Solène Breton

Juliette Jouhet

Ulysse Guyet

Valérie Gros

Justine Pittera

David Demory

Frédéric Partensky

Hugo Doré

Morgane Ratin

Eric Maréchal

Ngoc An Nguyen

Laurence Garczarek

Christophe Six
email: christophe.six@sb-roscoff.fr

Unveiling membrane thermoregulation strategies in marine picocyanobacteria

Keywords: cyanobacteria, marine Synechococcus, membrane lipids, temperature ecotype, adaptation

The wide latitudinal distribution of marine Synechococcus cyanobacteria partly relies on the differentiation of lineages adapted to distinct thermal environments. Membranes are highly thermosensitive cell components and the ability to modulate their fluidity can be critical for the fitness of an ecotype in a particular thermal niche.

. We compared the thermophysiology of Synechococcus strains representative of major temperature ecotypes in the field. We measured growth, photosynthetic capacities and membrane lipidome variations. We carried out a metagenomic analysis of stations of the Tara Oceans expedition to describe the latitudinal distribution of the lipid desaturase genes in the oceans.

. All strains maintained efficient photosynthetic capacities over their different temperature growth ranges. Subpolar and cold temperate strains showed enhanced capacities of lipid monodesaturation at low temperature thanks to an additional, poorly regiospecific 9desaturase. In contrast, tropical and warm temperate strains displayed moderate monodesaturation capacities but high proportions of double unsaturations in response to cold, thanks to regiospecific 12-desaturases. The desaturase genes displayed specific distributions directly related to latitudinal variations in ocean surface temperature.

. This study highlights the critical importance of membrane fluidity modulation by desaturases in the adaptive strategies of Synechococcus cyanobacteria during the colonization of novel thermal niches.

Introduction

Marine Synechococcus are among the smallest photoautotrophs on Earth but are thought to be responsible for about 17% of the global marine net primary production (Flombaum et al., 2013). They constitute a much diversified cyanobacterial radiation, most of them being gathered in the phylogenetic subcluster 5.1 sensu [START_REF] Herdman | Form-genus XIII. Synechococcus. In: Bergey's Manual of Systematic Bacteriology[END_REF] that, according to analyses based on the petB gene marker, includes 15 clades and 28 subclades [START_REF] Mazard | Multi-locus sequence analysis, taxonomic resolution and biogeography of marine Synechococcus[END_REF]. A number of phylogeographic studies have shown that five clades (I, II, III, IV and CRD1) predominate in the ocean, occupying different environmental niches. Clades I and IV co-occur in nutrient-rich, temperate and cold regions, whereas clades II and III are usually found in warmer waters, with clades II et III dominating in N and P-depleted areas, respectively [START_REF] Zwirglmaier | Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes[END_REF][START_REF] Sohm | Cooccurring Synechococcus ecotypes occupy four major oceanic regimes defined by temperature, macronutrients and iron[END_REF][START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF][START_REF] Paulsen | Synechococcus in the Atlantic Gateway to the Arctic Ocean[END_REF][START_REF] Kana | Effect of irradiances up to 2000 μE m-2 s-1 on marine Synechococcus WH7803-I. Growth, pigmentation, and cell composition[END_REF]. More recently discovered, the fifth major clade, CRD1, appears to be specialized to iron-limited environments, such as the so-called High Chlorophyll Low Nutrients areas of the South Pacific Ocean [START_REF] Sohm | Cooccurring Synechococcus ecotypes occupy four major oceanic regimes defined by temperature, macronutrients and iron[END_REF][START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF][START_REF] Kana | Effect of irradiances up to 2000 μE m-2 s-1 on marine Synechococcus WH7803-I. Growth, pigmentation, and cell composition[END_REF].

The wide latitudinal distribution of marine Synechococcus, spanning from the equator to polar circles [START_REF] Zwirglmaier | Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes[END_REF][START_REF] Huang | Novel lineages of Prochlorococcus and Synechococcus in the global oceans[END_REF], suggests that the different lineages have developed efficient strategies to colonize distinct thermal environments. Several studies have shown that representative strains of specific clades exhibit thermal preferenda that are in good agreement with the temperature of their isolation sites [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF][START_REF] Mackey | Effect of temperature on photosynthesis and growth in marine Synechococcus spp[END_REF][START_REF] Varkey | Effects of low temperature on tropical and temperate isolates of marine Synechococcus[END_REF][START_REF] Moore | Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity[END_REF]. These genetically distinct lineages, physiologically adapted to distinct niches, thus constitute 'temperature ecotypes', a.k.a. 'thermotypes', a concept previously defined for the other dominant marine cyanobacterium Prochlorococcus [START_REF] Johnson | Partitioning Among Prochlorococcus Ecotypes Along Environmental Gradients[END_REF][START_REF] Coleman | Code and context: Prochlorococcus as a model for cross-scale biology[END_REF]. The physiological bases that underpin the process of thermal niche partitioning of marine picocyanobacteria remain, however, poorly known. Several studies suggest that the ability to adapt the photosynthetic mechanisms to different temperatures could constitute a bottleneck for the competitiveness in different thermal environments [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF][START_REF] Pittera | Adaptive thermostability of light-harvesting complexes in marine picocyanobacteria[END_REF]. In particular, it has been shown that the photosystem II of different Synechococcus temperature ecotypes is differentially affected by thermal stress [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF], and that their photosynthetic antenna, called the phycobilisome, display

Accepted Article

This article is protected by copyright. All rights reserved differential functional stability consistent with the temperature preferenda of the strains [START_REF] Pittera | Adaptive thermostability of light-harvesting complexes in marine picocyanobacteria[END_REF]. Another important component that may directly impact the smooth running of the photosynthetic apparatus at different temperatures is the lipid matrix of thylakoid membranes. It is indeed well known that membranes are among the most thermosensitive cell components, because temperature drastically affects their fluidity and permeability and therefore the activity of all the membrane-embedded proteins [START_REF] Mikami | Membrane fluidity and the perception of environmental signals in cyanobacteria and plants[END_REF]. The ability to modulate the fluidity of membranes, notably the thylakoids, can thus be critical for the fitness of photosynthetic organisms in specific thermal niches.

Cyanobacterial membranes are composed of three main glycolipids, mono-and digalactosyldiacylglycerol (MGDG and DGDG) and a charged sulfolipid, sulfoquinivosyldiacylglycerol (SQDG). In addition, smaller amounts of a phospholipid, phosphatidyldiacylglycerol (PG) are usually observed [START_REF] Murata | Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria[END_REF]. Whereas a number of studies have dealt with the membrane thermoregulation of freshwater cyanobacteria, the composition of the membranes and the impact of temperature in their marine counterparts remain poorly studied [START_REF] Merritt | A comparison of the major lipid classes and fatty acid composition of marine unicellular cyanobacteria with freshwater species[END_REF]Van Mooy et al., 2009;[START_REF] Varkey | Effects of low temperature on tropical and temperate isolates of marine Synechococcus[END_REF]. A recent study has shown that the model marine strain Synechococcus sp. WH7803 contains membrane lipids with shorter fatty acid chains than freshwater cyanobacteria and is able to adjust its membrane fluidity to different growth temperatures [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. Indeed, in response to cold, the WH7803 strain induces the shortening of the fatty acid chains at the sn-1 position of the galactolipids, leading to membrane thinning, and activates fatty acid desaturation at specific sites of the three glycolipids [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF].

Unsaturations can be dynamically inserted in fatty acid chains by lipid desaturase enzymes.

The catalytic site of these enzymes comprises histidine-rich boxes, including a non-heme iron center whose activity requires electrons and oxygen [START_REF] Los | Structure and expression of fatty acid desaturases[END_REF]. Genomic studies of the fatty acid desaturase gene content in marine Synechococcus have shown the presence of four main distinct genes in the genomes, encoding two 9-(desC3 and desC4) and two 12desaturases (desA2 and desA3; [START_REF] Chi | Comparative analysis of fatty acid desaturases in cyanobacterial genomes[END_REF][START_REF] Varkey | Effects of low temperature on tropical and temperate isolates of marine Synechococcus[END_REF]. A closer look at the phyletic profiles of these four genes unveiled that the marine Synechococcus strains representative of the four dominant clades (I through IV) exhibit distinct desaturase gene contents, suggesting that the

Accepted Article

desaturation capacities could be part of the physiological strategies underlying the specialization of these lineages to different thermal environments [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. However, it is not yet known how these differential gene contents affect the composition and thermoacclimation processes of the membrane lipidome in the different temperature ecotypes.

In this study, we analyzed the composition of the membrane lipidome of a selection of four strains representative of each of the four dominant marine Synechococcus clades (I-IV) in natural communities and we unveiled marked differences in the respective regulation processes adjusting membrane fluidity in response to temperature. Furthermore, we show that the global oceanic distribution of the desaturase genes is fully consistent with their differential distribution among the ecotypes, demonstrating the significance of these thermoadaptation strategies in thermal niche partitioning of Synechococcus in situ. We discuss these findings in the context of the important role of marine Synechococcus in the ocean primary productivity.

Materials and methods

Growth conditions and experimental design

Four marine Synechococcus strains retrieved from the Roscoff Culture Collection (http://roscoff-culture-collection.org/) were used in this study (Table 1): MVIR-18-1 (clade I), A15-62 (clade II), WH8102 (clade III) and BL107 (clade IV), of which only WH8102 was axenic.

Strains were grown in PCR-S11 culture medium [START_REF] Rippka | Prochlorococcus marinus Chisholm et al. 1992 subsp. pastoris subsp. nov. strain PCC 9511, the first axenic chlorophyll a2/b2-containing cyanobacterium (Oxyphotobacteria). International Accepted Article This article is protected by copyright[END_REF][START_REF] Moore | Culturing the marine cyanobacterium Prochlorococcus[END_REF] supplemented with 1 mM sodium nitrate under low irradiance (20 µmol photons m -2 s -1 ), in order to ensure the presence of several thylakoidal lamellae in the cells [START_REF] Kana | Effect of irradiances up to 2000 μE m-2 s-1 on marine Synechococcus WH7803-I. Growth, pigmentation, and cell composition[END_REF].

Continuous light was provided by multicolor LED systems (Alpheus, France). The axenicity of Synechococcus sp. WH8102 cultures and minimal contamination levels of the other strains (<20%) was regularly checked by flow cytometry using SYBR-Green staining [START_REF] Marie | Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples[END_REF].

Cultures of the four cyanobacteria were acclimated for several weeks to a range of temperatures (10 to 30°C) in temperature-controlled chambers (Lovibond, Germany), monitored by flow cytometry and sampled during the exponential growth phase to measure photosynthetic parameters and analyze the membrane lipid structure in acclimated state. To study the dynamics

Accepted Article

This article is protected by copyright. All rights reserved of the temperature-induced remodeling of the membranes, we also carried out temperature shift experiments. Three liters of exponentially growing cultures (at 1-3 10 7 cell mL -1 ) maintained at 22°C were split into subcultures and transferred to either 13°C or 30°C, under identical light conditions. One subculture per condition was harvested every day during four days. All experiments were repeated three to four times.

Flow cytometry, in vivo fluorimetry and pigment analyses

Aliquots of cultures were preserved using 0.25% (v/v) glutaraldehyde final concentration (grade II, Sigma Aldrich, St Louis, MO, USA) and stored at -80°C until analysis. Cell concentrations were determined using a flow cytometer (FACS Canto II, Becton Dickinson, San Jose, CA, USA) as described previously [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF]. Growth rates were calculated on cultures preacclimated to a range of temperatures as the slope of a ln(Nt) vs. time plot function, where Nt is the cell concentration at time t. The growth models of [START_REF] Bernard | Validation of a simple model accounting for light and temperature effect on microalgal growth[END_REF] and [START_REF] Boatman | A key marine diazotroph in a changing ocean: The interacting effects of temperature, C0 2 and light on the growth of Trichodesmium erythraeum IMS101[END_REF] were used to model the optimal temperature for growth (T OPT ) and the temperature limits for growth (T MIN and T MAX ; for more details, see Notes S1).

The photosystem II quantum yield (F V /F M ) was measured using a Pulse Amplitude Modulation fluorometer (PhytoPAM I, Walz Effeltrich, Germany) in the presence of 100 µM of the PSII blocker 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), following a previously described procedure [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF]. The quantum yield was calculated as:

F V /F M = (F M -F 0 )/F M
where F 0 is the basal fluorescence level, F M the maximal fluorescence level and F V the variable fluorescence [START_REF] Campbell | Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation[END_REF][START_REF] Ogawa | Estimation of photosynthesis in cyanobacteria by pulse-amplitude modulation chlorophyll fluorescence: problems and solutions[END_REF].

For pigment analyses, 50 to 100 mL of culture were harvested by centrifugation during 10 min, at 4°C and 20,000 x g (Eppendorf 5804R) in the presence of 0.01% (v/v) pluronic acid (Sigma Aldrich, St Louis, MO, USA). The cell pellets were covered with 200 µL of cold methanol (HPLC Grade, Sigma Aldrich, St Louis, MO, USA), vortexed until complete resuspension and incubated for 30 min on ice. The pigment extracts were vortexed again and then centrifuged as described above (Eppendorf 5417R). The supernatants were transferred and supplemented with 10% (v/v) distilled water final volume, in order to avoid peak distortion. Pigments were measured by high

Accepted Article

This article is protected by copyright. All rights reserved pressure liquid chromatography using an HPLC 1100 Series System equipped with a photodiode array (Hewlett-Packard, St Palo Alto, CA, USA) as previously described [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF].

Membrane lipidome analyses

The global detailed procedure has been previously published in [START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF][START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. For each condition, 400 mL of culture were harvested by two successive centrifugation steps of 10 min, at 20,000 x g and 4°C (centrifuges Beckman Avanti J-25 and Eppendorf 5804R) and stored at -80°C until analysis. Membrane lipids were extracted in glass hardware following a modified version of the [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] procedure [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF].

Fatty acid regiolocalization -We first identified the positional distribution of the fatty acids esterified to the four main glycerolipids of the four Synechococcus strains samples. Each strain was grown at three different temperatures spanning their respective thermal growth range. The glycerolipids were then extracted and separated by 2-dimensional thin layer chromatography as previously described [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. Glycerolipids spots were revealed under UV light in the presence of malvidine-3-O-galactoside (primuline, 0.2% in pure methanol, Sigma Aldrich, St Louis, MO, USA) and scraped off the plate. Each separated lipid class were recovered from the silica by extraction in glass hardware and dried under nitrogen gas. The position of the different fatty acid molecular species esterified to the glycerol backbone of the purified glycerolipids was determined by MS/MS analyses as described in [START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF].

Lipid quantification -The quantity of fatty acids in the samples was first determined by gas chromatography coupled with a flame ionization detector (GC-FID). The lipid samples were first methanolyzed and analyzed following a previously described procedure [START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF]. The lipid samples were then prepared in order to inject 25 nmol of total fatty acids dissolved in 100 µL of chloroform/methanol [2/1, (v/v)] containing 125 pmol of each internal standard. The membrane lipids were separated by HPLC (Agilent 1200) equipped with a diol column (150 mm x 3 mm x 5 µm; Macherey-Nagel) at 40°C and quantified by MS/MS with a triple quadrupole mass spectrometer equipped with a jet stream electrospray ion source (Agilent 6460) [START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF][START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. Mass spectra were processed with the Agilent MassHunter Workstation software for lipid identification and quantification. Lipid amounts (pmol) were

Accepted Article

This article is protected by copyright. All rights reserved corrected for response differences between internal standards and endogenous lipids and by comparison with a qualified control (QC). QC extract corresponded to a known lipid extract from Arabidopsis cell culture qualified and quantified by TLC and GC-FID as described in [START_REF] Jouhet | LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation[END_REF].

In situ metagenomic analyses

Accepted Article

of the four desaturase clusters of likely orthologous genes (CLOGs) from the Cyanorak database based on their best hit sequence and read counts were normalized to the average gene length of each CLOG. In order to study the presence/absence of lipid desaturases with regard to thermal environments, only stations dominated by major Ecologically Significant Taxonomic Units (ESTU)

of Synechococcus clades I to IV, namely ESTUs IA, IIA, IIIA or IVA-C sensu [START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF], were used. Furthermore, to ensure sufficient coverage for the results to be reliable, only stations with a minimum of 2,500 distinct CLOGs, corresponding to the average number of genes in Synechococcus genomes, were considered. Altogether 52% (33 out of 63) of the Tara Oceans stations have been retained for the analysis. Reads assigned to genes encoding the four lipid desaturases (desC3, desC4, desA2, desA3) were then extracted and their respective abundances were normalized to the corresponding petB reads abundance, retrieved from [START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF].

Results

Thermal preferenda and photosynthetic activity

We acclimated four Synechococcus strains isolated from different thermal environments to a global range of temperatures spanning from 10 to 32°C. The strains displayed thermal preferenda that significantly differed with regard to their thermal growth limits, especially at cold temperatures, and less so by their optimal growth temperature (Fig. 1A). The subpolar clade I strain MVIR-18-1 showed significant growth down to 9-10°C but could not cope with temperatures higher than 26°C. The cold-temperate clade IV strain BL107 grew between 14°C and 28°C, while the warm temperate clade III strain WH8102 and the tropical clade II strain A15-62 grew from 16°C to more than 32°C. Growth vs. temperature models were used to estimate the growth parameters (Table 1, Fig. S1). Although some differences in these parameters could be observed depending on the model used (Tables S1,S2), the subpolar and cold-temperate strains displayed lower estimates of T MIN and T MAX than the warm-adapted strains (see Notes S1).

The quantum yield of the photosystem II reaction center (F V /F M ) remained generally high over the whole growth temperature ranges (Fig. 1B). The values were however somewhat lower

Accepted Article

at cold growth temperatures (16°C) for strains WH8102 and A15-62, and at warm growth temperatures for the subpolar MVIR-18-1 and cold temperate BL107 strains.

The β-carotene to chl a ratio increased at high growth temperature and this trend was more pronounced for the warm temperature strains WH8102 and A15-62 (Fig. 1C). For all strains, the zeaxanthin to chl a mass ratio was high at low growth temperature and lower at high temperature. This ratio was globally higher in the subpolar strain MVIR-18-1 and in the temperate strain BL107 than in the two other strains (Fig. 1D). These variations probably mostly originated from an increase in the zeaxanthin cell content with decreasing temperature, as suggested by the cold-induced increase of the zeaxanthin to β-cryptoxanthin ratio, the direct precursor of zeaxanthin in its biosynthetic pathway (Fig. S2). It is also likely that the chl a cell content decreased in response to cold.

Membrane lipidomic analyses

General composition of the membrane lipidome -We analyzed the composition of the total membrane lipidome, which in cyanobacteria includes a very high proportion of the thylakoidal lipids. The 2-dimensional thin layer chromatography (TLC) analyses revealed four major spots corresponding to the lipid classes usually observed in cyanobacteria: MGDG, DGDG, SQDG and PG. In total, there were about 30 different molecular species of complex lipids in the four studied strains of marine Synechococcus. The two galactolipids (MGDG and DGDG) were generally the major components of membranes and the fatty acids bound to these lipids were mostly C14, C16

and traces of C18 fatty acid chains. The number of unsaturations on these chains never exceeded 2. The global composition of the fatty acids bound to the four different lipids in these strains, reported in detail in Tables S3-6 and on Figs. S3-6 (see Notes S2), was overall similar to Synechococcus sp. WH7803 [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. The sn-1 position most often bound C16 chains, whereas the sn-2 bound exclusively C14 chains on the galactolipids (Figs. S3-4) and a mix of C14 and C16 chains on SQDG (Fig. S5).

PG is present in heterotrophic bacteria, complicating the interpretation of the PG data of the 3 non-axenic strains. In cyanobacteria, this minor lipid is thought to play roles in the stabilization of photosystems [START_REF] Itoh | Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone QBin photosystem II[END_REF][START_REF] Mizusawa | The role of lipids in photosystem II[END_REF]Boudière et al., 2014) rather than being directly involved in membrane thermoacclimation processes, as supported by the results

Accepted Article

This article is protected by copyright. All rights reserved for the axenic strains WH8102 and WH7803 [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]; Fig. S6). Hereafter we therefore compare the major mechanisms involved in thermoacclimation only for the three major membrane glycolipids only present in cyanobacteria.

Membrane thickness variations in response to temperature -As palmitic (C16:0) and myristic (C14:0) fatty acid chains were dominant in all glycolipids of all strains, we used the C14:C16 molar ratio as a proxy for assessing the variations of the membrane thickness. In the four strains, such variations may occur at the sn-1 position of the two most abundant lipids, MGDG and DGDG, as already observed in Synechococcus sp. WH7803 [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF], and at the sn-2 position of SQDG. The results showed an increase of the C14:16 ratio at the sn-1 position of MGDG and DGDG ratio in response to low growth temperatures only for the subpolar strain MVIR-18-1 and the tropical strain A15-62 (Fig. 2). This process was more pronounced for the MGDG than for to the DGDG. In contrast, BL107 and WH8102 strains showed a slight increase of the fatty acid length at these positions in response to low growth temperature. MVIR-18-1 was additionally capable of decreasing the length of the fatty acid bound to the sn-2 position of the SQDG in response to growth temperature lower than 14°C (Fig. S5). In the cultures grown at 22°C and shifted to either 13 or 30°C, only little variations of the C14:C16 ratio were observed (Figs. S7-14).

Mono-desaturation processes on galactolipids -MGDG showed the highest mono-unsaturation levels, with high proportions of 14:1 and 16:1 fatty acid chains. At the sn-1 position, the proportion of C16:1 chains increased in response to low growth temperature, especially in the subpolar strain MVIR-18-1 and the cold temperate strain BL107 (Fig. 3A-B). In contrast, the warm-adapted strains WH8102 and A15-62 showed much lower variations of the C16:1 relative level.

The proportion of C14:1 chains at the MGDG sn-1 position also increased at lower temperatures in all strains except for the warm temperate WH8102, for which the C14 species were low at this position. The largest C14:1 to C14:0 amplitude changes were achieved by the subpolar strain MVIR-18-1. It is likely that this resulted from the replacement of C16 chains by C14:1 chains in de novo synthetized MGDG molecules, rather than active desaturation of C14:0 chains by desaturase enzymes, inducing the abovementioned chain shortening. This conclusion is

Accepted Article

This article is protected by copyright. All rights reserved supported by the fact that no rapid complementary decrease of C14:0 was observed in acclimated cultures (Fig. 3) nor during thermal shift experiments (Fig S7 ,S11).

The proportion of C14:1 chains increased at the MGDG sn-2 position in the subpolar MVIR-18-1 and in both temperate strains in response to low temperature, with WH8102 showing the largest changes (Fig. 4A). Complementary changes between C14:0 and C14:1 chains were observed during the shift experiments, suggesting a 9-desaturation activity at this position (Fig. S7 andS11).

The DGDG showed overall similar variations to MGDG at both sn-1 (Fig. 3C,D) and sn-2 (Fig. 4B) positions, but the global C16 and C14 mono-unsaturation levels were however lower than for MGDG. This suggests the occurrence of an active C16:0 mono-desaturation process at the sn-1 site, especially for the subpolar strain MVIR-18-1, as also observed in the cold (Fig. S8) and warm (Fig. S12) shift experiment results. The insertion of de novo synthetized C14:1 chains at the DGDG sn-1 (Fig. 3D) and sn-2 (Fig. 4B) positions in cultures acclimated to low temperature were observable for all strains, but were weaker than for MGDG.

Mono-desaturation processes on SQDG -As already observed in Synechococcus sp. WH7803, SQDG showed a quite different fatty acid composition from the two galactolipids [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. In the four strains, the sn-1 position bound quasi exclusively C16 chains (Fig. S5) and was seemingly the site of an active 9-mono-desaturation activity in cultures acclimated to low temperature (Fig. 3E,F), as well as in cultures subjected to temperature shifts (Fig. S9,S13). The sn-2 position bound C14 and C16 chains, often in comparable proportions (Fig. S5). Active C16:0 mono-desaturation was induced in cultures acclimated to low temperature in the four strains, again with greater amplitudes in the subpolar strain MVIR-18-1 and the cold-temperate strain BL107, along with a weak increase in the C14:1 proportion (Fig. 4D, S9, S13).

Double desaturation processes -We used the C16:2 to C16:1 ratio to study the level of double unsaturation of the fatty chains of the different lipids (Fig. 5, S3-S6). Interestingly, double unsaturations occurred exclusively on C16 chains, at the sn-1 position of the two galactolipids.

The global level of double unsaturation was quite high in the strains adapted to warm environments, WH8102 and A15-62, whereas C16:2 chains were hardly detected in the coldtemperate and subpolar strains BL107 and MVIR-18-1 (Fig. 5). Noteworthy, strain WH8102

Accepted Article

This article is protected by copyright. All rights reserved responded to low temperature by a large induction of 16:2 at the sn-1 of MGDG and, to a lesser extent, DGDG. The capacity for efficient double desaturation activity of strain WH8102 was also observable during the cold shift experiments (Fig. S7).

Metagenomics of lipid-desaturase genes

Previous studies have shown that marine Synechococcus temperature ecotypes display different sets of genes encoding lipid-desaturases, including the 9-desaturases DesC3 and DesC4 and the 12-desaturases DesA2 and DesA3 (Notes S3; [START_REF] Varkey | Effects of low temperature on tropical and temperate isolates of marine Synechococcus[END_REF][START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. In order to complement the results obtained on strains representative of cold-(I and IV)

and warm-adapted (II and III) clades, we used the Tara Oceans dataset to study the global distribution and prevalence of the four desaturase genes along natural thermal gradients (Fig. 6).

The prevalence of the desC3 gene was close to 1 at all stations, independently of the seawater temperature and ESTU composition (Fig. 6B). This shows that this desaturase gene is present in all Synechococcus cells in situ in a single copy, just like petB. By contrast, the desC4 gene was virtually absent from waters warmer than 20°C (Fig. 6C). Below this threshold, the prevalence of this gene increased quasi linearly as seawater temperature decreased, reaching values close to 1 in 13°C waters. This gene was most abundant in populations colonizing waters below 17°C, dominated by Synechococcus ESTUs IA and IVA-C [START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF].

The prevalence of the desA2 gene, which encodes a 12-desaturase inserting a double unsaturation, showed its maximal value of c.a. 1 for temperatures higher than 20°C (Fig. 6D).

Below this temperature, the prevalence progressively decreased down to zero for seawater temperatures lower than 14°C. In this dataset, the desA2 gene is mostly present in populations dominated by Synechococcus ESTUs IIA and IIIA.

The other 12-desaturase, desA3, showed a similar global pattern to the desC4 gene with a marked shift in the relative abundance of this gene between populations dominated by Synechococcus ESTUs IA and IVA-C, colonizing waters below 18°C and those dominated by ESTU IIA, thriving in waters above this temperature. In contrast, it is worth noting that in Mediterranean Sea stations dominated by Synechococcus ESTU IIIA, the desA3 gene prevalence remained high independently from temperature (Fig. 6E), consistently with the fact that desA3 is

Accepted Article

This article is protected by copyright. All rights reserved present in the genome of all strains representative of clade III, while it is generally absent from most clade II strains [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF].

Discussion

Photosynthesis regulation in cold-and warm-adapted Synechococcus ecotypes

Previous studies have shown that adaptation to temperature in marine Synechococcus relies on the existence of physiologically specialized lineages that have colonized different thermal environments [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF][START_REF] Varkey | Effects of low temperature on tropical and temperate isolates of marine Synechococcus[END_REF]. Strains used in the present study have been isolated in water masses with contrasted temperatures, from tropical to subpolar habitats, and belong to the four major Synechococcus clades (Table 1). The study of their thermal preferenda showed that the two strains isolated from warm environments, Synechococcus spp.

A15-62 (clade II) and WH8102 (clade III), were able to grow at temperatures higher than 30°C.

This result is consistent with previous studies on the Sargasso Sea strains WH8103 (clade III) and WH7803 (clade V; [START_REF] Moore | Phytoplankton in the ocean use non-phosphorus lipids in response to phosphorus scarcity[END_REF][START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF][START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. In contrast, BL107 (clade IV) and MVIR-18-1 (clade I) strains, isolated from temperate and subpolar environments respectively, could not cope with these high temperatures but were able to grow down to 12°C or less (Fig. 1A). These results are in good agreement with previous observations that clades I and IV cells predominate in relatively cold environments, whereas clades II and III preferentially thrive in warmer waters [START_REF] Zwirglmaier | Global phylogeography of marine Synechococcus and Prochlorococcus reveals a distinct partitioning of lineages among oceanic biomes[END_REF][START_REF] Sohm | Cooccurring Synechococcus ecotypes occupy four major oceanic regimes defined by temperature, macronutrients and iron[END_REF][START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF][START_REF] Paulsen | Synechococcus in the Atlantic Gateway to the Arctic Ocean[END_REF][START_REF] Kana | Effect of irradiances up to 2000 μE m-2 s-1 on marine Synechococcus WH7803-I. Growth, pigmentation, and cell composition[END_REF]. Moreover, it worth noting that these differences in thermal preferenda would be likely even more apparent with cultures grown under higher light irradiance, as discussed in previous studies [START_REF] Pittera | Connecting thermal physiology and latitudinal niche partitioning in marine Synechococcus[END_REF][START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF].

All four strains managed to maintain fairly high photosystem II quantum yield over their whole temperature growth range, showing an efficient acclimation of their photosynthetic apparatus to different temperatures (Fig. 1B). Pigment analyses suggest that this ability partly relies upon changes in the relative proportions of major pigments in thylakoid membranes, which differed between strains. Synechococcus spp. WH8102 and A15-62 showed a marked increase of the β-carotene to chl a ratio in response to high growth temperature, whereas this increase was

Accepted Article

This article is protected by copyright. All rights reserved moderate in cold-adapted strains (Fig. 1C). Such variations might originate from a change in the photosystem (PS) ratio since these complexes bind the two pigments in fixed but very different proportions. As the β-carotene to chl a molar ratio is 0.23 in PSI [START_REF] Xu | Function and Structure of Cyanobacterial Photosystem I[END_REF] and 0.31 in PSII monomers [START_REF] Umena | Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å[END_REF], our results thus suggest an increase of the PSII to PSI ratio in response to high temperatures. This would lead to decrease the cyclic electron transport around PSI and increase the transthylakoidal pH gradient, then enhancing the ATP production in the cytosol of the warm adapted ecotypes at high growth temperature. In addition, β-carotene and chl a molecules have recently been found to be bound to High Light Inducible Proteins (HLIPs) in Synechocystis sp. PCC 6803, which are stress-inducible proteins allowing dissipation of excess light [START_REF] Dolganov | Cyanobacterial protein with similarity to the chlorophyll a/b binding proteins of higher plants: evolution and regulation[END_REF][START_REF] Komenda | Cyanobacterial high-light-inducible proteins -Protectors of chlorophyllprotein synthesis and assembly[END_REF]. The characterized proteins, whose dissipative activity is temperature-dependent, have a 0.33 (HliD) and 0.5 (HliC) β-carotene to chl a ratio [START_REF] Staleva | Cyanobacterial ancestor of plant antenna proteins[END_REF][START_REF] Shukla | Binding of pigments to the cyanobacterial high-light-inducible protein HliC[END_REF]. Even though such proteins have never been biochemically characterized in marine Synechococcus, their genomes contain many hli genes, which likely encode similar pigmented proteins [START_REF] Bhaya | Analysis of the hli gene family in marine and freshwater cyanobacteria[END_REF][START_REF] Scanlan | Ecological genomics of marine picocyanobacteria[END_REF].

Therefore, the regulation of such proteins might also be involved in temperature acclimation in marine Synechococcus, especially in the warm-adapted strains WH8102 and A15-62.

In all strains, the zeaxanthin to chl a ratio increased in response to low temperature (Fig. 1D).

Moreover, this ratio was globally higher in the cold-adapted strains MVIR-18-1 and BL107.

Variations of the zeaxanthin to chl a ratio likely originated partly from a decrease of the chl a cell content, a classical cold-induced response, in order to regulate light utilization in slow growth conditions [START_REF] Inoue | Acclimation to the growth temperature and the high-temperature effects on photosystem II and plasma membranes in a mesophilic cyanobacterium, Synechocystis sp. PCC 6803[END_REF]. However, an upregulation of the zeaxanthin synthesis was also most likely involved, as suggested by the increase of the zeaxanthin to β-cryptoxanthin ratio (Fig. S2), the latter pigment being directly converted to zeaxanthin by the β-carotene hydroxylase (CrtR). Even though the localization and function of zeaxanthin have never been formally demonstrated in marine Synechococcus, it could have a role in photoprotection by dissipating excess light under low temperature conditions, a process seemingly more prevalent in coldadapted strains (Fig. 1). On another hand, it is well known that the integration of polar xanthophylls into biological and model membranes strongly affects their properties (for a review see [START_REF] Popova | Carotenoid-Lipid Interactions[END_REF]. If zeaxanthin occurs as a free molecule in marine Synechococcus thylakoids, it is expected to decrease the membrane fluidity at low temperature,

Accepted Article

This article is protected by copyright. All rights reserved as shown in Antarctic bacteria [START_REF] Jagannadham | Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum[END_REF]. Yet, this potentially deleterious increase in membrane rigidity at low temperature seems to be counteracted by modifications of the membrane composition in order to maintain proper membrane fluidity.

Marine Synechococcus thermoregulate the membrane composition differently

Analysis of all molecular species of membrane lipids in four phylogenetically and physiologically distinct strains, as well as comparisons with our previous data on the model strain WH7803 [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF], allowed us for the first time to draw a broad picture of the composition of the membranes in marine Synechococcus. These five strains exhibit general features that seem to be specific of this group, including notably the variability of the chain bound to the SQDG sn-2 position and the relative insensitivity of the composition of PG to temperature [START_REF] Murata | Acyl-lipid desaturases and their importance in the tolerance and acclimatization to cold of cyanobacteria[END_REF][START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. Also in sharp contrast to freshwater strains, all five marine Synechococcus strains contain very low relative concentrations of C18 chains and are C14-rich. Therefore, while freshwater strains have C18 and C16 at the sn-1 and sn-2 positions respectively, marine Synechococcus lipids preferentially bind C16 and C14 at these positions, respectively. This fatty acid composition implies that marine Synechococcus thylakoids are probably thinner and globally more fluid than those of freshwater strains. This is also in good agreement with the much smaller cell volume of these picocyanobacteria compared to their freshwater counterparts. In addition, it is worth noting that C14:0 is usually one of the major fatty acids in samples from surface ocean regions (see e.g. [START_REF] Wakeham | Organic geochemistry of particulate matter in the eastern tropical North Pacific Ocean: Implications for particle dynamics[END_REF]Van Mooy and Fredricks, 2010). Thus, the finding that C14 dominates in all marine Synechococcus strains investigated so far further confirms that marine picocyanobacteria are a major source of this lipid in the oceans.

Some strains have however the capacity to modify the C16:C14 ratio, and thus to modulate the membrane thickness. Indeed, our results and a previous study show that, whereas it slightly increased in BL107 (clade IV) and WH8102 (clade III) in response to cold temperature, strains MVIR-18-1 (clade I), A15-62 (clade II) and WH7803 (clade V) were able to significantly decrease the membrane thickness by synthetizing de novo shorter lipids, therefore increasing its fluidity (Fig. 2; [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]. As these strains inhabit very different thermal environments, the capacity to carry out this process does not appear to be related to the thermotype

Accepted Article

This article is protected by copyright. All rights reserved differentiation. In order to better understand these different responses, future research should investigate the influence of other abiotic parameters, on a larger number of strains.

In response to temperature variations, the Synechococcus strains deployed a panel of different desaturation activities. Our result show that the cold-adapted strains MVIR-18-1 (clade I) and BL107 (clade IV) can carry out extensive desaturation of fatty acid mono-desaturation, on both C16 and C14 chains and at both sn positions of the three membrane glycolipids (Figs. 3,4).

Noteworthy, the primary 9 monounsaturations are expected to have larger membrane fluidifying effects than the secondary 12 unsaturations [START_REF] Hyvonen | Inequivalence of single CHa and CHb methylene bonds in the interior of a diunsaturated lipid bilayer from a molecular dynamics simulation[END_REF][START_REF] Hyvonen | Molecular dynamics simulations of unsaturated lipid bilayers: effects of varying the numbers of double bonds[END_REF][START_REF] Ollila | Polyunsaturation in Lipid Membranes : Dynamic Properties and Lateral Pressure Profiles[END_REF]. These large 9 monodesaturation capacities are thus likely critical for tolerance to low temperature. The warm-adapted strains A15-62 (clade II) and WH8102 (clade III) were overall able to carry out lower monodesaturation activities (Figs. 3,4).

Nevertheless, the tropical A15-62 strain showed stable and relatively high 16:2 content in the galactolipids and WH8102 grown at low temperature (Figs. 5 andS7) induced the highest contents of double unsaturations, whereas these were only detected at trace levels in the coldadapted strains. Thus, in contrast to cold-adapted strains, strains adapted to warm environments seem to display much higher relative contents of double insaturated fatty acids in their membranes.

Synechococcus thermotypes use different lipid-desaturase enzymes

The five strains of marine Synechococcus studied to date in the context of membrane lipid thermoacclimation capacities possess different sets of desaturase genes [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF] Table 1). The lipidomic analyses showed that all strains were able to induce 9monodesaturations, which is consistent with the presence of desC3 in all strains. The lipidic profiles of the A15-62 and WH8102 strains (Figs. 3,4), which only possess DesC3 to synthesize monounsaturated chains, show that this enzyme can incorporate 9 double bonds on the three glycolipids, at the two sn positions and on both palmitic (C16:0) and myristic (C14:0) chains.

Therefore, it appears to be a poorly regiospecific enzyme, responsible for the basal (but essential) monodesaturation capacity of all marine Synechococcus. The cold-adapted strains MVIR-18-1 and BL107, which contain an additional 9-desaturase gene (Table 1), desC4, showed increased monodesaturation capacities. This gene, thought to have derived from a recent gene

Accepted Article

This article is protected by copyright. All rights reserved duplication event [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF], could then provide additional monodesaturation capacity in order to cope with lower temperatures. However, on the lipids of these cold-adapted strains, we could not evidence sites of monodesaturation that would be specific of the DesC4 enzyme.

Therefore, our results suggest that the two DesC enzymes in marine Synechococcus are rather generalist monodesaturases of the C16 and C14 chains of glycolipids, i.e. display a relatively weak regiospecificity. This contrasts with the DesC1 and DesC2 enzymes of freshwater strains, which show differential site specificities [START_REF] Chintalapati | A novel Delta9 acyl-lipid desaturase, DesC2, from cyanobacteria acts on fatty acids esterified to the sn-2 position of glycerolipids[END_REF]. Future work should aim at studying the possible induction of the desC4 gene by low temperature, which would provide further evidence of a monodesaturation booster function of the DesC4 enzyme in marine Synechococcus.

Double unsaturations were detected mostly in A15-62 and WH8102, which like all other warm-adapted ecotypes sequenced to date, possess a specific 12-desaturase, DesA2 (Table 1).

In contrast to monounsaturations, the double unsaturations are located specifically on C16 chains at the sn-1 of the galactolipids binding a C14:1 chain at the sn-2 site, indicating a high level of regiospecificity of DesA enzymes in marine Synechococcus. It is worth noting that the WH8102 strain possesses two 12-desaturases, DesA3 and DesA2. This likely allows this strain to induce the strong double desaturation activities that were observed in response to low temperature (Fig. 5, Fig. S7H,K). The role of DesA3 in cold-adapted strains remains enigmatic since very few double unsaturations were detected in these strains both in acclimated cultures and temperature shift experiments. It is possible that, in these strains, this enzyme is activated in response to other environmental factors, such as high irradiance.

The lipid-desaturase genes show differential distributions in the Ocean

In order to provide more insights about the physiological strategies used by Synechococcus temperature ecotypes to thermoregulate their membranes, we studied the distribution of the four lipid desaturase genes at 33 widespread stations along the Tara Oceans transect (Fig. 6A). As expected, the core gene desC3 displayed a temperature-independent distribution. In contrast, the 9-desaturase gene desC4, which is specific of cold-adapted strains (Table 1), was found exclusively in waters colder than 20°C, consistent with its hypothesized role of monodesaturation enhancer enabling growth in cold thermal environments.

Accepted Article

This article is protected by copyright. All rights reserved

The desA2 distribution displayed a mirror pattern with regard to desC4, confirming that this 12-desaturase is specific of warm environments, dominated by either clade II or clade III Synechococcus cells. Like desC4, the 12-desaturase gene desA3 was abundant in waters colder than 20°C. This gene was also abundant between 17 and 25°C, but specifically in assemblages dominated by clade III strains, consistent with the presence of this gene in all clade III but no clade II strains sequenced thus far (Table 1). Clade III Synechococcus are most abundant in oligotrophic, P-limited areas such as the Mediterranean Sea [START_REF] Mazard | Is the distribution of Prochlorococcus and Synechococcus ecotypes in the Mediterranean Sea affected by global warming?[END_REF][START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF], which is subjected to a strong seasonality, being warm in summer (up to 30°C) but cold in winter, with minimal temperatures as low as 13°C in the northwestern basin. Our results thus suggest that the presence of both desC2 and desC3 genes confers fitness to clade III strains, which are subjected to much stronger seasonal variability over the year than their (sub)tropical clade II counterparts.

Conclusion and perspectives

Our study shows that marine Synechococcus temperature ecotypes can manage, via different mechanisms, to regulate their photosynthetic apparatus in order to maintain optimal photosynthetic capacity over most of their respective temperature growth range. We also show that marine Synechococcus have evolved different physiological strategies based on the presence of specific lipid desaturase gene sets, allowing them to cope with changes in the fluidity of photosynthetic membrane in different thermal niches. Even though more work such as mutant characterization is necessary, our data allow to propose functions and ecological roles for the four Synechococcus desaturases. DesC3 is a constitutive 9-desaturase with weak regiospecificity and its activity is probably mostly temperature-independent. Synechococcus strains belonging to clades I and IV use an additional enzyme, DesC4, which provide them with enhanced 9 monodesaturation capacities and allow to greatly increase the membrane fluidity in cold environments. The occurrence of the desC4 gene, likely by duplication of its common ancestor with desC3 [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF], has probably been an important event for the colonization of high latitude environments by marine Synechococcus. In warm environments, there is little need to perform strong variations of the membrane fluidity and marine Synechococcus have seemingly

Accepted Article

This article is protected by copyright. All rights reserved adopted a different strategy. Clade II strains, which live in warm and stable environments, have globally poor desaturation capacities. However, they constitutively contain membrane lipids with more double unsaturation thanks to DesA2, a 12-desaturase specific of warm environments. In warm-temperate environments, where temperature is more variable, clade III strains use the combination of two 12-desaturases DesA2 and DesA3. They confer high capacities of double desaturations, which probably help tolerating the low winter temperatures in this niche. The preferential use of double unsaturations, which have less influence on the membrane fluidity than the monounsaturations, likely allows a finer tuning of this physical property in the thermal environments of clade II and III strains than their cold-adapted counterparts. An alternative hypothesis is that 12-desaturases might be most efficient at high temperature, while 9desaturases were selected in colder environments during the evolution of marine Synechococcus.

In conclusion, this study strongly suggests that the capacity to greatly modulate membrane fluidity has been critical for the colonization of different thermal niches by marine Synechococcus, leading to the differentiation in physiologically distinct temperature ecotypes. It furthermore shows that lipid-desaturase genes are interesting markers for monitoring the dynamics of ocean microbial communities in the global change context. and [START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF], respectively. The average seawater temperature is the annual mean temperature at the isolation site of the strain over the 2000-2010 period, as derived from the NASA Sea Surface Temperature data (https://oceandata.sci.gsfc.nasa.g). The modelled values of thermal limits of growth were calculated using the phytoplankton growth model defined by [START_REF] Boatman | A key marine diazotroph in a changing ocean: The interacting effects of temperature, C0 2 and light on the growth of Trichodesmium erythraeum IMS101[END_REF]. Presence of a desaturase gene in a strain is indicated by grey color.

List of the tables and legends

Notes S3: Fatty acid desaturase Metagenomics

Table S1: Parameters calculated with the BR model.

Table S2: Parameters calculated with the Boatman model.

Table S3: Variations of the acyl chains esterified on the MGDG, DGDG, SQDG and PG of Synechococcus sp.

MVIR-18-1 grown at different temperatures.

Table S4: Variations of the acyl chains esterified on the MGDG, DGDG, SQDG and PG of Synechococcus sp.

BL107 grown at different temperatures.

Table S5: Variations of the acyl chains esterified on the MGDG, DGDG, SQDG and PG of Synechococcus sp.

WH8102 grown at different temperatures.

Table S6: Variations of the acyl chains esterified on the MGDG, DGDG, SQDG and PG of Synechococcus sp.

A15-62 grown at different temperatures.

Accepted Article

This article is protected by copyright. All rights reserved

Accepted Article

This article is protected by copyright. All rights reserved

Functional

  assignment of metagenomics data -Reads were first recruited against a database of 155 protein sequences of the four lipid desaturases (DesC3, DesC4, DesA2, DesA3) extracted from the 53 Synechococcus/Cyanobium genomes of the information system Cyanorak v2 (http://sb-roscoff.fr/Phyto/cyanorak;[START_REF] Pittera | Thermoacclimation and genome adaptation of the membrane lipidome in marine Synechococcus[END_REF]; Dataset S1) using BLASTX analyses (v 2.7.1+;[START_REF] Altschul | Basic Local Alignment Search Tool[END_REF] with default parameters, limiting the results to one target sequence (--max_target_seqs 1), and considering only the results with an e-value lower than 0.001. The recruited reads were then mapped with BLASTN (with the same options as above) against a database including the 155 above mentioned Synechococcus/Cyanobium desaturases as well as many sequences used as outgroups including 117 Prochlorococcus fatty acid desaturase sequences (Dataset S2), 185 outgroup cyanobacterial genomes from Cyanobase plus 537 genomes from proGenomes (Dataset S3), 6,092 additional non-picocyanobacterial fatty acid desaturase sequences retrieved from NCBI (Dataset S4) as well as all marine picocyanobacterial desaturases from Cyanorak. Reads showing a best hit to outgroup sequences or with less than 90% of their sequence aligned were removed and the remaining reads were assigned to Synechococcus according to their BLASTN best hit. Reads were then functionally assigned to one

Figure 1 :

 1 Figure 1: Variations of growth rates (A), photosystem II quantum yield (F V /F M ; B) and membrane pigment mass ratios (C, D) in Synechococcus sp. MVIR-18-1 (blue circles), BL107 (green diamonds), WH8102 (orange triangles) and A15-62 (red squares), acclimated to a range of temperatures spanning from 10°C to 30°C. Zea: zeaxanthin; Chl a: chlorophyll a; β-car: βcarotene. Measurements are average of four replicates with error bars ± SD.

Figure 2 :

 2 Figure 2: Variations of the C14 to C16 molar ratio, related to the average length of the fatty acid bound to the sn-1 position of the monogalactosyldiacylglycerol (MGDG, A) and the digalactosyldiacylglycerol (DGDG, B) for the four marine Synechococcus strains: MVIR-18-1 (blue circles), BL107 (green diamonds), WH8102 (orange triangles) and A15-62 (red squares), acclimated to a range of temperatures spanning from 10°C to 30°C. Measurements are average of three to four replicates with error bars ± SD.

Figure 3 :

 3 Figure 3: Variations of the mono-desaturation level, expressed as the C16:1 to C16:0 (left) and C14:1 to C14:0 (right) molar ratios, of the fatty acid bound to the sn-1 position of the monogalactosyldiacylglycerol (MGDG, A and B), digalactosyldiacylglycerol (DGDG, C and D) and sulfoquinovosyldiacylglycerol (SQDG, E and F) for the four marine Synechococcus strains: MVIR-18-1 (blue circles), BL107 (green diamonds), WH8102 (orange triangles) and A15-62 (red squares), acclimated to a range of temperatures spanning from 10°C to 30°C. Measurements are average of three to four replicates with error bars ± SD.

Figure 4 :

 4 Figure 4: Variations of the mono-desaturation level, expressed as the C16:1 to C16:0 (left) and C14:1 to C14:0 (right) molar ratios, of the C14 fatty acid bound to the sn-2 position of the Accepted Article

Figure 5 :

 5 Figure 5: Variations of the double-desaturation level, expressed as C16:2 to C16:1 molar ratios, of the fatty acid bound to the sn-1 position of the monogalactosyldiacylglycerol (MGDG, A), digalactosyldiacylglycerol (DGDG, B), for the four marine Synechococcus strains acclimated MVIR-18-1 (blue circles), BL107 (green diamonds), WH8102 (orange triangles), and A15-62 (red squares) acclimated to a range of temperatures spanning from 10°C to 30°C. Measurements are average of three to four replicates with error bars ± SD.

Figure 6 :

 6 Figure 6: Localization of the stations of the Tara Oceans expedition used in this study (A), andprevalence of the 9 lipid-desaturase genes desC3 (B), desC4 (C) and the 12 lipid-desaturase genes desA2 (D) and desA3 (E), as a function of seawater temperature at these stations. This was determined using metagenomic data from the Tara Oceans expedition for stations dominated by major Ecologically Significant Taxonomic Units (ESTUs) sensu[START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF] within Synechococcus clades I to IV. At each station, the relative abundance of each desaturase gene was normalized by the relative abundance of the core, single-copy gene petB The LOESS regression model was used as curve fitting method with a span α = 0.8 using the ggplot2 R package (https://cran.r-project.org/web/packages/ggplot2/index.html). The grey shading areas correspond to the 95% confidence interval.Accepted Article

Figure S1 :

 S1 Figure S1: Growth rate vs. temperature modelled curves with the BR and Boatman models.

Figure S2 :

 S2 Figure S2: Variations of the zeaxanthin to β-cryptoxanthin mass ratio function of growth temperature.

Figure S3 :

 S3 Figure S3: Variations in acyl chains esterified on the MGDG for the four Synechococcus strains grown at different temperatures.

Figure S4 :

 S4 Figure S4: Variations in acyl chains esterified on the DGDG for the four Synechococcus strains grown at different temperatures.

Figure S5 :

 S5 Figure S5: Variations in acyl chains esterified on the SQDG for the four Synechococcus strains grown at different temperatures.

Figure S6 :

 S6 Figure S6: Variations in acyl chains esterified on the PG for the four Synechococcus strains grown at different temperatures.

Figure S7 :

 S7 Figure S7: Variations in acyl chains esterified on the MGDG for the four Synechococcus strains in response to a temperature shift from 22°C to 13°C.

Figure S8 :

 S8 Figure S8: Variations in acyl chains esterified on the DGDG for the four Synechococcus strains in response to a temperature shift from 22°C to 13°C.

Figure S9 :

 S9 Figure S9: Variations in acyl chains esterified on the SQDG for the four Synechococcus strains in response to a temperature shift from 22°C to 13°C.

Figure S10 :

 S10 Figure S10: Variations in acyl chains esterified on the PG for the four Synechococcus strains grown in response to a temperature shift from 22°C to 13°C.

Figure S11 :

 S11 Figure S11: Variations in the acyl chains esterified on the MGDG for the four Synechococcus strains in response to a temperature shift from 22°C to 30°C.

Figure S12 :

 S12 Figure S12: Variations in the acyl chains esterified on the DGDG for the four Synechococcus strains in response to a temperature shift from 22°C to 30°C.

Figure S13 :

 S13 Figure S13: Variations in the acyl chains esterified on the SQDG for the four Synechococcus strains in response to a temperature shift from 22°C to 30°C.

Figure S14 :

 S14 Figure S14: Variations in the acyl chains esterified on the PG for the four Synechococcus strains in response to a temperature shift from 22°C to 30°C.

Table 1 :

 1 Characteristics of the four Synechococcus strains used in this study. Taxonomic assignment at the clade level and desaturase gene content are derived from[START_REF] Farrant | Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria[END_REF]

Breton et al., Table 1

	Strain name RCC # Clade	Isolation site	Coordinates	Average seawater temperature (°C)	T MIN (°C) T MAX (°C)	Lipid-desaturase gene content 9-desaturase 12-desaturase desC3 desC4 desA2 desA3
	MVIR-18-1	2385	I	North Sea	+61°0', +1°59'	10.4 ± 0.3	1.0	28.8
	BL107	515	IV	Mediterranean Sea +43°43', +3°33'	17.9 ± 0.3	10.37	28.4
	A15-62	2374	II	Offshore Mauritania +17°37', -20°27'	23.7 ± 0.4	9.0	30.8
	WH8102	539	III	Caribbean Sea	+22°29', -65°3'	27.1 ± 0.1	13.9

Acknowledgements

This work was funded by the French programs EC2CO-Microbien (METALIC), ANR CINNAMON (ANR-17-CE2-0014-01) and EMBRC France (INFRA-2010-2.2.5). Solène Breton and Ulysse Guyet were supported by the Région Bretagne and the French Ministry of Higher Education and Research. Juliette Jouhet and Eric Maréchal are supported by the French National Research Agency (ANR-10-LABEX-04 GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology; ANR-11-BTBR-0008 Océanomics; ANR-15-IDEX-02 GlycoAlps Cross DisciplinaryProject).

We are grateful to the Roscoff Culture Collection for maintaining the Synechococcus strain used in this study. We thank the ABIMS Platform (Station Biologique de Roscoff) for their help in the metagenomics data computing. We also thank the support and commitment of the Tara Oceans coordinators and consortium, Agnès B. and E. Bourgois, the Veolia Environment Foundation, Region Bretagne, Lorient Agglomeration, World Courier, Illumina, the EDF Foundation, FRB, the

Accepted Article

This article is protected by copyright. All rights reserved Prince Albert II de Monaco Foundation, and the Tara schooner and its captains and crew. Tara Oceans would not exist without continuous support from 23 institutes (oceans.taraexpeditions.org). We certify that there is no conflict of interest associated with this manuscript.

Accepted Article

This article is protected by copyright. All rights reserved influence of light and temperature on growth, pigments, fluorescence and absorptive properties. Marine Ecology Progress Series 116: 259-275. Van Mooy BAS, Fredricks HF. 2010. Bacterial and eukaryotic intact polar lipids in the eastern subtropical South Pacific: Water-column distribution, planktonic sources, and fatty acid composition. Geochimica et Cosmochimica Acta 74: 6499-6516. Van Mooy B a S, Fredricks HF, Pedler BE, Dyhrman ST, Karl DM, Koblízek M, Lomas MW, Mincer TJ,

Accepted Article

This article is protected by copyright. All rights reserved

Author contribution

CS, LG and FP designed the research. SB, JJ, UG, VG, JP, DD, FP, HD, MR, EM, NAN, LG, CS contributed to the performance of this work and the analysis of the data. CS, LG, FP, NN, JJ, EM, UG and DD worked on the writing of the manuscript.

Accepted Article

Supplementary information


Eric Merlet

Kevin Moreno

Arnaud Tron

Nathan Mcclenaghan

Brice Kauffmann

Yann Ferrand
email: y.ferrand@iecb.u-bordeaux.fr

Céline Olivier
email: celine.olivier@u-bordeaux.fr

Aromatic oligoamide foldamers as versatile scaffolds for induced circularly polarized luminescence at adjustable wavelengths

Keywords:

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

The development of molecular architectures exhibiting strong circularly polarized luminescence (CPL) is highly desirable. 1 CPL active molecules have sparked growing interest due to the resolution provided by the circular polarization of light which holds promising potential applications for advanced technologies such as 3D optical displays, 2 spintronic devices 3 or bio-imaging. 4 However, circularly polarized light is often obtained from a non-polarized light source through filters, and generally results in brightness loss and complex device architectures. Thus, strong incentives exist to develop systems or devices that can emit CP light directly. 5 In this context the design of organic chiral (macro)molecules and supramolecular systems showing strong CPL activity is essential. While extensively developed, CPL-active small organic molecules usually exhibit small levels of circular polarization and the scope of chiral designs remains limited. 6 At the molecular level, a helical pattern is arguably the architecture that best expresses chirality. Helicenes 7 and other organic helical structures such as polymers or short oligomers of optically-active subunits 8,9 are prominent examples for the generation of CPL from helical backbones.

Among organic helical structures, aromatic oligoamide foldamers represent a versatile class of synthetic oligomers that adopt highly stable and predictable folded conformations in solution due to stabilizing intramolecular interactions (e.g. hydrogen bonds, electrostatic repulsions) between adjacent units (Fig. 1a). 10 In particular, conformational studies of 8amino-2-quinolinecarboxylic acid oligomers have been thoroughly investigated. 11 The parent amino-acid monomers bear substituents oriented at 60° thus giving rise to helices with 2.5 units per turn and a pitch of 3.5 Å. A complete handedness preference can be induced through N-terminus functionalization using a camphanic acid moiety 12 resulting in either the right-handed P or the left-handed M enantiomers that show strong chiroptical properties. 13 A key feature of aromatic foldamers is their sequential nature which allows synthetic flexibility and provides highly modular structures that can be readily decorated by peripheral substituents (e.g. fluorophores) with atomic precision. Additionally, aromatic foldamers have been shown to promote efficient photoinduced charge transfer over long distances through multi-dimensional charge transport mechanisms. 14 Herein, we report the design, preparation and chiroptical characterisation of helically folded organic CPL emitters based on an aromatic oligoamide backbone. These helical oligomers provide a chiral scaffold, which was functionalized by a range of fluorophores which display induced CPL in the visible range, primarily from inherently non-chiral fluorophores.

Optically pure tetramers 1-4 were prepared using convergent synthetic strategies and derivatized at the Nterminus using either (1S)-() or (1R)-() camphanyl moieties to provide either (1S)-P or (1R)-M enantiomeric helices, respectively. 12 Sequence alignment of P/M 1-4 is shown in Fig. 1b. Quinoline tetramer 1 without additional fluorophore was designed to serve as a reference compound. 15 For the purpose of this study, three new monomers were prepared by functionalizing position 4 of the 8-amino-2-quinolinecarboxylic acid skeleton. Q DP , Q DF and Q Cz were equipped with 4,4'dimethoxy-diphenylamine, bis(9,9-dimethyl-9H-fluoren-2yl)amine and phenylcarbazole fluorophores, respectively. Tetramers 2, 3 and 4 were then prepared by inserting Q DP , Q DF and Q Cz units in the second position of their respective sequences (Fig. 1c). The synthesis of the newly designed monomers and oligomers are described in detail in the supporting information. The molecular model of oligomers P-1, P-2, P-3 and P-4 have been obtained using molecular mechanics calculations and shown in Fig. 2. The single crystal X-ray structures of the racemic precursors of 2 and 4 (i.e. before derivatization with camphanic acid) were also obtained via the slow diffusion of hexane into a solution of the helix in chloroform. Interestingly, the solid state structure and the model revealed a good match thus validating the accuracy of the MM calculation. The opto-electronic properties of the eight tetramers P/M 1-4 were assessed through UV-visible absorption and fluorescence emission spectroscopies in solution. The electronic absorption and emission spectra of the P-helices in chlorinated solvents are represented in Fig. S1 andS2, and the corresponding data is regrouped in Table 1. Additional solution studies are reported in the ESI (Fig. S4, Table S1). Tetramer 1 shows intense absorption bands of the quinoline oligomers in the 250-400 nm range, 14c that can be ascribed to * transitions within the quinoline cores. Additionally, tetramers 2 and 3 show a less intense absorption band located in the visible range which is ascribed to the absorption of the appended chromophore. The new absorption bands are located atmax = 430 nm for 2 (bearing a dimethoxydiphenylamine group) and atmax = 440 nm for 3 (bearing a bis(dimethylfluorenyl)amine group). Tetramer 4 shows similar features as 1, although with slightly red-shifted absorption bands, the lowest-energy band being located at 360 nm. The luminescence spectra of the oligoquinolines show one non-structured emission band. The maximum emission wavelength of 1 is located at 479 nm with fluorescence quantum yield lum = 0.014. On the other hand, the emission of 2 and 3 lies much further towards the red. This is attributed to electronic energy transfer to low-lying chromophore excited states, both tetramer-conjugates displaying a broad emission band in the range 500-750 nm, with maximum intensity at 630 nm and 594 nm, for 2 and 3, respectively. The fluorescence quantum yield of 2 is in the same range as 1 (lum = 0.012) whereas that of 3 is greatly increased (lum = 0.16) due to the fluorenyl moiety. The emission of 4 is similar to 1 with an emission band centred at 471 nm and a fluorescence quantum yield lum = 0.01. Time-resolved luminescence decay measurements provided the luminescence lifetime of the tetramers (Fig. S3). The lifetimes of weakly emissive 1, 2 and 4 are sub-nanosecond, whereas a much longer value was measured for 3. The 7.2 ns luminescence lifetime of 3 mirrors the measured enhanced emission quantum yield.

The chiroptical properties of tetramers 1-4 were first investigated by circular dichroism (CD) spectroscopy. The electronic CD absorption spectra of their P and M enantiomers are shown in Fig. 3. Oligomer 1 shows intense absorption bands in the 230-430 nm range, characteristic of the oligoquinoline chromophoric scaffold. 12,13 By analogy with previously reported quinoline oligoamides, the positive sign of the bands observed around 400 nm for the enantiomer bearing the (1S)-(-)- The chiroptical emission properties of the tetramers were investigated by CPL spectroscopy in order to scrutinize the chirality of the fluorophores in the excited state. The CPL and total photoluminescence spectra recorded in chloroform are shown in Fig. 4. The four tetramers show intense chiroptical activity and opposite signals are observed for the two enantiomers of each aromatic oligoamide. The wavelength ranges of circularly-polarized emission perfectly matches the total photoluminescence. Hence, 1 and 4 show CPL activity in the 400-500 nm region, while 2 and 3 show strongly red-shifted CPL signal with emission wavelengths ranging from 500 to 700 nm, the CPL signal reaching a maximum intensity at = 583 nm for 2 and  = 550 nm for 3. The optical activity in this region indicates that the chirality transfer from the helical scaffold to the inherently achiral fluorophore, as observed by CD at the ground-state for 2 and 3, is conserved in the excited-state, and thereby, induced CPL is produced at longer wavelengths, compared to the parent oligoquinoline. In addition, the sign of the signals follows the same trend as the CD absorption spectra, specifically P-1 and P-4 show positive signals while P-2 and P-3 lead to a negative signal (and conversely for the M helices). The magnitude of CPL is quantified by the luminescence dissymmetry factor, glum, which is given by the equation glum = 2(IL -IR) / (IL + IR), where IL and IR are the intensities of the leftand right-handed circularly polarized emissions, respectively. It is worth to note that intrinsically chiral non-aggregated small organic molecules with axial, planar or helical chirality typically afford glum factors in the range of 10 -3 -10 -5 . 6 The calculated luminescence dissymmetry factors of tetramers 1-4 and corresponding maximum wavelengths are listed in Table 1. The first observation concerns the benchmark tetramer 1 which affords remarkable glum values of +0.011/-0.012 at 424 nm and +0.007/-0.007 at 445 nm, for the P/M helices, respectively. These glum are about one order of magnitude higher than standard dissymmetry factors reported for helicenes, 6,7 and thus, through these values, quinoline-derived oligoamides proved to be an excellent tool to obtain a high degree of circular polarization of emitted light. Moreover, during the course of our study Jiang et al. reported native chiral emission from a series of fluorophore-free oxazolylaniline-derived quinoline oligoamide foldamers. 16 The study led to similar results thus confirming our findings for the bare tetrameric helix 1. More interestingly, the glum factors estimated at the vicinity of the maximum emission wavelength for 2 and 3 are -0.0013/+0.0013 and -0.0012/+0.0012 at 583 nm and 550 nm, respectively, for the P/M helices. Thus, although one order of magnitude lower than those of 1, the luminescence dissymmetry factor of 2 and 3 remains relatively high and comparable to other organic CPL emitters. [6][7][8] The tetramer 4

gives strong glum values of +0.0047/-0.0046 at 438 nm and +0.0039/-0.0037 at 455 nm, however the absorption and emission of the carbazole group occur in the same wavelength range as the quinoline oligomer (see above) and thus it was not possible to conclude about the origin of the signal in this case. Overall the high magnitude of glum values observed upon chiral induction from the helical scaffold to the appended fluorophores proves that a strong electronic coupling exists between the quinoline oligoamide foldamer and the fluorophore, thereby allowing effective chirality transfer through functionalization of the foldamers.

In conclusion, a series of eight fluorescent quinoline oligoamide tetramers was readily synthesized for assessment of their chiroptical properties. Tetrameric helices appended with inherently non-chiral fluorophores were derivatized with a camphanyl chiral inducer for complete handedness induction, leading to P-and M-helices selectively. As expected, strong chiroptical properties were observed as proven by CD and CPL spectroscopies. Most strikingly, induced CPL signals with high luminescence dissymmetry factors were observed in the visible range at wavelengths corresponding to the emission of the fluorophore. We therefore demonstrated that chirality transfer occurs from the helical scaffold to the appended fluorophore, even at the excited-state. Overall, the high modularity of the foldamer backbones can afford readily adjustable CP-emission wavelengths. Further rational molecular design as well as theoretical calculations are currently in progress in view of increasing the CPL response of such systems.

We thank Drs R. Oda and Y. Okazaki for assistance with CPL data collection.

There are no conflicts to declare.

Notes and references

Fig. 1

 1 Fig. 1 a) Structure of quinoline-derived oligoamide tetramers with camphanic chiral inducer at the N-terminus. b) Schematic representation of quinoline (Q) oligoamide tetramers 1, 2, 3 and 4 prepared for this study. c) Structural formulas of the chiral inducers (camph*) and of the substituents (R) diverging from the quinoline monomers.

Fig. 2

 2 Fig. 2 Side views of energy minimized structures (Maestro version 6.5 using MMFFs force field) of: a) P-1, b) P-2, c) P-3 and d) P-4. X-ray crystal structure (top view) of the P helix of e) QQ DF QQ (precursor of 3) and f) QQ Cz QQ (precursor of 4). The helices are shown in stick representation and color-coded as in Fig. 1. The non-polar hydrogens are omitted for clarity. Red spheres denote the position of isobutoxy side chains.

a

  Maximum absorption wavelength in CHCl3 (C = 2.5 x 10 -5 M). b Emission wavelength maximum in CH2Cl2 (1 and 4 : exc = 370 nm; 2 and 3 : exc = 440 nm). c Fluorescence quantum yield. d Luminescence dissymmetry factor and corresponding wavelength.

  a P handedness. Conversely, the negative sign of the bands observed for the enantiomer bearing the (1R)-(+)-camphanyl group implies M handedness.11b,12 The CD spectra of 2 and 3 similarly show strong absorption bands in the 230-430 nm range although Cotton effects are observed in this region. The change in chiroptical activity in this region, in comparison with 1, indicates a slight perturbation of the electronic states of the oligoquinoline scaffold upon functionalization. More interestingly, an additional absorption band is observed in the CD spectra of 2 and 3 in the visible region,  = 440 nm, corresponding to the absorption of the diarylamine chromophores. The chiroptical activity in this region confirms that the ground state energy level of the inherently achiral chromophores is effectively perturbed by the helical scaffold on which they are grafted. As expected from the UV-vis absorption properties described above, the CD spectra of 4 and 1 are similar.

Fig. 3

 3 Fig. 3 CD spectra recorded in CHCl3 (C = 100 µM, 298K) for (a) P-1 (black, plain), M-1 (black, dashed); (b) P-2 (orange, plain), M-2 (orange, dashed); (c) P-3 (green, plain), M-3 (green, dashed); (d) P-4 (purple, plain), M-4 (purple, dashed).

Fig. 4

 4 Fig. 4 CPL spectra (top raw) and total photoluminescence spectra (bottom raw) recorded in CHCl3 (C = 25 µM, 298K) for (a) P-1 (black, plain), M-1 (black, dashed); (b) P-2 (orange, plain), M-2 (orange, dashed); (c) P-3 (green, plain), M-3 (green, dashed); (d) P-4 (purple, plain), M-4 (purple, dashed). exc = 370 nm.

Table 1

 1 Absorption and luminescence properties of 1-4.

		abs/nm a	em.	lum	glum x 10 3 (/nm) d
		(/M -1 .cm -1 )	/nm b	/% c	P-Helix	M-Helix
	1	323 (32 600) 350 (28 700)	479	1.4	+11.3 (424) +7.2 (445)	-12.2 (424) -7.3 (445)
		325 (27 500)
	2	349 (24 000)	630	1.2	-1.3 (583)	+1.3 (583)
		430 (8 700)
	3	344 (56 300) 440 (12 900)	594	16.4	-1.2 (550)	+1.2 (548)
	4	328 (25 800) 343 (26 400) 360 (24 700)	471	1.0	+4.7 (438) +3.9 (455)


F Bavedila

Etienne Okada

Jean-Francois Lampin

Emilien Guillaume Ducournau

LT-GaAs-based photomixers with >2 mW

come

1 Abstract-It is shown in this communication that a LT-GaAs photomixer based on an optically resonant cavity is able to generate peak output powers above 2 mW up to 320 GHz when it is driven by optical pulses of 5 ns width at a repetition rate of 10 MHz.

he generation of microwaves by optical heterodyne of coherent sources on a photodetector, also called photomixing, has been proposed and demonstrated in the 1950s by A. Forrester et al. through the generation of 10 GHzfrequency microwaves thanks to the beating on a photocathode of Zeeman lines of a Hg202 source [START_REF] Forrester | Photoelectric Mixing of Incoherent Light[END_REF]. After the few attempts made in the 1960s following the invention of the laser [START_REF] Svelto | Photoconductive Mixing in CdSe Single Crystals[END_REF], the interest for this photonics-based CW source has been revived in the late 1990s when Brown et al. demonstrated the generation of electromagnetic waves from 0.1 THz up to 3.8 THz [START_REF] Brown | Photomixing up to 3.8 THz in low-temperature-grown GaAs[END_REF], using an ultrafast photoconductor coupled to a wideband antenna and pumped by two 0.8 µm-wavelength lasers. Its frequency tunability is indeed limited only by the properties of the photomixer, and not by the optical sources. This is because a negligible relative variation in the frequency of the infrared lasers is translated in a great relative variation in the microwave or terahertz frequency range. From a practical point of view, it allows, unlike electronic sources such as frequency multipliers, to generate waves continuously from dc to a few terahertz with the same optical sources mixed in only one wide band or several narrow band photomixers. Some years ago, we have demonstrated by means of on-wafer measurements that up to 1.8 mW around 250 GHz could be generated by a LT-GaAs vertical resonant photoconductor (see Fig. 1), thanks to a metallic mirror-based Fabry-Pérot cavity (FP) and to a deep submicron electrode spacing resulting in high carrier and current densities [START_REF] Peytavit | CW Source Based on Photomixing With Output Power Reaching 1.8 mW at 250 GHz[END_REF]. Since this demonstration, a number of other teams have proposed photomixers with mW level peak output powers in the THz frequency range thanks to the modulation of the pump optical power [START_REF] Berry | Plasmonics enhanced photomixing for generating quasi-continuouswave frequency-tunable terahertz radiation[END_REF]. Here we show that the resonant cavity photoconductor is able to generate peak power between 2 and 5 mW in the 220-325 GHz band when it is pumped by a modulated optical power at a frequency of 10 MHz and a duty cycle of 5%. The peak output power (P peak ) as a function of the beatnote frequency generated by is shown in Fig. 2. It has been deduced from the average output power measured by the powermeter (P avg ) and from the duty cycle (d cycle ) of the electrical pulses driving the modulator: P peak = P avg /d cycle . The temporal shape of the THz pulses has been also measured by means of a 220-325 zero-bias Schottky diode allowing us for using this relation between the average power and the peak power. It is worth to notice, that the oscillations in the power spectrum come mainly from the standing waves created by the reflections inside the coplanar probes. It can be seen that peak powers reaching 5 mW at 220 GHz and around 3 mW at 315 GHz has been obtained with only 31 mW of average optical power which is, to the best of our knowledge, the highest power ever reported for a single photomixer in the 220-325GHz Band.

F. Bavedila, E. Okada, J-F. Lampin, G. Ducournau and E. Peytavit IEMN, UMR CNRS 8520, Université de Lille , 59652, Villeneuve d'Ascq, France LT-GaAs-based photomixers with >2 mW peak output power up to 320 GHz T

Fig. 1 :

 1 Fig. 1: SEM picture of a Fabry-Pérot cavity photoconductor couple to a thin film microstrip line

Fig. 2 :

 2 Fig. 2: Peak Output power as a function of frequency