov., reclassification of Stanierella latercula (Lewin 1969) as Aquimarina latercula comb. nov. and Gaetbulimicrobium brevivitae (Yoon et al. 2006) as Aquimarina brevivitae comb. nov. and emended description of the genus Aquimarina[END_REF]. To our knowledge, the only description of iridescence was given for Tenacibaculum discolor isolated from a sole surface: "On marine agar 2216 (Difco), colonies also appear bright yellow from the front, but appear bright green when viewed at an angle of 30-45°C" [START_REF] Piñeiro-Vidal | a) Tenacibaculum discolor sp. nov. and Tenacibaculum gallaicum sp. nov., isolated from sole (Solea senegalensis) and turbot (Psetta maxima) culture systems[END_REF]. Mentions of optical effects were available for other Tenacibaculum strains: greenish glistening for T. litoreum [START_REF] Choi | Tenacibaculum litoreum sp. nov., isolated from tidal flat sediment[END_REF] and T. aestuarii [START_REF] Jung | Tenacibaculum aestuarii sp. nov., isolated from a tidal flat sediment in Korea[END_REF] and glistening and pale yellow for T. lutimaris [START_REF] Yoon | Tenacibaculum lutimaris sp. nov., isolated from a tidal flat in the Yellow Sea, Korea[END_REF]. No description was given for the other species [START_REF] Piñeiro-Vidal | a) Tenacibaculum discolor sp. nov. and Tenacibaculum gallaicum sp. nov., isolated from sole (Solea senegalensis) and turbot (Psetta maxima) culture systems[END_REF][START_REF] Piñeiro-Vidal | Tenacibaculum soleae sp. nov., isolated from diseased sole (Solea senegalensis Kaup)[END_REF][START_REF] Wang | Tenacibaculum aiptasiae sp. nov., isolated from a sea anemone Aiptasia pulchella[END_REF]. The iridescence phenomenon in marine bacteria, and more generally in prokaryotes, was generally omitted and it remains therefore still poorly known from a taxonomic point of view. Our data suggest that iridescence is potentially widespread and represents an interesting criterion for new microorganisms.

Gliding motility was observed for most iridescent strains isolated. Moreover, fast gliding (fast spreading edges) was typical of intensely iridescent colonies. A strong link between gliding motility and iridescence could therefore exist in several members of the Bacteroidetes phylum. As recently proposed [START_REF] Kientz | Glitter-like iridescence within the Bacteroidetes especially Cellulophaga spp.: optical properties and correlation with gliding motility[END_REF], gliding motility may be involved in the establishment, in time and space, of the necessary periodic structures responsible for the iridescent appearance. Such structures are still uncharacterized but they are under investigation by electron microscopy in our laboratory. Since mostly green and red iridescences were observed for the isolated strains, similar periodic structures, with minor variations of dimensions and periodicity, are likely inferred to be responsible for the iridescent appearances observed.

Flavobacteriales are common in the marine environment [START_REF] Bernardet | An introduction to the family Flavobacteriaceae[END_REF]. We found that iridescent strains were particularly abundant on rocky shore (53%). This biotope comprises a higher biodiversity of organisms than muddy environment. The presence of iridescent bacteria in rocky shore ecosystem that combined stressful conditions from solar exposure, desiccation, and great fluctuations in salinity, may give preliminary indications on the possible biological roles of the iridescence (thermoregulation, UV protection, light filtering and desiccation prevention [START_REF] Doucet | Iridescence: a functional perspective[END_REF][START_REF] Seago | Gold bugs and beyond: a review of iridescence and structural color mechanisms in beetles (Coleoptera)[END_REF]). Moreover, we recently showed that iridescence of C. lytica CECT 8139 was conserved under environmental stress conditions such as low temperature and high salinity [START_REF] Kientz | Effect of abiotic factors on the unique glitterlike iridescence of Cellulophaga lytica[END_REF]. Interestingly, the majority of isolated species were in association with the marine flora (macroalgae) and fauna (mollusks, cnidarians). For several macroorganisms, recruitment of an epibiontal community with protection activities has been proved [START_REF] Goecke | Chemical interactions between marine macroalgae and bacteria[END_REF][START_REF] Patel | Specificity in the settlementmodifying response of bacterial biofilms towards zoospores of the marine alga Enteromorpha[END_REF].

Iridescence may therefore provide selective advantages for the bacterial population itself and/or their hosts. Moreover, several strains and especially strains of Cellulophaga sp. were isolated from the surfaces of red macroalgae. For the isolates belonging to the genus Aquimarina and showing a red pigmentation, the source was only the surface of Rhodophytae (i.e. red macroalgae). These observations might be of a major importance since a few red macroalgae have been previously described to exhibit iridescence. Indeed, Chondrus crispus and Irideae spp. possess iridescent multilayered fronds [START_REF] Craigie | Cuticules from Chondrus crispus (Rhodophyta)[END_REF][START_REF] Gerwick | Structural, chemical and ecological studies on iridescence in Iridaea (Rhodophyta)[END_REF]. Moreover, iridescence of Irideae has been supposed to prevent dessication [START_REF] Gerwick | Structural, chemical and ecological studies on iridescence in Iridaea (Rhodophyta)[END_REF]. It can therefore be hypothesized that iridescent epibiontic bacteria might play beneficial roles in some red malcroalgae. More generally, iridescent marine Bacteroidetes might have biological or ecological functions and play important roles in rocky shore ecosystems. These hypotheses have to be examined in the future.

Figures legends

Fig. 1. Maps of the Charente Maritime coast in France and of the different sampling sites (see details in the Table 1) Gliding motility can be identified as the spreading zone from the colony center.

Agarolysis corresponds to the dark halo visible on colony edges. 5. Phylogenetic tree constructed 16S rDNA genes of isolates and related species of CFB group. The tree was built with the maximum likelihood method. Bar indicated 1% dissimilarity sequence. Selected sequences from the Rikenellaceae family of CFB bacteria were used to root the tree. Bootstrap percentages are obtained using 1000 replicates, and values >50% indicated at the nodes. * indicated branches that were also found using the neighbor-joining and the maximum parcimony methods Fig. 6. Iridescence of the isolated marine bacteria after 1 day (left) and 3 days (right) of culture at 20°C on CYT medium. Both OTU 2 (a,b) were isolated from Chondrus crispus red macroalgae. OTU 3 were isolated from (c) Pterocladia sp. red macroalgae, (d) an unknown hydroid and (e) a seastar Asterina gibosa. OTU 4 were from (f) a decomposed oyster, (g) seawater sample and (h) a limpet. OTU 5, 6 and 7 were respectively isolated from Fucus vesiculosus (i), a winkle (j), and from seawater (k). OTU 8 were isolated from (l) Pterocladia sp. red macroalgae, (m) an unattached laminaria, (n) sand, (o) Corallina officinalis red macroalgae, (p) worms sand formation, (q) seawater and (r) crab. OTU 9, 10 and 11 were respectively isolated from a Zostera marina (s), a decomposed oyster (t) and an unattached Saccharina sp. brown macroalgae.

Tables

Fig. 2

 2 Fig.2

Fig. 3

 3 Fig.3

Fig. 4

 4 Fig.4

Fig. 6

 6 Fig.6

Fig. 5

 5 Fig. 5

Fig. 2 .

 2 Fig. 2. Observation under direct illumination of CYT isolation plates incubated for three days

Fig. 3 .

 3 Fig. 3. Glitter-like iridescence appearance under direct illumination of different Cellulophaga

Fig. 4 .

 4 Fig. 4. Distribution of all isolated iridescent strains within the diverse biotopes and samples.

Fig. 5 .Fig. 6 .FiguresFig. 1 .Fig. 2 .

 5612 Fig. 5. Phylogenetic tree constructed 16S rDNA genes of isolates and related species of

Fig. 3 .

 3 Fig. 3. Glitter-like iridescence appearance under direct illumination of different Cellulophaga affiliated isolates (OTU 1) after one day (up line) and four days (bottom line) of incubation at 20°C on CYT medium. Strains were isolated from (a) dry sand at Ronce les bains, (b) rhodophyta Palmaria palmata at Whales lighthouse, (c) phaeophyta Fucus vesiculosus at Ronce les bains, (d) chlorophyta Ectocarpus siliculosis at Chassiron lighthouse.Gliding motility can be identified as the spreading zone from the colony center. Agarolysis corresponds to the dark halo visible on colony edges.

Fig. 4 .

 4 Fig. 4. Distribution of all isolated iridescent strains within the diverse biotopes and samples.

Fig.

  Fig.5. Phylogenetic tree constructed 16S rDNA genes of isolates and related species of CFB group. The tree was built with the maximum likelihood method. Bar indicated 1% dissimilarity sequence. Selected sequences from the Rikenellaceae family of CFB bacteria were used to root the tree. Bootstrap percentages are obtained using 1000 replicates, and values >50% indicated at the nodes. * indicated branches that were also found using the neighbor-joining and the maximum parcimony methods

Table 1 .

 1 Sampling sites with specified biotopes.

	Site	Location	Sampling	Nature of biotope	Number of
			date		samples *
	1 Mus du loup beach	Ronce les bains, La Tremblade	29.09.11	Sand and rocks	6
	2 Whales lighthouse	St Clément des Baleines, Ré Island	24.10.11	Rocks	51
	3 Eelgrass park	Loix, Ré Island	3.11.11	Silt with eelgrass	10
	4 Arnérault Beach	La Flotte, Ré Island	3.11.11	Sand and rocks	7
	5 Belvedère	Chef de Baie, La Rochelle	3.11.11	Sand and rocks	9
	6 Chassiron lighthouse	Chassiron, Oléron Island	15.11.11	Rocks	31
	7 Smoke peak	Fouras	29.11.11	Silt and sand with	25
				oysters

* Total number of samples: 139

Table 2 .

 2 Affiliation of isolated iridescent strains.

	OTU	Representative	Accession	Number (%)	-Closest relative strain	%	Accession
		strain	number	of isolates	-Closest relative Type Strain (T)	sequence	number of the
						similarity	closest relative
	OTU 1 Strain 14	JX454529	34 (35.1%)	-Cellulophaga lytica	99 %	AB032512
					-Cellulophaga lytica DSM7489 T	98%	CP002534
	OTU 2 Strain 46	JX454530	5 (5.2%)	-Aquimarina latercula	98 %	AB681002
					-Aquimarina latercula	97%	M58769
					ATCC23177 T
	OTU 3 Strain 35	JX454522	18 (18.6 %)	-« Flexibacter aurantiacus subsp.	99 %	AB078044
					Copepodarum »
					-Tenacibaculum aiptasiae a4 T	98%	EF416572
	OTU 4 Strain 116	JX454523	6 (6.2 %)	-« Flexibacter aurantiacus subsp.	98 %	AB078044
					Copepodarum »
					-Tenacibaculum aiptasiae a4 T	97%	EF416572
	OTU 5 Strain 66	JX454528	2 (2.1%)	-Tenacibaculum ovolyticum	97 %	AB681005
					-Tenacibaculum ovolyticum IFO	97%	AB078058
					15947 T
	OTU 6 Strain 70	JX454527	2 (2.1%)	-Tenacibaculum soleae	99 %	AM989479
					-Tenacibaculum soleae LL04	99%	AM746476
					12.1.7 T
	OTU 7 Strain 103	JX454524	1 (1.0 %)	-Tenacibaculum discolor	97 %	JQ231117
					-Tenacibaculum aiptasiae a4 T	96%	EF416572
	OTU 8 Strain 68	JX454521	19 (19.6 %)	-Tenacibaculum gallaicum A37.1 T	99 %	AM746477
	OTU 9 Strain 4	JX454520	1 (1.0 %)	-« Flexibacter aurantiacus subsp.	96 %	AB078044
					Copepodarum »
					-Tencibaculum litopenaei B-I T	95%	DQ822567
	OTU 10 Strain 82	JX454526	1 (1.0 %)	-Tenacibaculum aiptasiae	98 %	NR044202
					-Tenacibaculum aiptasiae a4 T	98%	EF416572
	OTU 11 Strain 105	JX454525	8 (8.2 %)	-Tenacibaculum aestuarii SMK-4 T	99 %	DQ314760

OTU: Operational Taxonomic Unit.


H J Van Gorkom
email: lavaud@biologie.ens.fr

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

The pool size of the xanthophyll cycle pigment diadinoxanthin in the diatom Phaeodactylum tricornutum depends on illumination conditions during culture. Intermittent light caused a doubling of the diadinoxanthin pool without significant change in other pigment contents and photosynthetic parameters, including the Photosystem II (PSII) antenna size. On exposure to high light intensity extensive de-epoxidation of diadinoxanthin to diatoxanthin rapidly caused a very strong quenching of the maximum chlorophyll fluorescence yield (F m , PSII reaction centers closed), which was fully reversed in the dark. The non-photochemical quenching of the minimum fluorescence yield (F o , PSII centers open)

decreased the quantum efficiency of PSII proportionally. For both F m and F o , the nonphotochemical quenching expressed as F/F"-1 (with F" the quenched level) was proportional to the diatoxanthin concentration. However, the quenching of F o relative to that of F m was much stronger than random quenching in a homogeneous antenna could explain, showing that the rate of photochemical excitation trapping was limited by energy transfer to the reaction center rather than by charge separation. The cells can increase not only the amount of diatoxanthin they can produce, but also its efficiency in competing with the PSII reaction center for excitation. The combined effect allowed intermittent light grown cells to downregulate PSII by 90 % and virtually eliminated photoinhibition by saturating light. The unusually rapid and effective photoprotection by the xanthophyll cycle in diatoms may help to explain their dominance in turbulent waters.

INTRODUCTION

Photosynthetic organisms have developed strategies to optimize light harvesting at low intensities while minimizing photoinhibitory damage due to excess energy at high light intensity. From hours onwards, they regulate the quantity and composition of the lightharvesting complexes and of a number of other components of their photosynthetic machinery (reviewed by [START_REF] Falkowski | Acclimation to spectral irradiance in algae[END_REF]Anderson et al., 1995). On shorter time scales, they react to an unbalance between light intensity and photosynthetic capacity (e.g. due to a change in light intensity, temperature, or nutrient supply) by rapid structural modifications within the light-harvesting complex of photosystem II (PSII) [START_REF] Horton | Regulation of light harvesting in green plants[END_REF]Bassi and Caffarri, 2000). These modifications lead to a decrease in the fluorescence yield designated as the "non-photochemical quenching" of chlorophyll a fluorescence (NPQ). NPQ is supposed to dissipate excess excitation energy through a harmless non-radiative pathway. The partitioning of absorbed energy between transfer to the reaction center and photoprotective non-radiative dissipation is controlled by the trans-thylakoid pH gradient (for a recent review see Müller et al., 2001) and by the reversible conversion of epoxidized to de-epoxidized forms of xanthophylls (the so-called xanthophyll cycle, [START_REF] Gilmore | Mechanistics aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves[END_REF]. The molecular mechanisms of photoprotection have mostly been studied in higher plants [START_REF] Demmig-Adams | The role of xanthophyll cycle carotenoids in the protection of photosynthesis[END_REF]. Several mutants of Arabidopsis and Chlamydomonas with modified violaxanthin (the epoxidated xanthophyll cycle pigment form) content and NPQ extent have been investigated and a specific role has been attributed to the PsbS protein (Niyogi, 1999, Müller et al., 2001).

However, the field remains controversial due to the obvious complexity of photoprotective mechanisms. In this sense, the use of diatoms to study the role of xanthophylls in photoprotection may have important advantages.

Diatoms are well known to flourish in turbulent waters [START_REF] Harris | Phytoplankton Ecology: Structure, Function, and Fluctuation[END_REF], where the amount of light available to the phytoplanktonic unicellular organisms is highly unpredictable.

The deep vertical mixing continuously sweeps them up and down, exposing the cells to very large short-term changes in light intensity on a time scale of minutes to hours. The organization of the photosynthetic apparatus in diatoms differs in many respects from that of higher plants. The thylakoids are loosely appressed and organized in extended bands of three, and the photosystems I and II are not segregated in different domains (Pyszniak and Gibbs, 1992). The light-harvesting complexes, which contain Chl a, Chl c, fucoxanthin and the xanthophyll cycle pigment diadinoxanthin (DD) [START_REF] Brown | Photosynthetic pigment organization in diatoms (Bacillariophyceae)[END_REF], are equally distributed among appressed and non appressed regions (Pyszniak and Gibbs, 1992) and there is no evidence of any state-transitions (Owens, 1986). The light-harvesting complexes sub-units are made of several highly homologous proteins encoded by a multigene family (Fucoxanthin Chlorophyll Proteins, FCPs, Bhaya and Grossman, 1993). The CP26 and CP29 subunits present in higher plants are not found in diatoms (Müller et al., 2001) and the existence of the PsbS protein has yet to be proven. The supramolecular organization of photosystem II within the membrane remains unknown. When the cells are suddenly exposed to high light intensity, a NPQ is rapidly developed [START_REF] Demers | Rapid light-induced changes in cell fluorescence and in xanthophyll-cycle pigments of Alexandrium excavatum (Dynophyceae) and Thalassiosira pseudonana (Bacillariophyceae): a photo-protection mechanism[END_REF]Ting and Owens, 1993;Arsalane et al., 1994;Olaizola et al., 1994;Olaizola and Yamamoto, 1994;[START_REF] Casper-Lindley | Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments[END_REF].

NPQ is associated with a xanthophyll cycle (the DD-cycle) which differs from that of higher plants. The DD cycle converts the mono-epoxide carotenoid DD into the de-epoxide form diatoxanthin (DT) under high light, and DT back into DD under low light or in darkness (Arsalane et al., 1994;[START_REF] Casper-Lindley | Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments[END_REF].

In diatoms, the DD content can be modulated by the light regime to which culture is exposed (Willemoës and Monas, 1991;Arsalane et al., 1994;[START_REF] Casper-Lindley | Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments[END_REF]Mouget et al., 1999). We were able to define an intermittent light regime in which cells of the diatom Phaeodactylum tricornutum had an increased DD content but unchanged PSII antenna size. By comparing cells with either a low or high DD content, we studied the influence of the DD pool size on the DD-cycle activity and NPQ formation under high light and investigated its functional role in protecting PSII from over-excitation. All results obtained demonstrate that a larger pool of DD leads to a more effective photoprotection.

RESULTS

Comparison of the photosynthetic apparatus of CL and IL cells.

Phaeodactylum tricornutum cells with different DD contents were obtained from cultures grown under different illumination regimes. "Continuous light" (CL) cultures were grown using a 16 h light / 8 h dark cycle. "Intermittent light" (IL) cultures were grown under a 5 min light / 55 min dark cycle. In both cases the light intensity was 40 E.m -2 s -1 , which was weak enough not to induce any de-epoxidation of DD. The growth rate under IL illumination was reduced by a factor of 10, which is nearly accounted for by the 8-fold lower total amount of light supplied.

The DD content of IL cells was two times higher than that of CL cells (Table I).

Otherwise no significant differences were found in their pigment contents. Cell size and chloroplast ultrastructure were similar for the two types of cells. It is important to note that the PSII antenna size was the same in the two types of cells, in their dark-adapted state, as indicated by their equal fluorescence yield rise-time upon illumination in the presence of DCMU (Table I). This was confirmed by the fact that equal flash intensities were required for half-saturation of the steady-state O 2 yield per flash with dark-adapted IL and CL cells (open symbols in Fig. 5, see below).

Non-photochemical quenching (NPQ) evaluated by SV o and SV m in CL and IL cells.

Figure 1 shows typical PAM-fluorometer recordings of the chlorophyll fluorescence yield changes induced by illumination of CL cells (A) and IL cells (B), as detected by a nonactinic modulated beam. The cells were illuminated for 5 min, indicated by up and down arrows, at an intensity of 450 µE.m -2 .s -1 that was just saturating for the rate of oxygen We will use this value to indicate F o " but note that its real value might be even somewhat lower because the presence of some remaining Q A -at this point in time cannot be ruled out.

The minimum of F o " variations is reached in less than two minutes. Some NPQ may already have disappeared but the corresponding F m " values show that NPQ relaxation is still small at this point. Recovery of the dark-adapted state took about 15 min in CL cells (Fig. 1A). In IL cells, NPQ induced by the same illumination was much more pronounced, appeared more rapidly, and disappeared more slowly than in CL cells (Fig. 1B).

The extent of NPQ is usually quantified as F/F"-1, which should be proportional to the quencher concentration if a simple Stern-Volmer relation applies (see materials and methods section). We will use the "Stern-Volmer coefficients" SV o and SV m to denote F o /F o -1 and F m /F m -1, respectively. The induction kinetics of SV o and SV m in the two types of cells is shown in Figure 2 for light intensities of 450 µE.m -2 .s -1 (A, just saturating light, Table I) and 2 mE.m -2 .s -1 (B, full sunlight, [START_REF] Long | Photoinhibition of photosynthesis in nature[END_REF]. The open and solid symbols refer to CL and IL cells, respectively, circles and triangles to SV o and SV m . In all conditions the kinetics were similar: the major fast phase already visible in Fig. 1 was followed by a much slower increase.

Figure 3 shows SV o and SV m induced by 5 min illumination as a function of light intensity.

CL cells required a somewhat higher light intensity for NPQ induction than IL cells, but for both a large part of the maximum NPQ was already induced at a light intensity of 0.5 mE.m - 2 .s -1 that was just saturating for oxygen evolution.

Correlation of NPQ and diatoxanthin accumulation.

The kinetics of DT accumulation was similar to that of NPQ formation. No DT could be detected in dark-adapted cells. The amount of DT formed by de-epoxidation of DD results from the competition between epoxidation and de-epoxidation (activated by the proton gradient). At the onset of light the first phase corresponds to the rapid transformation of DD to DT. The equilibrium reached at the end of the five minutes illumination depends on the light intensity: at low light epoxidation competes efficiently with de-epoxidation then deepoxidation becomes more efficient with increasing light intensities and a quasi steady state is reached when the equilibrium is strongly shifted in favor of DT.

The maximum amount of DT formed, measured after 1 h illumination at 2 mE.m -2 .s -1 , was 5 and 12.5 mol DT.(100 mol Chl a) -1 for CL and IL cells, respectively, corresponding to the de-epoxidation of 50 and 65 % of the total DD pool. As shown in Figure 4, SV o and SV m were found to be proportional to the DT concentration for both types of cells.

Decrease of the quantum efficiency of PSII by NPQ.

Fig. 4 first mention

Since NPQ dissipates part of the excitation energy, it is expected to decrease the quantum efficiency of PSII. This was verified by measurements of the amount of oxygen produced in short flashes as a function of flash energy (Fig. 5). The saturation curves of darkadapted CL and IL cells (open symbols) were almost identical, confirming that the PSII antenna size was the same in both culture conditions. The flash illumination used, 20 single turn-over flashes in 10 s, corresponds to a very low continuous intensity and did not induce any NPQ. After exposure to high light intensity the saturation curves for flash-induced oxygen evolution were indeed shifted to higher flash energies (solid symbols). These measurements were taken 7 min after the light treatment (the time required for sedimentation on the et al., 1994). As predicted by Arsalane et al., 1994, the larger the DD content, the longer time is needed to reach the compensation point (not shown).

An additional method for evaluation of photoinhibition was used. Fig. 7 shows the decrease of PSII activity measured as the steady-state O 2 yield per flash (Y SS ) after illumination at 0.45 or 2 mE.m -2 .s -1 followed by 7 min dark-adaptation. Photoinhibition kinetics were indeed faster for CL cells (open symbols) than for IL cells (solid symbols), especially during the first 5 min. After 1 h of illumination, the remaining percentage of active PSII centers was lower for CL cells under the two light intensities. The difference between IL and CL cells is actually larger than Fig. 7 suggests, because especially for IL cells the recovery from NPQ is much slower than 7 min (Fig. 1) and saturation of Y SS could not be reached with the available flash energy. Since 85 or 90 % of the energy was dissipated by NPQ after 1 h at 0.45 or 2 mE.m -2 .s -1 , respectively (Fig. 6), the flash saturation curve for dark-adapted cells in Figure 5 indicates that only 87 or 79 % saturation of Y SS could be reached. The exact correction has not been done because combining the information from the different data in Figures 5, 6 and 7 in this way seems too daring, but the corrections may obviously be large for IL cells. The correction would leave essentially no evidence for photoinhibition of IL cells at 0.45 mE at all. For CL cells no such correction applies because NPQ largely disappeared in 7 min and even at 65 % energy dissipation (Fig. 6) the flashes would still be more than 95 % saturating (Fig. 5). The fast phase of photoinhibition seen during the first few minutes at high light intensity correlates with the time required for accumulation of DT and NPQ and illustrates the importance of the photoprotective effect. Such a short delay in the build-up of to the effect of intermittent illumination on higher plants which leads to a drastic reduction of PSII antenna size. Intermittent light grown peas are devoid of all LHCII proteins with the exception of CP26 one of the minor inner antenna chlorophyll binding proteins [START_REF] Jahns | Thylakoids from pea seedlings grown under intermittent light: biochemical and flash-spectrophotometric properties[END_REF]. This way a specific increase in DD content could be induced without significant change in the amounts of other pigments, PSI/PSII ratio, or PSII antenna size and quantum yield.

Upon exposure to high light intensity, a substantial de-epoxidation of DD occurred, largely within a few minutes. This in agreement with DD-cycle activity previously reported (Arsalane et al., 1994;Olaizola et al., 1994;[START_REF] Casper-Lindley | Fluorescence quenching in four unicellular algae with different light-harvesting and xanthophyll-cycle pigments[END_REF]. The larger pool of DD in IL cells, and its more extensive de-epoxidation, led to much larger accumulation of DT during high light illumination. The maximal DT accumulation is of 5 mol DT.(100 mol Chl a) -1 in CL cells and 12.5 mol DT.(100 mol Chl a) -1 in IL cells. In higher plants, Ruban et al., (1999) have estimated that each LHCII monomer can bind at least one violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest deepoxidation state was found for the main LHCII components and the lowest for CP29. Very little is known about the nature of the subunits of LHC in diatoms (Bhaya and Grossman, 1993) and it seems premature to speculate on their DT binding sites at this stage.

Correlation of light-induced DT accumulation and NPQ.

The Stern-Volmer parameters SV o and SV m relate the non-photochemical quenching on F o and F m to the quencher concentration [START_REF] Bilger | Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis[END_REF][START_REF] Gilmore | Zeaxanthin formation and energy-dependent fluorescence quenching in pea chloroplasts under artificially mediated linear and cyclic electron transport[END_REF]Olaizola and Yamamoto, 1994). Random quenching in a homogenous antenna would produce a normalized fluorescence yield as depicted by equation 2 (see materials and methods section). For the quenching of F m , DT is the only quencher present in the light-harvesting complex of PSII, the photochemical quenching being totally suppressed.

In that case, the slope of SV m is the same for IL and CL cells. (SV m ) IL can reach values higher than (SV m ) CL , the difference being only due to the difference in the maximal concentration of DT which can be formed (see Fig. 4). For the F o level, the photochemical quencher (Q) is Evidence for the induction of an electron transport cycle around PSII that competes with oxygen evolution at high light intensity will be presented elsewhere (Lavaud et al., submitted).

The phenomena reported here are probably not restricted to P. tricornutum. Other marine planktonic diatoms like Chaetoceros (Olaizola et al., 1994), Thalassiosira [START_REF] Demers | Rapid light-induced changes in cell fluorescence and in xanthophyll-cycle pigments of Alexandrium excavatum (Dynophyceae) and Thalassiosira pseudonana (Bacillariophyceae): a photo-protection mechanism[END_REF]), Haslea (Mouget et al., 1999) and freshwater planktonic diatoms like Nitzschia (Willemoës and Monas, 1991) are able to synthesize large amounts of de-epoxidizable xanthophylls. It seems likely that the large and adjustable capacity of DT accumulation, and the fast and efficient photoprotection by the associated NPQ, are general properties of planktonic diatoms and play an important role in their successful adaptation to the strongly fluctuating light intensity in their natural habitat [START_REF] Harris | Phytoplankton Ecology: Structure, Function, and Fluctuation[END_REF].

MATERIALS AND METHODS

Cells cultivation and preparation.

Phaeodactylum tricornutum Böhlin cells were grown photoautotrophically 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]. Cultures of 300 mL were incubated [START_REF] Gilmore | Zeaxanthin formation and energy-dependent fluorescence quenching in pea chloroplasts under artificially mediated linear and cyclic electron transport[END_REF][START_REF] Bilger | Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis[END_REF][START_REF] Gilmore | Zeaxanthin formation and energy-dependent fluorescence quenching in pea chloroplasts under artificially mediated linear and cyclic electron transport[END_REF]. We use k IC : rate constant of intersystem crossing to the Chl a triplet state + rate constant of internal conversion to the ground state. [DT]: rate constant of non-photochemical quenching.

k Q [Q]: rate constant of photochemical quenching. [DT] [Q] with [*] ] * [ DT Q IC F k k k k k k dt d        k DT

The fluorescence yield is

In the absence of non-photochemical quenching, Q is the only quencher and and when Q is at its maximal concentration then,

In the presence of the quencher DT, if Q = 0, In the presence of both quenchers Then, O 2 yield per flash.

The relative O 2 yield produced per flash during a sequence of single-turnover saturating flashes at a frequency of 2 Hz was measured with a rate electrode described by [START_REF] Lemasson | Photo-inactivation of system II centers by carbonyl cyanide m-chlorophenylhydrazone in Chlorella pyrenoidosa[END_REF]. Short (5 µs) saturating flashes were produced by a Strobotac (General Radio Co., Concord, MA). O 2 evolution was monitored at 18C. For the control

k k F   F [Q] k' k k k Q IC F Q      1 [Q] 1 F F o m 0 Q when F m     k' k k k F IC F F (1) 1 - ' F F SV and 1 ' F F m m m m m [DT] k' [DT] k' DT DT     [DT] k' k' [DT] k' [Q] k' DT DT DT Q m o o m m o o o o m o m F F 1 ' F F F F ' F F and F F 1 ' F F           (2) SV F F F F 1 - ' F F SV m m o m o o o o    [DT] k' DT ] Q [ F o Q IC F F k k k k   
sequences, cells were first dark-adapted during 20 min and then deposited on the electrode.

Both control and illuminated samples taken from the PAM-fluorometer were allowed to settle on the electrode for 7 min in darkness before measurement. The steady-state O 2 yield per flash (Y SS ) was taken to be the average yield of the last 4 flashes of a series of twenty flashes when the classical four-step oscillations due to the S-states cycle [START_REF] Kok | Cooperation of charges in photosynthetic O2 evolution-I. A linear four step mechanism[END_REF]

[DT] k [Q] k k k [Q] k DT Q IC F Q      Q (3) ' F F I I o o Q 0] [DT Q 0] [DT 1/2 1/2             [Q] k k k [DT] k [Q] k k k Q IC F DT Q IC F Arsalane W,

  Fig. 1 first mention

  Fig. 2 first mention

  electrode) and were compared to the fluorescence yield measured at the same time after illumination, which is already substantially less quenched by NPQ than 2 min after illumination (c.f. Fig.1). The remaining quenching of F o at this time was equal to the decrease of the quantum yield of O 2 evolution as indicated by the reciprocal of the half-saturating flash intensity (inset, Fig.5). This result is consistent with equation 3 in Materials and Methods and may indicate that PSI fluorescence does not significantly contribute to F o or is similarly quenched by NPQ.If NPQ decreases the quantum yields of fluorescence and O 2 evolution equally also at stronger quenching, when the available flash energies were insufficient to determine the halfsaturating intensity, the energy dissipation measured by 1-F o /F o equals the down-regulation of PSII by NPQ. Fig.6shows the development of the energy dissipation in the antenna of open PSII centers for the two types of cells as a function of illumination time at 0.45 and at 2 mE.m -2 .s -1 . The energy dissipation reached values up to 90 % and was much higher for IL cells than for CL cells at both light intensities, especially after short illumination times (enlarged in the inset).

  Fig. 5 first mention

  Fig. 7 first mention

  present (PSII centers are open) and the two types of quenchers compete for the trapping of the excitation. If the quenchers were randomly competing in a homogeneous antenna, the slope of SV o should be F o /F m times the slope of SV m (see equation 2 in Material and Methods). It is not the case: the observed SV o /SV m ratios were much larger than F o /F m , especially for IL cells. The discrepancy is far too large to be substantially affected by possible errors in the determination of the extent of photochemical quenching due quenching by the plastoquinone pool or incomplete oxidation of Q A -after illumination. Our results provide an additional indirect proof that excitation transfer to the open PSII reaction centers becomes rate-limiting for trapping by open PSII reaction centers, causing a heterogeneity of the Chl fluorescence yield in the PSII antenna. The selective increase of SV o indicates that the Chls involved contribute a larger fraction of the emission at F o than at F m .Functional significance of energy dissipation by DT and photoinhibition.Most studies on NPQ consider only the quenching of F m . While the quenching of F m presumably helps to diminish singlet oxygen production in the antenna by reducing the yield of Chl triplet formation, the quantitative significance of this protective effect under physiological conditions is probably negligible. The triplet yield of Chl a is twice the fluorescence yield and in an aerobic environment its very fast transfer to carotenoids followed by intersystem crossing to the ground state is essential anytime. Only the reaction center Chl itself cannot be protected this way(van Gorkom and Schelvis, 1993), but at F m the PSII reaction center does not quench fluorescence(Thielen and van Gorkom, 1981). Hence the probability that the triplet state is formed there, rather than in the antenna, is as small as the fraction of Chl a contained in the reaction center. At F o , on the other hand, most excitations cause a charge separation. If that cannot be stabilized by secondary electron transfer reactions due to saturation of electron transport, it will most likely recombine to the reaction center triplet state(van Gorkom, 1985; van Gorkom, 1986). Any mechanism to avoid that would be physiologically significant and could bring a decisive advantage under continually and widely changing light intensity. Our results show that the xanthophyll cycle in P. tricornutum provides such a mechanism. The quenching of F o is a direct measure of the extent of down-regulation of PSII electron transport resulting from NPQ. We have shown that energy dissipation by DT equally decreases the quantum yield of O 2 evolution and the quantum yield of fluorescence emission when the PSII centers are open. The corresponding effect in higher plants may be small (c.f.Santabarbara et al., 2001), but our results clearly confirm the large decrease of PSII quantum efficiency by NPQ in P. tricornutum, reported by[START_REF] Koblizek | On the relationship between the nonphotochemical quenching of the chlorophyll fluorescence and photosystem II light harvesting efficiency. A repetitive flash fluorescence induction study[END_REF]. Moreover, this unusual capacity for down-regulation of PSII is adjusted to the illumination conditions during growth.Finally, we note an intriguing observation: after 1 h at 2 mE.m -2 .s -1 , CL cells lost up to 70 % of their O 2 evolving PSII reaction centers, and yet no deviation from the proportionality between SV o , SV m and [DT] is seen in the inset of Fig. 4. Apparently there is nothing special about the fluorescence yield, including the variable fluorescence, of this "inactivated PSII". This indicates that these diatoms are not photoinhibited in the classical sense and in fact Olaizola et al., 1994 already mentioned the absence of D1 photodamage in P. tricornutum.

  Photosystem I reaction center (P 700 ) concentration.

  the "Stern-Volmer parameters" SV o and SV m to indicate NPQ of F o and NPQ of F m , respectively. The fluorescence yields F o , F m , F o " and F m " are indicated in Figure 1A. The following formalism was used to relate the Stern-Volmer equations with the amounts of photochemical (Q for quinones) and non-photochemical (DT) quenchers. The decay rate of the excited state of Chl a molecules in PSII is where k F : rate constant of fluorescence emission.

  was almost damped. Y SS was used to assess the relative concentration of O 2 evolving PSII reaction centers. For the saturation curves of Y SS , the intensity of the flashes was varied with neutral density filters. Samples were taken from the PAM-fluorometer after illumination and deposited on the electrode. For each flash intensity a new sample was used. In parallel, fluorescence yield was measured with the PAM set-up at the same time after illumination. The reciprocal of the half-saturating flash intensity as a measure of the antenna size was expressed as follows. The quantum yield of photochemistry is given by the equation: and should be inversely proportional to the amount of light needed to activate a given fraction of the reaction centers, so that: with I 1/2 : the light intensity needed for half-saturation of Y SS in presence of DT and I 1/2 [DT=0] : the light intensity needed for half-saturation of Y SS in absence of DT. LITERATURE CITED Anderson JM, Chow WS, Park Y-I (1995) The grand design of photosynthesis: acclimation of the photosynthetic apparatus to environmental cues. Photosynth Res 46: 129-139 Arsalane W, Paresys G, Duval J-C, Wilhelm C, Conrad R, Büchel C (1993) A new fluorometric device to measure the in vivo chlorophyll a fluorescence yield in microalgae and its use as a herbicide monitor. Eur J Phycol 28: 247-252

Figure 1 .

 1 photoinhibition. Photochem Photobiol 60: 237-243

Figure 2 .

 2 Figure 2. Non-photochemical quenching as a function of illumination duration for two light intensities: 450 µE.m -2 .s -1 (A), and 2 mE.m -2 .s -1 (B). Quenching of F m (circles, SV m ) and of F o

Figure 3 .

 3 Figure 3. NPQ as a function of light intensity for a fixed illumination duration of 5 min. SV m

Figure 4 .

 4 Figure 4. Correlation of NPQ and DT concentration. SV m (circles) and SV o (triangles) for CL

Figure 5 .Figure 6 .

 56 Figure 5. Steady-state O 2 yield per flash (Y SS ) as a function of flash intensity. Dark-adapted

Figure 7 .

 7 Figure 7. Photoinhibition kinetics of PSII as a function of illumination duration. CL cells (open symbols) and IL cells (solid symbols) at 450 µE.m -2 .s -1 (squares) and 2 mE.m -2 .s -1

Figure

  Figure 1: Lavaud, Rousseau, van Gorkom and Etienne

ACKNOWLEDGEMENTS

We thank Prof. J.-C. Duval for experimental assistance and helpful discussions, C.

Lichtlé for electron microscopic photographs and Dr K. Niyogi and A. Pascal for critical reading of the manuscript.

Lavaud J. was supported by a grant from the French Ministry of National Education, Research, and Technology. H. J. van Gorkom was supported by an invited professorship from