Andre ´s Ritter

Martin Ubertini

Sarah Romac

Fanny Gaillard

Ludovic Delage

Aaron Mann

J Mark Cock

Thierry Tonon

Juan A Correa

Dr Philippe Potin
email: potin@sb-roscoff.fr

Copper stress proteomics highlights local adaptation of two strains of the model brown alga Ectocarpus siliculosus Ã

Keywords: 2-DE / Algae / Bromoperoxidase / Copper / Plant proteomics / Stress glutathione, LHC, light harvesting complex, OEC, oxygen evolving complex, PC, phytochelatin, PMF, protein mass fingerprint, SAM, S-adenosyl methionine, SHMT, serine hydroxymethyl transferase, vBPO, vanadium

Ectocarpus siliculosus is a cosmopolitan brown alga with capacity to thrive in copper enriched environments. Analysis of copper toxicity was conducted in two strains of E. siliculosus isolated from (i) an uncontaminated coast in southern Peru (Es32) and (ii) a copper polluted rocky beach in northern Chile (Es524). Es32 was more sensitive than Es524, with toxicity detected at 50mg/L Cu, whereas Es524 displayed negative effects only when exposed to 250 mg/L Cu. Differential soluble proteome profiling for each strain exposed to sub-lethal copper levels allowed to identify the induction of proteins related to processes such as energy production, glutathione metabolism as well as accumulation of HSPs. In addition, the inter-strain comparison of stress-related proteomes led to identify features related to copper tolerance in Es524, such as striking expression of a PSII Mn-stabilizing protein and a Fucoxanthine chlorophyll a-c binding protein.

Es524 also expressed specific stress-related enzymes such as RNA helicases from the DEAD box families and a vanadium-dependent bromoperoxidase. These observations were supported by RT-qPCR for some of the identified genes and an enzyme activity assay for vanadium-dependent bromoperoxidase. Therefore, the occurrence of two different phenotypes within two distinct E. siliculosus strains studied at the physiological and proteomic levels strongly suggest that persistent copper stress may represent a selective force leading to the development of strains genetically adapted to copper contaminated sites.

Introduction

The release of heavy metals, including copper, by a wide range of human activities, remains a major threat for marine ecosystems, impacting benthic flora and fauna assemblages [START_REF] Correa | Copper, copper mine tailings and their effect on the marine algae in Northern Chile[END_REF][START_REF] Livingstone | Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms[END_REF][START_REF] Gledhill | The toxicity of copper (II) species to marine algae, with particular reference to macroalgae[END_REF]. Copper is a micronutrient essential for all forms of life, acting as cofactor for many enzymatic systems and participating in crucial physiological processes including photosynthesis and respiration. However, copper is extremely toxic at high concentrations, particularly affecting photosynthetic organisms that suffer effects on chlorophyll structure, synthesis and function, on fatty acid metabolism, and on carbohydrate synthesis [START_REF] Maksymiec | Effect of copper on cellular processes in higher plants[END_REF][START_REF] Yruela | Photoinhibition of photosystem II from higher plants. Effect of copper inhibition[END_REF][START_REF] Fernandes | Biochemical, physiological, and structural effects of excess copper in plants[END_REF]. In addition, copper can catalyze the synthesis of the highly reactive hydroxyl radical in presence of H 2 O 2 via the Fenton reaction, a process that causes oxidative stress [START_REF] Halliwell | Biologically relevant metal ion-dependent hydroxyl radical generation. An update[END_REF][START_REF] Halliwell | Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life[END_REF]. Some local geographic areas that have been chronic recipients of wastes containing heavy metals (i.e. mine wastes) constitute a persistent stressful environment that acts as a selective force leading, in some cases, to the generation of tolerant strains genetically adapted [START_REF] Baker | The influence of cadmium, copper, lead, and zinc on the distribution and evolution of metallophytes in the British Isles[END_REF][START_REF] Macnair | The genetics of metal tolerance in vascular plants[END_REF][START_REF] Pauwels | When population genetics serves genomics: putting adaptation back in a spatial and historical context[END_REF][START_REF] Medina | Micro-evolution due to pollution: possible consequences for ecosystem responses to toxic stress[END_REF]. In northern Chile, copper mine wastes discharges were, for almost 70 years, a major and only source of coastal pollution at Chan ˜aral bay and surroundings, where local disturbances included the creation of large tailing beaches and the disappearance of most of the seaweeds and invertebrates [START_REF] Correa | Copper, copper mine tailings and their effect on the marine algae in Northern Chile[END_REF][START_REF] Medina | Biodiversity of rocky intertidal benthic communities associated with copper mine tailing discharges in northern Chile[END_REF]. The process of biological recovery, expected to follow after tighter legislation, is far from being complete and many key ecological species, such as the brown kelp Lessonia nigrescens [START_REF] Medina | Biodiversity of rocky intertidal benthic communities associated with copper mine tailing discharges in northern Chile[END_REF], are still absent. Among the few species recorded in this area, the filamentous brown alga Ectocarpus siliculosus is a persistent inhabitant, thriving in tide pools [START_REF] Medina | Biodiversity of rocky intertidal benthic communities associated with copper mine tailing discharges in northern Chile[END_REF]. Early studies on E. siliculosus have identified copper tolerant populations, isolated from the hulls of ships treated with copper-based antifouling paints [START_REF] Hall | Copper accumulation in copper-tolerant and non-tolerant populations of the marine fouling alga Ectocarpus siliculosus (Dillw.) Lyngbye[END_REF][START_REF] Hall | Heavy metal Co-tolerance in a copper tolerant population of the marine fouling alga, Ectocarpus siliculosus (Dillw.) Lyngbye[END_REF][START_REF] Russel | Copper tolerance in marine fouling alga Ectocarpus siliculosus[END_REF][START_REF] Morris | Inter-specific differences in response to copper by natural populations of Ectocarpus[END_REF]. Although copper exclusion mechanisms were proposed for explaining this differential tolerance, analytical methods did not allow identifying genetically distinct population traits and/or characterizing the physiological or molecular bases leading to this differential tolerance. Nowadays, major advances have been made in vascular plants toward understanding copper homeostasis and tolerance [START_REF] Puig | Copper and iron homeostasis in Arabidopsis: responses to metal deficiencies, interactions and biotechnological applications[END_REF][START_REF] Upper | Heavy metal uptake by plants and cyanobacteria[END_REF][START_REF] Hall | Cellular mechanisms for heavy metal detoxification and tolerance[END_REF]. Vascular plants have evolved a number of general detoxification mechanisms, the best known being complexation by strong ligands such as phytochelatins (PCs) and metallothioneins [START_REF] Cobbett | Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis[END_REF]. Other plant chelating mechanisms employ organic acids such as histidine, proline or nicotianamine [START_REF] Upper | Heavy metal uptake by plants and cyanobacteria[END_REF][START_REF] Hall | Cellular mechanisms for heavy metal detoxification and tolerance[END_REF][START_REF] Clemens | Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants[END_REF]. Active metal exclusion mediated by P-type ATPases may also act for detoxification of copper in Arabidopsis [START_REF] Andre ´s-Cola ´s | The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots[END_REF]. Compared to the large body of literature available for vascular plants, much less is known about the mechanisms responsible for copper detoxification in brown algae. In the genus Fucus, metallothioneins and PCs were induced by copper excess [START_REF] Pawlik-Skowronska | Concentrations of phytochelatins and glutathione found in natural assemblages of seaweeds depend on species and metal concentrations of the habitat[END_REF][START_REF] Morris | Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus[END_REF]. Phenolic compounds have been proposed as metal chelators, but results seem contradictory among species [START_REF] Toth | Lack of phlorotannin induction in the brown seaweed Ascophyllum nodosum in response to increased copper concentrations[END_REF][START_REF] Salgado | Localization of specific monosaccharides in cells of the brown alga Padina gymnospora and the relation to heavy-metal accumulation[END_REF][START_REF] Sueur | Measurement of the metal complexing ability of exudates of marine macroalgae[END_REF][START_REF] Karez | Metal contents in polyphenolic fractions extracted from brown alga Padina gymnospora[END_REF]. In addition, charged sulfated polysaccharides of the cell wall could play an important role acting as primary ion filter of algal cells [START_REF] Salgado | Localization of specific monosaccharides in cells of the brown alga Padina gymnospora and the relation to heavy-metal accumulation[END_REF][START_REF] Andrade | Role of Padina gymnospora (Dictyotales, Phaeophyceae) cell walls in cadmium accumulation[END_REF]. Once inside the cell, it is well documented that metals in general, and copper in particular, activate antioxidant mechanisms in seaweeds [START_REF] Contreras | Antioxidant responses in Scytosiphon lomentaria (Phaeophyceae) inhabiting copper-enriched coastal environments[END_REF][START_REF] Pinto | Heavy metal-induced stress in algae[END_REF]. Moreover, Cu-induced ROS production in brown algae seems to trigger oxylipin signaling pathways that could be related to detoxification mechanisms [START_REF] Ritter | Copper stress induces biosynthesis of octadecanoid and eicosanoid oxygenated derivatives in the brown algal kelp Laminaria digitata[END_REF]. Brown algae (class Phaeophyceae) belong to the division Heterokonta, which evolved as an independent lineage more than a billion years ago [START_REF] Baldauf | The deep roots of eukaryotes[END_REF]. Based on their particular evolutionary history, it is likely that original physiological mechanisms have evolved in these organisms in order to adapt to the various and highly fluctuating habitats they colonize. Consistent with this, a recent study underlined a unique antioxidant system based on iodide metabolism in the kelp Laminaria digitata [START_REF] Upper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF]. Because of the particular evolutionary history of the Division, a consortium of laboratories initiated a project in 2004 to sequence the genome of E. siliculosus. At present, the annotation has been achieved, and this will allow large-scale genome explorations [START_REF] Cock | The Ectocarpus genome and the independent evolution of multicellularity in the brown algae[END_REF] The pertinence of using E. siliculosus as a facultative metallophyte, its easy handling in the laboratory and the current efforts to develop this species as a model organism for Phaeophyceae [START_REF] Charrier | Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research[END_REF] make it an attractive candidate for studying metal tolerance through global approaches such as proteomics.

At the proteomic level, whereas several studies on plant responses to heavy metal stress exist, they are mostly restricted to the exposure to arsenic, cadmium, and zinc [START_REF] Requejo | Proteome analysis of maize roots reveals that oxidative stress is a main contributing factor to plant arsenic toxicity[END_REF][START_REF] Sarry | The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses[END_REF]. Fewer reports on this domain concern copper stress. Some proteomic studies have focused on the glutathione-S-transferase family regulation caused by copper excess [START_REF] Smith | Proteomic analysis of Arabidopsis glutathione S-transferases from benoxacor-and copper-treated seedlings[END_REF], and the copper-binding proteins of Arabidopsis roots and seedlings have been explored by IMAC-MS [START_REF] Kung | Proteomic survey of copper-binding proteins in Arabidopsis roots by immobilized metal affinity chromatography and mass spectrometry[END_REF]. Recently, the molecular effects of copper on the root proteome of Cannabis sativa was investigated by 2-DE-based analysis [START_REF] Bona | Proteomic characterization of copper stress response in Cannabis sativa roots[END_REF]. At the present time, no meaningful proteomic study does exist in marine algae. This is probably due to the fact that until recently no adequate techniques for obtaining high-quality protein extracts was available for these organisms [START_REF] Contreras | Two-dimesional gel electrophoresis analyses of brown algal protein extracts[END_REF]. In this work, copper tolerance of two E. siliculosus strains, originating from habitats with contrasting histories of copper levels, was analyzed by a comparative 2-DE proteomic approach. Patterns of differentially expressed proteins were compared within and between the two strains to better identify several cellular processes potentially involved in copper stress responses and tolerance.

Materials and methods

Plant material and cultivation treatments

E. siliculosus (Ectocarpales, Phaeophyceae) unialgal strains 32 (Culture Collection of Algae and Protozoa accession 1310/4, origin San Juan de Marcona, Peru 15122 0 S, 75110 0 W) and 524 (Culture Collection of Algae and Protozoa accession 1310/333, origin Caleta Palito, Chile 26115 0 S, 70140 0 W) were cultivated in 10-L plastic flasks in a culture room at 141C using 0.22 mm filtered seawater enriched with Provasoli nutrients [START_REF] Starr | UTEX-the culture collection of algae at the university of Texas at Austin 1993 list of cultures[END_REF]. Light was provided by Philips daylight fluorescence tubes at a photon flux density of 40 mmol photon/m 2 /s for 14 h per day. Cultures were aerated with filtered (0.22 mm) compressed air to avoid CO 2 depletion.

Two days before exposing the algae to copper excess, tissues were transferred into 1 L of seawater free of organic matter prepared with 0.22 mm filtered natural SW treated overnight with 0.2 g/L activated charcoal (Merck, Germany). Treated water was then filtered at 0.45 mm to remove charcoal excess. Culture flasks used during the experiments were washed overnight with 1% HCl to limit copper adsorption to glass. Copper stress was triggered by changing the algae to fresh seawater free of organic matter and enriched with CuCl 2 (Merck, Germany) at nominal final concentrations of 50, 150, 250 and 500 mg/L. The term ''acute stress'' was employed for conditions of exposure to high copper concentrations (250-500 mg/L) during short periods of time (4-8 h). On the other hand, ''chronic stress'' defines the exposure to lower sub-lethal copper concentrations (50-150 mg/L) during longer periods of time (6-10 days). No nutrients were added during the experiments. Culture media were renewed every 2 days. For each kinetic point, three replicate algal samples were harvested. At the end of the experiment, thalli were frozen in liquid nitrogen and kept at À801C for analysis.

In vivo fluorescence measurements and microscopic observations

To monitor the intensity of the stress in the algal tissues caused by the copper treatments we measured the maximum quantum yield of PSII (dark adapted) as F v /F m , using a Walz Phyto-PAM (Waltz, Germany). After 5 min of dark adaptation, samples were illuminated with modulated excitation light, provided by a combination of photodiodes emitting at 470, 520, 645 and 665 nm, at a frequency of 25 Hz, in order to define the basal fluorescence level F 0 . After signal stabilization, a light pulse was triggered by a red light emitting photodiode array (3500 mmol photon/m 2 /s intensity for 200 ms, 655 nm), which allowed measuring maximal fluorescence level F m . The emitted fluorescence is detected at a wavelength range above 710 nm. The PSII quantum yield was then calculated as F v /F m 5 (F m -F 0 )/F m . Since photosynthesis is stress sensitive, the quantum yield decreased under suboptimal (stressful) conditions. Simultaneously to PAM measurements, loss of chlorophyll autofluorescence, accounting for cell damage, was monitored using an Olympus BX60 (Olympus, Japan) epifluorescence microscope. Thalli were observed with a 460-500 nm excitation filter and a band pass emission filter centered at 585 nm. Images were obtained using a digital camera (DIAGNOSTIC instruments model 2.1.1). The number of fluorescent cells was estimated and represented as percentage of fluorescent cells.

Protein extraction and 2-DE separation

Global soluble proteome extraction followed the protocol developed by Contreras et al. [START_REF] Contreras | Two-dimesional gel electrophoresis analyses of brown algal protein extracts[END_REF]. A total of 300 mg of tissue (fresh weight) was rinsed with Milli-Q water, blotted dry and fast frozen in liquid nitrogen. After tissue grinding in liquid nitrogen, powder was re-suspended in 5 mL of extraction buffer (1.5% w/v PVP, 0.7 M sucrose, 0.1 M KCl, 0.5 M Tris-HCl, pH 7.5, 250 mM EDTA, complete protease inhibitor cocktail (Roche, Switzerland), 2% v/v b-mercaptoethanol and 0.5% w/v CHAPS) and homogenized at 41C for 20 min. Then, 10 mL of Tris-HCl pH 7.5 saturated phenol was added and the mixture was re-homogenized for 20 min at 41C. The mixture was centrifuged at 10 000 Â g for 20 min and the upper phenol phase was removed. Proteins in the phenol phase were precipitated by addition of five volumes of 0.1 M ammonium acetate dissolved in methanol and incubated at À201C for 3 h. The extract was centrifuged at 10 000 Â g for 20 min, the supernatant was discarded and the protein pellet was rinsed in ammonium acetate (0.1 M in methanol) for 20 min at À201C. Subsequently, the protein pellet was rinsed four times in four volumes of 80% ice-cold acetone.

Image analysis

Gel images obtained using an Image scanner UMAX Powerlook III (UMAX Technologies, USA) at 300 dpi resolution were analyzed with the Melanie version 5.0 software (Swiss Institute of Bioinformatics, Switzerland). Manual editing and normalization were applied after automated spot detection and matching. Spot quantification was based on spot volume (integration of spot density over spot area) as percentage of the total spot volumes of the gel to normalize for possible staining differences between gels. Gel annotations and matching fidelity were checked manually to eliminate matching errors caused by the software. Three gels representing independent biological samples were analyzed for each condition. All spots selected for MS analyses presented statistically significant variation (po0.05), ratioo1.5 and an average fold changeo2 (at least in one of the stressed conditions). Further information regarding gel analyses is available at the MIAPE-GI information sheet: http://www.sb-roscoff.fr/UMR7139/ectocarpus/proteomics/ 001/MIAPE/MIAPE GI sheet.htm.

Protein mass fingerprints (PMF)

Coomassie blue-stained protein-bearing gel slices were cut into small pieces, washed with distilled water and de-stained with ACN. The cysteine residues were reduced by 100 mL of 10 mM DTT at 561C and alkylated by 150 mL of 55 mM iodoacetamide at room temperature. The iodoacetamide solution was replaced by 100 mL of 100 mM NH 4 HCO 3 and gel dehydration was achieved with ACN. After evaporation in Speed-Vac (Thermo, USA), proteins were digested overnight at 371C in a solution containing 0.9 mg of a modified sequencing grade bovine trypsin (Roche, Germany) prepared in 25 mM NH 4 HCO 3 . Finally, a double extraction followed, first with 5% v/v formic acid and subsequently with 100% v/v ACN. The resulting peptide mixture was extracted and vacuum-dried, re-suspended in 1% formic acid and desalted using a ZipTip (Millipore, USA) C-18 reverse phase microcolumn. After evaporation, the desalted peptide mixture was re-suspended in 10 mL of 1% formic acid. PMF by MALDI-TOF-MS was performed using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, USA). One microliter of tryptic digest was mixed with an equal volume of CHCA matrix (Sigma; 10 mg/L in 50% ACN, 0.1% TFA) and spotted onto the MALDI target. Spectra were acquired in positive ion reflector mode under 20 kV accelerating voltage and a mass range of 800-4000 Da. Internal calibration was performed using trypsin autolysis fragments at m/z 1433.70, 2163.05 and 2289.1. The monoisotopic masses of tryptic peptides were used to query the specific E. siliculosus protein database deduced from the version 2 of the genome annotation (genome accessible at http://bioinformatics.psb.ugent.be/webtools/bogas) using Mascot version 2.1. Search conditions included an initially permissive mass window of 200 ppm for external calibration, followed by 100-50 ppm applied for internal calibration with trypsin autodigestion fragments (when trypsin peptides were detected), allowing double missing cleavage, modification of cysteines by iodoacetamide, methionine oxidation and N-terminal pyroglutamylation as variable modifications. Only significant scores were selected for publication. Further information regarding the MS procedures is accessible at the MIAPE-MS information sheet: http://www.sb-roscoff.fr/UMR7139/ectocarpus/proteomics/ 001/MIAPE/MIAPE MS sheet.htm.

RNA extraction and RT-qPCR analysis

The RNA extraction, quantification, cDNA synthesis and RT-qPCR reactions followed the protocol described by Le Bail et al. [START_REF] Bail | Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus[END_REF]. For each gene, a specific pair of oligonucleotide primers in the 3 0 coding sequence (Supporting Information Table S1) was designed using Beacon designer 5.00 (Premier Biosoft International, USA). The qPCR reactions were done in a Chromo4 apparatus (Biorad) with a SYBR Green reaction mix AB-1162/B from ABgene (UK). A dilution series ranging from 91 to 121 312 copies of the E. siliculosus genome was prepared. The Dynein gene was used as internal control as previously described [START_REF] Bail | Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus[END_REF]. Relative variations in gene expression were calculated as x-fold changes compared with the appropriate control treatment.

Bromoperoxidase activity assay

Total protein extracts were obtained from of 0.2-0.5 g of fresh weight tissue, grounded in liquid nitrogen and mixed with 0.4 mL of extraction buffer containing 25 mM MOPS, pH 7.2, 15 mM MgCl 2 , 15 mM EGTA, 1 mM DTT, 0.5% PVP and anti-proteases cocktail at a concentration specified by the manufacturer (Roche, France). Extracts were homogenized for 1 h at 41C, centrifuged at 10 000 Â g for 30 min and the supernatant collected. Vanadate was added to the extracts at 1 mM final concentration, after which they were incubated at 41 C for at least 1 h prior to the analysis. The total amount of proteins was quantified using the Bradford assay [START_REF] Bradford | Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding[END_REF], and 20 mg were loaded on the gel. Bromoperoxidase enzymatic assay was conducted on non-denaturing gels as described elsewhere [START_REF] Colin | Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases[END_REF]. The activity levels using band density in the gels were assessed by the ImageQuant v5.2 software (Molecular Dynamics, GE Healthcare, USA). The relative changes among conditions were calculated as Volume Intensity and quantified in arbitrary units (AU).

Statistical analysis

The statistical significances of the differences among treatments were determined by the non-parametric Mann-Whitney U test run on Statistica version 5.1 software (StatSoft, USA). All conclusions are based on at least 5% level of significance (po0.05).

Results

Copper toxicity tests in strains Es32 and Es524

The toxicity of copper was assessed in the two strains by in vivo measurements of chlorophyll fluorescence and by epifluorescence microscopy observations. The dose-response relationships between Cu concentrations and cell-mortality differed between both strains after 10 days of exposure to copper excess (Fig. 1A andB). In Es32, a significant drop to 70% of chlorophyll autofluorescence was observed under 10 days of incubation with 50 mg/L Cu, indicating the occurrence of cell death. On the other side, Es524 displayed a lower decay of cell-autofluorescence accounting for the deleterious effects only at 250 mg/L Cu or higher. In agreement with these observations, the F v /F m was negatively affected by Cu in both strains, but at different thresholds (Fig. 1C). After 10 days, a significant decline in the yield was observed in strain Es32 when copper concentration was higher than 50 mg/L, with F v /F m decreasing in a dose-dependent manner. After the same time of incubation, Es524 displayed significant decrease in F v /F m only for Cu concentrations of 500 mg/L. These results allowed to select the best concentrations to expose each isolate to non-lethal conditions of copper stress, treatments required for differential soluble proteome analyses.

Analyses of the proteins induced by chronic copper stress in Es32

To identify proteins specifically regulated under chronic copper stress conditions, the most sensitive strain (Es32) was exposed to 10 days at 50 mg/L nominal Cu. The 2-DE profiles of soluble proteome fractions were compared between control and treated thalli. Coomassie blue staining provided an average yield of 790 spots per gel (Supporting Information Fig. S1). Image analysis of the gels produced over 560 reproducible spot groups considering the two experimental conditions (Fig. 2A), with 58 statistically variable spots (ratio41.5). Among these spots, two downregulated and eight upregulated proteins were selected for MS identification because they presented enough intensity to be located and excised manually (Fig. 2A). PMF spot identifications are described in Table 1. For most of the identified spots, the 2-DE observed molecular mass and pI values are in agreement with the theoretical values obtained from the corresponding protein hits in the genomic database. Among the identified enzymes, there was a transketolase belonging to the pentose phosphate metabolism. Several enzymes involved in amino acid metabolism were also identified. For instance, copper excess enhanced the accumulation of serine hydroxymethyl transferase (SHMT) and cysteine synthase involved in the biosynthesis of glycine and cysteine, respectively. Related to this, the nucleotide metabolism enzyme S-adenosyl methionine synthetase (SAM) also appeared increased. Protein folding was involved as well since a HSP10 and the HSP cochaperone Immunophilin FKBP 11 were more abundant in stressed individuals. Finally, the chaperone calreticulin was downregulated by copper excess.

Analyses of the proteins induced by chronic copper stress in Es524

The tolerant strain Es524 was exposed during 10 days to 50 and 150 mg/L Cu. Protein extracts from Es524 were analyzed with identical criteria as for Es32. The analysis of gel images obtained under the control condition and after treatment with 50 mg/L resulted in over 423 matched spot groups. Spot intensity analysis showed 32 differentially expressed spots (ratio41.5). Among these spots, 20 were up-regulated and 12 down-regulated. On the other hand, comparison of control thalli with those exposed to 150 mg/L of copper produced 494 reproducible groups, from which 58 presented significant changes (Fig. 2B). For PMF, a first group of six spots corresponding to proteins accumulated under both treatments with copper excess was considered (638, 734, 745, 941, 1174 and 1176). Then, a second group was selected with nine spots only regulated by exposure to 150 mg/L, corresponding to seven upregulated (814, 897, 584, 1089, 768 and 1184) and one downregulated (1252) proteins. PMF results are presented in Table 2. One enzyme involved in the pentose phosphate pathway, the G6PD, was identified. As for Es32, copper excess caused Classes histograms with the mean volume% and mean squared deviation (MSD) are presented in Fig. S3. a) http://bioinformatics.psb.ugent.be/genomes/view/Ectocarpus-siliculosus.

Proteomics 2010, 10, 1-15 differential expression of enzymes involved in amino acid biosynthesis and nucleotide metabolism related enzymes, such as GOGAT and SAM. Copper excess clearly enhanced protein folding processes through the accumulation of the chaperone HSP70, and the HSP cochaperone stress-inducible protein (STI 1). Cell cycle may also be regulated by copper, as a hypersensitive-induced reaction protein and a CDC-ATPase accumulated under stress. Interestingly, among up-regulated spots in Es524, we found two RNA helicases from the DEAD box family, both related to the cell rescue process. In addition, one specific brown algal antioxidant-related enzyme, the vanadium-dependent bromoperoxidase (vBPO), was also identified in this set of proteins.

Inter-strain differences in protein expression caused by copper excess

Finally, patterns of proteome expression were compared between Es32 and Es524 exposed to 50 mg/L of copper after 10 days. Over 406 spots matched on reproducible groups between both strains, whereas 93 proteins were differentially expressed with a ratio higher than 2 (Fig. 2C). From these, in Es524 we identified nine clearly increased and two decreased spots, which were subsequently identified (PMF identification in Table 3). Two spots, corresponding to the same locus Esi0002_0327 and encoding for a potential pentose phosphate related transketolase, showed contrasting regulation in the two strains: increased in Es524 and decreased in Es32. Several other glycolysis and pentose phosphate metabolism-related enzymes were also markedly increased in Es524: a phosphoribulokinase, a glyceraldehyde-3-phosphate dehydrogenase and a fructose 1,6-bisphosphate aldolase (SMALDO). Concerning the amino acid metabolism, a GS appeared highly increased in Es524. Photosynthesis-related processes were also stimulated in this strain. For instance, the PSII Mn-stabilizing protein of the oxygen evolving complex (OEC33) was markedly overexpressed in Es524, while it was not detectable in Es32. Similar results were obtained for a light harvesting complex (LHC) protein Fucoxanthine chlorophyll a-c binding protein (Fcp).

Expression analysis of selected key genes by RT-qPCR under acute and chronic stress conditions

Four genes, encoding for proteins identified through proteomic analyses, vBPO, HSP70, HSP10 and SAM, were chosen for RT-qPCR experiments in order to compare changes in their level of expression during acute and chronic stress (Fig. 3A andB). Acute stress was induced by exposing algal cultures to 250 and 500 mg/L of copper for 4 and 8 h. For chronic stress, final concentrations of 50 and 150 mg/L of Cu were applied for 6 and 10 days. Interestingly, vBPO is the gene whose expression level was the most altered under stress. Its expression was induced under all the tested conditions in both strains, and more specifically, this gene was the most upregulated under chronic stress. Accumulation of HSP70 transcripts was observed under all acute stress conditions in Es32 (Fig. 3A) and Es524 (Fig. 3B). However, during chronic stress, the transcription of this gene was up-regulated only in Es524 exposed to 150 mg/L for 10 days. Significant changes in the expression of the HSP10 gene were recorded only under acute stress, mainly in Es32 (Fig. 3A). Slight induction of this gene was observed after incubation of Es524 for 4 h in presence of 250 mg/L copper. SAM gene was down-regulated in most of the conditions tested (Fig. 3A andB).

Bromoperoxidase activity assay

To assess the importance of vBPO in the regulation of the oxidative stress created by Cu in Es32 and Es524, changes in enzymatic activity were followed on native PAGE gels. No visible activity was detected in Es32 under the control condition (Fig. 4A), while an intense band appeared under stress conditions, with an estimated molecular mass of $140 kDa. Quantification of the band intensity showed an increase of 4E16 AU in stressed Es32 (Fig. 4B). Protein extract from Es524 incubated in absence of copper showed only a slight coloration, indicating a low basal bromoperoxidase activity (Fig. 4C andD). After treatment with 50 or 150 mg/L, a band similar to that observed in extracts from stressed Es32 was identified, demonstrating an increase in bromoperoxidase activity. The quantification of this band was estimated at 3E16 AU on all individuals and for both copper treatments. Similar results were obtained when replacing potassium bromide by potassium iodide during gel development, indicating that the protein identified in E. siliculosus exhibits a true vBPO activity (data not shown).

Discussion

Studies of differential protein expression mapping are still uncommon in marine algal models. Proteomics allowed us to analyze responses to chronic stress in two strains in the marine brown alga E. siliculosus isolated from habitats contrasting in their history of copper enrichment. Exposure of these isolates to chronic conditions of copper stress allowed the identification of proteins and genes that help to support the hypothesis that isolate Es524 from Caleta Palito is a copper tolerant ecotype, which likely evolved after more than 60 years of exceedingly high levels of copper in its normal habitat. Protein accumulation analysis in both strains shows regulation of a high number of proteins directly implicated in various mechanisms of stress control, which validates the pertinence of this approach.

Common protein expression features of the two isolates exposed to copper

Surprisingly, the marked differences in tolerance to copper between the two isolates were not accompanied by major Proteomics 2010, 10, 1-15 differences in protein expression patterns. Exposure to sublethal copper concentrations induced the expression of proteins common to both strains, underlining the crucial importance of certain metabolic pathways during the response to stress. Copper induced the accumulation of proteins involved in pathways related to energy production such as the pentose phosphate pathway. These observations are in agreement with the correlation established between alteration of the photosynthesis and the ATP pool depletion in the diatom Phaeodactylum tricornutum [START_REF] Cid | Copper toxicity on the marine microalga Phaeodactylum tricornutum: effects on photosynthesis and related parameters[END_REF]. Also, as in vascular plants [START_REF] Sarry | The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses[END_REF], heavy metal stress induced the onset of repairing and detoxification systems, which represent an additional energetic cost for the cells, and therefore, the accumulation of enzymes involved in energy production in both isolates should not come as a surprise. In addition, the NADPH coming from the pentose phosphate shunt is one of the main cellular sources of reducing power, which is very important for detoxification, i.e. restoration of the reduced glutathione pool. In relation to this, copper stress activates enzymes involved in amino acid metabolism. These enzymes encompassed SHMT and cysteine synthase, linked to the biosynthesis of glycine and cysteine respectively. These two enzymes, plus the GOGAT/GS, are essential for the production of amino acid precursors used during the biosynthesis of glutathione (GSH). In this context, GSH plays a pivotal role in protecting living organisms from environmental stresses [START_REF] Xiang | Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis[END_REF]50] by detoxifying ROS and toxins [START_REF] Halliwell | Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life[END_REF]50,[START_REF] Alscher | Biosynthesis and antioxidant function of glutathione in plants[END_REF]. The GSH polymers PCs are fundamental for chelating metal excess in vascular plants [START_REF] Cobbett | Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis[END_REF]. Recently, concentrations of PCs and GSH in the brown algae Fucus vesiculosus and F. serratus have been correlated with the history of copper contamination of the sampling sites [START_REF] Pawlik-Skowronska | Concentrations of phytochelatins and glutathione found in natural assemblages of seaweeds depend on species and metal concentrations of the habitat[END_REF]. The enhanced GSH biosynthesis could also be related to the activity of the detoxification enzymes glutathione-S-transferases. Theses enzymes are particularly abundant in E. siliculosus, and some of them have been recently shown to be induced by copper stress (strain Es32) [START_REF] De Franco | Whole genome survey of the glutathione transferase family in the brown algal model Ectocarpus siliculosus[END_REF]. Theses findings, together with our results, highlight the involvement of thiol peptides in maintaining the homeostasis in brown algae under metal stress, as well as in detoxification processes. In addition, SAM, which synthesizes S-adenosyl methionine, was also up-regulated in both strains. Previous proteomic work in vascular plants and animals demonstrated that this enzyme was up-regulated under a condition of heavy metal stress [START_REF] Requejo | Proteome analysis of maize roots reveals that oxidative stress is a main contributing factor to plant arsenic toxicity[END_REF][START_REF] Lee | Proteomic evaluation of cadmium toxicity on the midge Chironomus riparius Meigen larvae[END_REF]. This enzyme belongs to the methyl cycle that interacts with the GSH biosynthetic pathway, specifically at the level of the SHMT enzyme. In vascular plants, SAM is the direct precursor for nicotianamine, involved in copper complexation [START_REF] Liao | Copper uptake and translocation in chicory (Cichorium intybus L. cv Grasslands Puna) and tomato (Lycopersicon esculentum Mill. cv Rondy) plants grown in NFT system. II. The role of nicotianamine and histidine in xylem sap copper transport[END_REF].

Both strains showed also up-regulation of enzymes involved in protein folding and stabilization, which are important players in recycling stress-damaged proteins. HSPs, known to be induced by a variety of stress conditions, and whose role is to protect cells against protein degradation [START_REF] Vierling | The roles of heat shock proteins in plants[END_REF][START_REF] Lindquist | The heat-shock proteins[END_REF], appeared increased in both strains. Indeed, both strains up-regulated stress-related HSPs co-chaperones such as the stress-induced protein STI 1 and the Immunophilin-FKBP 11. These co-chaperones are needed to create functionally active protein complexes by establishing interactions between HSPs [START_REF] Pearl | Structure and mechanism of the Hsp90 molecular chaperone machinery[END_REF]. Additional data obtained through gene expression analysis in Es32 showed a strong upregulation of HSP70 and HSP10 in response to acute copper stress. In contrast, level of expression of HSP10 was not regulated in Es524, which is in agreement with the proteomic results. In both strains, HSP 70 was mainly induced under acute exposures to copper excess, with a higher expression in Es32 than in Es524, suggesting an involvement in rapid stress responses rather than in adaptation.

The protein chaperone calreticulin was downregulated in Es32, whereas Es524 upregulates hypersensitive-induced reaction and CDC-ATPase proteins. These proteins participate in cell cycle control and apoptosis in several organisms, under a variety of stress, including metal ions [START_REF] Panaretakis | The co-translocation of ERp57 and calreticulin determines the immunogenicity of cell death[END_REF][START_REF] Nadimpalli | Prohibitins, stomatins, and plant disease response genes compose a protein superfamily that controls cell proliferation, ion channel regulation, and death[END_REF][START_REF] Braun | Mechanisms of Cdc48/VCP-mediated cell death -from yeast apoptosis to human disease[END_REF]. Further studies should now be carried out to fully characterize the stress responses of Ectocarpus.

Strain-specific responses

Several individual proteins exhibited inter-strain qualitative or quantitative differences in expression. An increased expression of the LHC component Fcp was observed in Es524. In brown algae, and as in other photosynthetic organisms, LHC is primarily damaged by a wide range of abiotic stresses [START_REF] Upper | Heavy metal uptake by plants and cyanobacteria[END_REF][START_REF] Rijstenbil | Effects of UVB radiation and salt stress on growth, pigments and antioxidative defence of the marine diatom Cylindrotheca closterium[END_REF][START_REF] Clendennen | Photosynthetic response of the giant kelp Macrocystis pyrifera (phaeophyceae) to ultraviolet radiation[END_REF]. Therefore, the enhanced turnover of Fcp could contribute to the maintenance of the LHC integrity in this strain. Copper excess can also damage directly the PSII, generating oxidative stress as a result of the PSII dysfunction [START_REF] Yruela | Photoinhibition of photosystem II from higher plants. Effect of copper inhibition[END_REF][START_REF] Halliwell | Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life[END_REF][START_REF] Halliwell | Oxygen toxicity, oxygen radicals, transition metals and disease[END_REF][START_REF] Yruela | Copper effect on the protein composition of photosystem II[END_REF][START_REF] Bernal | Copper effect on cytochrome b559 of photosystem II under photoinhibitory conditions[END_REF]. In agreement with this, a marked increase in OEC33 was observed in Es524 when compared with Es32. This could account for an enhanced turnover of OEC33, which constitutes a major copper toxicity target in PSII [START_REF] Yruela | Copper effect on the protein composition of photosystem II[END_REF][START_REF] Henmi | Release and reactiveoxygen-mediated damage of the oxygen-evolving complex subunits of PSII during photoinhibition[END_REF]. From these observations, it seems that differences in the expression of proteins related with the photosynthetic apparatus can explain, at least partly, the different response to Cu stress displayed by the two strains used in this study. However, further research is necessary to fully address the issue of differential copper tolerance.

The enzymes involved in cell detoxification, DEAD box helicases and vBPO, appeared increased under copper stress in Es524 but not in Es32. RNA helicases from the DEAD box family are found in almost all organisms and have important roles in RNA metabolism [START_REF] Rocak | DEAD-box proteins: the driving forces behind RNA metabolism[END_REF]. Moreover, these enzymes have been reported to play important roles in resistance to abiotic stress in vascular plants [START_REF] Sanan-Mishra | Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield[END_REF][START_REF] Luo | Expression of a putative alfalfa helicase increases tolerance to abiotic stress in Arabidopsis by enhancing the capacities for ROS scavenging and osmotic adjustment[END_REF]. Two DEAD box helicases accumulated in copper-stressed Es524, which suggests their involvement in copper tolerance. On the other hand, vBPOs are peroxidases unique to brown and red algae, and are absent in vascular plants or metazoans [START_REF] Leblanc | Iodine transfers in the coastal marine environment: the key role of brown algae and of their vanadium-dependent haloperoxidases[END_REF]. They catalyze the oxidation of bromide, as well as iodide, in the presence of hydrogen peroxide, to generate oxidized halogens which are thought to be related to iodine uptake [START_REF] Upper | Iodine uptake in Laminariales involves extracellular, haloperoxidase-mediated oxidation of iodide[END_REF]. Recent studies in the kelp L. digitata showed that iodine was mainly stored in the apoplastic region as iodide [START_REF] Upper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF][START_REF] Verhaeghe | Microchemical imaging of iodine distribution in the brown alga Laminaria digitata suggests a new mechanism for its accumulation[END_REF]. K .

upper et al. presented iodide as a powerful scavenger for a wide variety of ROS because of its high reducing capacity [START_REF] Upper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF]. Furthermore, these authors suggested that vBPO could catalyze this process. Gene expression analysis showed a strong induction of vBPO in both strains under all conditions of copper excess, which highlights the importance of vBPO in stress responses. In agreement with these results, the in-gel activity assay clearly showed the increase of vBPO activity during chronic stress. Iodide metabolism is not well characterized in E. siliculosus and therefore this is, to our knowledge, the first study describing vBPO and its regulation under stress in this species. Based on our results and on previous work in brown algae, it is likely that vBPOs play a mayor role in the processes leading to ROS detoxification [START_REF] Upper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF][START_REF] Roeder | Identification of stress gene transcripts in Laminaria digitata (phaeophyceae) protoplast cultures by Expressed Sequence Tag analysis[END_REF]. In addition, if significant amounts of I À are released upon stress, it could be possible that complexation with Cu 21 occurs, leading to a nonbioavailable form of copper. However, additional research is required to test this hypothesis.

Conclusions

In this work, we have characterized two strains of E. siliculosus presenting different copper tolerance (Es32 and Es524). The availability of these different strains paves the way for future studies on the genetic bases of adaptative traits in seaweeds. Global proteome profiling led to identification of several pathways involved in copper tolerance and helped to better understand the differential tolerance to the metal observed between strains. In this context, photosynthesis-related proteins seem to be crucial for copper tolerance. In addition, we suggest the occurrence of an original antioxidant response of brown algae, based on halide metabolism and involving a vBPO.

Figure 1 .

 1 Figure 1. Physiological effects of copper toxicity on E. siliculosus strains Es32 and Es524. (A) Comparison of epifluorescence and transmission microscopic photographs after 10 days of copper treatment. Top images represent the green cut-off filter fluorescence images with their corresponding transmission images at the bottom. For each strain, the images are organized as follows: control condition (left) and 250mg/L of copper (right). Bars represent 0.1 mm. (B) Effect of different copper concentrations on the percentage of remaining auto-fluorescent cells during 10-day trials. Control seawater (black), and different concentrations of copper: 50 mg/L (blue), 150 mg/L (green), 250 mg/L (red) and 500 mg/L (orange). Values represent means of three independent replicates and bars represent the SE. Black-filled symbols represent a significantly different condition from the control group (po0.05). (C) Changes in the photosynthetic yield during 10-day trials. Control seawater (black), and different concentrations of copper: 50 mg/L (blue), 150 mg/L (green), 250 mg/L (red), 500 mg/L (orange). Values represent means of three independent replicates and bars represent the SE. Black-filled symbols represent a significantly different condition from the control group (po0.05).

Figure 2 .

 2 Figure 2. Proteomic profiling of E. siliculosus strains Es32 and Es524 in response to copper stress. (A) Representative 2-DE gels of total soluble proteome fractions of Es32 under control condition (left gel) and 50 mg/L of nominal Cu, for 10 days (right gel) using a non-linear gradient of pI 4-7. The identified differentially expressed spots are highlighted on the bottom panel. Spot identification numbers are listed on the left side of the images. (B) Representative 2-DE gels using a non-linear gradient of pI 4-7 of total soluble proteome fractions of Es524 under control condition (left gel) and 150 mg/L nominal Cu, for 10 days (right gel). Differentially expressed spots are highlighted on the bottom panel. (C) Representative 2-DE using a non-linear gradient of pI 4-7 gels comparing total soluble proteomes between Es32 and Es524 submitted to 50 mg/L nominal Cu for 10 days. Differentially expressed proteins are highlighted on the bottom panel.

Figure 3 .

 3 Figure 3. Changes in gene expression in E. siliculosus strains Es32 and Es524 exposed to acute and chronic stresses monitored by RT-qPCR. (A) Expression profile of vanadium bromoperoxidase (vBPO), heat shock protein 70 (HSP70), heat shock protein 10 (HSP10) and S-adenosyl methionine synthase (SAM) genes in Es32 submitted to acute Cu stresses (250 and 500 mg/L for 4 and 8 h) at the left and chronic Cu stresses (50 mg/L for 6 and 10 days) at the right. (B) Expression profile of vBPO, HSP70, HSP10 and SAM genes from Es524 exposed to acute Cu stresses (250 mg/L and 500 mg/L for 4 and 8 h) at the left and chronic Cu stresses (50 and 150 mg/L for 6 and 10 days) at the right. Relative gene expression ratios were calculated as 2n-fold variation as described in Section 2. Results are means7SE for two determinations from three independent biological samples. Ã Significantly different from the control group (po0.05).

Figure 4 .

 4 Figure 4. Native-PAGE bromoperoxidase (BPO) activity staining assay from E. siliculosus strains Es32 and Es524 exposed to 10 days of copper excess. Twenty microgram of each total protein extract were loaded into the gel for testing BPO activity as described in Section 2. (A) Lines 1-3: Es32 controls; lines 4-6: Es32 treated with 50 mg/L nominal copper. (B) Image band quantification of Es32 controls and 50 mg/L Cu treated individuals. Values (arbitrary units, A.U.) represent the mean of the three replicates and SE. Ã Significantly different from the control group (po0.05). (C) Lines 7-9: Es524 controls; lines 10-12: Es524 treated with 50 mg/L copper; lines 13-15: Es524 treated with 150 mg/L nominal copper. (D) Image band quantification of Es524 controls, 50 and 150 mg/L Cu-treated individuals. Values (arbitrary units, A.U.) represent the mean of the three replicates and SE. Ã Significantly different from the control group (po0.05).

  This work has been partially funded by Marine Genomics Europe NoE 7 (EU contract no. GOCE-CT-2004-505403) and by the French Embassy and the CONICYT of Chile through PhD fellowships to A. R. This work was supported by the Laboratoire International Associe ´''Dispersal and Adaptation of Marine Species'' (LIA DIAMS) PUC, Chile and CNRS-UPMC, France. Additional support came from FONDAP 1501-0001 (Program 7) and ICA research grant, both to J. A. C. We are especially grateful to Jessica Beltra ´n and Santiago Andrade for valuable suggestions.

Table 1 .

 1 Proteins identified in Es32 for which the level of expression changed significantly (po0.05) under exposure to copper excess(50

	mg/L for 10 days)

a) http://bioinformatics.psb.ugent.be/genomes/view/Ectocarpus-siliculosus.

Table 2 .

 2 Proteins identified in Es524 for which the level of expression changed significantly (po0.05) under exposure to copper excess(50 and 150

	mg/L for 10 days)

a) http://bioinformatics.psb.ugent.be/genomes/view/Ectocarpus-siliculosus.

Table 3 .

 3 Proteins identified in Es524 proteins whose expression levels under copper excess(50

	mg/L for 10 days) changed significantly compared to Es32

Proteomics 2010, 10, 1-15

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

A. Ritter et al. Proteomics 2010, 10, 1-15 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

These authors have declared no conflict of interest.


Aliou Ly

Gilles Bailly

Arnaud Fernandez

Olivier Llopis

Chromatic Dispersion measurement of short optical fibers by sinusoidal phase modulation of a monochromatic light

Keywords: chromatic dispersion, optical fiber, phase modulator, microwave optics, optoelectronic oscillator I

We report a simple method to measure the chromatic dispersion of short length optical fibers using phase modulation of a continuous-wave light. The results obtained were exploited to design a coupled optoelectronic oscillators with optimization of its intracavity dispersion.

INTRODUCTION

Chromatic dispersion (CD) in single mode optical fibers is caused by two effects: the material dispersion and the waveguide dispersion of the fiber. The former is due to the wavelength dependence of the refractive index of glass. The latter is due to the fiber's guiding properties. In both cases, it is well known that chromatic dispersion has detrimental effects in high data rate optical fiber communications as well as in radio over fiber transmission systems [START_REF] Meslener | Chromatic dispersion induced distortion of modulated monochromatic light employing direct detection[END_REF]. As a result, many and well established techniques have been proposed for precise measurement of CD. Among them one can mention the phase shift based method, optical interferometry or the microwave interference effect [START_REF] Han | A new method for fiber chromatic dispersion measurement with microwave interference effect[END_REF]. However, it is worth noticing all those methods were implemented to measure CD of optical fibers with lengths of tens of kilometers because of the above mentioned applications. Thus, it is quite striking to see that CD measurement of short length optical fibers (hundreds of meters typically) have gained less attention whereas there are some applications in which this is necessary. This is the case for the coupled optoelectronic oscillator (COEO). Indeed, the spectral purity of the RF signal generated by this device strongly depends on its cavity dispersion [START_REF] Matsko | Theory of coupled optoelectronic microwave oscillator II: phase noise[END_REF]. A recent work aiming at measuring this dispersion has been reported [START_REF] Lelièvre | In-situ dispersion measurement of a coupled optoelectronic oscillator[END_REF].

Nevertheless, in a primary step of designing a COEO, it would be advantageous to precisely measure the CD of its optical fiber loops. In this frame, we present here the measurement of short optical fibers CD by mean of a sinusoid ally phase modulated continuous-wave (cw) light. To the best of our knowledge, only one work using this approach has been reported to date and it was applied to very long optical fibers [START_REF] Yamamoto | Simple and precise chromatic dispersion measurement using sinusoidally phase-modulated CW light[END_REF].

II. CD MEASUREMENT PRINCIPLE AND THEORY

A. CD measurement principle

The experimental setup for CD measurement is depicted in Fig. 1. The cw laser is phase modulated by a 10 GHz wideband phase modulator (MPZ-LN Photline). A polarization controller ensures that the incident light enters the modulator with the right polarization. The phase modulated light next travels a dispersive medium, the fiber under test (FUT) before being detected by a photodiode (Discovery DSC30S). The modulation depth θm will be weak enough to assume that the laser spectrum at the output of the modulator will consist mainly in the carrier and two optical sidebands which are 180° out of phase. The FUT is characterized by its geometrical length L, its complex propagation constant β, its CD per unit length of fiber D in (ps/nm/km) and its accumulated CD over the length L, Dtot in (ps/nm). The two optical sidebands will be affected differently by the dispersion of the FUT when propagating through it. This will cause the amplitude of the recovered modulation to vary with modulating frequency as a result of a phase to intensity modulation conversion that takes place inside the FUT. Thus, by measuring this amplitude variation one can extract the CD of the FUT. To do so, a vector network analyzer (VNA, ZVB20 Rohde&Schwarz) is used as a RF signal generator and receiver. The RF signal frequency fm is swept from 10 MHz to 20 GHz and the gain of the S21 parameter is measured. The next paragraph will show how the measurement of this gain can be processed to extract the CD of the FUT.

B. Theory

To see how the phase modulated light behaves after propagating through the FUT, let's start with the usual expression for a sinusoidally phase modulated light:

( ) = 2 ( ) + ( ) ( ) + ( ) ( ) + . . (1)
Where c.c. stands for complex conjugate to take into account that the electric field is real, ωc is the carrier angular frequency and ωm corresponds to the angular modulation frequency. In this equation we assume weak modulation so as the phase modulated light can be expanded into Bessel functions of first kind by only keeping the order +1 and -1. At the output of the FUT, the expression of the electric field becomes:

( ) = 2 ( ) + ( ) ( ) + ( ) ( ) + . . (2)
The imaginary part of the complex propagation constant β leads to attenuation of the modulated light through the fiber and its real part causes chromatic dispersion. It can be expanded in Taylor series as follows:

( ) = ( ) + ( )( -) + 2 ( )( -) (3)
The third term in Eq. ( 3) is responsible for the distortion and the broadening of the optical signal. Indeed, it is related to CD per unit length by the following expression:

= - 2 ( ) (4)
The photocurrent produced at the output of the photodiode can then be written as:

( ) = ɳ ( ) cos( ) + ( ) cos(( + ) + ) + ( ) cos(( + ) + ) (5)
Where = is the FUT attenuation which is assumed to be the same for all frequency components, ɳ the response of the photodiode in A/W and φm is given by:

= - (6)
It corresponds to the phase distortion induced by the dispersion of the FUT.

The photocurrent in Eq. ( 5) has a DC component and contains terms oscillating at the modulation frequency and high order harmonics. The DC current allows the measurement of the FUT attenuation by comparing its value with and without the FUT. After expanding Eq. ( 5) and applying the appropriate trigonometric formula, the sum of the oscillating terms at ωm converted into a voltage at the VNA input is:

( ) = ɳ ( ) ( ) sin sin(2 ) (7 )
With RL the 50 Ω load resistance of the VNA. It can be clearly seen in this equation the process of dispersion induced phase distortion conversion to amplitude modulation. The S21 gain measured at the VNA is related to the amplitude of the recovered modulation in Eq. ( 7) as follows:

| | = 20 log ( ) - (8)
Where Pin in (dBm) is the RF power at the phase modulator input. Thus, from the measurement of the VNA, one can determine the amplitude of the recovered modulation which evolves sinusoidally versus the square of the modulating frequency. A way to subsequently retrieve the CD per unit length D is to look at the frequency at which the sine cancels. This was done in [START_REF] Yamamoto | Simple and precise chromatic dispersion measurement using sinusoidally phase-modulated CW light[END_REF], but, as stated earlier, it requires long fiber lengths for moderate modulation frequencies. In our case, we will just take advantage of the linear part of this sine function. By fitting its linear part, one can extract the total dispersion Dtot of the FUT from the obtained slope. However, since this slope depends on parameters that may fluctuate, a prior calibration is necessary before characterizing the dispersion of short optical fibers.

III. CD MEASUREMENT OF SMF FIBERS

The results below are all obtained under the following experimental conditions: the laser operating wavelength was set at 1552 nm with an output power of 13 dBm and the RF power from the VNA was fixed at Pin =7 dBm. The slope of the linear part of the amplitude of the sine function in Eq. ( 7) can be written as:

A. Calibration with a 155 m SMF-28 fiber

= ɳ ( ) ( ) ≡ (9)
The calibration consists then in determining the value of the constant κ. Indeed, once its value is known, the total dispersion for any FUT with an attenuation of αFUT is given by:

= ( 10
)
To this aim, we use a 155 m length SMF-28 fiber as a calibration FUT with a known total dispersion of 2.64ps/nm and a known attenuation α of 0.796. Fig. 2. gives the result obtained. The presence of ripples might come from residual amplitude modulation into the phase modulator even if a polarization controller is employed. Anyway, it can be clearly seen that this amplitude evolves linearly between 6.5 GHz and 15.8 GHz. Thus, extracting this portion and performing a linear fit yields a value of κ of 4.55x10 -23 V.nm/ps/Hz 2 . Fig. 3 shows the result of this calibration. The standard error introduced by the linear fitting leads to an error in the determination of κ of 1.18x10 -2 . This will corresponds to an error of 0.01 ps/nm only in the calculation of CD using Eq. (10).

B. CD measurement of short optical fibers

Once the calibration performed, the total CD of three optical fibers with lengths of 288 m, 413 m and 2 km has been measured. The evolution of the recovered modulation amplitude for these fibers is plotted in Fig. 4.

By measuring the slope of the linear part of the curves above as well as the attenuation introduced by each fiber, we were able to extract their CD using Eq. (10). The results are reported in table 1. The coupled optoelectronic oscillator (Fig. 5) is an efficient way to generate pure microwave signals and optical combs. However, the microwave phase noise performances of the COEO are strongly dependent on to the optical pulse temporal width. Indeed, the shorter this width the lower the pulse jitter and, consequently, the lower the phase noise [START_REF] Matsko | Theory of coupled optoelectronic microwave oscillator II: phase noise[END_REF]. Thus, in order to generate ultra-high frequency and spectrally pure signals at harmonic frequencies of the COEO fundamental signal, the cavity dispersion of this latter must be carefully optimized. With the approach in Fig. 5 we have demonstrated recently a 90 GHz signal generator (harmonic mixing from a 30 GHz COEO) with an ultra-low phase noise level of -104 dBc/Hz at 1 kHz offset frequency [START_REF] Ly | Highly spectrally pure 90 GHz signal synthesis using a coupled optoelectronic oscillator[END_REF]. The results are displayed on Fig. 6. V. CONCLUSION

IV. APPLICATION TO THE OPTIMIZATION OF A COEO

We have demonstrated a simple method to measure the chromatic dispersion of optical fibers with length in the range of one hundred meters. The resolution of this system is better than 1 ps/nm. We have taken advantage of these results to compensate the chromatic dispersion in an optoelectronic oscillator using a chirped fiber Bragg filter. This oscillator produces high spectral purity reference signals in the millimeterwave range.

Fig. 1 .

 1 Fig. 1. Experimental setup for CD measurement: PC: polarization controller, PM: phase modulator, FUT: fiber under test, PD: photodiode, VNA: vector network analyzer.

Fig. 2 :

 2 Fig. 2: Evolution of the recovered modulation amplitude for a 155 m fiber.

Fig. 3 :

 3 Fig. 3: Extracted linear portion of the recovered modulation amplitude and the corresponding linear fit in green.

Fig. 4 :

 4 Fig. 4: Evolution of the recovered modulation amplitude for short optical fibers of different length.

Fig. 5 :

 5 Fig. 5: COEO experimental setup, including the fiber spools and the chirped Bragg filter (CFBG) which have been combined to get a close to zero dispersion in the optical cavity.

Fig. 6 :

 6 Fig. 6: Phase noise measurements at 90 GHz with a cross correlation technique (correlation factor of 300). Green: phase noise floor of the signal source analyzer at 90 GHz.

Table 1 :

 1 Measured total CD of three short optical fibers.

		2 )	Dtot (ps/nm)
	288 m fiber	19.6x10 -23	4.66
	413 m fiber	23.4x10 -23	6.46
	2 km fiber	96.4x10 -23	29.55

ACKNOWLEDGMENTS

The research on the COEO is supported by French MoD, Délégation Générale à l'Armement.


Blaise Mtopi Fotso

Maryvonne Dulmet
email: mdulmet@ens2m.fr

Eric Bonjour

Design of product families based on a modular architecture

Our purpose is to present a methodology concerning the design of product families. From the allocation of the overall function into sub-function, we linked the functional model with the structural model of the product. Our methodology is illustrated by an academic example.

Introduction and context

During the last fifteen years, some essential characteristics of the market have changed. In order to remain competitive, companies must meet consumer's needs and expectations. The fast reactivity and adaptation of firms to this changing environment is strongly related to an advanced control of their internal mechanisms. They compete in five dimensions: price, quality, flexibility, delivery and service. In addition to cost and quality criteria, a tendency to decrease the life cycle of product and increase product diversity is observed. In response to these market changes, companies develop mass customization strategies. Mass customization strategy is designed to support mutually contradicting priorities of price and variety wished by the consumer.

Two linked questions are put in light: what degree of variety is need and how to manage and produce this variety with low costs of design and production. The approaches and strategies of design of product families for mass customization is well know [START_REF] Jiao | Product family modeling for mass customization[END_REF], [START_REF] Siddique | On the Applicability of Product Variety Design Concepts to Automotive Platform Commonality[END_REF]. Multiple products are developed based on product families sharing a common platform. Many firms are pursuing modular product architecture design strategies in order to shorten new product development lead-time and to introduce quickly new product variants at reduced cost. This paper is organized into four sections. Next section presents a short literature review, section 3 describes the functional approach and section 4 develops the structural approach, section 5 establishes a link between these two approaches 2 Literature review

Architecture and modularity

The literature gives a significant amount of work focusing on developing methodologies for the design of product families, [START_REF] Erens | The Synthesis of Variety: Developing Product Families[END_REF], [START_REF] Kusiak | Decomposition in Concurrent Design, Engineering[END_REF], [START_REF] Suh | The Principles of Design[END_REF]. We can find several design and manufacturing strategies to offer some variety that begin with commonality metrics. Similar systems produced by firms have different product architecture design is due to different design and technologies choices, [START_REF] Jiao | Generic bill of materials and Operations for high-variety production management[END_REF], [START_REF] Ulrich | Fundamentals of product modularity[END_REF]. "Product architecture" is the arrangement of functional elements of a product into several physical building blocks, including the mapping from functional elements to physical components, and the specification of the interfaces among interacting physical components ; Ulrich [START_REF] Ulrich | The Role of Product Architecture in the Manufacturing Firm[END_REF] defines product architectures as "the scheme by which the function of a product is allocated to physical components". A key feature of product architecture is the degree to which it is modular or integrative. In modular architectures, functional models of the product map one-to-one to its physical components. On the other hand, in integrative architectures a large subset of the products functional models map to a single or small number of components. Eppinger [START_REF] Eppinger | Patterns of product development interactions[END_REF] claims that the product architecture is also the scheme by which "the chunks of a product interact." Product architecture choice depends on the innovation and standardization policy of the firm. A typology of product architecture may be given as follows: architecture can be integral or modular and can be split into 3 sub cases to the way in which modules are interconnected with one another, [START_REF] Ulrich | Fundamentals of product modularity[END_REF]. An ideal architecture is one that splits the product into practical and useful modules. Modules as subsystems of a product are bounded as an unit which presents identifiable functions. In complex products, the chunks of physical components are complex systems as well.

Product Family

A product family is defined as a set of distinguished products whose main functions are similar, it is a set of final products composed of a large number of constituents [START_REF] Ulrich | Fundamentals of product modularity[END_REF]. It is possible to identify similarities and differences in the individual products of the family. Variety is manifested in a set of generic items and a set of variants involving changes and their relationships [START_REF] Liu | Product family and variants: Definition and models[END_REF]. Product families' modeling includes two aspects: representation of a generic structure and dynamic of the process variant derivative. Previously, some works were developed in Laboratoire d'Automatique de Besancon [START_REF] Djemel | Contribution à la conception des systèmes flexibles dans le cas mult -produits[END_REF], [START_REF] Stadzisz | Contribution à une méthodologie de conception intégrée des familles des produits pour l'assemblage[END_REF]. A product family may find its origin in a differentiation process of a base product or in an aggregation process of distinct products. In the first case, the family represents a product series with different technologies, optional parts and functions due to the product evolution and demand for diversity. In the second case, the family represents the standardization of a set of products whose functions and main components are similar. In both cases, the goal is to form a group of products to reduce their variability and, therefore, to decrease investment and production costs, [START_REF] Erens | The Synthesis of Variety: Developing Product Families[END_REF], [START_REF] Erens | Architectures for product families[END_REF], [START_REF] Siddique | On the Applicability of Product Variety Design Concepts to Automotive Platform Commonality[END_REF]. Product design is an evolutionary process from high level of abstraction to detailed low levels of our description. The family description consists of a functional model and a structural model. As every system, a product family has a hierarchical organization. At the functional and the physical levels, the product is decomposable in sub -systems, them-selves composed of modules or constituents.

Our work focuses on the design of product family based on a modular architecture and it is founded on previously developed works in Laboratoire of Besancon. The objective of this paper is to specify the articulation between the functional domain and the organic domain. Our methodology will be illustrated by an academic example: a car wheel family.

Functional approach

Functional modeling, also know as functional decomposition is the process of breaking the overall function of a product into smaller easily solvable sub functions. Pahl & Betz [START_REF] Siddique | On the Applicability of Product Variety Design Concepts to Automotive Platform Commonality[END_REF] provide an overview for such a functional decomposition process. The functional model of a family describes the organization of the functions of each of its products. A product function (also called functional requirement) is the product aptitude for fulfilling an user's desired task or for satisfying an user's expectation. We refer to this kind of functions as main functions (or service function) to differentiate from internal functions of the products and specify the intent of components and subassemblies in the product. In other words, internal functions represent the decomposition of the main functions into subfunctions. Thus, products in a family have the same base set of main functions with eventual, optional main functions, which are specific to some products. Subsequent functional breaking up may produce differentiable or similar sub-functions for products in the family. The functional model is a hierarchical description of the function decomposition. At the highest level, there are the main functions that define the product intents, and at the other levels there are the sub-functions, which establish how main functions will be achieved by components and subassemblies [START_REF] Ulrich | The Role of Product Architecture in the Manufacturing Firm[END_REF]. Associated with the functions, we have functional constraints. These constraints establish the boundary on acceptable solutions and describe dependencies between functions and/or sub-functions. In the multi-product context, constraints also specify diversity of the functions.

In our example, the wheel is composed of the rim and the wheel center. The family offers the choice of two types of rims (tube type and tubeless) two rim diameters (13 and 16 inches) and two kinds of flange (B or J), so we obtain a family of 8 products. Figure 1 illustrates the functional decomposition. A relation of membership bounds these different levels between the functions Figure 1: Functional structure of a wheel family On this figure, the relations of membership between functions are highlighted. We can now establish a typology of the functions in the setting of the families of products, that allows us to distinguish three types of functions: the constant function, the variant function and the partial function [16]. Constant Function: the function is identically applied on products with the same parameters and the same constraints : F 11 , F 12 , F 22, Variant Function: F 1 , F 2 , F 21 F 211 , F 212 , these functions are realized in the eight types of wheels, but the parameters are variable (type of assembly, diameter, type of flanges) . Optional Function: function, which are only present of some product F 11 : concerns only wheel with dismantled wheel centre , F 12 : concerns only wheel with fixed wheel centre F 22 : concerns only rim with tubeless tire This functional decomposition is also translated in the physical domain (components, connections, subassemblies). The more similar products are, the more unified or standardized the assembly system will be. Therefore, the main objective in designing a product family and its corresponding assembly system is to develop a set of products which correspond to functional requirements through the most standardized set of components and production processes.

Structural Approach

All family of products contains a set of products possessing some similarities between them. These similarities express themselves as well at the functional level (as we presented it to the previous paragraph) as at the level of their material constitution (components and common or similar links).

In this section, we are interested in the generic nomenclature concept and in the establishment of a components typology and of the links in a same product family. This typology, as we will further see, is essential to the realization of a model of representation of product families. A composed product is a set of components manufactured in accordance with some assembly process. Independently of the order of the components assembly, a composed product can be considered as a functional arrangement of constituents and of sets of constituents. Djemel in [START_REF] Djemel | Contribution à la conception des systèmes flexibles dans le cas mult -produits[END_REF] qualifies the description of such an arrangement of nomenclature of product. All product variants of the family share a common structure. Once the distinctive structure for each product in the family has been developed, they must reunited into a generic structure. So we obtain a hierarchy comprising constituents of the family [START_REF] Jiao | Product family modeling for mass customization[END_REF]. At the first level we can see (AND tree) a structure, which reveals the topology of the family. At the second level, we placed variety parameters, which represent different instances of a particular component, and at the third level, we put in light variant parameters of the component. The analysis of the functional equivalence relations of the elements of the eight types of wheels leads to the constitution of nine equivalence classes materialized by the following matrix: Table1. Matrix relation between the F i functions and the classes of equivalence

! F 0 F 1 F 11 F 12 F 2 F 21 F 22 F 211 F 212 " # $ $ $ $ $ $ $ $ $ $ $ $ % & ' '
The F i functions are those presented on the figure 1. {S 0 } achieves the F 0 function: overall function of a wheel family, and S 0 is the set of the 8 variants.

In order to better structure and to permit a better exploitation of the matrix, we identified one generic constituent by functional equivalence class. this definition is founded on the hypothesis that in a product family, material differentiation is justified by its functional model, (S 2 S 3 S 6).

Variant component: In this case, one of the functions which the component achieves is variable, the others are constant functions. This type of component is present in all the types of product family. Optional component: generic component is qualified of optional if at least one of the function which it achieves is optional, the other may be constant or variant, (S 2 S 3 S 6).

This example puts in light how it is possible to structure technological solutions obtained from the functional analysis. But we think that this kind of results is strongly linked to the knowledge on the product. And this aspect needs further developments about traceability, project memory and knowledge management.

Conclusion

In this paper we present a methodology to pass through functional requirement to technological solutions in the case of product families; The tree structure provides a way to characterize variant derivation at different levels. The analysis of the functional equivalence relations between these elements leads to the constitution of equivalence classes materialized by a matrix and after that we define a diagonal block matrix (transformation matrix) which links functional characteristics and generic components. This method provides a means to describe variants of the product family. It can be used to present both final products and components.

Figure 2 :

 2 Figure 2: Two examples of distinctive structures of wheels

Figure 3 :

 3 Figure 3: A generic structure representing variety in a wheel family

Cl 1 =

 1 {Wheel centre } = {S 1 } Cl 2 = { Screw Nut}) = {S 2 } }; Cl 3 = ={Unique Fixation =} = {S 3 } Cl 4 = { Rim } = {S 4 } Cl 5 ={ Tube type rim, Tubeless rim } = {S 5 }; Cl 6 ={ Tubeless rim } = {S 6 }; Cl 7 = { Diam_13_B, Diam_16_B, Diam_13_J, Diam_16_J} = {S 7 }; Cl 8 = { Diam_13_B, Diam_16_B, Diam_13_J, Diam_16_J} = {S 8 }; Every element of an equivalence class achieves a function and we can establish some membership relations between components classes. {S 8 typology of functions, we define three kinds of generic constituents of a family: constant component, variant component and optional component Constant component: A generic component is qualified of constant if all the functions it achieves are constant;

6

But the geometrical links between the components do not remain identical in the set of the types of product. Because the components are liable to change from a type of product to another, some links can also be different according to the type of the product in the family.