Martin Laviale
email: martin.laviale.bio@gmail.com

Alexandre Barnett

João Ezequiel

Bernard Lepetit

Silja Frankenbach

Vona Méléder

João Serôdio

Johann Lavaud

# Silja Frankenbach

Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal

Introduction

Estuarine tidal flats are one of the most productive ecosystems on Earth, largely owing to the photosynthetic productivity of benthic microalgae communities, or microphytobenthos (MPB) [START_REF] Underwood | Primary production by phytoplankton and microphytobenthos in estuaries[END_REF]. MPB communities are often dominated by motile pennate diatoms [START_REF] Haubois | Spatiotemporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oleron Bay, France[END_REF][START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf bay, France)[END_REF][START_REF] Ribeiro | Structure and diversity of intertidal benthic diatom assemblages in contrasting shores: A case study from the Tagus estuary[END_REF]. In fine sediment habitats, they undergo rhythmic vertical migration patterns in the upper layers of the sediment following tidal/dial cycles [START_REF] Saburova | Diatom activity within soft sediments: behavioural and physiological processes[END_REF][START_REF] Consalvey | The ups and downs of life in a benthic biofilm: Migration of benthic diatoms[END_REF][START_REF] Coelho | Endogenous versus environmental control of vertical migration by intertidal benthic microalgae[END_REF]. During daylight emersion, the upward migration of cells results in the formation of transient photosynthetic biofilms which are thus periodically exposed to variable and extreme environmental conditions, due to the complex interplay of timing of tidal exposure and weather conditions [START_REF] Admiraal | The ecology of estuarine sediment inhabiting diatoms[END_REF][START_REF] Paterson | Microphytobenthos in contrasting coastal ecosystems: Biology and dynamics[END_REF]. Excessive light exposure generates intra-cellular oxidative stress [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB radiation and salinity[END_REF][START_REF] Waring | Lightinduced responses of oxygen photoreduction, reactive oxygen species production and scavenging in two diatom species[END_REF] which can lower photosynthetic efficiency (i.e. photoinhibition; [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF][START_REF] Cartaxana | Photoinhibition in benthic diatom assemblages under light stress[END_REF] and ultimately community-level primary productivity [START_REF] Blanchard | Quantifying the short-term temperature effect on light-saturated photosynthesis of intertidal microphytobenthos[END_REF][START_REF] Guarini | Modelling the dynamic of the microphytobenthic biomass and primary production in European intertidal mudflats[END_REF]. Furthermore, benthic diatom's photosynthesis often operates under the combined action of high light and other potentially stressful environmental factors such as extreme temperature [START_REF] Blanchard | Seasonal effect on the relationship between the photosynthetic capacity of intertidal microphytobenthos and temperature[END_REF][START_REF] Serôdio | Fortnightly light and temperature variability in estuarine intertidal sediments and implications for microphytobenthos primary productivity[END_REF]. Although photosynthesis is known to be highly sensitive to temperature [START_REF] Mathur | Photosynthesis: Response to high temperature stress[END_REF], effects of high temperature on MPB productivity, either alone or in combination with excess light have received little attention [START_REF] Grant | Sensitivity of benthic community respiration and primary production to changes in temperature and light[END_REF]Vieira et al., 2013 and references therein). However, recent results showed implications on both dial and seasonal scales [START_REF] Hancke | Effects of temperature and irradiance on a benthic microalgal community: A combined two-dimensional oxygen and fluorescence imaging approach[END_REF]. On a longer term perspective, the cumulative effects of light and temperature could be relevant as well, as climate change is expected to influence not only average values but also the frequency and the intensity of extreme events such as heat waves [START_REF] Schär | The role of increasing temperature variability in European summer heatwaves[END_REF][START_REF] Stott | Human contribution to the European heatwave of 2003[END_REF][START_REF] Solomon | Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change[END_REF].

The actual photoinhibitory effects depend on the photoadaptive strategy of the diatoms of MPB, i.e. the operating efficiency of a range of mechanisms which include 'behavioural' and 'physiological' photoprotective processes [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF]. The 'behavioural photoprotection' consists of a strong negative phototaxis resulting in downward migration under supersaturating light intensities [START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF][START_REF] Perkins | Vertical cell movement is a primary response of intertidal benthic biofilms to increasing light dose[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. The vertical migration of cells within the steep light gradient of the sediment photic zone [START_REF] Paterson | Microphytobenthos in contrasting coastal ecosystems: Biology and dynamics[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF] has been hypothesized to allow for the optimization of light harvesting and the avoidance of excess light [START_REF] Admiraal | The ecology of estuarine sediment inhabiting diatoms[END_REF]. Strong evidence for a photoprotective role of vertical migration was provided by the use of the diatom motility inhibitor Latrunculin A (Lat A) on undisturbed biofilms [START_REF] Perkins | Vertical cell movement is a primary response of intertidal benthic biofilms to increasing light dose[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. 'Physiological photoprotection' mainly includes the excess energy-dissipative non-photochemical quenching of chlorophyll (Chl) a fluorescence (NPQ). NPQ is mainly controlled by the presence of the xanthophyll pigment diatoxanthin (DT) and of Lhcx proteins in the light-harvesting complexes of photosystem II (PSII) [START_REF] Depauw | Exploring the molecular basis of responses to light in marine diatoms[END_REF][START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF][START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and macrophytic brown algae. In Non-Photochemical Fluorescence Quenching and Energy Dissipation In Plants, Algae and Cyanobacteria[END_REF]. In diatoms, DT is produced by the (dark/low light) reversible lightdependent conversion of the xanthophyll diadinoxanthin (DD) in the so called xanthophyll cycle (XC) [START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. Besides its involvement in NPQ, it seems to have another function as antioxidant [START_REF] Lepetit | Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms[END_REF].

Despite the crucial role of NPQ and XC in the photoprotective response of diatoms to excessive fluctuating light regimes, especially in field situations [START_REF] Brunet | Can the xanthophyll cycle help extract the essence of the microalgal functional response to a variable light environment?[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF][START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and macrophytic brown algae. In Non-Photochemical Fluorescence Quenching and Energy Dissipation In Plants, Algae and Cyanobacteria[END_REF], they have only scarcely been studied in MPB.

They show a relation to diurnal and tidal cycles, season, latitude [START_REF] Serôdio | Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos[END_REF][START_REF] Van Leeuwe | Photoacclimation in microphytobenthos and the role of xanthophyll pigments[END_REF][START_REF] Chevalier | In situ photosynthetic activity and xanthophylls cycle development of undisturbed microphytobenthos in an intertidal mudflat[END_REF], to vertical position of diatoms within the sediment [START_REF] Jesus | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF] or along the intertidal elevation gradient [START_REF] Jesus | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF], with significant differences between the main growth forms (i.e. epipelic and epipsammic: [START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF][START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF]. As for the implication of Lhcx proteins in diatom NPQ, its characterization has been limited to few model species [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF][START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF]. Lhcx1 is involved probably at least by binding DT [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF][START_REF] Büchel | Fucoxanthin-chlorophyll-proteins and non-photochemical fluorescence quenching in diatoms. In Non-Photochemical Fluorescence Quenching and Energy Dissipation In Plants, Algae and Cyanobacteria[END_REF]. It is present at low light and its transcript level/synthesis is slightly enhanced under high light (HL) [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF][START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF].

While the transcript levels/synthesis of other examined isoforms (i.e. Lhcx2, Lhcx3, Lhcx6) strongly increase during HL [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF] and temperature stress (for Lhcx6; [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF], their exact physiological role remains unknown, although Lhcx3 and Lhcx6 are suspected to be involved in the binding of de novo synthesized DT and/or the sustain part of NPQ (i.e. qI) during a prolonged HL stress [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF].

This work intended to study the combined effects of high light (HL) and high temperature on the photophysiology of intertidal diatom-dominated MPB communities. Light stress-recovery experiments (LSRE) were performed to distinguish physiological photoprotection efficiency from photoinhibitory effects, and to quantify behavioural photoprotection [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. Until now, these processes have been characterized only to the extent of chlorophyll fluorescence measurements and XC pigments analysis. The present study combines chlorophyll fluorescence imaging, pigment analysis and for the first time on intact biofilms, lipid peroxidation quantification and immunodetection of Lhcx proteins. LSRE were performed on MPB from two locations in France and Portugal with different solar exposure and temperature regimes, likely to support the establishment of diatom communities with contrasting photo-and thermo-adaptive strategies. Moreover, the potential role of temperature in modulating the HL-induced stress was investigated by carrying out LSRE at two experimental temperature conditions ('optimal' vs. high temperature). In parallel, the estimated photosynthetic and protective indices were further compared with field situation at the two sites during a typical summer diurnal emersion.

Results

Environmental conditions, in situ photosynthetic activity and taxonomic composition of microphytobenthos in PT and FR sites

Environmental conditions and MPB photosynthetic activity were assessed during typical summer diurnal emersion at both Portuguese (PT) and French (FR) intertidal flats. The Photosynthetic Active Radiation (PAR) reached its daily maximum (2000 µmol photons m -2 s - 1 ) at both sites (Fig. 1A-B) with a comparable light dose over the emersion (~19 to 23 mol photons m -2 , Table S1). Temperature was higher at PT site (Fig. 1A-B). Average temperatures at the sediment surface were 37.5°C and 28.8°C for PT and FR sites, respectively.

Temperature decreased with sediment depth with a stronger effect in PT site (-7°C and -6°C in the first 0.5 cm for PT and FR sites, respectively: Table S1, Fig. 1A-B).

At the beginning of emersion, the effective PSII quantum yield (ΦPSII) was close to its At both sites, MBP communities were dominated by epipelic (i.e. motile) benthic diatoms of the genus Navicula (Table S2). Navicula phyllepta sensu lato [START_REF] Vanelslander | Ecological differentation between sympatric pseudocryptic species in the estuarine benthic diatom Navicula phyllepta (Bacillariophyceae)[END_REF] was the dominant species at PT site (relative abundance: 44 %) while FR site was co-dominated by N. spartinetensis (33 %) and N. phyllepta s.l. (28 %).

Comparison between in situ and experimental conditions

The two temperature treatments used for the laboratory experiments during the LSREs were 27.4 ± 1.2°C and 37.3 ± 0.7°C (thereafter named 27°C and 37°C temperature treatments, respectively), which was close to the average temperatures monitored at the sediment surface for both sites (see above and Table S1). The light exposure of 3 h of 1200 µmol photons m -2 s - 1 represented a light dose of 12.96 mol photons m -2 [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. This light dose was reached after 2 h 53 min and 3 h 34 min in PT and FR sites, respectively (Table S1).

LSRE-induced photoinhibition and NPQ in sediment from FR site

ΦPSII remained constant in samples maintained under LL throughout the experiment (0.66 ± 0.02, Fig. 2). In comparison, samples exposed to HL exhibited dramatic changes over time (time: P < 0.001, see Table S3a for detailed statistical outputs). After 3h HL, ΦPSII decreased down to values between 0.13 ± 0.03 (37°C +Lat A) and 0.34 ± 0.02 (27°C Ctrl). It increased during LL recovery but did not reach back its initial level (0.46 ± 0.05 (37°C +Lat A) and 0.61 ± 0.01 (27°C Ctrl). The differences observed between temperature and chemical treatments were significant (temperature: P < 0.001; chemical: P < 0.01; Table S3a), and they both increased with time (time × temperature: P < 0.001; time × chemical: P < 0.05; Table S3a). Similarly, NPQ of samples maintained under LL remained constant (0.38 ± 0.04, Fig. 3) and changed over time according to light (time: P < 0.001, Table S3b): it increased under HL (between 4.4 ± 0.5 and 3.4 ± 0.4 from 37°C +Lat A and 27°C Ctrl treatments, respectively) but only partially relaxed during the subsequent 15 min LL incubation (between 1.9 ± 0.1 and 1.1 ± 0.1; Fig. 3). The effect of temperature was highly significant (temperature: P < 0.001, Table S3b) and the observed differences increased with time (time × temperature: P < 0.001; Table S3b). In comparison, the effect of lat A was less significant (chemical: P < 0.05 and time × temperature: P = 0.11; Table S3b).

LSRE-induced allomerization of Chl a and xanthophyll cycle in sediment from FR site

All samples exhibited typical diatom pigment signatures, including Chl a, Chls c, fucoxanthin, the carotenoid ß, ß carotene (data not shown) and the xanthophyll cycle pigments DD and DT. The allomer form of Chl a (Chl a-allo) accumulated during the LSRE as illustrated by the Chl a allomerization molar ratio (AR= Chl a-allo / total Chl a × 100, Fig. 4A). AR increased ~ 2-fold under HL and remained stable thereafter (time: P < 0.001; Table S3c). No significant changes were observed with temperature (temperature: P = 0.48; Table S3c), whereas AR was significantly higher in +Lat A samples than in Ctrl (chemical: P < 0.01; Table S3c), with mean maximum values of 11.9 ± 5.0% and 8.4 ± 2.1%, respectively (Fig. S1). AR did not significantly change in samples maintained in LL during 3 h (5.9 ± 2.7% on average).

The pool of DD+DT (in mol. 100 mol Chl a -1 ) increased ~ 3-fold under HL and remained stable during the LL recovery (Fig. 4B, time: P < 0.001; Table S3d). Overall, it reached higher values at 37°C than at 27°C (temperature: P < 0.01; Table S3d) but it was comparable in Ctrl and +Lat A samples (P = 0.58; Table S3d). At 37°C, DD+DT increased even in the samples maintained under LL for 3 h (from 15.0 ± 4.0 to 23.8 ± 6.6 mol. 100 mol Chl a -1 , Tukey HSD: P < 0.05, Fig. S2). DD de-epoxidation (DES = DT / DD+DT × 100) correlated well with light whatever the temperature in both Ctrl and Lat A-treated samples (P < 0.001) (Fig. 4C-D). DES increased under HL and decreased during LL recovery while it remained stable in samples maintained in LL for 3 h (14.0 ± 3.2% on average) (Fig. S3). DES was significantly higher in +Lat A samples than in Ctrl ones (chemical: P < 0.001; Table S3d), and at 37°C than at 27°C (temperature: P < 0.001; Table S3d) with maxima of 67.2 ± 7.5% and 33.8 ± 7.5% at 37 °C and 27 °C, respectively, in +Lat A samples (Fig. S3). DES recovered much faster at 27°C than at 37°C, especially for Ctrl samples. In contrast to AR and the DD+DT pool, interaction between time and temperature was significant for DES (time × temperature: P < 0.001; Table S3d). Moreover, NPQ and DES were significantly positively correlated according to the chemical treatment (Ctrl/+Lat A), i.e. the slope of the NPQ vs. DES linear regression was 2.5 lower in +Lat A samples (Fig. S4) while for both treatments the origin of the regression was similar and non-nul (Ctrl: 17.6 ± 0.4; +Lat A: 16.4 ± 0.4), i.e. some DT was synthesized without NPQ development.

LSRE-induced Lhcx synthesis in sediment from FR site

The anti-FCP6 (i.e. Lhcx1) revealed a clear Lhcx isoform in FR samples (Fig. 5A). Its size of about 24 kDa was slightly higher than the one of Lhcx 3 in Phaeodactylum tricornutum and its amount increased after 3 h HL exposure especially in +Lat A samples. Interestingly, N. phyllepta s.l., the dominant species of the MPB assemblage, showed an Lhcx pattern similar to P. tricornutum so that three isoforms (Lhcx1, Lhcx2 and Lhcx3) could be identified. The anti-Lhcx6 from Thalassiosira pseudonana revealed two Lhcx isoforms in FR samples, the size of which was different from Lhcx6 and was neither present in HL exposed P. tricornutum cells (Fig. 5B). N. phyllepta showed two isoforms, one which size was slightly higher than Lhcx6 and the other one with a similar size to the 33-50 kDa isoform from FR samples. With such size, this isoform could be a hypothetic Lhcx dimer (given that the available sizes of Lhcx monomers deduced from the genomes of P. tricornutum, T. pseudonana and Fragilariopsis cylindrus range between 21 and 29 kDa) but different than in P. tricornutum.

The 17-23 kDa isoform showed a size similar to Lhcx 1 in P. tricornutum and N. phyllepta (see Fig. 5A). Interestingly, the two isoforms reacted to light, temperature and Lat A in different ways: while the 17-23 kDa isoform appeared not to react to any of the treatments, the 33-50 kDa isoform positively reacted to HL and 37°C with an effect enhanced by Lat A at 37°C (Fig. 5C).

LSRE-induced lipid peroxidation in sediment from FR site

Common markers of membrane lipid peroxidation (TBARS-thiobarbituric acide reactive substances) were quantified. In Lat A treated samples, TBARS content normalized to the surface microphytobenthos biomass (I diat biomass index) did not change significantly over time and was not significantly affected by chemical or temperature treatments (P = 0.71, 0.99 and 0.19, respectively; Table S3f), exhibiting a mean value of 1.19 ± 0.29 10 -6 nmol eq MDA mL -1 (Fig. 6).

Comparison between FR and PT sites: NPQ, photoinhibition, lipid peroxidation and Lhcx.

ΦPSII and NPQ measured on PT samples at T0 (0.67 ± 0.02 and 0.40 ± 0.05, respectively)

were comparable to the one measured on FR ones (Fig. 2 and 3: 0.66 ± 0.01 and 0.39 ± 0.01, respectively), and they exhibited the same general trend under HL stress. However, PT samples were less affected by the HL treatment, as ΦPSII recovered faster under LL (site: P < 0.001, Table S4a), regardless of temperature or Lat A application (Fig. 7A). Moreover, NPQ did not relax entirely, but the level of this sustained NPQ was lower than in FR samples (site: P < 0.01, Table S4b), although post-hoc multiple comparison tests did not show any statistical differences between sites (Tukey HSD: P > 0.05; Fig. 7B).

For each site and temperature, the extent of photoinhibition and the contribution of vertical migration to the overall photoprotection capacity of MPB were estimated as in Serôdio et al.

(2012) (Table 1). Photoinhibition was higher in FR than PT site but it increased similarly (~3 fold) with temperature. The contribution of vertical migration to photoprotection remained below 15 % but was always lower in PT biofilms (< 5%).

In Lat A treated samples, TBARS content normalized to surface biomass did not change significantly at PT site whatever the temperature (Fig. S5). Mean value was 3.26 ± 0.66 10 -6 nmol eq MDA mL -1 , which was significantly higher than for FR samples (t-test: P < 0.001).

While the anti-FCP6 (i.e. Lhcx1) revealed two clear Lhcx isoforms in PT samples (Fig. 8), the anti-Lhcx6 did not bring any signal probably due to its unspecificity as regards to the species composing the microphytobenthic community of PT site. Their respective size were close with one at about 23 kDa, the size of Lhcx 3 in P. tricornutum and N. phyllepta, and the other one slightly higher (about 24 kDa) which was also present in FR samples (Fig. 5A). Both isoforms (although the 24 kDa reacted in a stronger way) were enhanced after 3 h of HL exposure as well as by the higher temperature; this effect was increased by the addition of Lat A. Interestingly, at 27°C, the 24 kDa isoform was not present under LL and HL exposure, it appeared with the addition of Lat A solely (LL 27°C +) and its content was increased by HL exposure (HL 27°C +). Similarly, the 23 kDa isoform positively reacted to Lat A to a level even similar to HL exposure without Lat A (compare 27°C LL+ and 27°C HL-).

Discussion

In this study, higher temperature significantly increased photoinhibition susceptibility of epipelic MPB biofilm. Communities from the Portuguese (PT) site were clearly more resistant to a coupled high light-temperature stress, in comparison to biofilms from the French (FR) site. At both site, photoprotection was based on the interplay between physiological (NPQ, DT and Lhcx proteins) and behavioural (vertical motility) response, nevertheless PT biofilms displayed a lower relative contribution of vertical migration and a stronger NPQ. The apparent latitudinal pattern observed at the community level ('MPB biofilm') might be driven by the individual response of the respective dominant diatom species/ecotypes in each site.

These different aspects are discussed below.

Evidence for a latitudinal adaptation in the response of epipelic MPB to a coupled lighttemperature stress

Our results suggest that PT MPB communities were adapted to a higher light and/or temperature environment than FR ones. Overall, PT samples were more resistant than FR ones to HL stress, regardless the effect of temperature. At 27°C, PT control samples recovered almost entirely from HL exposure (97 %), and photoinhibition increased only from 3 to 10 % at 37°C. This trend was also obvious in situ: the time course and level of ΦPSII were comparable between sites despite the potentially more stressful conditions at PT site (similar light conditions but 8-9°C higher average surface temperature). One of the reasons for this differential latitudinal adaptation might lie in the photoprotective strategies the two MPB communities exhibited. PT MPB communities showed higher NPQ max average values, an indication of a strong(er) physiological photoprotective response [START_REF] Serôdio | Non-photochemical quenching of chlorophyll fluorescence and operation of the xanthophyll cycle in estuarine microphytobenthos[END_REF]. PT NPQ max was already high right from the beginning of the emersion although light intensity was still moderate and it remained relatively stable along the emersion. Consequently, vertical migration contributed less to the overall photoprotection capacity (< 5 %) in comparison to FR biofilms. FR NPQ max tended to follow the fluctuations of light intensity along the emersion which would be in agreement with a more dynamics MPB response, regarding both motility and physiology. Only few studies have examined the link between seasonal and/or latitudinal photoacclimation and the coupling between behavioural and physiological photoprotection in MPB communities. A similar increase of migration contribution was reported for communities acclimated to lower irradiances (i.e. seasonal acclimation [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. Another study provided evidence for the differential role of migration vs. XC along a latitudinal gradient (Ireland, UK and Portugal), although the role of sediment type (i.e. mud vs. sand) could not be ruled out [START_REF] Van Leeuwe | Photoacclimation in microphytobenthos and the role of xanthophyll pigments[END_REF].

In contrast, the contribution of thermal adaptation is difficult to ascertain. Although PT samples were more resistant and resilient, higher temperature leads to comparable effects: photoinhibition increased to about 3-times (i.e. from 3 to 10 % and from 8 to 22 %, in PT and FR sites, respectively) and lipid peroxidation was comparable. The pattern of the detected Lhcx isoforms was also similar, although no quantification was possible. Average surface and -0.5 cm temperature conditions experienced by the FR and PT communities were clearly different. The fact that this difference was lower than the observed amplitude over an emersion could explain the apparent lack of temperature latitudinal pattern. This is in agreement with other comparative studies on short-term (i.e. over few hours) temperature effects on MPB photosynthesis [START_REF] Grant | Sensitivity of benthic community respiration and primary production to changes in temperature and light[END_REF][START_REF] Blanchard | Seasonal effect on the relationship between the photosynthetic capacity of intertidal microphytobenthos and temperature[END_REF][START_REF] Hancke | Temperature effects on respiration and photosynthesis in three diatom-dominated benthic communities[END_REF]. Instead, the different average temperature conditions at PT and FR sites recorded in this study, which are representative of typical summer conditions in these areas (Guarini et al., 1997;[START_REF] Lillebo | Daily and inter-tidal variations of Fe, Mn and Hg in the water column of a contaminated salt marsh: Halophytes effect[END_REF], might have played a role on a longer time scale. As temperature and light adaptations rely on similar strategies [START_REF] Davison | Environmental effects on algal photosynthesis -Temperature[END_REF] this study, see below), it is likely that the difference in average temperatures influenced the susceptibility of MPB to photoinhibition, directly and/or via its photoacclimation status, together with e.g. daily light dose, day length and/or UV exposure.

The latitudinal pattern in ecophysiological characteristics observed at the community level coincided with a shift in the diatom species composition. Although it is difficult to separate 'community' response from 'individual species' response within the biofilm, it is possible that the observed responses at each site were driven by the few dominant species (representing c.a. 50% of the total abundance). The two dominant species, N. phyllepta s.l. and N.

spartinetensis, are known to be widespread along the European Atlantic and Northwestern coasts [START_REF] Vanelslander | Ecological differentation between sympatric pseudocryptic species in the estuarine benthic diatom Navicula phyllepta (Bacillariophyceae)[END_REF][START_REF] Witkowski | Morphology and distribution of a little known but widespread diatom (Bacillariophyceae), Navicula spartinetensis Sullivan et Reimer[END_REF]. Epipelic diatoms generally exhibit a low NPQ, in comparison to non-motile ones [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF]. This does not preclude the existence of interspecific variability between organisms sharing the same life style, considering both behavioural (Underwood et al., 2005;[START_REF] Du | Effect of light and sediment grain size on the vertical migration of benthic diatoms[END_REF][START_REF] Du | Migratory responses of benthic diatoms to light and temperature monitored by chlorophyll fluorescence[END_REF] and physiological photoprotective mechanisms [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF]. Furthermore, the occurrence of N. phyllepta s.l. at both sites can be explained by the pseudocryptic feature of this taxonomic group. It comprises different closely related species morphologically indistinguishable but exhibiting important ecophysiological differences and environmental preferences [START_REF] Vanelslander | Ecological differentation between sympatric pseudocryptic species in the estuarine benthic diatom Navicula phyllepta (Bacillariophyceae)[END_REF]. To which extent the differences observed between PT and FR sites (i.e. stronger NPQ max in PT assemblage regardless of the stress treatment; different Lhcx patterns) can be explained by the differential relative abundances of N. phyllepta s.l. vs. N. spartinetensis and/or pseudopcryptic species or ecotypes of N. phyllepta s.l is a question that remains to be answered.

Higher temperature increases photoinhibition susceptibility in epipelic MPB

To our knowledge, this is the first study which shows that on intact migratory MPB biofilms high temperature and HL acts synergistically in decreasing photosynthesis, a feature so far reported only for individual species [START_REF] Salleh | The effects of temperature on the photosynthetic parameters and recovery of two temperate benthic microalgae, Amphora cf. coffeaeformis and Cocconeis cf. sublittoralis (Bacillariophyceae)[END_REF]. Other works have shown that temperature alone impacts photosynthesis in benthic diatoms, in monospecific cultures [START_REF] Morris | Influence of temperature on the relationship between oxygen-and fluorescence-based estimates of photosynthetic parameters in a marine benthic diatom (Cylindrotheca closterium)[END_REF][START_REF] Yun | Photosynthetic activity of benthic diatoms in response to different temperatures[END_REF], MPB suspensions [START_REF] Blanchard | Seasonal effect on the relationship between the photosynthetic capacity of intertidal microphytobenthos and temperature[END_REF], and intact MPB biofilms [START_REF] Vieira | Effects of short-term changes in sediment temperature on the photosynthesis of two intertidal microphytobenthos communities[END_REF]. In conditions where photosynthesis is lightsaturated, benthic diatom photosynthesis can respond to a transient temperature increase if it remains within a 25-35°C range. The high temperature used here (37.3  0.7°C) falls slightly outside this range, a situation likely to occur regularly in situ in summer, as confirmed by our field data.

In situ measurements showed that ΦPSII correlated well with irradiance during the emersion period. During LSRE, a strong decrease was also observed after 3 h of HL exposure.

Although the applied light dose was lower than in situ, our results show that it was strong enough to induce photoinhibition at the community level. The incomplete recovery of ΦPSII after 15 min LL coincided with a sustained phase of NPQ which was induced during the prolonged HL exposure. This can be partly attributed to photoinhibition and illustrates the physiological response to harsh stress [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF][START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]. At optimal temperature (27°C; see [START_REF] Blanchard | Seasonal effect on the relationship between the photosynthetic capacity of intertidal microphytobenthos and temperature[END_REF] and Experimental procedures section), photoinhibition remained below 20 %, which is consistent with a previous study using a similar experimental approach [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. Photoinhibitory effects may be attributed to the progressive accumulation of reactive oxygen species (ROS, [START_REF] Roncarati | Photosynthetic performance, oxidative damage and antioxidants in Cylindrotheca closterium in response to high irradiance, UVB radiation and salinity[END_REF][START_REF] Waring | Lightinduced responses of oxygen photoreduction, reactive oxygen species production and scavenging in two diatom species[END_REF], as evidenced by the increase of AR at the end of LSRE. Indeed, ROS have been shown to be directly involved in Chl a allomerization (Hynninen et al., 2010 and references therein). This is also consistent with the accumulation of Chl a allomers in response to oxidative stress in the diatom P. tricornutum [START_REF] Cid | Copper toxicity on the marine microalga Phaeodactylum tricornutum -Effects on photosynthesis and related parameters[END_REF]. When superimposed to HL stress, significantly increased photoinhibition (c.a. 3-times increase in the controls from 27 to 37°C) which is not surprising as PSII is the most thermosensitive component of the photosynthetic apparatus [START_REF] Mathur | Photosynthesis: Response to high temperature stress[END_REF]. Nevertheless, the amount of MDA and other aldehydes (TBARS) were not affected, suggesting that antioxidant activities were sufficient to prevent from substantial ROS-mediated membrane damage during the 3 h stress exposure, even when migration was inhibited. In such short-term experiments (i.e. < 24 h), it seems that only stronger oxidative stress such as UV-or herbicides-induced leads to noticeable lipoperoxidation [START_REF] Rijstenbil | UV-and salinity-induced oxidative effects in the marine diatom Cylindrotheca closterium during simulated emersion[END_REF]Wang et al., 2011).

New evidences on the interplay between behavioural and physiological response to lighttemperature stress in epipelic microphytobenthos

In response to HL alone, diatoms which were free to move (i.e. in the controls) could reach deeper layers of sediment with lower light conditions, potentially closer to their photosynthetic optimum [START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF]. This downward migration contributed to a reduction of the deleterious effects of HL observed in Lat A treated samples (i.e. immobilized cells at the surface of sediment) such as an enhanced accumulation of Chl a allomers and a slower ΦPSII recovery. Our results are in agreement with previous works using Lat A on natural biofilms [START_REF] Perkins | Vertical cell movement is a primary response of intertidal benthic biofilms to increasing light dose[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. Similarly, the motile diatoms could reach a depth with lower and more stable temperature, as shown by the in situ temperature depth profiles monitored here. Nevertheless, our measurements were not refined enough to conclude about the steepness of the vertical temperature gradient in muddy sediment, as for light [START_REF] Paterson | Microphytobenthos in contrasting coastal ecosystems: Biology and dynamics[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF]. Additionally, high temperature alone is known to directly affect the motility of MPB diatoms, i.e. over 35°C cell motility significantly drops-down [START_REF] Cohn | The effect of temperature and mixed species composition on diatom motility and adhesion[END_REF][START_REF] Du | Migratory responses of benthic diatoms to light and temperature monitored by chlorophyll fluorescence[END_REF]. Such feature might partly explain the stronger photoinhibition observed at 37°C in Lat A free samples, together with a direct impact of temperature on physiological mechanisms.

NPQ developed in the same range of mean values (i.e. 3-4) in situ than during LSRE with HL stress alone. Concomitantly, the DD+DT pool size as well as DES increased. DT synthesis was based not only on DD de-epoxidation (as illustrated by DES) but also by additional DT de novo synthesis [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF] which enlightened the high level of stress the cells were exposed to. Whatever the source, additional DT provides higher physiological photoprotection via higher NPQ and/or via higher prevention of oxidative lipid peroxidation [START_REF] Lepetit | Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]. DT synthesis (DD+DT and DES) is enhanced by the high temperature stress alone [START_REF] Salleh | The effects of temperature on the photosynthetic parameters and recovery of two temperate benthic microalgae, Amphora cf. coffeaeformis and Cocconeis cf. sublittoralis (Bacillariophyceae)[END_REF], and this effect was even stronger in conditions of a coupled HL and high temperature stress.

Specifically, DD+DT and DES showed a slower recovery which could represent a feed-back reaction (i.e. related to HL/high temperature impairment of the Calvin cycle activity, [START_REF] Mathur | Photosynthesis: Response to high temperature stress[END_REF] for the maintenance of a photoprotection capacity to cope with prolonged stressful temperature conditions, even under LL [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF]. When the diatom motility was inhibited by Lat A, DES significantly increased independently of the temperature, and its recovery was slower. This was apparently not due to a higher DT de novo synthesis as illustrated by the similar DD+DT pool size with and without Lat A. Higher DT synthesis in presence of Lat A had no significant effect on NPQ, except for its increased sustainability when in conjunction with high temperature. Higher sustained NPQ (i.e. qI) is known to be related with both sustained DT and photoinhibition in conditions of a strong stress [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF]. As a consequence, the DT quenching efficiency (i.e. the slope of the NPQ-DT relationship) was lower in presence of Lat A, likely due to the differential involvement of DT molecules in NPQ [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]: in presence of Lat A, the additional DT molecules instead most probably acted as ROS scavengers in prevention of lipid peroxidation [START_REF] Lepetit | Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms[END_REF] arguing for a pronounced stress (i.e. HL + hight temperature + inhibition of motility) level.

Although, as expected, the Lhcx pattern in sediment samples was not as clear as the one of monocultures of N. phyllepta, one of the dominant species common to the FR and PT MPB communities, the confirmation of the presence of some of the known isoforms was possible.

While an isoform with a similar size than Lhcx1 apparently did not react to any of the treatments, we found that the synthesis of two isoforms were enhanced by HL, high temperature and Lat A: one isoform with a size similar to Lhcx3, and one isoform with the size of a dimer which especially positively reacted to high temperature and Lat A treatment.

Interestingly, these two isoforms were also present in N. phyllepta. A third isoform (of about 24 kDa), which was not detected in N. phyllepta, was synthetized in all treatments. Based on our knowledge of the synthesis and roles of Lhcx proteins during prolonged stress, it is reasonable to argue that these isoforms participated, at least in part, to the above described NPQ and XC patterns during the coupled HL and high temperature stress. Interestingly, the Lhcx3 and 24 kDa isoforms strongly reacted to Lat A out of any stress and to a level comparable to HL stress. It suggests that the inhibition of motility might generate a strong preventive physiological answer in diatom cells blocked at the surface of sediment (which can occur in situ during sediment desiccation events for instance), an additional hint that physiological and behavioural photoprotection are closely related in epipelic diatoms. This compensation, together with a stronger NPQ induction and XC activity, was nevertheless not strong enough to prevent immobilized cells from additional photoinhibition, as ΦPSII recovery was always lower in Lat A treated samples than in migratory biofilms.

Conclusions and perspectives

By using a number of methodological novelties in MPB research (a new cryo-sampling method, lipid peroxidation markers for photooxidative stress-TBARS, photosynthetic protein markers for physiological protection-Lhcx), the present study clearly strengthens the previous statement of a coupled and complementary action of physiological and behavioural processes in protecting the photosynthetic activity of MPB diatoms in stressful environmental conditions [START_REF] Perkins | Vertical cell movement is a primary response of intertidal benthic biofilms to increasing light dose[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF][START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. Moreover, our results bring a physiological basis to the previously reported depression of MPB photosynthetic productivity in summer [START_REF] Guarini | Modelling the mud surface temperature on intertidal flats to investigate the spatio-temporal dynamics of the benthic microalgal photosynthetic capacity[END_REF]. They emphasize the joint role of temperature and light in limiting, at least transiently (i.e. during emersion), the photosynthetic activity of MPB biofilm in situ. They confirm that high temperature, together with high light, is an important environmental driver which supports seasonal, spatial and potentially latitudinal MPB biodiversity and biomass distribution at the scale of intertidal mudflats along the Atlantic coast of Southern Europe [START_REF] Guarini | Modelling the mud surface temperature on intertidal flats to investigate the spatio-temporal dynamics of the benthic microalgal photosynthetic capacity[END_REF][START_REF] Brito | Seasonality of microphytobenthos revealed by remote-sensing in a South European estuary[END_REF].

Moreover, temperature being expected to increase due to global change [START_REF] Solomon | Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change[END_REF], further longer-term experiments are required in order to question its importance in modulating MPB photosynthetic activity. Our results support previous works suggesting that MPB gross photosynthesis will most probably not be affected by a small increase (even if significant, i.e. +1°C to +4°C for the most extreme previsions; [START_REF] Solomon | Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change[END_REF] of average temperature [START_REF] Vieira | Effects of short-term changes in sediment temperature on the photosynthesis of two intertidal microphytobenthos communities[END_REF][START_REF] Hancke | Effects of temperature and irradiance on a benthic microalgal community: A combined two-dimensional oxygen and fluorescence imaging approach[END_REF]. However, it does not preclude a potential change in the community structure, as observed for associated invertebrate assemblages [START_REF] Hicks | Impact of biodiversity-climate futures on primary production and metabolism in a model benthic estuarine system[END_REF], which might be of importance in driving the whole community response as reported here. Further experiments should rather focus on the cumulative effects of successive extreme events such as heat waves experienced during spring/summer [START_REF] Schär | The role of increasing temperature variability in European summer heatwaves[END_REF][START_REF] Stott | Human contribution to the European heatwave of 2003[END_REF], with an emphasis on the 'species/individual' vs. 'community/biofilm' response.

Experimental procedures

Study areas and experimental design

Two intertidal mudflats were sampled along the Atlantic Coast of Southern Europe, in France and Portugal (FR and PT sites, respectively): a macrotidal one in the Baie de l'Aiguillon near Esnandes (FR, 46°15.36'N, 1°8,55'W) and a mesotidal estuary in the Ria de Aveiro near Vista Alegre (PT, 40°35' N, 8°41' W). Both sites are composed of fine muddy sediments (dominant particle size < 63 µm) and are known to be colonized by epipelic diatom-dominated MPB throughout the year [START_REF] Haubois | Spatiotemporal structure of the epipelic diatom assemblage from an intertidal mudflat in Marennes-Oleron Bay, France[END_REF]Serôdio et al., 2012, respectively).

All experiments were performed consecutively in early summer (weeks 26-27, June-July 2012). For each site, natural migratory MPB was studied in situ over one diurnal emersion period. In parallel, light stress-recovery experiments (LSRE) were carried out in controlled conditions in the laboratory at two temperatures using freshly collected samples (see section below). During LSRE, samples were sequentially exposed to (i) low light (LL) level to determine pre-stress reference state; (ii) high light (HL) intensity to induce photoinhibitory effects; (iii) LL to monitor recovery from HL stress [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF].

In situ measurements

For each site, the day for carrying out the experiment was selected so that the emersion period matched with maximum light and temperature exposure, i.e. noon/early afternoon low tides.

Photosynthetic Active Radiation (PAR) and temperature at the surface of the sediment were assessed every 30 s with a universal light-meter and data logger (ULM-500, Walz Effeltrich Germany) equipped with a plane light/temperature sensor (accessory of the ULM-500) and a plane cosine quantum sensor (Li-COR). Depth temperature profiles were measured with Hobo sensors (Hobo Pro V2, Massachussets, USA) fixed on a home-made stick that was vertically inserted into the sediment to position the sensors at four (-0.5 cm, -2 cm, -5 cm, -10 cm). The effective photosystem II quantum yield (ΦPSII) and the non-photochemical quenching of chlorophyll a (Chl a) fluorescence (NPQ) of MPB was assessed with a Water-PAM (microfiber version, Walz GmbH, Effeltrich, Germany) as described in [START_REF] Lefebvre | Duration of rapid light curves for determining the photosynthetic activity of microphytobenthos biofilm in situ[END_REF]. ΦPSII was calculated as ∆F / F m ' = (F m -F) / F m '. Rapid light curves (RLCs) with 30 s light steps were used to measure NPQ vs irradiance (NPQ-E) curves (as recommended in [START_REF] Lefebvre | Duration of rapid light curves for determining the photosynthetic activity of microphytobenthos biofilm in situ[END_REF]. NPQ-E curves were fitted with the model by [START_REF] Serôdio | A model for describing the light response of the nonphotochemical quenching of chlorophyll fluorescence[END_REF] in order to estimate NPQ max where NPQ = (F m -F m ') / F m . F m and F m ' are the maximum Chl a fluorescence levels measured in dark-adapted and illuminated (at the end of each RLCs light steps) MPB, respectively, while F is the steady-state Chl a fluorescence level measured at the end of each RLC light step, just before F m ' was assessed. F was measured with the application of a non-actinic modulated beam of 455 nm, and F m and F m ' with the application of oversaturating (about 4000 µmol photons m -2 s -1 ) light pulses (800 ms). Because in in situ as well as in laboratory (see below) conditions (i.e. light-responsive migratory biofilm) it is virtually impossible to measure a true F m level, for the calculation of NPQ, F m was in reality the maximum F m ' of the respective RLC (i.e. F m ' max ~ F m , see [START_REF] Lefebvre | Duration of rapid light curves for determining the photosynthetic activity of microphytobenthos biofilm in situ[END_REF].

Light stress-recovery experiments (LSRE)

LSRE were carried out following a protocol adapted from [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. The top 1 cm of sediment was collected at the beginning of low tide and immediately transported to the laboratory where it was sieved (0.5 mm mesh) to remove debris and macrofauna. Sediment was thoroughly mixed and spread in trays, forming a 2-4 cm thick slurry, and it was covered with seawater from the sampling site and left undisturbed overnight in the dark at room temperature (~20-25°C). The next morning, the slurry was homogenized and transferred to 24-well plates using a 100 mL syringe, filling the wells completely (ca. 3 mL). The well plates were then exposed to constant LL of 50 µmol photons m -2 s -1 , provided by three slide projectors (Reflecta Diamator AF, Reflecta GmbH, Rottenburg, Germany) containing halogen lamps (Quartzline DDL 150W, General Electric, USA) to induce the upward migration of MPB. Once the MPB biofilm was formed (typically after 3 h), the plates were transferred to a water bath of constant temperature of 25°C or 40°C and exposed to constant LL for 30 min. These two experimental temperatures were defined based on a previous study (Blanchard et al., 1997) which defined 25°C as an optimum and 35-40°C as an extreme maximum for the photosynthetic productivity of MPB. A fixed volume (200 µL) of Lat A solution (10 µM final concentration) or of filtered sea water (Control samples: Ctrl) was added to each well. Lat A solution was freshly prepared daily from a 1 mM stock solution of purified Lat A (Sigma-Aldrich) dissolved in dimethylsulfoxide and stored at -20°C [START_REF] Cartaxana | Inhibiting diatom motility: a new tool for the study of the photophysiology of intertidal microphytobenthic biofilms[END_REF].

After 15 min waiting for the inhibitor to diffuse, the plates were exposed to HL (1200 µmol photons m -2 s -1 , 3 h). This light dose (12.96 mol photons m -2 ) was previously shown to be excessive for MPB photosynthesis [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. After 3 h, samples were placed under LL for 15 min. In parallel, additional samples (for both Ctrl and +Lat A treatments) were maintained during 3 h under continuous LL. Temperature of the sediment in the top 5 mm was regularly checked using the same sensor previously described. The duration of each step described above was always the same, but the timing was adjusted so that HL period always started at the low tide maximum expected in situ.

For each chemical and light treatment (Ctrl and +Lat A; HL or continuous LL), several endpoints were assessed on 3-6 independent samples at three times: just before the HL stress (T 0 ), at the end of the HL stress and after 15 min of LL recovery (Recov). For the experiments with FR samples, Chl a fluorescence was measured using an Imaging-PAM fluorometer (Maxi-PAM M-series, Walz GmbH, Effeltrich, Germany) and samples were collected for further quantification of photosynthetic pigments, lipid peroxidation, and Lhcx proteins. For PT samples, Chl a fluorescence was measured with a FluorCAM 800MF, open version (PSI, Brno, Czech Republic, see [START_REF] Serôdio | A method for the rapid generation of non-sequential light-response curves of chlorophyll fluorescence[END_REF]. Samples were collected for the same analyses than for FR site, except that pigments were not quantified. Samples were collected for the same analyses than for FR site, except that pigments were not quantified. ΦPSII and NPQ were calculated as previously described [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF].

For the sediment sampling in wells, a contact-corer specially designed was used to collect rapidly (< 30 s) the uppermost layer of sediment (0.5 mm thick, 1.5 cm 2 area) in each well. It is based on the cryo-sampling of the biofilm, through the contact of the sediment surface with a metal cylinder of known length previously cooled in liquid nitrogen (Laviale et al., submitted). Each core was immediately frozen in liquid nitrogen and stored at -80°C until further analysis.

Pigment analysis

Pigment extraction and HPLC analysis were carried out following [START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF], with the exception of the first extraction step which was performed after 24 h lyophilisation of the sediment cores (3 mL of 4°C mixture extraction buffer: 90% methanol/0.2 M ammonium acetate (90/10 vol/vol) + 10% ethyl acetate). Extracted samples were analyzed with a Hitachi LaChrom Elite HPLC system equipped with a 10°C-cooled autosampler and a Nucleosil 120-

Lipid peroxidation

Lipid peroxidation generated by oxidative stress was estimated by quantifying thiobarbituric acide reactive substances (TBARS) according to [START_REF] Kwon | Malonaldehyde in aqueous solution and its role as measure of lipid oxidation in foods[END_REF]. Each sample was resuspended in 0.8 mL of 10% trichloroacetic acid (TCA, Sigma-Aldrich) and 500 mg L -1 butylated hydroxytoluene (BHT, Sigma-Aldrich) and sonicated (U200S control; IKA Labortechnik, Ika-Werke GmbH, Staufen, Germany) on ice 5 times during 30 s (cycle: 1, amplitude: 30%) with intervals of 30 s to prevent overheating. After extraction, 0.8 mL of 0.5% thiobarbituric acid (TBA, Sigma-Aldrich) in 10% TCA was added. The mixture was heated for 30 min at 90 °C and immediately put on ice to stop the reaction. Samples were then centrifuged (15 min, 10000 g) and 250 µL of supernatant were transferred in a 96 well-plate (Sarsted). TBARS were measured by subtracting the nonspecific turbidity (at 600 nm) from the TBARS absorption maximum (532 nm) using a Synergy HT absorbance microplate reader (BioTek Instruments Inc., Vermont, USA). Blanks consisted of the same mixture without sediment sample. TBARS concentration was expressed in malondialdehyde (MDA) equivalent (nmol mL -1 ) using an extinction coefficient of 155 mM -1 cm -1 . As it was not possible to obtain paired measurements of Chl a and MDA, values were normalized to the microphytobenthos biomass present at the sediment surface (measured by spectral reflectance: see below). As migration was susceptible to occur in Ctrl samples, only Lat A-treated samples could be compared.

MPB surface biomass

MPB biomass present in the surface layers of the sediment was estimated by spectral reflectance analysis with a fiber optic spectrometer (USB2000-VIS-NIR, grating #3, Ocean Optics, Duiven, The Netherlands) according to [START_REF] Serôdio | Effects of chlorophyll fluorescence on the estimation of microphytobenthos biomass using spectral reflectance indices[END_REF]. In particular, measurements were carried out under LL (50 µmol photons m -2 s -1 ) supplied with an halogen lamp (120W, Plusline ES, Philips Lighting) positioned with a 45° angle to the sediment surface while the sensor was at a fixed distance (~2 cm) perpendicularly to it. Reflectance measurements at 550, 600 and 675 nm ( 550 ,  600 and  675 , respectively) were used to calculate the normalized index I diat as follows: I diat = 2  600 / ( 550 +  675 ) (modified from [START_REF] Kazemipour | Microphytobenthos biomass mapping using the optical model of diatom biofilms: Application to hyperspectral images of Bourgneuf Bay[END_REF]. It is based on the spectral peak centered at 600 nm between the absorbance bands due to the fucoxanthin at 550 nm and the Chl a at 675 nm. In comparison to other MPB spectral indices available [START_REF] Méléder | Hyperspectral imaging for mapping microphytobenthos in coastal areas[END_REF], it was shown to be less affected by water or detritical organic matter contents, thus allowing a better comparison between sites (data not shown).

Lhcx protein analysis

Protein extraction from sediment cores, SDS-PAGE, Western-blot and ECL immunodetection were carried out following the protocol by [START_REF] Coesel | Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity[END_REF] modified by [START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF]: In order to improve the proteins extraction, sediment samples were exposed to eight freeze thaw cycles in the extraction buffer (500 µL lysis buffer + 500 µL protease inhibitor, Sigma-Aldrich, USA) and then incubated at RT (total extraction was 90 min). Chl a concentration of extracted samples was determined according to [START_REF] Jeffrey | New spectrophotometric equations for determining chlorophylls a, b c1 and c2 in higher-plants, algae and natural phytoplankton[END_REF] and samples corresponding to an amount of 0.5 µg Chl a were loaded on the gel.

Correct blotting was verified by correct transfer of pre-stained protein markers on the PVDF membrane (Amersham Hybond-P, GE Healthcare life Sciences, USA) and by staining gels with Coomassie R-250 (Pierce Imperial Protein stain, Thermo scientific, Rockford, USA).

Anti-FCP6 (Lhcx1) from Cyclotella cryptica [START_REF] Westermann | Localisation of fucoxanthin chlorophyll a/c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy[END_REF] and anti-Lhcx6 from T. pseudonana [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF] were applied in a 1:1000 dilution for LR samples overnight (respective incubations with the secondary antibody were 2 h and 1 h). For PT samples, the anti-Lhcx6 antibody did not yield a good signal; anti-FCP6 was used at a dilution of 1:1000 (incubation with the secondary antibody, 1 h). Note that because of the species-specific immunodetection pattern no quantification per se was possible. However, the comparison with the planktonic models P. tricornutum [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF] and T. pseudonana [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF] enabled to possibly distinguish different isoforms including some for which behaviour under HL and temperature stress and their role is known or suspected (see Introduction; [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF][START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF][START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF][START_REF] Büchel | Fucoxanthin-chlorophyll-proteins and non-photochemical fluorescence quenching in diatoms. In Non-Photochemical Fluorescence Quenching and Energy Dissipation In Plants, Algae and Cyanobacteria[END_REF]. For P. tricornutum, T. pseudonana and N. phyllepta samples, the procedure was as in [START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF]: exponentially growing cells were exposed to HL stress (2 h at 2000 µmol photons. m -2 . s -1 ) to induce protein expression of several Lhcx isoforms which were separated on the LDS-gel.

Taxonomic composition

For each site, the first cm of sediment was collected and stored at -20 °C until analysis.

Extraction from sediment of the organic fraction, including microalgae cells, was done following [START_REF] Méléder | Spatiotemporal changes in microphytobenthos assemblages in a macrotidal flat (Bourgneuf bay, France)[END_REF]. Preliminary observations of the organic fraction using optical microscopy (Olympus AX70 using 200-fold magnification) were performed to confirm the absence of other classes of microalgae than diatoms. Permanent slides of cleaned diatom frustules (cremation: 2 h at 450 °C) were then prepared in a high refractive index medium (Naphrax; Northern Biological Supplies Ltd, Ipswich, UK). The taxonomic composition was determined to the species level by examining 200-300 frustule valves at 500 fold magnification on the basis of morphological criteria using reference works (Ribeiro et al., 2013, and references therein). Scanning electron microscopy was used to confirm the analysis (JEOL JSM 7600F reaching 50 000-fold magnification).

Statistics

Data presented are mean ± one standard deviation (SD). Each LSRE experiment was performed twice. Analyses of variance (ANOVA), Tukey's honestly significant difference (HSD) tests, Student's t-tests and linear regressions were performed using the R statistical computing environment (v 2.15.1, Ihaka and Gentleman, 1996). In particular, potential interactions between factors were tested considering data set from each site individually, with time (T 0 , after 3 h of HL, after 15 min recovery under LL), temperature (27°C and 37°C) and chemical (Ctrl or +Lat A) as fixed factors. The fact that Chl a fluorescence measurements were made on the same samples over the experiment was also taken into account using the sample name as random effect in a linear mixed effect model (lme function of the nlme package: [START_REF] Pinheiro | Mixed-models in S and S-PLUS[END_REF]. Comparisons between sites (FR, PT) were performed on data gathered at the end of the LSRE (after recovery), expressed as % of T 0 . Site, temperature and chemical were considered as fixed factors. Data normality (Shapiro-Wilk test) and homoscedasticity (Bartlett test) were checked using the residuals. (-). HL, after 3 h HL (1200 µmol photons m -2 s -1 ); LL, after 3 h of LL (50 µmol photons m -2 s -1 ). All samples were normalized to the same Chl a concentration (15 µg Chl a mL -1 ). A-Lhcx proteins were detected using a monoclonal anti-FCP6 (Lhcx1). This antibody detects, similarly as the anti-LHCSR of C. reinhardtii [START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF], also Lhcx2 and Lhcx3 in P. tricornutum (strain 'P.t.1') used here as a control. N. phyllepta s.l. (N.p.), the dominant species of the MPB community, was also tested. B-and C-Lhcx proteins were detected using a monoclonal anti-Lhcx6 from T. pseudonana (T.p.) [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF] which also potentially detect Lhcx3 and an Lhcx dimer in P. tricornutum ; C-is a special focus on the band between 33-50 kDa in 37°C exposed samples. Revelation times were: A-30 s; B-15 s for the bands between 17-23 kDa, 45 s for the band between 33-50 kDa; C-30 s;. Note that for A-all samples exposed at 37°C did not yield satisfying signal; and for B-, the sample HL+ 27°C was lost. s -1 ) following 3 h of HL exposure (1200 µmol photons m -2 s -1 ), *** significant difference between sites (P < 0.001, Tukey HSD). Values are mean ± SD (n=6) .

Figure 8

Western-blot of the Lhcx proteins of microphytobenthos harvested at the Portuguese site during light stress recovery experiments performed on sediment exposed at two temperatures (27°C or 37°C) and with an inhibitor of cell motility (+) or without (-). HL, after 3 h HL (1200 µmol photons. m -2 . s -1 ) treatment; LL, after 3 h of LL exposure. All samples were normalized to the same Chl a concentration (15 µg Chl a mL -1 ). Lhcx proteins were detected using a monoclonal anti-FCP6 (Lhcx1). This antibody detects, similarly as the anti-LHCSR of C. reinhardtii [START_REF] Lepetit | High light Acclimation in the secondary plastids containing diatom Phaeodactylum tricornutum is triggered by the redox state of the plastoquinone pool[END_REF], also Lhcx2 and Lhcx3 in P. tricornutum (strain 'P.t.1') used here as a control. Note that i) the 27°C and 37°C samples were not loaded on the same gel but the revelation time was the same (30 s), ii) the use of the monoclonal anti-Lhcx6 from T. pseudonana did not yield any signal with these samples.

  optimum at both sites (~ 0.70: Fig. 1C-D, open squares). It decreased/increased opposite to the PAR evolution over the emersion period, reaching values as low as ~ 0.40. The maximal non-photochemical quenching of Chl a fluorescence (NPQ max ) was about 4-5 at PT site and lower than 1 at FR site at the beginning of emersion (Fig. 1C-D, closed circles). It decreased continuously at PT site to reach values around 3 at the end of the emersion, while it covaried with PAR at FR site, reaching a maximum value of about 6 (matching the sudden increase in PAR level) and values of 3-4 at the end of the emersion.

5

  C18 column (Macherey-Nagel). The allomerization of Chl a was calculated as the ratio AR = Chl a-allo / tot Chl a × 100 where tot Chl a = (Chl a-allo + Chl a' + Chl a) and Chl a-allo and Chl a' are the allomer and epimer forms of Chl a, respectively. The pigment molar concentrations (expressed as mol. 100 mol Chl a -1 ) for the xanthophyll cycle (XC) pigments diadinoxanthin (DD) and diatoxanthin (DT) were used to calculate the DD de-epoxidation state (DES) as follows: DES = [DT / (DD + DT)] × 100.

  Figure legends

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Figure 4

 4 Figure 4

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6

Figure 7

 7 Figure 7

  Figure 1_Laviale et al.

Acknowledgements

The present study was supported by the Egide/Campus France-PHC Pessoa exchange program (n°27377TB, JL and JS), the Centre National de la Recherche Scientifique-CNRS (JL), the University of La Rochelle-ULR and the Contrat Plan Etat Région-CPER 'Littoral' (JL, BL post-doc grant), the Region Poitou-Charentes (AB Ph.D. grant), the Deutscher Akademischer Austausch Dienst-DAAD (BL post-doc grant), European Funds through COMPETE, the Portuguese Science Foundation (FCT) through projects PEST (PEst-C/MAR/LA0017/2013) and MigROS (PTDC/MAR/112473/2009: JS, ML post-doc grant and SF research assistant grant), and through grants SFRH/BSAB/962/2009 (JS), SFRH/BD/ 44860/2008 (JE Ph.D. grant). The authors thank Dr. E. Rhiel (Univ. Oldenburg, Germany) and Prof. B. Green (Univ. British Columbia, Canada) for the kind gifts of the anti-FCP6 and of the anti-Lhcx6, respectively, Prof. B. Green for her help analyzing the Lhcx6 Western-blot, C. Pais for her technical assistance with the TBARS quantification, Dr L. Ribeiro (Univ.

Lisboa, Portugal) for his help in confirming the identification of N. phyllepta s.l., and Sr Armindo do Evaristo for his cheerful support. We thank the two anonymous reviewers for their constructive comments on the manuscript.


Jean-Charles Cadoret

Raphael Demoulière

Johann Lavaud

Hans J Van Gorkom

Jean Houmard

Anne-Lise Etienne
email: aetienne@biologie.ens.fr

Dissipation of excess energy triggered by blue light in cyanobacteria with CP 43' (IsiA)

The stress-induced peripheral chlorophyll antenna in cyanobacteria is found to confer a new type of photoprotection that is triggered by blue light.

The chlorophyll-protein CP43' induced by stress conditions in cyanobacteria with the isiA gene is shown to serve as an antenna for photosystem II, in addition to its known role as an antenna for photosystem I. At high light intensity, however, it is converted to an efficient trap for chlorophyll excitations that protects system II from photoinhibition. In contrast to the 'energy-dependent non-photochemical quenching' in chloroplasts, this photo-protective energy dissipation in cyanobacteria is triggered by a blue light receptor. The induction is proportional to light intensity. Induction and decay of the quenching have the same large temperature-dependence.

Sunlight is the driving force for photosynthesis. At the level of individual cells, the available light intensity varies unpredictably. Photosynthetic organisms can adjust to changes in average light intensity in various ways, together called photo-acclimation. They also have to cope with light intensity fluctuations that are too large or too fast for photoacclimation to occur (1). This is especially clear in the case of the oxygen-evolving photosystem II (PSII), where over-saturating excitation leads to a rapid inhibition when destruction of the reaction center protein D1 becomes faster than its replacement (2). In the chloroplasts of algae and higher plants, excessive illumination induces a change in the chlorophyll binding antenna proteins that accelerates thermal dissipation of the excitation energy, as conveniently monitored by a decrease of the chlorophyll fluorescence yield (2,3). This 'non-photochemical quenching' (NPQ) appears to be triggered by excessive acidification of the thylakoid lumen when consumption of the proton gradient by ATP synthesis cannot keep up with its generation by electron transport. It involves deepoxidation of xanthophylls bound to the antenna proteins (4,5) and requires specific polypeptides, like PsbS in higher plants (6,[START_REF] Aspinall-0'dea | Proc. Natl. Acad. Sci. USA[END_REF]. In some cases NPQ can reduce the excitation rate of PSII reaction centers by an order of magnitude [START_REF] Lavaud | [END_REF].

A comparable photo-protective mechanism has not been reported for cyanobacteria, whose shorter generation time allows for a more rapid photo-acclimation (9). They normally have phycobilisomes (PBS) instead of chlorophyll binding antenna proteins.

Under stress conditions, however, many cyanobacteria such as Synechococcus PCC 7942 and Synechocystis PCC6803 synthesize a chlorophyll-binding polypeptide called CP43', the product of isiA (10,11,12). CP43' is a homolog of the PSII core antenna protein CP43 (PsbC) and of Pcb in Prochlorococcus and Prochloron, which serves as a peripheral antenna for both photosystems [START_REF] Bibby | Proc. Natl. Acad. Sci[END_REF][START_REF] Bibby | [END_REF]. CP 43' was recently shown to be an antenna for PSI (15,16,17,18). A role in light-harvesting for PSII has also been proposed (19).

However, the effective cross-section of PSII was actually found to be smaller in the presence of CP43' in Synechococcus PCC 7942, suggesting that CP43' is an efficient quencher of chlorophyll excitations that protects PSII from over-excitation (20,21).

It seems unlikely that the same protein could serve both as an antenna and as an excitation sink, unless it can exist in a non-quenching as well as in a quenching form. Here we demonstrate that the accumulation of CP 43' is, in fact, accompanied by the appearance of a reversible, light-induced NPQ that provides an effective protection against photoinhibition of PSII. A first characterization of induction and decay of this cyanobacterial NPQ reveals that its regulation is completely different from the analogous phenomenon in chloroplasts of photosynthetic eukaryotes.

Iron-starvation of Synechocystis PCC 6803 (22a) leads to gradual changes in the relative concentrations of CP43', PSI and PSII over generations. Especially the iron-rich PSI is severely down-regulated, while CP43' is up regulated (11). To monitor the changing photosystem composition, fluorescence emission spectra were measured at 77 K with preferential chlorophyll excitation (22a). The PSI peak was shifted from 723 to 719 nm and decreased relative to the amplitude of the PSII emission peaks at 685 nm and 695 nm, reflecting a decrease of the fraction of chlorophyll associated with PSI. The F685/F695 ratio increased from less than 1 in the control cells to 1.5 after 4 days of iron starvation and up to 2 after 10 days. CP43' not only contributes to PSI excitation but also to PSII and ultimately accumulates in a form that does not efficiently transfer excitation energy to either photosystem and fluoresces at 685 nm.

At room temperature, the fluorescence normally comes from PSII. Its yield can vary between F 0 , at maximum photochemical quenching, and a maximum level F m , when accumulation of the reduced electron acceptor Q A -prevents photochemical activity. To estimate the effective antenna size of PSII, we measured the rise time of the light-induced fluorescence increase in the presence of DCMU (22a), which blocks electron transfer beyond Q A [START_REF] Duysens | Studies on Microalgae and Photosynthetic Bacteria, special issue of Plant Cell Physiol[END_REF]. When chlorophylls were excited at an intensity that produced a half-rise time of 80 ms in control cells, the half-rise time was 28 ms in iron-starved cells. Iron starvation caused a nearly 3-fold increase of the chlorophyll antenna size of PSII. In control cells (Figure 1A), the NPQ only quenches F m and persists after illumination. These properties are characteristic of the quenching by PSII centers that are damaged by photo-inhibition (known as 'qI' ( 24)). In iron-starved cells (Figure 1B andC), the NPQ appears much faster, quenches both F 0 and F m , and disappears after illumination.

These properties are qualitatively similar to the 'energy dependent' NPQ of the PSII antenna in chloroplasts (known as 'qE' [START_REF] Krause | [END_REF]). The difference between Fig. 1B and 1C is in the extent of iron starvation, 4 days and 10 days, respectively. Iron deficiency causes an increase of F 0 and F m because the fraction of Chl associated with the low-fluorescent PSI decreases. At a later stage F m increases to a lesser extent than F 0 , due to the increasing contribution of CP43' fluorescence. The large NPQ reached in Figure 1C shows that it also quenches the fluorescence of CP43' that is not functionally connected to either photosystem. This indicates that the NPQ is associated with CP 43' and does not depend on its binding to PSII (or, presumably, to PSI).

The relative concentration of the non-photochemical quencher in Fig. 1A andB, quantified as F m /F m ' -1, with F m ' denoting the quenched F m level [START_REF] Lavaud | [END_REF], is plotted in Figure 1D. In control cells, NPQ steadily increased during illumination, reaching a value of 0.8 at 15 min, and did not decrease after illumination. In iron-starved cells, the NPQ rapidly reached 1.4 during the illumination, and decayed afterwards to 0.2, showing that a much smaller fraction of PSII centers was photo-inhibited than in the control cells. The decrease of the excitation rate of PSII centers by NPQ was confirmed by measurements of the initial slope of the saturation curve of oxygen evolution before and after NPQ induction by a continuous illumination (22a). The slope was decreased by 40 % by the presence of a NPQ of 1.5 (for F m ), in agreement with the observed quenching of F 0 [START_REF] Lavaud | [END_REF]. We conclude that NPQ in iron-starved cells protects PSII considerably from photo-inhibition. The initial rate of NPQ induction by blue light is proportional to light intensity; no saturation was found at intensities up to 1 mE.m -2 s -1 (Fig. 3A). The decay rate after illumination is mono-exponential, independent of the illumination used. Both induction and decay were strongly temperature-dependent. Fig. 3B shows that their Arrhenius plots were linear and parallel in the measured range of 5 -35 °C, with an activation enthalpy of 46 kJ/mole. These findings suggest that the quenching state is formed with a temperaturedependent quantum yield from the excited state of the blue light receptor and spontaneous reversal to the non-quenching state requires thermal activation to the same rare conformation of the local molecular environment that is required for the light-induced reaction to succeed.

Ferrous iron is an essential constituent of photosynthetic reaction centers.

Qualitatively, the observed responses of Synechocystis to iron starvation may be explained by the need to economize on reaction center production. To compensate for their decreased abundance, the remaining reaction centers are pushed to higher activity: The CP43' proteins added to PSI and PSII increase the absorption cross section and hence the average excitation rate per reaction center. In photo-acclimated cells however, such an increase would lead to photo-destruction at times of saturating light intensity. In this perspective, it is not surprising that the accumulation of CP43' coincides with the appearance of a powerful regulation mechanism that causes dissipation of the energy absorbed by CP43' at high light intensity.

Without CP43', cyanobacterial PSII contains chlorophyll only in the core complex, about 35 chlorophylls/PSII. We found that CP43' could increase the PSII chlorophyll antenna size nearly three times (80/28), so, around 70 chlorophylls per PSII were added. If CP43' contains 13 chlorophylls, like its homolog CP43, up to 5 CP43' proteins can be associated with each PSII core. The same number of Pcb proteins, another homolog of CP43', is constitutively associated with PSII in Prochloron and the structure of that complex (13) may serve as a model for that of PSII in iron-starved Synechocystis.

Apparently PSII cannot bind more than 5 CP43' units. Additional CP43' accumulated during prolonged iron starvation was found to be highly fluorescent, independent of the redox state of Q A .

Blue light converts CP43' to an efficient sink for excitation energy that reduces the rate of PSII photo-inhibition in strong light substantially. The conversion rate is proportional to light intensity, to the thermal population of a conformational state required for the conversion, and to the fraction of un-converted CP43' remaining. The quenching state is unstable and decays in minutes, independent of light intensity, but dependent on the same thermal activation. Compared to the photoprotective NPQ in chloroplasts, this simple two-state equilibrium of CP43'-associated NPQ seems relatively unsophisticated. As argued above, however, its primary function may not be to cope with rapid light intensity fluctuations, but to adjust the extra excitation rate provided by CP43' to an already photoacclimated system. .s -1 ) A

Figure 1

 1 Figure 1 shows recordings by a pulse amplitude modulated (PAM) fluorometer of

Figure 2

 2 Figure 2 compares the fluorescence yield changes induced in iron-starved cells by

Figure 1 Chlorophyll

 1 Figure 1

Figure 2 Fluorescence yield changes of 4 -

 24 Figure 2

Figure 3 Light

 3 Figure 3

  Figure 1 Cadoret, Demoulière, Lavaud, van Gorkom, Houmard and Etienne

Photoprotective quenching by CP43' in cyanobacteria triggered by blue light

Jean-Charles Cadoret, Raphael Demoulière, Johann Lavaud, Hans J. van Gorkom, Jean Houmard, Anne-Lise Etienne

Supplemental Online Material

Materials and Methods

Synechocystis PCC 6803 (Pasteur collection) was grown photo-autotrophically in 100-mL batch cultures at 32 °C in BG11 medium at a light intensity of 70 µE.m -2 s -1 in a thermostated shaker (Gallenkamp) with a CO 2 enriched atmosphere. For iron starvation, cells in their late exponential phase were diluted five times in iron-deficient medium. They continued to grow exponentially, and were diluted in fresh iron-deficient medium every other day, up to 10 days after onset of starvation. The growth rate slowed down during iron starvation (0.7 of the initial rate after 4 days down to 0.3 after 10 days).

Fluorescence emission spectra at 77 K were measured in a Hitachi spectrofluorometer with excitation at 440 nm. 20 µL samples were rapidly vacuum-sucked onto a paper filter that was plunged in liquid nitrogen immediately after.

Light-induced chlorophyll fluorescence yield changes at room temperature were measured using a PAM-101 Walz fluorometer (Walz, Effletrich, Germany) (S1). Samples at a concentration of 5 (µg chlorophyll).mL -1 were dark-adapted for 10 minutes before measurement. Fluorescence induction kinetics in the presence of DCMU were measured with the setup described in (S2).