domain: sde sdv sdu.envi

text:
Lander Blommaert

Marie J J Huysman

Wim Vyverman

Johann Lavaud

Koen Sabbe
email: koen.sabbe@ugent.be

Contrasting NPQ dynamics and xanthophyll cycling in a motile and a non-motile intertidal benthic diatom

Keywords: photoprotection, xanthophyll cycle, microphytobenthos, diatom, non-photochemical quenching

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Introduction

Diatoms are dominant primary producers in areas characterized by pronounced fluctuations in light conditions [START_REF] Armbrust | The life of diatoms in the world's oceans[END_REF][START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF]. Rapid changes in light climate in well mixed waters or on intertidal flats challenge planktonic and benthic diatoms, respectively, to adjust light harvesting to what can be safely used for photosynthesis. As periods of high light conditions can result in oxidative damage to, in particular, the photosystem II (PSII) core, diatoms possess various mechanisms to deal with high light stress: [START_REF] Serôdio | Analysis of variable chlorophyll fluorescence in microphytobenthos assemblages: implications of the use of depth-integrated measurements[END_REF] avoid excess light energy absorption by decreasing cell pigment content [START_REF] Macintyre | Photoacclimation of photosynthesis irradiance response curves and photosynthetic pigments in macroalgae and cyanobacteria[END_REF]; (2) dissipate excess excitation energy as heat in a process called Non-Photochemical Quenching of chlorophyll a (Chl a) fluorescence (NPQ) [START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF], and/or by engaging alternative electron cycling pathways [START_REF] Wagner | Photosystem II cycle activity and alternative electron transport in the diatom Phaeodactylum tricornutum under dynamic light conditions and nitrogen limitation[END_REF]); (3) scavenge reactive oxygen species (ROS) [START_REF] Janknegt | Oxidative Stress Responses in the Marine Antarctic Diatom Chaetoceros Brevis (Bacillariophyceae) During Photoacclimation 1[END_REF](Janknegt et al. , 2009a;;b;[START_REF] Waring | Light-Induced Responses of Oxygen Photoreduction, Reactive Oxygen Species Production and Scavenging in Two Diatom Species[END_REF]); (4) repair damaged PSII cores, mainly by replacing the D1 protein of the PSII reaction centre [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF][START_REF] Lavaud | Photosystem II repair in marine diatoms with contrasting photophysiologies[END_REF]); (5) behavioural down regulation through vertical cell movement (microcycling and bulk migration) [START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF][START_REF] Serôdio | Analysis of variable chlorophyll fluorescence in microphytobenthos assemblages: implications of the use of depth-integrated measurements[END_REF]). Of the above mechanisms, especially NPQ is able to track fast light fluctuations experienced in the natural habitat [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 | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF].

In land plants, three NPQ components have been distinguished, based on the relaxation kinetics after high light exposure: the rapidly relaxing component qE (seconds to minutes), the slower state transitions qT (tens of minutes), and the so-called 'photoinhibitory' quenching qI, which relaxes in the range of hours [START_REF] Horton | Studies on the induction of chlorophyll fluorescence in isolated barley protoplasts. IV. Resolution of non -photochemical quenching[END_REF]. In diatoms, however, only two of these have been observed [START_REF] Owens | Light-Harvesting Function in the Diatom Phaeodactylum tricornutum II. distribution of excitation energy between photosystems[END_REF]. Energy dependent quenching (qE) is the main component and is controlled by [START_REF] Serôdio | Analysis of variable chlorophyll fluorescence in microphytobenthos assemblages: implications of the use of depth-integrated measurements[END_REF] the build-up of a proton gradient (ΔpH) across the thylakoid membrane, (2) the (reversible) de-epoxidation of diadinoxanthin (Ddx) to diatoxanthin (Dtx) in the xanthophyll cycle (XC) and (3) the presence of Light-Harvesting Complex X (LHCX) proteins, homologs of the Light-Harvesting Complex Stress-Related (LHCSR) proteins found in green algae [START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF]. The origin of the second component (qI), however, is less clear. Besides PSII photoinactivation and damage, Dtx and some LHCX isoforms might be involved in this sustained quenching mechanism [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]. It is increasingly referred to as NPQs (for sustained NPQ) or 'dark NPQ' as it persists even under prolonged dark acclimation, particularly in intertidal benthic diatoms [START_REF] Perkins | The application of variable chlorophyll fluorescence to microphytobenthic biofilms[END_REF][START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF].

A molecular mechanism of qE in diatoms has been recently proposed [START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF]. qE is hypothesized to be based on two quenching sites within the LHC antenna of PSII: 1) Q2 which is localized in a part of the LHC that remains attached to the PSII and which directly depends on the synthesis and activation of Dtx, and 2) Q1 which is localized in a part of the LHC that detaches from PSII upon Dtx activation at Q2 and which forms an energy sink that amplifies Q2 quenching. It is believed that the persistence of Dtx, even in the dark, is responsible for keeping both quenching sites active and especially Q1, i.e. as long as Dtx is present at Q2 site, FCP (Fucoxanthin Chlorophyll a/c binding Protein) oligomers cannot reconnect to PSII which generates part of the sustained qI/NPQs [START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF].

Marked differences in NPQ capacity and kinetics were discovered between planktonic diatom species and even between ecotypes isolated from habitats experiencing different degrees of average irradiances and/or light fluctuations. These differences have been attributed either to variation in XC kinetics and/or the amount of LHCX proteins [START_REF] Lavaud | Photoprotection capacity differs among diatoms : Possible consequences on the spatial distribution of diatoms related to fluctuations in the underwater light climate[END_REF][START_REF] Dimier | Photoprotection and xanthophyll cycle activity in three marine diatoms[END_REF][START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF][START_REF] Petrou | Heterogeneity in the photoprotective capacity of three Antarctic diatoms during short-term changes in salinity and temperature[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]Lepetit et al. in press).

The emerging picture from these reports is that a higher/faster Dtx synthesis supports a faster NPQ induction and a higher NPQ capacity in species/ecotypes adapted to habitats characterized by strong light fluctuations and/or average higher irradiance [START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF]. One of the specificities is the de novo Dtx synthesis, which in case of prolonged stress light conditions helps to amplify photoprotection via enhanced NPQ and/or ROS scavenging [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].

Species thriving in fluctuating light conditions, for example, exhibit high de novo synthesis of Dtx molecules which correlates well with NPQ development during strong light conditions. Species experiencing a more stable light climate in their natural habitat also synthetize Dtx molecules de novo when shifted to high light conditions but these are probably not involved in NPQ but may rather have an antioxidant function [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]. In addition to a high NPQ capacity and fast Dtx production during oversaturating light conditions, fast relaxation of NPQ in low light conditions is key to track changes in irradiance [START_REF] Lavaud | Photoprotection capacity differs among diatoms : Possible consequences on the spatial distribution of diatoms related to fluctuations in the underwater light climate[END_REF]. As Dtx molecules have to be epoxidized back to Ddx to switch the antenna system from an energy dissipating to a light harvesting mode, diatoms with a high Dtx epoxidation rate dissipate NPQ faster compared to diatoms with a lower epoxidation rate (Goss et al. 2006;[START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. A difference in NPQ capacity can also be attributed to differences in LHCX protein content. The low amount of LHCX1 protein, for instance, explains limited NPQ capacity in a high latitude Phaeodactylum tricornutum ecotype isolated from a supralitoral rockpool (P.t.4) which experiences lower average light intensity, and less drastically fluctuating light conditions, compared to other ecotypes [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF]. Whereas the LHCX1 gene is already maximally expressed in low light conditions, several other LHCX family members of both centric and pennate diatoms are highly and rapidly upregulated when exposed to high light [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF][START_REF] Park | Dynamic response of the transcriptome of a psychrophilic diatom, Chaetoceros neogracile, to high irradiance[END_REF][START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress[END_REF]Lepetit et al. 2013, in press) and other stressful environmental conditions that impair photosynthetic capacity [START_REF] Taddei | Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum[END_REF]). These proteins might either confer higher NPQ capacity by binding newly synthetized Dtx molecules and/or be involved in NPQs after prolonged high light exposure [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress[END_REF]Lepetit et al. 2013, in press).

While our knowledge of NPQ regulation is mostly based on studies of planktonic diatoms, whose light climate is mostly governed by water column turbulence, far less attention has been paid to NPQ regulation in benthic diatoms thriving in, and on, the sediments of intertidal flats [START_REF] Jesus | Adaptations of microphytobenthos assemblages to sediment type and tidal position[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]Cartaxana et al. , 2016a;;b;[START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF][START_REF] Lavaud | The peculiar features of the non-photochemical fluorescence quenching in diatoms and brown algae[END_REF][START_REF] Ezequiel | Photoacclimation state determines the photobehaviour of motile microalgae: The case of a benthic diatom[END_REF][START_REF] Pniewski | Photoregulation in microphytobenthos from intertidal mudflats and non-tidal coastal shallows[END_REF][START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled lighttemperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]. Like terrestrial plants, these diatoms can experience fast light fluctuations, not buffered by a water column, during low tide. The tidal rhythm, furthermore, can change the light climate drastically as no or very little light reaches the sediments in turbid estuaries when submerged [START_REF] Underwood | Primary production by phytoplankton and microphytobenthos in estuaries estuaries[END_REF].

NPQ capacity of intertidal benthic diatoms is mainly defined by their ability or inability to avoid excess light energy [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][START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF]. Diatoms belonging to the raphid clade possess a raphe system that allows movement by secreting extracellular polymer substances (EPS) through the raphe slit. Raphid diatoms can thus migrate vertically into the sediment matrix to a more optimal light climate [START_REF] Consalvey | The ups and downs of life in a benthic biofilm: Migration of benthic diatoms[END_REF]). In addition, microcycling of motile diatoms within the top layers of the sediments was proposed with algae migrating down to avoid photoinhibition being replaced by others [START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF][START_REF] Serôdio | Analysis of variable chlorophyll fluorescence in microphytobenthos assemblages: implications of the use of depth-integrated measurements[END_REF]. Such sequential turnover at the species level was indeed observed in laboratory mesocosms [START_REF] Paterson | The migratory behaviour of diatom assemblages in a laboratory tidal micro-ecosystem examined by low temperature scanning electron microscopy[END_REF] and during an in situ emersion period [START_REF] Underwood | Patterns in microphytobenthic primary productivity: Species-specific variation in migratory rhythms and photosynthesis in mixed-species biofilms[END_REF].

In contrast, diatoms living attached or in close association with single sand grains (epipsammic diatoms) are immotile or only capable of limited movement and therefore need to rely on physiological photoprotection [START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF]). This can explain a higher deepoxidation of the Ddx-Dtx cycle pigments in epipsammic communities [START_REF] Jesus | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF]. [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF] experimentally demonstrated higher NPQ values, coupled with higher Dtx content, in epipsammic diatoms. A more comprehensive comparison between the regulation and kinetics of the NPQ mechanism of both motile and non-motile diatoms, however, has so far not been made.

In this study we demonstrate fast irradiance tuning of NPQ, coupled with fast XC kinetics in the immotile epipsammic diatom species Opephora guenter-grassii. We show that this species, in addition to Dtx, also accumulates considerable amounts of the de-epoxidized xanthophyll zeaxanthin (Zx) during a short period (1 h) of high light exposure, a feature so far only observed in planktonic diatoms after prolonged (up to 6 h) periods of oversaturating light conditions [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF]. As the high de novo synthesis of de-epoxidized xanthophylls in this species is not paralleled by an equal increase in NPQ, these xanthophylls are not expected to be directly involved in the NPQ mechanism. In contrast, an epipelic species, Seminavis robusta, shows a less dynamic NPQ, despite concerted de novo synthesis of both Dtx molecules and LHCX proteins.

Our findings add to the physiological underpinning of the differential response of motile and nonmotile diatom species [START_REF] Juneau | Combined effect of high light and high salinity on the regulation of photosynthesis in three diatom species belonging to the main growth forms of intertidal flat inhabiting microphytobenthos[END_REF][START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF] and of benthic diatom communities in sediment [START_REF] Pniewski | Photoregulation in microphytobenthos from intertidal mudflats and non-tidal coastal shallows[END_REF][START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled lighttemperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]Cartaxana et al. 2016b) to their environment.

Materials

Culture conditions

Strains were obtained from the diatom culture collection (BCCM/DCG) of the Belgian Coordinated Collection of Micro-organisms (http://bccm.belspo.be), accession numbers Seminavis robusta (DCG 0105) and Opephora guenter-grassii (DCG 0448), and grown in semi-continuous batch culture in 1.8 l glass Fernbach flasks (Schott) in a day/night rhythm of 16/8 hour with a Photosynthetically Available Radiation (PAR) of 20 µmol photons m -2 s -1 . Cells were cultured in Provasoli's enriched f/2 seawater medium using Tropic Marin artificial sea salt (34.5 g l -1 ) enriched with NaHCO3 (80 mg l -1 final concentration). Cultures were acclimated to these culturing conditions for at least 2 weeks. Chlorophyll a (Chl a) was measured daily 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] to monitor growth.

High light exposure

Cultures in exponential growth were concentrated to 10 mg Chl a l -1 by centrifugation (Eppendorf 5810 R) at 4000 RCF for 5 min in 50 ml falcons. The cultures were again acclimated to their growth conditions for 2 h before exposure to high light. Immediately before the experiment started NaHCO3 (4 mM, final concentration) was added from a 2 M stock to prevent carbon limitation during the experiment. Four 65 W white light energy-saving lamps (Lexman) were used to provide high light (HL) conditions (2000 µmol photons m -2 s -1 ) for one hour. Cells were then allowed to recover for one hour in low light (LL, 20 µmol photons m -2 s -1 ), provided by one 20 W Lexman energy saving lamp. All light conditions were measured as PAR with a spherical micro quantum sensor (Walz) submerged in the centre of a 10 mg Chl a l -1 diatom suspension, thus corresponding to the concentrations used during the experiments. Cells were continuously stirred in a glass test tube to obtain a homogenous cell suspension. This glass test tube was cooled in a custom-made glass cooler by a water bath at 20°C.

LHCX protein detection

Sampling was conducted as described 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]. Samples were taken immediately before light exposure (T0), after one hour HL and after one subsequent hour of LL recovery.

Protein extraction, SDS-PAGE, Western-blot and ECL immunodetection were carried out as published by [START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled lighttemperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]. Both an FCP6 antibody (dilution 1/10000), 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 an anti-LHCSR3 (dilution 1/20000) from Chlamydomonas reinhardtii [START_REF] Bonente | Analysis of LhcSR3, a protein essential for feedback deexcitation in the green alga Chlamydomonas reinhardtii[END_REF] were tested. Anti-PsbB (CP47, Agrisera) was used as a loading control. 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] was not usable for the two investigated species. Phaeodactylum tricornutum CCAP 1055/1 (P.t.) samples exposed to HL for 3 hours were analysed at the same time as a control.

Pigment analyses

Diatom suspensions were rapidly filtered onto Isopore 1.2 µm RTTP filters (Merck Millipore), immediately frozen in liquid nitrogen and stored at -80°C. Samples were freeze-dried before adding -20°C cold 1.4 ml extraction buffer (90% methanol/0.2 M ammonium acetate (90/10 vol/vol) and 10% ethyl acetate). Pigment extraction was enhanced by adding glass beads (diameter 0.25-0.5 mm, Roth) and vortexing for 30 s. The extracts were sonicated for 30 s on ice at 40% amplitude with 2 s pulse, 1 s rest and filtered over a 0.2 µm filter. One hundred µl were immediately injected into the HPLC system (Agilent). Samples were analysed according to [START_REF] Van Heukelem | f phytoplankton pigments[END_REF]. As buffered extraction medium was used, no additional TBAA buffer was injected. All pigment concentrations (chlorophyll c (Chl c), fucoxanthin (Fx), diadinoxanthin (Ddx), diatoxanthin (Dtx), violaxanthin (Vx), antheraxanthin (Ax), zeaxanthin (Zx), chlorophyll a (Chl a) and β-carotene (β-car)) were calculated by comparison with pigment standards. All standards were obtained from DHI, with exception of Chl a which was obtained from Sigma-Aldrich.

Pulse Amplitude Modulated (PAM) Fluorometry

Chlorophyll fluorescence was measured using a Diving PAM fluorometer (Walz). Saturating flashes (0.4 s, 3600 µmol photons m -2 s -1 ) were provided by the internal halogen lamp to measure photosynthetic parameters (see [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF] for a complete overview of parameters). The duration of 0.4 s for saturating pulses was tested as the best setting for measurement of the maximum photosynthetic efficiency of PSII (Fv/Fm) and effective quantum efficiency of PSII photochemistry (ΔF/Fm'). When applied longer, the maximal fluorescence yield (Fm) is underestimated which artificially lowers Fv/Fm and ΔF/Fm'. This is most probably due to the high energy delivered by the halogen lamp of the Diving-PAM fluorometer (different from the LEDs used for most other PAM fluorometers). We have applied these settings before (see [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF] and it provided reliable results. To avoid interference from the HL setup, the lights were switched off immediately before firing a saturating pulse (see [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]). The photosynthetic efficiency of PSII (ΔF/Fm') was calculated as (Fm'-F')/Fm' and expressed as a percentage, taking the maximal photosynthetic efficiency (Fv/Fm), measured immediately before HL onset as 100%. Non-Photochemical Quenching (NPQ) was calculated as (Fm-Fm')/Fm'.

Rate estimation and statistics

The ΔF/Fm' recovery rate constant (k) was calculated by fitting an exponential decay function: ΔF/Fm'(t) = ΔF/Fm'rec + [ΔF/Fm'(0) -ΔF/Fm'rec]e -kt where t represents time (in min) during recovery and ΔF/Fm'(0) and ΔF/Fm'rec represents ΔF/Fm' (expressed in percentage from the ΔF/Fm' before HL onset) at the start of the recovery period and after 30 min of recovery in LL respectively [START_REF] Serôdio | Efficiency of photoprotection in microphytobenthos: role of vertical migration and the xanthophyll cycle against photoinhibition[END_REF]. Ddx de-epoxidation, Dtx and Zx epoxidation and the XC de novo synthesis rates were calculated as in [START_REF] Lavaud | General Features of Photoprotection By Energy Dissipation in Planktonic Diatoms (Bacillariophyceae)[END_REF] using exponential decay functions for epoxidation and de-epoxidation rate constants (k). The Ddx epoxidation, for instance, was fitted as the decrease of Ddx with the exponential decay function:

Ddx (t) = Ddxminimal + (Ddxinitial -Ddxminimal)e -kt
where t represents time (in min), and Ddxinitial and Ddxminimal represent the highest and lowest observed concentrations, respectively. Linear functions were fitted for xanthophyll de novo synthesis rates. Statistical analyses were conducted using the statistical software package SAS 9.4. Species parameters (3 replicates per species) were compared using the general linear model PROC GLM. In case of unequal variances, a Welch's t-test was performed.

Results

General characteristics

The epipelic diatom Seminavis robusta and epipsammic diatom Opephora guenter-grassii were grown under low light (LL) conditions; resulting in a XC pigment pool (Ddx + Dtx) of 4.94 ± 0.45 mol (100 mol Chl a) -1 for S. robusta and 9.88 ± 0.59 mol (100 mol Chl a) -1 for O. guentergrassii. The maximal PSII quantum yield ΔF/Fm, measured immediately before HL exposure and without dark adaptation, was 0.685 ± 0.031 for S. robusta and 0.665 ± 0.017 for O. guentergrassii and did not differ significantly (p = 0.099) between the two species. This indicates that the cells were in an unstressed condition prior to HL exposure, which is also supported by the absence or negligible concentrations of diatoxanthin (Dtx).

PSII quantum yield and NPQ

Both O. guenter-grassii and S. robusta were exposed to HL for one hour, after which they were allowed to recover in LL conditions. The quantum yield of PSII (ΔF/Fm') of both species dropped during HL (Fig. 1a), but was significantly higher for O. guenter-grassii at the end of the HL period in comparison with S. robusta (p = 0.049, Welch-test). During the subsequent low light conditions ΔF/Fm' of O. guenter-grassii recovered about 90 % of its value before HL exposure whereas S. robusta recovered less than 75 %. The ΔF/Fm' recovery rate constant was more than double the rate constant for the epipsammic species (0.096 min -1 ± 0.009 compared to 0.040 min - 1 ± 0.004 for S. robusta). At each time point during HL, NPQ was higher in O. guenter-grassii, compared to S. robusta (Fig. 1b). During the start of the LL period, O. guenter-grassii showed very rapid NPQ relaxation, with about half of its NPQ relaxing within 2.5 minutes. Both fast NPQ relaxation and recovery of ΔF/Fm' slowed down when O. guenter-grassii was placed in darkness instead of LL (Fig. 2a&b). NPQ dissipation in S. robusta occurred more gradually and was incomplete after one hour of LL, with a remaining NPQ of 0.504 ± 0.07 compared to 0.188 ± 0.002 for O. guenter-grassii.

Xanthophyll cycle characteristics

The higher Ddx-Dtx pool of O. guenter-grassii (p = 0.000) resulted in higher Dtx concentrations after 5 minutes of HL (p = 0.0447) (Fig. 3a&b, Table 1). The de-epoxidation state (DES, calculated as Dtx/(Ddx + Dtx)), however, was not significantly different between the species during the HL period (Fig. 4). From 15 minutes onwards the total Ddx + Dtx pool increased (due to de novo synthesis of xanthophylls) with similar rates in both species (Fig. 3a&b, Table 1).

During one hour of HL treatment each species synthetized an additional 2 mol Ddx + Dtx (100 mol Chl a) -1 . At the end of the HL period O. guenter-grassii contained significantly (p = 0.040) more Dtx (5.69 ± 1.41 mol (100 mol Chl a) -1 ) than S. robusta (3.15 ± 0.42 mol (100 mol Chl a) -1 ) (Table 1). Due to the lower amount of Dtx originating from de-epoxidation of Ddx in S. robusta and similar de novo Dtx synthesis as O. guenter-grassii, the relative contribution of de novo synthetized Dtx was about two-thirds of the accumulated Dtx in S. robusta, whereas the de novo contribution was only one third in the case of O. guenter-grassii.

During the LL recovery period, Dtx was rapidly epoxidized by O. guenter-grassii. Its Dtx epoxidation rate constant in LL was about 5 times higher than in S. robusta (p = 0.003) (see Table 1), with most Dtx being epoxidized to Ddx within the first 5 min of LL recovery.

Epoxidation occurred more gradually in S. robusta (Fig. 3a&b). Differences in epoxidation rate resulted in significant differences in de-epoxidation state at 5 (p = 0.022) and 15 min (p = 0.019) during the LL recovery period (Fig. 4). At the end of the recovery period however, nearly all Dtx had disappeared in both species (Fig. 3a&b, Table 1). The fast Dtx epoxidation by O. guenter-grassii in LL was not observed in darkness (Fig. 2c). In both species, an increase in the Ddx + Dtx pool was recorded during LL treatment. O. guenter-grassii gained 2.26 ± 0.21 mol Ddx + Dtx (100 mol Chl a) -1 , whereas in S. robusta the increase was 1.84 ± 1.10 mol Ddx + Dtx (100 mol Chl a) -1 (Table 1).

Besides the Dtx cycle pigments, pigments of the violaxanthin (Vx) cycle were detected in both species during HL (Fig. 3c&d, Table 1). O. guenter-grassii accumulated about 2 mol (100 mol Chl a) -1 Vx cycle pigments during the HL period (Table 1). At the end of the HL treatment, 1.34 ± 0.19 mol (100 mol Chl a) -1 of the de-epoxidized pigment Zeaxanthin (Zx) was detected in O.

guenter-grassii. The intermediate between Zx and Vx, antheraxanthin (Ax), was also detected during HL (Fig. 3c). In comparison, S. robusta accumulated significantly less (p = 0.001) Vx cycle pigments (Fig. 3d

Correlation between Dtx accumulation and NPQ

NPQ correlated well with Dtx mol (100 mol Chl a) -1 for both species (Fig. 5). They showed similar slopes (0.7-0.8) until about 3 Dtx mol (100 mol Chl a) -1 , after which less NPQ was developed per mol Dtx for O. guenter-grassii. The relationship remained true for S. robusta during the course of the experiment. Its Dtx content, nevertheless, did not exceed the threshold at which the curve slope changed in O. guenter-grassii. The y-axis intercept differed from zero for S. robusta, as was reported earlier [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF].

LHCX presence during HL

For immunodetection of LHCX-isoforms in O. guenter-grassii (Fig. 6a) the best results were obtained using the anti-FCP6 antibody, as less a-specific binding occurred in comparison with the LHCSR3 antibody. Only one LHCX isoform could be detected with a molecular weight close to that of Phaeodactylum tricornutum LHCX3 (22.24 KDa). This isoform was apparent in LL acclimated cells and increased in abundance during the one hour of HL and the subsequent hour of recovery in LL.

In S. robusta (Fig. 6b) only the anti-LHCSR3 antibody revealed LHCX isoforms. One isoform, with a molecular weight equal to P. tricornutum LHCX3 (22.24 KDa) and another more faint band with an equal size to P. tricornutum LHCX2 (24.73 KDa) were present in LL acclimated cells. The former increased in abundance during HL and subsequent recovery in LL. After one hour of HL, moreover, two additional LHCX isoforms could be detected. An LHCX isoform of about 23 KDa was clearly present after one hour of HL and after the additional recovery period.

The second one, about the size of LHCX1 in P. tricornutum (21.95 KDa), became visible after one hour of HL.

Discussion

In this study we demonstrate marked differences in irradiance tuning of NPQ and associated XC pigment and LHCX protein dynamics between a motile and a non-motile marine benthic diatom.

The non-motile species (O. guenter-grassii) exhibits a dynamic and strong high-energy quenching (qE), coupled to fast XC kinetics and pronounced synthesis of de-epoxidized xanthophylls, including zeaxanthin. In this species, strong physiological photoprotection may compensate for its lack of motility as a way to avoid oversaturating light conditions. The motile species (S. robusta) on the other hand exhibited an overall lower qE capacity, even though NPQ increased during the light period, possibly due to de novo synthesis of both Dtx and LHCX proteins.

Prior to the experiments, both species were acclimated to low light conditions, to avoid the presence of Dtx and NPQ in cultures, as much as possible, which could bias the measurement of Fm. These growth conditions resulted in similar XC content as observed by [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF] under identical light conditions for the same benthic species used in this study, and also as observed for a range of planktonic species grown in a PAR of 40 µmol photons m -2 s -1 [START_REF] Lavaud | General Features of Photoprotection By Energy Dissipation in Planktonic Diatoms (Bacillariophyceae)[END_REF]). The non-motile epipsammic species O. guenter-grassii showed higher NPQ during high light exposure, compared to the epipelic diatom S. robusta. As reported by [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF], higher NPQ values coincided with a higher overall Dtx content de-epoxidized from a larger initial Ddx pool, rather than a higher de-epoxidation state (DES) or a higher involvement of Dtx molecules in the NPQ mechanism [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]. Indeed, we did not observe a difference in DES between O. guenter-grassii and S. robusta, nor a difference in the slopes of the NPQ/Dtx plots. Note that [START_REF] Jesus | Adaptations of microphytobenthos assemblages to sediment type and tidal position[END_REF], working on natural epipelic and epipsammic communities, did observe a difference in DES between both, but this may have been due to high light avoidance by vertical migration and/or microcycling in the epipelic communities, which was impeded in our study.

Accumulation of Dtx, independent from Ddx de-epoxidation, was observed for both species during high light exposure as reported for planktonic diatoms [START_REF] Lavaud | General Features of Photoprotection By Energy Dissipation in Planktonic Diatoms (Bacillariophyceae)[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]) and natural epipelic communities [START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled lighttemperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]. The rate constant of this de novo Dtx synthesis was similar for both species and in the range of planktonic diatoms exposed to the same HL conditions [START_REF] Lavaud | General Features of Photoprotection By Energy Dissipation in Planktonic Diatoms (Bacillariophyceae)[END_REF], resulting in the same increase of the Ddx + Dtx pool. The XC pool at the end of the HL period, nonetheless, was still relatively low for both species, as up to 26 mol Ddx + Dtx (100 mol Chl a) -1 were observed by [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF] in Cyclotella meneghiniana and even up to 30-40 Ddx + Dtx (100 mol Chl a) -1 in Chaetoceros socialis [START_REF] Dimier | Photoprotection and xanthophyll cycle activity in three marine diatoms[END_REF]). These large XC pools, however, required a prolonged exposure (i.e., several hours) to high irradiances, whereas in this study the HL period was relatively short (1 h).

In the diatoms Plagiogramma staurophorum and Brockmanniella brockmannii, nonetheless, acclimation to a PAR of 75 µmol photons m -2 s -1 resulted in XC pools higher than 25 Ddx + Dtx (100 mol Chl a) -1 [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF].

The involvement of de novo synthetized Dtx differed between both species. Whereas the NPQ-Dtx relationship remained true for S. robusta, de novo synthetized Dtx in O. guenter-grassii did not contribute equally to the NPQ mechanism, as shown by a decline in the NPQ-Dtx relationship. Part of this additionally synthetized Dtx is possibly present in the lipid matrix of the thylakoid membrane [START_REF] Schumann | Investigation of the quenching efficiency of diatoxanthin in cells of Phaeodactylum tricornutum (Bacillariophyceae) with different pool sizes of xanthophyll cycle pigments[END_REF]) to prevent 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]). It should be noted, however, that the total Dtx values observed for S. robusta during our experiments remained rather low compared to values recorded for other species using a similar setup [START_REF] Lavaud | General Features of Photoprotection By Energy Dissipation in Planktonic Diatoms (Bacillariophyceae)[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 | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF] and might be due to a small Ddx pool before HL onset [START_REF] Lavaud | General Features of Photoprotection By Energy Dissipation in Planktonic Diatoms (Bacillariophyceae)[END_REF]. A stable NPQ/Dtx slope during de novo synthesis of Dtx, as observed in S. robusta, nonetheless, may indicate synthesis of new Dtxbinding proteins such as LHCXs [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 fast epoxidation of Dtx is crucial to switch the light harvesting system from an energy dissipation state to a light harvesting state, we monitored NPQ relaxation and Dtx epoxidation during low light following high light exposure. O. guenter-grassii displayed very rapid Dtx epoxidation coupled with an equally fast NPQ relaxation, but not during dark recovery, as previously reported (Goss et al. 2006b). This is also demonstrated by the fast recovery of PSII quantum yield in low light, which is severely restricted in darkness as the epoxidation reaction is possibly slowed down by NADPH depletion (Goss et al. 2006b). The fast reversal of NPQ and nearly complete recovery of PSII quantum yield, moreover, indicate that the observed high NPQ values comprise mostly qE while qI/NPQs is virtually absent.

A fast switch from energy dissipation to light harvesting after high light exposure was not observed in the epipelic diatom S. robusta, where Dtx epoxidation and coupled NPQ relaxation occurred more gradually. Together with an incomplete and slower recovery of PSII quantum yield our data demonstrate a higher susceptibility to photoinhibition during prolonged high light as has been shown for species isolated from habitats lacking strong light fluctuations (Goss et al. 2006b;[START_REF] Su | the Impact of Nonphotochemical Quenching of Fluorescence on the Photon Balance in Diatoms Under Dynamic Light Conditions[END_REF][START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF]. The observed NPQ values, increasing during the high light treatment, therefore comprise not only qE but also a significant fraction of qI due to PSII photoinactivation and damage (since Dtx is almost fully converted back to Ddx).

Even though both S. robusta and O. guenter-grassii accumulated similar amounts of newly synthetized Dtx within one hour of high light, the latter synthetized the same amount of Vx cycle and Dtx cycle pigments, including Zx. The presence of a parallel Vx-Ax-Zx cycle has been demonstrated in several algae possessing the Ddx-Dtx cycle, including the diatom species Cyclotella meneghiniana and Phaeodactylum tricornutum [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF]. Zx accumulation in these species, however, required prolonged (up to 6 hours) high light exposure (Lohr andWilhelm 1999, 2001) and has never been reported in studies on P. tricornutum using similar PAR and exposure time (i.e. within one hour, 2000 µmol photons m -2 s -1 ) as used in this study [START_REF] Lavaud | General Features of Photoprotection By Energy Dissipation in Planktonic Diatoms (Bacillariophyceae)[END_REF][START_REF] Domingues | Response of the diatom Phaeodactylum tricornutum to photooxidative stress resulting from high light exposure[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 | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF].

Epoxidation of Zx in low light was as fast as Dtx epoxidation, while the second epoxidation step occurred more slowly, resulting in a transient peak in the intermediate Ax. This transient peak in Ax has been reported before for the green alga Chlorella (Goss et al. 2006a). The Vx cycle pool of O. guenter-grassii declined during the one hour recovery period and to a lesser degree also in S. robusta, whereas a similar Vx cycle pool decline in P. tricornutum was not observed within one hour of low light recovery [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF]. The high amount of Vx cycle pigments synthetized by O. guenter-grassii in high light may have been converted to Ddx during the recovery period, as the decline in Vx cycle pigments was paralleled by an equal increase in Ddx + Dtx. A pathway from Vx to Ddx through the intermediate neoxanthin has been proposed by [START_REF] Dambek | Biosynthesis of fucoxanthin and diadinoxanthin and function of initial pathway genes in Phaeodactylum tricornutum[END_REF]. In S. robusta, however, more Ddx + Dtx accumulated during the LL period than was lost from the Vx cycle pool. This might be due to additional synthesis of Ddx cycle pigments during low light, even though additional de novo synthesis in low light conditions is considered to be low [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF]. According to Lohr andWilhelm (1999, 2001), the primary role of Vx cycle pigments in diatoms is not photoprotection as they mainly serve as intermediates in Ddx and fucoxanthin production. Increasing the light intensity, nonetheless, changes the allocation of newly synthesized xanthophylls to the Vx-Ax-Zx pool in P. tricornutum [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF]. Moreover, Vx cycle pigments are mostly detected in algae displaying high de novo xanthophyll synthesis combined with high de-epoxidase activity. This fits with our observations of O. guenter-grassii, de novo synthetizing substantially more xanthophylls (considering both Ddx and Zx cycle pigments) and de-epoxidising more Ddx to Dtx during HL than S. robusta. We do not expect Zx to be directly involved in the NPQ mechanism of O.

guenter-grassii as the NPQ/Dtx relationship decreased during de novo synthesis of both xanthophylls. In higher plants, Zx can dissolve in the thylakoid membrane lipids instead of being protein bound [START_REF] Jahns | Mechanism and regulation of the violaxanthin cycle: the role of antenna proteins and membrane lipids[END_REF], scavenging reactive oxygen species with a higher capacity than other xanthophylls found in higher plants [START_REF] Havaux | Zeaxanthin has enhanced antioxidant capacity with respect to all other xanthophylls in Arabidopsis leaves and functions independent of binding to PSII antennae[END_REF], or possibly regulating membrane fluidity [START_REF] Havaux | Heat and light induced chlorophull a fluorescence changes in potato leaves containign high or low levels of the carotenoid zeaxanthin: indications of a regulatory effect of zeaxanthin on thylakoid fluidity[END_REF].

As LHCX proteins play a central role in the NPQ mechanism of diatoms [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], we compared LHCX synthesis for the first time between an epipsammic and an epipelic diatom. We could detect only one LHCX isoform (~22 kDa) in the epipsammic model O. guenter-grassii using the FCP6 antibody. It did not strongly react to a shift to high light and was more abundant in subsequent low light. The epipelic diatom S. robusta, however, revealed two out of four isoforms which strongly reacted to HL: one isoform with MW ~23 kDa and a second one with MW ~19 kDa. Interestingly, in epipelic communities, a 23 kDa isoform was shown to positively reacts to high light, high temperature and motility inhibition [START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled lighttemperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]. The two isoforms already present in low light might provide benthic diatoms with a basic NPQ to rapidly cope with sudden changes in light climate, as has been demonstrated for LHCX1 of P. tricornutum [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF]. However, the S. robusta genome does not contain a close homolog to the P. tricornutum LHCX1 gene at the sequence level (L. Blommaert et al., data not shown). LHCXs which are strongly upregulated during high light have been suspected to either bind de novo synthetized Dtx, conferring higher NPQ and/or participate in a sustained component of NPQ (NPQs) after prolonged high light exposure [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress[END_REF]Lepetit et al. 2013, in press). As S. robusta accumulates novel Dtx during HL while its NPQ increases, the two observed light-responsive LHCX isoforms may be responsible for Dtx binding, as suggested by [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress[END_REF], and Lepetit et al. (2013, in press). However, other FCP proteins may be responsible as for instance the LHCR6 and LHCR8 (Light-Harvesting Complex Red lineage) genes are strongly upregulated in P. tricornutum, upon a shift to high light [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF].

Ecological implications

A fast and strong irradiance-tuning of NPQ is to be expected in immotile epipsammic diatoms as they live attached to sand grains [START_REF] Ribeiro | Structure and diversity of intertidal benthic diatom assemblages in contrasting shores: a case study from the Tagus estuary[END_REF] and are unable to move away from oversaturating light conditions. Furthermore, sandy sediments are characterized by strong light scattering in the uppermost layers, thereby increasing the average incident irradiance to which these diatoms are exposed [START_REF] Kuhl | Light penetration and light intensity in sandy marine sediments measured with irradiance and scalar irradiance fiber-optic microprobes[END_REF]Cartaxana et al. 2016b). Even though our epipsammic species was acclimated to low PAR (20 µmol photons m -2 s -1 ) it was able to cope with a sudden change to a light intensity similar to full sunlight. Similar transitions from low to full sunlight (and vice versa) can be common in sandy sediments during low tide [START_REF] Hamels | Organisation of Microbenthic Communities[END_REF]. Given the prolonged harsh light conditions in these sediments, epipsammic diatoms probably demonstrate a high de novo synthesis of photoprotective xanthophylls in situ, including Zx. This can also explain the previously observed discrepancy between high Zx content and absence of colonial cyanobacteria (containing Zx) in sandy sediments, as reported in [START_REF] Hamels | Organisation of Microbenthic Communities[END_REF]. Taken together, our results suggest that epipsammic diatoms use a combination of distinct photoprotective strategies described by [START_REF] Lavaud | An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms[END_REF] to cope with the light climate of sandy intertidal sediments: (1) a strong and fast reversible qE to track light fluctuations, combined with (2) high de novo synthesis of de-epoxidized xanthophylls, probably unbound to the LHC antenna system, which may fulfil an anti-oxidant function during prolonged light conditions. Even though our study was performed on only one epipsammic representative, a strong qE and a relatively higher XC pool (compared to epipelic species) seem to be general for epipsammic species [START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF].

The epipelic model S. robusta displayed a lower NPQ consisting partly of photoinhibition (qI). This is expected as epipelic diatoms use vertical migration and/or microcycling as their primary photoprotection mechanism when motility is allowed [START_REF] Kromkamp | Determination of microphytobenthos PSII quantum efficiency and photosynthetic activity by means of variable chlorophyll fluorescence[END_REF][START_REF] Serôdio | Analysis of variable chlorophyll fluorescence in microphytobenthos assemblages: implications of the use of depth-integrated measurements[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][START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled lighttemperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF]. Furthermore, vertical migration is fast enough to reduce the amount of absorbed photons and can operate simultaneously with NPQ induction [START_REF] Laviale | The importance of being fast: comparative kinetics of vertical migration and non-photochemical quenching of benthic diatoms under light stress[END_REF]. Both synthesis of new Dtx pigments and LHCX proteins, nonetheless, have been shown in epipelic communities under high light conditions [START_REF] Laviale | Response of intertidal benthic microalgal biofilms to a coupled lighttemperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe[END_REF], which is in line with our findings. Furthermore, our data suggests the involvement of light-regulated LHCX proteins during harsh light conditions, allowing epipelic species to acclimate to prolonged higher light conditions [START_REF] Ezequiel | Photoacclimation state determines the photobehaviour of motile microalgae: The case of a benthic diatom[END_REF][START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF].

Hence, although adapted to a habitat with more cohesive sediments, characterized by a strongly attenuated photic zone (Cartaxana et al. 2016b), epipelic diatoms still possess the ability to increase their low basal photoprotective ability. The fact that epipelic species have been shown to emerge at the sediment surface at different times during tidal emersion suggests that they have different species-specific light niches [START_REF] Paterson | The migratory behaviour of diatom assemblages in a laboratory tidal micro-ecosystem examined by low temperature scanning electron microscopy[END_REF][START_REF] Underwood | Patterns in microphytobenthic primary productivity: Species-specific variation in migratory rhythms and photosynthesis in mixed-species biofilms[END_REF]. As a result, their capacity for physiological photoprotection is also expected to differ between species. Future studies, therefore, should focus on the interspecific differences in the balance between behavioural and physiological photoprotection.

Figure legends

  ) and both Zx and Ax were only present in trace amounts. During the LL recovery period epoxidation of Zx started immediately in O. guenter-grassii, resulting in a short peak of Ax (at time point 2.5-5 min) and an increase in Vx. The total Vx cycle pool (Vx + Ax + Zx) decreased markedly for both species during LL with a decrease of 1.62 ± 0.48 mol (100 mol Chl a) -1 for O. guenter-grassii and a smaller decrease of 0.07 ± 0.02 mol (100 mol Chl a) -1 for S. robusta. Notably, in O. guenter-grassii, Vx cycle pigments decreased as fast during the LL period as new Ddx cycle pigments were synthetized (0.28 mmol (mol Chl a) -1 min -1 ± 0.08 and 0.36 mmol (mol Chl a) -1 min -1 ± 0.05 respectively). During the course of the experiment no notable changes in Fx, Chl c and β-car were observed (Data not shown).

Fig. 1a&b

 1a&b Fig. 1a&b Photophysiological measurements

Fig. 2

 2 Fig. 2 Dark recovery of O. guenter-grassii after HL exposure

Fig. 3a ,

 3a Fig. 3a,b,c&d Xanthophyll cycle kinetics

Fig. 4

 4 Fig. 4 De-epoxidation state of O. guenter-grassii and S. robusta O. guenter-grassii (filled circles) and S. robusta (open circles) were exposed to one hour of HL (2000 µM photons m -2 s -1 ) and one subsequent hour of recovery in low light (LL, 20 µM photons m -2 s -1 ). De-epoxidation state (DES) was calculated as 100[Dtx/(Ddx + Dtx)].

Fig. 5a&b

 5a&b Fig. 5a&b Relationship between NPQ and Dtx

Fig. 6a&b

 6a&b Fig. 6a&b Western blot of LHCX proteins

Figures

  Figures

Fig

  Fig 2a,b,c&d

  Fig. 4a&b

O

  Fig. S3a&b

Table 1 Xanthophyll cycle characteristics

 1

	Tables
		S. robusta	O. guenter-grassii
	Ddx + Dtx content	4.942	9.880
	[mol (100 Chl a) -1 ]	±0.479	±0.594
	Dtx after 60 min HL	3.146	5.685
	[mol (100 Chl a) -1 ]	±0.424	±1.413
	Ddx deepoxidaton rate	0.081	0.164
	[min -1 ]	±0.017	±0.0521
	De novo synthetized Dtx	1.976	2.138
	[mol (100 Chl a) -1 ]	±0.422	±0.612
	Dtx de novo synthesis rate	0.341	0.444
	[mmol (mol Chl a) -1 min -1 ]	±0.035	±0.065
	De novo synthetized Vx cycle pigments	0.131	2.043
	[mol (100 Chl a) -1 ]	±0.010	±0.216
	De novo synthesis rate Vx cycle pigments	0.0185	0.35
	[mmol (mol Chl a) -1 min -1 ]	±0.009	±0.025
	Dtx epoxidation rate in LL	0.08	0.406
	[min -1 ]	±0.01	±0.144
	Zx epoxidation rate in LL	n.d.	0.311
	[min -1 ]		±0.060

Acknowledgements

The authors would like to thank the Research Foundation Flanders (FWO project G.0222.09N), Ghent University (BOF-GOA 01G01911) and the Egide/Campus France-PHC Tournesol (n128992UA) exchange program for their financial support. JL also thanks the CNRS and the French consortium CPER Littoral for their financial support. M.J.J.H. acknowledges a Postdoctoral Fellowship of the Research Foundation Flanders.

Abbreviations: Chl a, Chlorophyll a; Ddx, diadinoxanthin; Dtx, diatoxanthin; Vx, violaxanthin; Zx, zeaxanthin. All pigments are expressed as mol (100 mol chlorophyll a) -1 . Epoxidation and deepoxidation rates are calculated by fitting exponential decay functions. De novo synthesis rates were fitted with linear functions. Values represent averages of three independent measurements ± standard deviations.

Supporting material