domain: sde sdv

text:
Bernard Lepetit
email: bernard.lepetit@uni-konstanz.de

Gautier Gélin

Mariana Lepetit

Sabine Sturm

Sascha Vugrinec

Alessandra Rogato

Peter G Kroth

Angela Falciatore

Johann Lavaud

The diatom Phaeodactylum tricornutum adjusts NPQ capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis

Keywords: diatoms, dynamic light, Lhcx, NPQ, Phaeodactylum tricornutum, photoprotection, xanthophyll cycle

Introduction

Diatoms are unicellular microalgae constituting one of the most important phytoplankton groups in terms of biodiversity [START_REF] Mann | An inordinate fondness? The number, distributions, and origins of diatom species[END_REF] and productivity (about 45 % of marine carbon fixation) [START_REF] Geider | Primary productivity of planet earth: biological determinants and physical constraints in terrestrial and aquatic habitats[END_REF]. They strongly participate in the biological carbon pump and the functioning of contemporary aquatic ecosystems [START_REF] Armbrust | The life of diatoms in the world's oceans[END_REF]. Due to their high productivity and high lipid content, diatoms could potentially replace American fossil oil consumption in the future [START_REF] Levitan | Diatoms: a fossil fuel of the future[END_REF] or be used for production of high quality plastics [START_REF] Roesle | Synthetic polyester from algae oil[END_REF]. A peculiar feature of diatoms is their ability to live in turbulent waters, where they can benefit from high nutrient availabilities [START_REF] Tozzi | Historical climate change and ocean turbulence as selective agents for two key phytoplankton functional groups[END_REF].

In such habitats light intensity changes over several orders of magnitude on the timescale of minutes [START_REF] Long | Photoinhibition of Photosynthesis in Nature[END_REF][START_REF] Macintyre | The effect of water motion on short-term rates of photosynthesis by marine phytoplankton[END_REF][START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: mechanisms, evolution and ecopysiology[END_REF], therefore flexible photosynthesis and efficient photoprotection mechanisms are necessary to avoid overexcitation of the photosynthetic apparatus which would lead to the generation of reactive oxygen species (ROS), eventually resulting in cell death [START_REF] Niyogi | Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis[END_REF]. Diatoms possess both, an unusual flexibility of photosynthetic productivity [START_REF] Wilhelm | The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae[END_REF][START_REF] Kroth | A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis[END_REF][START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF][START_REF] Bailleul | Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms[END_REF], and effective photoprotection mechanisms which include: 1) a fast operating PSII electron cycle (Lavaud et al., 2002c;[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], 2) a tuneable amount of membrane dissolved xanthophylls diadinoxanthin (Dd) and diatoxanthin (Dt) acting as antioxidants [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], and 3) a high capacity for dissipation of excess excitation energy, illustrated by non-photochemical fluorescence quenching (NPQ) [START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF]. NPQ in plants and green algae is divided into three to four subtypes, which are not similarly well defined in diatoms [START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF]. Here we will refer to NPQ as a photoprotective mechanism whose induction depends upon three regulatory components: (1) The proton gradient generated between the thylakoid lumen and the chloroplast stroma during light exposure (ΔpH), (2) a fast operating xanthophyll cycle (XC) through enzymatic conversion of Dd into Dt in the presence of the ΔpH and the back conversion in its absence (i.e. typically in the dark), and (3) chloroplast located, but nuclear encoded antenna proteins of the Light-Harvesting Complex (LHC) superfamily. While Lhcf proteins build up the peripheral light harvesting antenna (called "FCP", likely predominantly associated with PSII in vivo) [START_REF] Grouneva | The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery[END_REF][START_REF] Gundermann | Identification of several sub-populations in the pool of light harvesting proteins in the pennate diatom Phaeodactylum tricornutum[END_REF][START_REF] Nagao | Comparison of oligomeric states and polypeptide compositions of fucoxanthin chlorophyll a/c-binding protein complexes among various diatom species[END_REF][START_REF] Schaller-Laudel | The diadinoxanthin diatoxanthin cycle induces structural rearrangements of the isolated FCP antenna complexes of the pennate diatom Phaeodactylum tricornutum[END_REF], Lhcr proteins form the PSI antenna [START_REF] Veith | Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana[END_REF][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] Grouneva | The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery[END_REF][START_REF] Ikeda | Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms[END_REF]Bina et al., 2016). In contrast, Lhcx proteins are involved in NPQ in the pennate diatom Phaeodactylum 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 the centric Thalassiosira pseudonana [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] Dong | Light harvesting proteins regulate non-photochemical fluorescence quenching in the marine diatom Thalassiosira pseudonana[END_REF]. Involvement of Lhcx proteins in photoprotection in other diatoms is also very likely [START_REF] Beer | Subunit composition and pigmentation of fucoxanthin-chlorophyll proteins in diatoms: Evidence for a subunit involved in diadinoxanthin and diatoxanthin binding[END_REF][START_REF] Park | Dynamic response of the transcriptome of a psychrophilic diatom, Chaetoceros neogracile, to high irradiance[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][START_REF] Ghazaryan | Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes[END_REF]. It is assumed that Lhcx proteins bind Dd and Dt [START_REF] Beer | Subunit composition and pigmentation of fucoxanthin-chlorophyll proteins in diatoms: Evidence for a subunit involved in diadinoxanthin and diatoxanthin binding[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 they apparently influence the supramolecular organisation of the antenna complexes [START_REF] Ghazaryan | Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes[END_REF]. The location of Lhcx within thylakoids remains ambiguous as based on contrasting reports of both FCP [START_REF] Beer | Subunit composition and pigmentation of fucoxanthin-chlorophyll proteins in diatoms: Evidence for a subunit involved in diadinoxanthin and diatoxanthin binding[END_REF][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] Grouneva | The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery[END_REF][START_REF] Nagao | Comparison of oligomeric states and polypeptide compositions of fucoxanthin chlorophyll a/c-binding protein complexes among various diatom species[END_REF][START_REF] Schaller-Laudel | The diadinoxanthin diatoxanthin cycle induces structural rearrangements of the isolated FCP antenna complexes of the pennate diatom Phaeodactylum tricornutum[END_REF] and PSI association [START_REF] Grouneva | The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery[END_REF]. The current NPQ model proposes two major quenching sites in diatoms (Miloslavina et al., 2009;[START_REF] Chukhutsina | Disentangling two nonphotochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77K[END_REF][START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF][START_REF] Derks | Diverse mechanisms for photoprotection in photosynthesis. Dynamic regulation of photosystem II excitation in response to rapid environmental change[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF]:

Quenching site 1 is mechanistically independent of Dt [START_REF] Chukhutsina | Disentangling two nonphotochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77K[END_REF] and is formed rapidly mainly by detached, oligomeric antenna complexes due to the build-up of the ΔpH, while quenching site 2 is located close to the PSII reaction centres and is directly dependent on Dt formation. Quenching site 1 also exists in P. tricornutum (Miloslavina et al., 2009), but in this species NPQ always relies on Dt (Lavaud et al., 2002a;[START_REF] Goss | The importance of a highly active and delta pH regulated diatoxanthin epoxidase for the regulation of the PSII antenna function in diadinoxanthin cycle containing algae[END_REF], except under special (i.e. artificial) conditions [START_REF] Lavaud | In diatoms, the transthylakoid proton gradient regulates the photoprotective non-photochemical fluorescence quenching beyond its control on the xanthophyll cycle[END_REF][START_REF] Eisenstadt | Changes in the photosynthetic reaction centre II in the diatom Phaeodactylum tricornutum result in non-photochemical fluorescence quenching[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 apparent contradiction could not been fully solved so far, but an indirect influence of Dt also on formation of quenching site 1 has been postulated [START_REF] Lavaud | The peculiar features of non-photochemical fluorescence quenching in diatoms and brown algae[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF].

In different diatoms, the NPQ capacity, in relation to the light environment of the respective planktonic and benthic habitat, can be rather variable. Diatoms that cope with sudden light exposures, e.g. coastal planktonic and immotile estuarine sediment-inhabiting diatoms, show a higher NPQ capacity than diatoms living in more stable water bodies (e.g. semi-enclosed bays, open ocean) and the motile and photophobic sediment-inhabiting forms [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 | Comparative ecophysiology of the xanthophyll cycle in six marine phytoplanktonic species[END_REF][START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF]. The same holds true for diatom species adapted to the seasonally successive polar habitats [START_REF] Petrou | Heterogeneity in the photoprotective capacity of three Antarctic diatoms during short-term changes in salinity and temperature[END_REF]. P. tricornutum is cosmopolitan, but prefers habitats where light climate is unstable and reaches punctual but regular high intensities, such as coasts, estuaries, or rocky pools [START_REF] Martino | Genetic and phenotypic characterization of Phaeodactylum tricornutum (Bacillariophyceae) accessions[END_REF]. Different P. tricornutum ecotypes have different and variable NPQ capacities (Lavaud & Lepetit, 2013) that largely depend on the amount of Lhcx1 [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF].

Additionally, there is growing evidence that two other light regulated isoforms, Lhcx2 and Lhcx3, may also participate to NPQ under prolonged light stress [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] Taddei | Multi-signal control of the expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum[END_REF].

For several diatom species the influence of near-natural light conditions on the photosynthetic performance and on growth has been thoroughly investigated [START_REF] Kromkamp | Effect of short-term variation in irradiance on light harvesting and photosynthesis of the marine diatom Skeletonema costatum: a laboratory study simulating vertical mixing[END_REF][START_REF] Litchman | Growth rates of phytoplankton under fluctuating light[END_REF][START_REF] Fietz | Acclimation of the diatom Stephanodiscus neoastraea and the cyanobacterium Planktothrix agardhii to simulated natural light fluctuations[END_REF][START_REF] Wagner | Balancing the energy flow from captured light to biomass under fluctuating light conditions[END_REF][START_REF] Jakob | A complete energy balance from photons to new biomass reveals a light-and nutrient-dependent variability in the metabolic costs of carbon assimilation[END_REF][START_REF] Van De Poll | Acclimation to a dynamic irradiance regime changes excessive irradiance sensitivity of Emiliania huxleyi and Thalassiosira weissflogii[END_REF][START_REF] Kropuenske | Photophysiology in two major Southern Ocean phytoplankton taxa: photoprotection in Phaeocystis antarctica and Fragilariopsis cylindrus[END_REF][START_REF] Su | The impact of nonphotochemical quenching of fluorescence on the photon balance in diatoms under dynamic light conditions[END_REF][START_REF] Jallet | Photosynthetic physiology and biomass partitioning in the model diatom Phaeodactylum tricornutum grown in a sinusoidal light regime[END_REF]. As P. tricornutum is one of the best characterized diatoms on the molecular level, we investigated its photophysiology during acclimation from stable low light to dynamic and potentially stressful light conditions with respect to the interplay between Dd+Dt synthesis, Lhcx expression and NPQ capacity. Sine light (SL) conditions simulated the rise and decline of the sun during a cloudless day in a stable water body. Fluctuating light conditions (FL) superimposed the effect of vertical cell movement along the water column in an idealized manner with a mixing from and to the aphotic zone via the surface within periods of 30 minutes. We also aimed at investigating the influence of the initial photoprotection capacity on the adjustment of NPQ capacity to dynamic light. Therefore, we used two P. tricornutum ecotypes with different initial NPQ capacities (i.e. low and high natural NPQ phenotypes, see [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF], but also strains with silenced or overexpressed Lhcx1 protein. Our results contribute to the better understanding of the molecular fine-tuning of NPQ capacity during acclimation to dynamic light conditions in pennate diatoms.

Materials and Methods

Cell culturing and light treatments

Experiments were performed in four Phaeodactylum tricornutum strains with different NPQ capacities: (1) P. tricornutum strain 1 (Pt1, CCAP 1055/1); (2) Pt1sil; Pt1 strain which contains an antisense construct against the Lhcx1 gene, leading to reduced Lhcx1 protein synthesis [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF]; (3) P. tricornutum strain 4 (Pt4, UTEX 646); (4) Pt4ov; Pt4 strain which overexpresses the Lhcx1 gene. The full length cDNA of the Lhcx1 gene (JGI ID: 27278) was cloned downstream of the FcpA (Lhcf1) promoter into the pPha-T1 transformation vector [START_REF] Zaslavskaia | Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes[END_REF]. Pt4 cells were biolistically transformed with this construct according to [START_REF] Kroth | Genetic transformation -a tool to study protein targeting in diatoms[END_REF]. Positive clones were selected on Zeocin TM containing solid medium plates. Lhcx1 overexpressing clones were screened based on their NPQ capacity and amongst several clones showing increased NPQ capacity the one with the highest NPQ was selected for the present experiments (Pt4ov). This clone has an identical photosynthetic yield as the wildtype under low light conditions (data not shown), but shows strongly increased Lhcx1 gene expression (Fig. S1). All four strains were grown in airlift tubes (4 cm diameter) at 20°C in a 16 h day/8 h night rhythm with a light intensity of 50 µmol photons m -2 s -1 (onset at 8:00) defined as low light (LL). Light was provided by computer controlled flora LED units (CLF Plant Climatics, Germany) with all LEDs (white, blue, red and far red) switched on. Cells were cultured in sterile Provasoli's enriched F/2 seawater medium. Chlorophyll a (Chl a) concentration was determined as described 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]. Cells in logarithmic phase were adjusted with fresh F/2 medium to a concentration of 1.4 µg Chl a mL -1 at 18:00 each day for four consecutive days. The 5 th day (day 0), sampling started (see "Sampling" below). The following three days (day 1 to day 3) two different dynamic light treatments were applied during the day phase, provided by the flora LED system: 1) SL with a maximum light intensity of 500 µmol photons m -2 s -1 reached at 16:00 (18.3 mol photons m -2 integrated daily light dose), 2) two different FL treatments with 32 light intervals in total, where maximum intensity was either 500 (FL 500, 4.5 mol photons m -2 integrated daily light dose) or 1000 µmol photons m -2 s -1 (FL 1000, 8.9 mol photons m -2 integrated daily light dose) (Fig. 1). Light intensities were measured with a spherical quantum sensor (US-SQS/L, Walz, Germany) in the centre of the airlift tubes. Due to the relatively large diameter of the airlift tubes and the relatively dense algal culture, light attenuation was steep, which, together with the bubbling of the cultures, led to a continuous micro-fluctuation superimposed on both SL and FL. The specific light intensities for all dynamic treatments are idealized and were calculated based on the formulas of [START_REF] Kroon | An algal cyclostat with computer-controlled dynamic light regime[END_REF] and as described in [START_REF] Wagner | Balancing the energy flow from captured light to biomass under fluctuating light conditions[END_REF], assuming a dense algal culture during FL conditions with exponential light attenuation in the water column. During the whole experiment (day 0 to day 3), Chl a concentration was determined daily at 18:00 and cultures were diluted with fresh F/2 medium to a concentration of 1.4 µg Chl a mL -1 , in order to prevent nutrient limitation and selfshading.

Sampling

Cells were harvested with a sterile syringe via a tube drawn in the airlift flask which was sealed except for sampling. For pigment analyses, 500 µl of cells were filtered each day at 11:00, 14:00 and 17:00 on an Isopore Polycarbonate filter 1.2 µm (Millipore, USA) and immediately frozen in liquid nitrogen. Cells exposed to FL were additionally harvested during the light maxima directly before the three indicated time points. For gene expression and protein analyses, 15 and 23 mL of cell suspension, respectively, were harvested each day at 14:00 and centrifuged for 4 min at 4°C and 4000 g. The precipitated cells were resolved in 1 mL ice cooled phosphate buffered saline and centrifuged at 14000 g for 1 min. The pellet was frozen in liquid nitrogen and stored at -80 °C until further analysis.

Fluorescence analyses

Cells harvested each day at 11:00, 14:00 and 17:00 were acclimated to 30 µmol photons m -2 s - 1 for 30 min before measuring the maximum photosynthetic efficiency of PSII as (F m -F o )/F m =F v /F m with an Aqua Pen (PSI Instruments, Czech Republic). In order to take into account slower relaxing NPQ processes and to assess the maximum NPQ capacity, rapid light curves, measured with a Water PAM and an Imaging PAM (Walz, Germany), were recorded after 45 min acclimation to 30 µmol photons m -2 s -1 , by applying thirteen steps of increasing light intensity up to 1250 µmol m -2 s -1 with a respective duration of 30 s at 455 nm. Before the onset of the actinic light and during each rapid light curve, an 800 ms pulse of 4000 µmol photons m -2 s -1 was applied to determine the maximum fluorescence levels F m and F m' , respectively. Maximum relative electron transport rates (rETR max ) and other photosynthetic and photoprotective parameters were obtained by fitting the obtained values according to [START_REF] Eilers | A model for the relationship between light intensity and the rate of photosynthesis in phytoplankton[END_REF] and Serodio & Lavaud (2011). A description of these parameters can be found in Table S1.

Pigment, transcript and protein analyses

Pigment extraction and HPLC analysis were performed as described 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].

The de-epoxidation state was calculated as DES = Dt/(Dd+Dt).

RNA extraction, cDNA synthesis, qPCR and quantification followed the protocol in Lepetit et al. (2013), except that RPS (ribosomal protein S1, JGI ID: 44451) was used as the reference gene instead of 18s due to a more stable transcript amount under our dynamic light conditions. The primer sequences for Lhcx1, Lhcx2 and Lhcx3 are listed 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]. For RPS we used 5'-AATTCCTCGAAGTCAACCAGG-3' and 5'-GTGCAAGAGACCGGACATAC-3' as forward and reverse primer, respectively, and for Lhcf2 the forward and reverse primer were 5'-GCCGATATCCCCAATGGATTT-3' and 5'-CTTGGTCGAAGGAGTCCCATC-3', respectively.

Protein extraction and Western blot analysis followed the protocol described in [START_REF] Coesel | Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity[END_REF], but using a 14% Lithiumdodecylsulfate PAGE for protein separation. Samples corresponding to an amount of 1 μg Chl a were loaded on the gel. Anti-FCP6 [START_REF] Westermann | Localisation of fucoxanthin chlorophyll a/c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy[END_REF] was kindly provided by Dr. Erhard Rhiel (University of Oldenburg, Germany).

This antibody detects all Lhcx isoforms in P. tricornutum [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][START_REF] Taddei | Multi-signal control of the expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum[END_REF], but based on its sequence it has the highest affinity for Lhcx3 and the lowest affinity for Lhcx2. Anti-FCP6 was applied in a 1:5000 dilution overnight. Accurate loading and blotting was verified by correct transfer of pre-stained protein markers (Roti-Mark BICOLOR, Roth, Germany) on the PVDF membrane (Amersham Hybond-P, GE Healthcare, USA), by staining gels with the Coomassie R-250 Pierce Imperial Protein stain (Thermo Fisher Scientific, USA) and by incubating the blot membrane with Anti-PsbB (CP47, Agrisera, Sweden). Anti-PsbB detection was only used as a proxy for correct loading and blotting, as the amount of CP47 has a high turnover under light stress conditions [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF]. Antibody signals were detected using the ECL Plus chemiluminescence system (GE Healthcare, USA) followed by x-ray film exposure. In order to compare relative Lhcx protein expression within the time-course of the experiments, protein samples of each P. tricornutum strain of either the SL or the FL 1000 experiment were loaded on a single gel. Antibody signal intensities were quantified using ImageJ (http://imagej.nih.gov/ij/). For each blot several films with different exposure and development times were produced, in order to avoid the saturation of the immunodetection signal for Lhcx1 (due to its high abundance) and Lhcx3 (due to its highest affinity to the anti-FCP6), while also obtaining a signal for the weakly visible Lhcx2 protein (due to its lowest affinity to anti-FCP6). Relative quantification of Lhcx1 and Lhcx3 referred on the corresponding signal at unstressed conditions (day 0). As Lhcx2 could not be detected at day 0, relative quantification was performed based on comparison of Lhcx2 to the Lhcx3 value at day 0. For each experimental point (SL and FL 1000), the mean of all strains was calculated ,except that Pt4ov was omitted from the analysis of Lhcx1 expression due to its artificial regulation by the overexpressing Lhcf1 promoter (see "Cell culturing and light treatments").

Statistics

Significance with a P value ≤ 0.05 was determined with Student's t-test calculated with SigmaPlot, but for gene expression we used the Pairwise Fixed Reallocation Randomization Test performed by REST according to [START_REF] Pfaffl | Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR[END_REF].

Results

NPQ capacity and photosynthetic parameters during SL and FL conditions

Before exposing cells to dynamic light conditions, we analysed their NPQ capacity under stable low light growth conditions. NPQ capacity was highest in the Lhcx1 overexpressing strain Pt4ov with values between 4 and 5 (Fig. 2, day 0). Pt1 showed an NPQ capacity of ~ 2.5, Pt1sil of ~2 and Pt4 of ~1.8 (Fig. 2). These different NPQ capacities were mainly due to different expression of the Lhcx1 gene (Fig. S1), as already shown in [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF].

The very high NPQ capacity of the Pt4ov strain was achieved by driving Lhcx1 gene expression by the Lhcf1 promoter, resulting in a more than 10 fold higher Lhcx1 transcript level (Fig. S1).

The rationale of working with P. tricornutum strains showing naturally or geneticallymanipulated differential NPQ capacities was to investigate whether these differences would influence their acclimation to dynamic light conditions. There was a strong continuous rise of NPQ capacity during the SL treatment (except in Pt4ov, see below), while under FL conditions NPQ increased at the first day similarly as under SL conditions, but then slowed down (Fig. 2). Importantly, the high NPQ strains (Pt1 and Pt4ov) still exhibited the highest NPQ at the end of both dynamic light treatments. Pt4 eventually reached a higher or similar NPQ as Pt1sil under SL and FL conditions, respectively. The Pt4ov strain behaved somewhat differently, as there was no increase of NPQ on day 1 under SL. This is most likely due to the pronounced decrease of Lhcx1 gene expression (Fig. S1), as its overexpressing Lhcf1 promoter is repressed under light stress [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF]. However, from day 2 on Pt4ov also started to increase NPQ capacity. Interestingly, although under stable LL conditions (i.e. day 0) Pt4ov had already higher NPQ values than the other strains at the end of FL treatment, it further increased NPQ during FL exposure (Fig. 2). All these results did not indicate a strong influence of initial NPQ capacity on the NPQ adjustment to dynamic light; instead, all strains responded in a similar manner by increasing their NPQ capacity. As the standard errors were rather high due to the dynamic nature of the experiment, in the following we combined results of the different strains in order to better reveal the specific response of P. tricornutum to the very distinct dynamic light conditions. This way, it became directly apparent that the doubling of light intensity under FL 1000 conditions led to no further increase of NPQ compared to FL 500 conditions. Actually, with a final value of ~ 3.2 the NPQ was almost identical at both FL conditions, while it was 5.8 under SL conditions (Fig. 3).

The maximum photosynthetic yield of PSII gradually decreased during SL conditions, especially during day 3, to 0.55 (20 % decrease), illustrating the appearance of photoinhibition (Fig. 4a). In contrast, F v /F m was maintained high (~ 0.65) during FL conditions. Again, no differences between FL 500 and FL 1000 conditions were observed (data not shown, but can be seen from the minimal error bars in Fig. 4a). Because also no major differences between FL 500 and FL 1000 conditions could be observed during the follow-up experiments, data were combined (unless otherwise noted), in order to highlight significant differences compared to SL.

Under SL conditions, rETR max (see Table S1 for the definition of the parameters) dropped during the first day and slightly increased over the next two days compared to LL (Fig. 4b). In contrast, rETR max did not change during the first day of FL, but strongly increased during day 2 and 3. α decreased by roughly 20 % already during the first day of SL and then stabilized (Fig. 4c). It didn't remarkably change under FL conditions. Interestingly, there was no major change in E k between SL and FL conditions (Fig. 4d). It increased by ~ 50 % during day 2 and stabilized over day 3. The apparent low E k values were likely due to the blue light of the Imaging-PAM excitation beam and are in line with previous E k values obtained the same way (Serodio & Lavaud, 2011). Huge differences were observed in NPQ Ek /NPQ max . It increased from ~7 % in LL to almost 20 % under SL conditions, while under FL it even decreased (Fig. 4e). In line with this, also E50 NPQ behaved differently: At first it decreased, but then increased under both conditions. However, while the final E50 NPQ value under SL conditions was similar as under LL, it was more than 1.5 fold higher under FL conditions (Fig. 4f).

Pigment stoichiometry during SL and FL

The Chl a increase per day per culture volume (µ Chla ) strongly differed under both dynamic conditions. Under SL conditions, µ Chla dramatically decreased by 50 % on day 1 (compared to LL) and by 80 % over the rest of the experiment (Fig. 5). In contrast, under FL conditions a slight decrease of µ Chla occurred during the first day, while already from the second day on the cultures produced as much Chl a per day as during LL conditions. For both, the Chl c to Chl a and the Fx to Chl a ratio, no remarkable differences between SL and FL conditions could be observed (Fig. S2).

In general, changes of the NPQ capacity were well reflected by changes in the amount of XC pigments. Dd+Dt increased 3.4 fold during SL treatment, reaching its maximum on the last day (Fig. 6). Although most of the increase already occurred during day 1, the XC pool size steadily increased during day 2 and 3 with repeated drops at night. In contrast, under FL conditions the increase of XC pigments was only 1.7 fold, and the largest part of XC pigments was also synthesized on day 1. The observed trend was identical in all strains under both light conditions, although Pt1 strains synthesized more Dd+Dt than Pt4 strains (Fig. S3).

The DES (Dt/(Dd+Dt)) reached values of around 50 % during the first day of SL at 14:00 (6 hours after light onset) and 17:00 (9 hours after light onset) (Fig. S4). During the following SL days and during all three days of FL conditions DES was lowest in the morning (11:00, 3 hours after light onset) and highest in the afternoon (17:00), in line with the respective light intensities). During the light intervals of FL conditions, DES reached similar values as under SL conditions, but always decreased below 10 % during the subsequent low light/dark phases, indicating that Dt epoxidation took place rapidly during the decline of light intensity.

NPQ versus Dd+Dt relationship

The ratio of NPQ versus Dd+Dt is a robust indicator for the efficiency of the XC pigments to confer NPQ (Lavaud & Lepetit, 2013;[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]. NPQ/(Dd+Dt) strongly decreased during the first day of SL treatment and recovered during the following days (Fig. 7). At the last time point (day 3-17:00), it became statistically indistinguishable from day 0, indicating that eventually Dt quenching efficiency reached the levels of LL acclimated cells. In contrast, NPQ/(Dd+Dt) remained high under FL conditions, highlighting a high quenching efficiency of Dt. During the second and third day it was statistically supported even higher at some time points than under LL conditions.

Lhcx gene expression and protein synthesis

In agreement with previous analyses for low light to high light shifts [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[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], cells of all P. tricornutum strains strongly increased the transcript amounts of Lhcx2 and Lhcx3 under both SL and FL conditions, while Lhcx1 was only slightly more transcribed (Fig. S1; note that Lhcx1 transcription in Pt4ov reacted differentially due to the regulation of the Lhcx1 gene by the Lhcf1 promoter). This became even more obvious, when comparing the mean values of all strains (Fig. 8a-c). Intriguingly, there was a strong difference in Lhcx2 and Lhcx3 transcript amounts depending on the light climate: Under SL conditions, Lhcx2 transcription was much more pronounced than under FL. In contrast, Lhcx3 transcript level was higher under FL conditions. Transcription of Lhcx2 and Lhcx3 increased throughout SL treatment, while under FL conditions the maximum transcript level was already reached on day 1 (but note the decrease of Lhcx3 transcription on day 2 and 3). Lhcf2 is one of the major classical light harvesting antenna proteins of the FCP under low light conditions [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] Grouneva | The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery[END_REF][START_REF] Gundermann | Identification of several sub-populations in the pool of light harvesting proteins in the pennate diatom Phaeodactylum tricornutum[END_REF], hence under stressful light conditions an expression pattern opposite to Lhcx genes was expected. Indeed, there was a strong Lhcf2 transcript reduction throughout the whole SL treatment (Fig. 8d). In contrast, under FL conditions Lhcf2 transcript levels dropped only during day 1, but reached almost initial levels already by day 2.

In order to investigate whether the differences in Lhcx transcription between SL and FL conditions were also reflected by the protein level, the Lhcx proteins were quantified. No significant changes could be observed for Lhcx1 compared to LL conditions neither in SL nor in FL (Fig. 9a). There was a gradual increase of Lhcx2 protein synthesis throughout the whole SL experiment (Fig. 9b and Fig. S5 for an example of the Western blots obtained for Pt4), while under FL conditions its level reached maximum values already on the second day and was much lower compared to SL conditions on day 2 and day 3. In contrast, Lhcx3 content similarly increased during day 1 under SL and FL, and declined during the following days (Fig. 9c). Hence, only the Lhcx2 protein content correlated with the respective amounts of transcripts, and both paralleled the increase in NPQ capacity under SL exposure (see Fig. 3). This is better illustrated by plotting the mean relative amount of Lhcx2 versus the mean increase of NPQ capacity under SL conditions of all strains (but Pt4ov due to its unusual NPQ behaviour caused by the Lhcx1 overexpressing Lhcf1 promoter), yielding a linear correlation with an R 2 of 0.997 (Fig. 10).

Discussion

Up-regulation of NPQ capacity is independent of initial NPQ capacity during acclimation to dynamic light conditions

We could not observe major differences in adjustment of NPQ capacity during dynamic light conditions in the low NPQ strains (Pt1sil and Pt4) compared to the high NPQ strains (Pt1 and Pt4ov). In line with this result, there was also no correlation between initial NPQ capacity and XC pigment synthesis, the latter being strain dependent: Pt1 and Pt1sil showed a stronger increase of Dd+Dt pool size than Pt4ov and Pt4 (Fig. S3). Furthermore, the Pt4ov strain, which under LL already possessed an NPQ capacity as high as Pt1sil at the end of the SL treatment (Fig. 2), similarly increased Lhcx2 and Lhcx3 gene expression as well as the Dd+Dt pool size (Fig. S1 and Fig. S3). The apparently low influence of the initial NPQ capacity on the subsequent NPQ adjustment during dynamic light conditions may be due to the fact that initial NPQ capacity under stable LL conditions is first determined by Lhcx1 and Dd+Dt amount [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF]. As reported here for dynamic light and before for prolonged high light conditions [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], a higher NPQ capacity is obtained by the combined increase of Dd+Dt pool size and primarily Lhcx2 expression.

Insights into the regulation of NPQ actors in diatoms have been gained recently, and clearly photoreceptor mediated processes influence NPQ capacity [START_REF] Schellenberger Costa | Blue light is essential for high light acclimation and photoprotection in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Brunet | Spectral radiation dependent photoprotective mechanism in the diatom Pseudo-nitzschia multistriata[END_REF]. In line with this, the Lhcx1 gene promoter has a binding motif for a blue light receptor, aureochrome 1a [START_REF] Schellenberger Costa | Blue light is essential for high light acclimation and photoprotection in the diatom Phaeodactylum tricornutum[END_REF]. The cryptochromes CPF [START_REF] Coesel | Diatom PtCPF1 is a new cryptochrome/photolyase family member with DNA repair and transcription regulation activity[END_REF] and CryP [START_REF] Juhas | A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light harvesting protein levels[END_REF] regulate expression of Lhcx genes.

Interestingly, in Chlamydomonas reinhardtii the cryptochrome aCRY, which is related to CryP, seems to react rather on light intensity than on light quality [START_REF] Beel | A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii[END_REF], hence CryP could modulate NPQ capacity in diatoms in response to different light intensities.

Besides the influence of photoreceptors on NPQ adjustment, we previously demonstrated that Dd+Dt increase and Lhcx2 expression rates are controlled by changes in the redox state of the PQ-pool, while Lhcx3 expression may be regulated via ROS [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] Lepetit | Light signaling in photosynthetic eukaryotes with 'green' and 'red' chloroplasts[END_REF]. In the present study Lhcx2 expression and Dd+Dt content correlated well under both dynamic light conditions, indicating the presence of a common trigger. Clearly, the expression of Lhcx2 and Lhcx3 differed under SL and FL, suggesting that both light conditions elicit two different regulation pathways, which is likely due to the different characteristics of SL and FL in combination with their different total light doses. The PQ-pool redox state responsive Lhcx2 and Dd+Dt reacted rather gradually to long lasting light stimuli under SL, which generated a higher cumulative photon amount per day. In contrast, Lhcx3 expression was regulated by short but intense light intervals, conditions which are expected to generate pronounced amounts of ROS. Because the NPQ capacity has an influence on energy flow into the electron transport chain and thus on the redox state of the PQ-pool and on ROS generation [START_REF] Triantaphylidès | Singlet oxygen is the major reactive oxygen species involved in photooxidative damage to plants[END_REF][START_REF] Kruk | Singlet oxygen and non-photochemical quenching contribute to oxidation of the plastoquinone-pool under high light stress in Arabidopsis[END_REF], the concentration of the reduced PQ-pool and ROS should be different in the four P. tricornutum strains under dynamic light conditions. Still, the four strains adjusted their NPQ capacity in a similar way under dynamic light conditions. The fact that NPQ capacity increases even in the highest NPQ strain (Pt4ov) illustrates that initial NPQ capacity was not sufficient to avoid partial overreduction of the electron transport chain. Sensitive PQ-pool redox state and ROS responding promoter elements may already react to a partly reduced PQ-pool and small amounts of ROS, so that minor differences in these triggers would hardly differentially affect the expression of Lhcx genes and the synthesis of Dd+Dt pigments. Moreover, the redox state of the PQ-pool can be influenced by changes of the metabolome [START_REF] Jungandreas | The acclimation of Phaeodactylum tricornutum to blue and red light does not influence the photosynthetic light reaction but strongly disturbs the carbon allocation pattern[END_REF][START_REF] Wilhelm | Light acclimation in diatoms: From phenomenology to mechanisms[END_REF][START_REF] Levitan | An RNA interference knock-down of nitrate reductase enhances lipid biosynthesis in the diatom Phaeodactylum tricornutum[END_REF]. Such influences would be fairly independent of the NPQ capacity and could also explain the similar NPQ response in the four P. tricornutum strains.

Lhcx2 in combination with the amount of XC pigments likely increases NPQ capacity under dynamic light

In diatoms, the increase of XC pigments does not necessarily lead to a higher NPQ [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]. Specific proteins must be synthesized to bind these pigments for an effective involvement in NPQ [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]. Lhcx1 does not increase significantly during dynamic light conditions (Fig. 9a), and hence confers only basal NPQ capacity. In Lhcx2 or Lhcx3 overexpression lines, both proteins provide additional NPQ capacity [START_REF] Taddei | Multi-signal control of the expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum[END_REF]. Lhcx2 content similarly increased on day 1 in SL and FL conditions, and remained stable during the following days in FL, while it increased in SL. These features were paralleled by the NPQ capacity. In fact, Lhcx2 amount was linearly correlated to the NPQ capacity increase under SL conditions (Fig. 10). Although this correlation is based only on a few data points, it suggests Lhcx2 being a major actor in modelling NPQ capacity under dynamic light conditions, together with the size of the XC pigment pool and the degree of deepoxidation.

The impact of Lhcx3 on NPQ capacity enhancement is difficult to deduce. Our data suggest that Lhcx3 was less responsible for NPQ increase during day 2 and 3 in SL and FL, as its protein content was rather decreasing (Fig. 9c). Instead, the prompt increase of Lhcx3 on day 1 under both dynamic conditions may provide a fast increase of photoprotection capacity, while Lhcx2 continues to add up during prolonged light stress conditions like under SL treatment. Interestingly, there was a significant difference in Lhcx3 transcript levels between FL and SL on day 1, which was not reflected by the protein level. This suggests that posttranscriptional control mechanisms, that specifically respond to fast light intensity fluctuations, partially prevent Lhcx3 protein synthesis. In line with this, it was recently shown in A. thaliana that high light regulation of several high light responsive target proteins often occurs differentially on the transcriptional and translational level [START_REF] Oelze | The link between transcript regulation and de novo protein synthesis in the retrograde high light acclimation response of Arabidopsis thaliana[END_REF]). An additional control point at the Lhcx3 translational level might be a strategy to acclimate the NPQ system to an average light intensity delivered by light fluctuations, while keeping a high Lhcx3 transcript reservoir in case of prolonged light stress. This would avoid a too strong down-regulation of photochemistry during low light periods, while ensuring sufficient excess energy dissipation during high light regular peak exposures, a feature in line with the fast on/off switch of the NPQ system [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] and the fine regulation of Dd+Dt synthesis versus the velocity of light fluctuations [START_REF] Giovagnetti | The velocity of light intensity increase modulates the photoprotective response in coastal diatoms[END_REF] in diatoms.

Besides Lhcx2, and to some extent Lhcx3, other proteins could contribute to additional NPQ capacity under dynamic light, especially to the slight increase of NPQ during FL on day 2 and day 3. The Lhcr gene family contains a phylogenetically separated subclade [START_REF] Nymark | Molecular and photosynthetic responses to prolonged darkness and subsequent acclimation to re-illumination in the diatom Phaeodactylum tricornutum[END_REF], which genes are transcriptionally up-regulated during high light stress [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF]. The corresponding proteins may be at least partially involved in modulating NPQ capacity. Another possible candidate is Lhcf15, which is the only Lhcf gene showing upregulation during short term light stress [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF], but which especially responds to red light [START_REF] Schellenberger Costa | Blue light is essential for high light acclimation and photoprotection in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Valle | System responses to equal doses of photosynthetically usable radiation of blue, green, and red light in the marine diatom Phaeodactylum tricornutum[END_REF][START_REF] Herbstová | Molecular basis of chromatic adaptation in pennate diatom Phaeodactylum tricornutum[END_REF].

Lhcf15 can build up specific antennae complexes with a red-shifted fluorescence emission [START_REF] Herbstová | Molecular basis of chromatic adaptation in pennate diatom Phaeodactylum tricornutum[END_REF], that could be correlated to the NPQ capacity (Lavaud & Lepetit, 2013).

FL triggers a very effective photoprotective response

Under SL conditions, the cells first synthesized much more Dd+Dt than could be used for NPQ. Eventually, the ratio NPQ/(Dd+Dt) was better adjusted towards a higher quenching efficiency of Dt due to a slowdown of Dd+Dt synthesis and a concomitant catch-up of Lhcx2 synthesis. Despite a strongly increased NPQ capacity as well as a massive reduction of Chl a synthesis and Lhcf2 transcription, decrease in photosynthetic efficiency could not be completely prevented. In contrast, cells under FL conditions kept the NPQ/(Dd+Dt) efficiency rather comparable to that of LL conditions, i.e. they synthesized only as many Dd+Dt as actually could be used for providing an optimal effective involvement of Dt in NPQ. Parallel to enhancing NPQ capacity, FL cells adjusted the reactivity of NPQ activation. The light intensity, for which 50 % of the maximum NPQ capacity was reached, was shifted to much higher values (Fig. 4f). Simultaneously, despite increasing E k , FL cells kept the level of NPQ activation at E k very low (i.e. a few % of NPQ max, Fig. 4e). This is noteworthy, because although E k increased similarly, SL cells were unable to adjust the threshold for NPQ onset and activated a pronounced NPQ already at E k . Ultimately, FL cells strongly increased rETR max , but kept α high as in LL cells, in line with previous results in Skeletonema costatum [START_REF] Kromkamp | Effect of short-term variation in irradiance on light harvesting and photosynthesis of the marine diatom Skeletonema costatum: a laboratory study simulating vertical mixing[END_REF]. All these changes enabled FL cells to use absorbed light efficiently until E k was reached and even beyond due to the moderate switch-on of NPQ. Hence, they exploited as much light as possible for photochemistry during the short light periods thanks to the adjustment of NPQ capacity and kinetics. Consequently, after one day of acclimation, µ Chla and Lhcf2 transcription reached similar values as under LL conditions. As FL cells developed a much higher NPQ capacity than LL cells, altogether FL acclimation does not correspond to either a low or high light type strategy, but shows peculiar characteristics. Such special fluctuating light acclimation strategy has also been observed in the diatom Stephanodiscus neoastraea [START_REF] Fietz | Acclimation of the diatom Stephanodiscus neoastraea and the cyanobacterium Planktothrix agardhii to simulated natural light fluctuations[END_REF].

We have to note that the fine-acclimation to FL conditions with different intensity maximums may be limited. Although FL 1000 cells faced much higher light intensities, steeper light gradients and the double daily photon dose, NPQ capacity increased similarly as in FL 500 cells. On the one hand, this very similar NPQ pattern may be co-initiated by an internal trigger such as the circadian clock. The influence of the circadian clock on the regulation of the XC pigments in P. tricornutum has already been demonstrated [START_REF] Ragni | Circadian variability in the photobiology of Phaeodactylum tricornutum: pigment content[END_REF].

On the other hand, [START_REF] Giovagnetti | The velocity of light intensity increase modulates the photoprotective response in coastal diatoms[END_REF] showed that light acclimation response in Pseudonitzschia multistriata is triggered by light intensity, but also by the velocity of the light increase. Moreover, in Skeletonema marinoi, NPQ capacity is not directly correlated to the total daily photon dose [START_REF] Orefice | Light-induced changes in the photosynthetic physiology and biochemistry in the diatom Skeletonema marinoi[END_REF]. Our results suggest that, below a certain duration threshold of regular light periods, different high light intensities only trigger an efficient on/off reaction of NPQ capacity adjustment, but no fine-tuned response. This is possibly due to the lack of the gradual PQ-pool signal. Interestingly, when the light intervals become even shorter than the dark intervals in a fluctuating light regime, P. tricornutum is able to generate a very high NPQ (Lavaud et al., 2002b;[START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF], which may not be triggered by light, but may be elicited by darkness instead. As only the transcription of the Lhcx4 isoform is stimulated in the dark [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] Nymark | Molecular and photosynthetic responses to prolonged darkness and subsequent acclimation to re-illumination in the diatom Phaeodactylum tricornutum[END_REF] and the respective protein can induce NPQ [START_REF] Taddei | Multi-signal control of the expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum[END_REF], Lhcx4, together with the increased Dd+Dt content (Lavaud et al., 2002b), may be responsible for this particular increased NPQ capacity.

Conclusion

Our study highlights the importance of investigating the influence of dynamic light conditions on NPQ in diatoms. Recently, it was shown that P. tricornutum is coping well with fluctuating light conditions by possessing a low-cost PSII repair cycle compared to diatoms living in the open ocean in more stable light conditions [START_REF] Lavaud | Photosystem II repair in marine diatoms with contrasting photophysiologies[END_REF]. A comprehensive study by [START_REF] Wagner | Balancing the energy flow from captured light to biomass under fluctuating light conditions[END_REF] demonstrated that in P. tricornutum absorbed photons are converted to a much higher extent into biomass in FL than in SL conditions, which is due to a strongly decreased amount of alternative electrons and a lowered quantum requirement per molecule oxygen evolved. The finely adjusted regulation of NPQ capacity by balanced Lhcx2/Lhcx3 and Dd+Dt synthesis under FL has three consequences which support the observations of [START_REF] Wagner | Balancing the energy flow from captured light to biomass under fluctuating light conditions[END_REF]: (1) Investment costs for photoprotection mechanisms are lower than in SL; (2) Photodamage is reduced, keeping costs for repair processes low; (3) Too much photoprotection, leading to a poor light energy to chemical energy conversion rate and hence to a high quantum requirement for carbon fixation, is prevented. This latter feature has been recently demonstrated to be of global importance in the upper ocean [START_REF] Lin | Phytoplankton. The fate of photons absorbed by phytoplankton in the global ocean[END_REF]. The better balanced photoacclimation strategy under FL compared to SL conditions may be one reason why diatoms dominate in habitats where the light climate is regularly punctuated with high intensity exposure periods, such as coastal waters and estuarine intertidal sediments [START_REF] Strzepek | Photosynthetic architecture differs in coastal and oceanic diatoms[END_REF][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 | Comparative ecophysiology of the xanthophyll cycle in six marine phytoplanktonic species[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] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF]. Figure 5: Chl a increase rate per day per culture volume (µ Chla ) of all four Phaeodactylum tricornutum strains under SL and FL 1000 conditions. Dynamic light conditions started on day 1 (indicated with an arrow). µ Chla (in day -1 ) was calculated as µ Chla = ln (Chl t n /1.4), where Chl t n refers to the Chl a content measured at 18:00 during the four days of experiment (day 0 to day 3) and 1.4 is the Chl a content (in µg mL -1 ) at which the cultures were adjusted to each day after Chl a determination. Because data for FL 500 conditions were not complete they were omitted, but, similarly to FL 1000 cells, FL 500 cells showed a higher µ Chla than SL 500 cells. Values represent the means ± SE of at least four biological replicates. Meaning of significance letters: a, µ Chla is significantly different compared to day 0 (p < 0.05); b, µ Chla is significantly different in cells exposed to FL 1000 conditions compared to cells exposed to SL conditions for the same day (p < 0.05).

Figure legends

Figure 1 :

 1 Figure 1: Dynamic light conditions used in the experiments. One sine light (SL) and two fluctuating light (FL) conditions with different intensities were applied during the daily phases of the light exposure and light intensity was measured every minute.

Figure 2 :

 2 Figure 2: Comparison of NPQ capacity in the four different Phaeodactylum tricornutum strains (Pt1, Pt1sil, Pt4, Pt4ov) under SL (a) and FL (b) conditions. Time is indicated as experimental day (0 to 3, separated with vertical bars/arrow) and the respective time of sampling. Dynamic light conditions started on day 1 (indicated with an arrow). FL values combine data from the FL 500 and FL 1000 treatment. Values represent the means ± SE of three to four different experiments.

Figure 3 :

 3 Figure 3: Mean NPQ values of Phaeodactylum tricornutum strains Pt1, Pt1sil and Pt4 underSL and FL conditions. Pt4ov was not included in the mean due to its much higher NPQ and its partly unusual characteristics (cf. Fig.2); this is exemplified by its SL 500 trace (dashed line). Time is indicated as experimental day (0 to 3, separated with vertical bars/arrow) and the respective time of sampling. Dynamic light conditions started on day 1 (indicated with an arrow). Values represent the means ± SE of at least six biological replicates (except Pt4ov-SL 500: three biological replicates).

Figure 4 :

 4 Figure 4: Mean photosynthetic and photoprotective parameters of all four Phaeodactylum tricornutum strains under SL and FL (500 and 1000 combined) conditions. a) Fv/Fm; b) the maximum relative electron transport rate (rETR max ); c) α, the slope of the relative electron transport rate versus light intensity under non-saturating light conditions; d) E k , the interception point between α and ETR max , a measure for the minimal light intensity to saturate photosynthesis; e) NPQ Ek /NPQ max , indicates the relative amount of NPQ at E k ; f) E 50 , the light intensity, at which half of NPQ max capacity is reached. Time is indicated as experimental day (0 to 3, separated with vertical bars/arrow) and the respective time of sampling. Dynamic light conditions started on day 1 (indicated with an arrow). Values represent the means ± SE of at least eight biological replicates. Meaning of statistical significance letters: a, values are significantly different compared to day 0 (p < 0.05); b, values from cells exposed to FL are significantly different compared to cells exposed to SL for the same time point (p < 0.05).

Figure 6 :

 6 Figure 6: Mean diadinoxanthin and diatoxanthin (Dd+Dt) pool size (in mol (100 mol Chl a) -1 ) of all four Phaeodactylum tricornutum strains under SL and FL (500 and 1000 combined) conditions. Time is indicated as experimental day (0 to 3, separated with vertical bars/arrow) and the respective time of sampling. Dynamic light conditions started on day 1 (indicated with an arrow). For SL conditions, data for each strain are available in Fig. S4. Values represent the means ± SE of eight biological replicates. Meaning of statistical significance letters: a, Dd+Dt pool size is significantly different compared to day 0 at 17:00 (p < 0.05); b, Dd+Dt pool size from cells exposed to the respective FL light treatment is significantly different compared to cells exposed to SL for the same time point (p < 0.05).

Figure 7 :

 7 Figure 7: Correlation of NPQ capacity versus Dd+Dt (in mol (100 mol Chl a) -1 ) under SL and FL (500 and 1000 combined) conditions. Values are taken from all Phaeodactylum tricornutum strains except Pt4ov due to its different characteristics in NPQ (cf. Fig. 2). Time is indicated as experimental day (0 to 3, separated with vertical bars/arrow) and the respective time of sampling. Dynamic light conditions started on day 1 (indicated with an arrow). Values represent the means ± SE of six biological replicates. Meaning of statistical significance letters: a, NPQ/(Dd+Dt) is significantly different compared to day 0 at 17:00 (p < 0.05); b, NPQ/(Dd+Dt) from cells exposed to FL is significantly different compared to cells exposed to SL for the same time point (p < 0.05).

Figure 8 :

 8 Figure 8: Relative transcript amounts of Lhcx1 (a), Lhcx2 (b), Lhcx3 (c) and Lhcf2 (d) underSL and FL (500 and 1000 combined) conditions. Gene expression was normalized on transcript amount of the RPS gene. For each gene, the expression was calculated as the mean transcript amount of all Phaeodactylum tricornutum strains at the specific time points and light conditions except for Lhcx1 where the values of Pt4ov were omitted due to the artificial Lhcf1 promoter in this strain (c.f. Fig.S1). Dynamic light conditions started on day 1 (indicated with an arrow). Values represent the means ± SE of eight biological replicates

Figure 9 :

 9 Figure 9: Mean of relative protein expression of Lhcx1 (a), Lhcx2 (b) and Lhcx3 (c) of allPhaeodactylum tricornutum strains under SL and FL 1000 conditions, respectively. For Lhcx1, protein expression data of Pt4ov were omitted due to the artificial regulation by the Lhcf1 promoter (c.f. Fig.S1). Dynamic light conditions started on day 1 (indicated with an arrow). Values represent the means ± SE of at least four biological replicates (except for Lhcx1 with at least three biological replicates). Meaning of significance letters: a, protein is significantly differentially expressed compared to day 0 (p < 0.05); b, protein from cells exposed to FL is significantly differentially expressed compared to cells at the corresponding time point exposed to SL (p < 0.05).

Figure 10 :Figures

 10 Figure 10: Correlation of relative Lhcx2 protein content versus NPQ increase under SL conditions. NPQ was calculated as NPQ day1,2,3 -NPQ day 0 , always measured at the 14:00 time point. The three data points correspond to the Lhcx2 protein amount versus NPQ on day 1, day 2 and day 3. Values for NPQ and Lhcx2 protein content are the means ± SE of all Phaeodactylum tricornutum strains except Pt4ov.

Acknowledgement

We thank Dr. Torsten Jakob (Leipzig) for help with installing the dynamic light conditions, Doris Ballert (Konstanz) for performing the biolistic transformation of the Pt4ov strain, Dr. Erhard Rhiel (Oldenburg) for providing the FCP6 antibody and Dr. Giovanni Finazzi (Grenoble) for valuable scientific discussion. We also thank the three reviewers for their constructive criticism. This work was supported by the German Academic Exchange Service-DAAD (postdoctoral grant to B.L.), an Marie Curie Zukunftskolleg Incoming Fellowship, University of Konstanz (grant no. 291784, to B.L.), a University of Konstanz Zukunftskolleg Interim grant (to B.L.), the Centre National de la Recherche Scientifique and the French consortium Contrat de Plan État-Région Littoral (grants to J.L.), the Deutsche Forschungsgemeinschaft (grant nos. LE 3358/3-1 to B.L., LA-2368/2-1 to J.L., and KR-1661/7-1 to P.G.K.), and the Marie-Curie ITN CALIPSO (ITN 2013 GA 607607, to A.F.).