Johann Lavaud
email: johann.lavaud@univ-lr.fr

Bernard Lepetit

An explanation for the inter-species variability of the photoprotective nonphotochemical chlorophyll fluorescence quenching in diatoms

Keywords: diatoms, diatoxanthin, ecophysiology, photoprotection, non-photochemical fluorescence quenching, xanthophyll cycle Chl a, Chlorophyll a, Chl a FIKs, Chl a fluorescence induction kinetics

An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms

Introduction

Diatoms are a major group of microalgae ubiquitous in all marine and freshwater ecosystems. They are among the most significant photosynthetic organisms contributing to about 40% of the aquatic primary production, especially in coastal ecosystems [START_REF] Kooistra | The origin and the evolution of the diatoms: Their adaptation to a planktonic existence[END_REF][START_REF] Armbrust | The life of diatoms in the world's oceans[END_REF].

Their biological characteristics have largely shaped the structure of contemporary aquatic ecosystems [START_REF] Kooistra | The origin and the evolution of the diatoms: Their adaptation to a planktonic existence[END_REF][START_REF] Falkowski | The evolution of modern phytoplankton[END_REF]. As for most microalgae, the photosynthetic productivity of diatoms strongly depends on the aquatic light climate [START_REF] Falkowski | Aquatic Photosynthesis[END_REF][START_REF] Macintyre | The effect of water motion on short-term rates of photosynthesis by marine phytoplankton[END_REF][START_REF] Kirk | Light and Photosynthesis in Aquatic Ecosystems[END_REF]. Planktonic as well as benthic diatoms tend to dominate ecosystems (coasts and estuaries) where they have to cope with a light climate with high-frequency irradiance fluctuations coupled with large amplitudes.

Depending on the rate of water mixing, on the tidal cycle, on the daily/seasonal changes of solar irradiation, diatoms can be exposed to punctual or chronic excess light, possibly generating stressful conditions that impair their photosynthetic efficiency and their productivity (i.e. photoinhibition/photoinactivation) [START_REF] Long | Photoinhibition of photosynthesis in nature[END_REF][START_REF] Olaizola | Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom[END_REF][START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF][START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF]. In order to prevent such situation, diatoms [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF][START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF], and to a larger extent microalgae and other photosynthetic organisms [START_REF] Li | Sensing and responding to excess light[END_REF][START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF][START_REF] Brunet | Pigments and photoacclimation processes[END_REF][START_REF] Jahns | The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II[END_REF][START_REF] Ruban | The photoprotective molecular switch in the photosystem II antenna[END_REF], have evolved fast regulatory physiological processes which compose a 'photoprotective network'. They help to safely dissipate the excess of absorbed light energy as heat and/or to balance the excitation energy within the photosynthetic apparatus thus preventing/lowering the potential oxidative damages to the photosynthetic machinery. Additional mechanisms help repairing the unavoidable photodamages [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF][START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF]. In diatoms, two fast processes are believed to be essential for the photoprotection: the electron cycle of the photosystem II reaction center (PS II CET) and the nonphotochemical quenching of chlorophyll a (Chl a) fluorescence (NPQ) [11-12, 14, 19].

NPQ is composed of three components, namely the high-energy state (qE), the state-transition (qT) and the 'photoinhibitory' (qI) quenchings [START_REF] Li | Sensing and responding to excess light[END_REF][START_REF] Ruban | The photoprotective molecular switch in the photosystem II antenna[END_REF]. In diatoms, qT does not exist and the origin of qI remains unclear [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF][START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF]. Therefore, the diatom NPQ mainly relies on qE, a quenching mechanism which is controlled by i) the build-up of the transthylakoidal proton gradient (ΔpH), ii) the conversion of the xanthophylls diadinoxanthin (DD) into diatoxanthin (DT) called 'xanthophyll cycle' (XC), and iii) the presence of specific polypeptides of the light-harvesting complex (LHC) antenna named Lhcx [11-12, 14, 20-23]. Additionally, there exists a PS II reaction center quenching which might be related to the PS II CET [START_REF] Lavaud | Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms[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]. These fast regulatory mechanisms proved to be important in field situation for maintaining the photosynthetic efficiency and productivity of diatoms [START_REF] Brunet | Pigments and photoacclimation processes[END_REF][START_REF] Brunet | Can the xanthophyll cycle help extract the essence of the microalgal functional response to a variable light environment ?[END_REF]. NPQ was shown to significantly participate in the cellular response under fluctuating light conditions [START_REF] Wagner | Balancing the energy flow from captured light to biomass under fluctuating light conditions[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] Mills | Photophysiology in two Southern ocean phytoplankton taxa: Photosynthesis of Phaeocystis antarctica (Prymnesiophyceae) and Fragilaria cylindrus (Bacillariophyceae) under simulated mixed-layer irradiance[END_REF]. Recently, an increasing number of reports suggested that the diatom/microalgae inter-species differences in NPQ are involved in the differential colonization of aquatic habitats depending on the rate of light fluctuations of the light climate [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[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] Mills | Photophysiology in two Southern ocean phytoplankton taxa: Photosynthesis of Phaeocystis antarctica (Prymnesiophyceae) and Fragilaria cylindrus (Bacillariophyceae) under simulated mixed-layer irradiance[END_REF][START_REF] Strzepek | Photosynthetic architecture differs in coastal and oceanic diatoms[END_REF][START_REF] Dimier | Photoprotective capacity as functional trait in planktonic algae: relationship between xanthophyll cycle and ecological characteristics in three diatoms[END_REF][START_REF] Dimier | Comparative ecophysiology of the xanthophyll cycle in six marine phytoplanktonic species[END_REF][START_REF] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF][START_REF] Petrou | Heterogeneity in the photoprotective capacity of three Antarctic diatoms during short-term changes in salinity and temperature[END_REF].

While the most recent advances in our knowledge of the physiological regulation of diatom photosynthesis, and especially of NPQ and the XC, were generated by the use of genetically modified cells of the model 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] Depauw | Exploring the molecular basis of responses to light in marine diatoms[END_REF][START_REF] Materna | First induced plastid genome mutations in an alga with secondary plastids: psbA mutations in the diatom Phaeodactylum tricornutum (Bacillariophyceae) reveal consequences on the regulation of photosynthesis[END_REF][START_REF] Lavaud | Silencing of the violaxanthin de-epoxidase gene in the diatom Phaeodactylum tricornutum reduces diatoxanthin synthesis and non-photochemical quenching[END_REF], the comparison of different species/strains [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF][START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric 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] 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 | Photoprotective capacity as functional trait in planktonic algae: relationship between xanthophyll cycle and ecological characteristics in three diatoms[END_REF][START_REF] Grouneva | The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and 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] together with the use of different growth conditions [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF][START_REF] Dimier | Photoprotective capacity as functional trait in planktonic algae: relationship between xanthophyll cycle and ecological characteristics in three diatoms[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] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF] has also proven to be useful. Indeed, the XC and NPQ extent and kinetics can differ with regards to the species [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF][START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric 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 | Photoprotective capacity as functional trait in planktonic algae: relationship between xanthophyll cycle and ecological characteristics in three diatoms[END_REF], and to the light [START_REF] Dimier | Photoprotective capacity as functional trait in planktonic algae: relationship between xanthophyll cycle and ecological characteristics in three diatoms[END_REF][START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF][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] and the temperature [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF] acclimations. In a previous study, we compared the photoprotective ability of P. triconutum and Skeletonema costatum [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]. One of the major differences concerned NPQ: its extent in S. costatum was approximately half the one in P. tricornutum. It was not due to a lower de-epoxidation rate of DD to DT, as reported before for diatoms and other related organisms [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF][START_REF] Meyer | Xanthophyll cycling in Phaeocystis globosa and Thalassiosira sp.: a possible mechanism for species succession[END_REF][START_REF] Rodrigues | A smaller impaired xanthophyll cycle makes the deep sea macroalgae Laminaria abyssalis (Phaeophyceae) highly sensitive to daylight when compared with shallow water Laminaria digitata[END_REF].

We suggested two explanations for such discrepancy [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]: 1) a different organization and FCP (fucoxanthin-chlorophyll protein) composition of the LHC antenna of PS II, including both Lhcf and Lhcx proteins, 2) a lower amount of DT molecules involved in the NPQ process; the two hypotheses not being exclusive from each other [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF]. The present work aimed at going further deep in the understanding of NPQ differences between diatom species/strains. For that purpose, we examined four species/strains currently used as models for NPQ investigations and known to show different NPQ amplitude and regulation [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[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] Grouneva | The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana[END_REF][START_REF] Grouneva | A new multi-component NPQ mechanism in the diatom Cyclotella meneghiniana[END_REF][START_REF] Miloslavina | Ultrafast fluorescence study on the location and mechanism of nonphotochemical quenching in diatoms[END_REF][START_REF] Gundermann | Factors determining the fluorescence yield of fucoxanthin-chlorophyll complexes (FCP) involved in non-photochemical quenching in diatoms[END_REF]: a low and a high NPQ strains of P. tricornutum (P.t.2 and P.t.4, respectively), S. costatum and Cyclotella meneghiniana. The three species are also representative of the two major groups of diatoms, the pennates (P. tricornutum) and the centrics (S. costatum and C. meneghiniana); the genome of P. tricornutum is available and C. meneghiniana is a close relative of Thalassiosira pseudonana whose genome is also published [START_REF] Tirichine | Decoding algal genomes: tracing back the history of photosynthesis life on Earth[END_REF]. Finally, the ecological niches where P.t.2, P.t.4 and S. costatum were isolated (temperate estuary, high latitude shore, Mediterranean semi-enclosed bay, respectively) show very different underwater light climates [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[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].

Material and Methods

Cultures

Phaeodactylum tricornutum Böhlin CCAP1052-CCAP1055/1 ('P.t.2 strain', Culture Collection of Algae and Protozoa, Oban, Scotland) and CCAP 1052/6 ('P.t.4 strain', Sammlung Algenkulturen Göttingen (SAG), Germany), Skeletonema costatum (Greville) Cleve (Laboratoire Arago algal collection, Banyuls-sur-Mer, France) and Cyclotella meneghiniana Kützing SAG 1020_1a (SAG, Germany) cells were grown photoautotrophically in sterile natural seawater F/2 medium [START_REF] Guillard | Studies of marin planktonic diatoms. 1. C. nana (Hustedt) and D. confervacea (Cleve)[END_REF]. Non-axenic cultures of 300 mL were incubated at 18C in airlifts continuously flushed with sterile air. They were illuminated at a light intensity of 40 µmol photons m -2 s -1 with white fluorescent tubes (Claude, Blanc Industrie, France) with a 16 h light/8 h dark photoperiod ('continuous light', CL cells, see [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF]). Intermittent light (IL) cells of P. tricornutum were grown in the same conditions except the light regime which was 5 min light/55 min dark cycle (see [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF]). Cells were harvested during the exponential or stationary phase of growth as specified in Table 1, centrifuged at 3000 g for 10 min and resuspended in their culture medium to a final concentration of 10 µg chlorophyll a (Chl a) mL -1 . The concentrated suspension was then continuously stirred at 18C under low light before use.

Pigment content

Pigment analyses were performed by HPLC as previously described [START_REF] Lavaud | Enrichment of the light-harvesting complex in diadinoxanthin and implications for the nonphotochemical quenching fluorescence quenching in diatoms[END_REF]. Cells collected from the Clark electrode adapted to the PAM-fluorometer (see below) were deposited on a filter and frozen in liquid nitrogen. Pigments were extracted with a methanol:acetone (70:30, v/v) solution. The xanthophyll de-epoxidation state (%) was calculated as (DT/(DD+DT)) x 100 where DD is the diadinoxanthin, the epoxidized form, and DT is the diatoxanthin, the de-epoxidized form.

Spectroscopy

Ambient temperature absorption spectra were performed with a DW-2 Aminco spectrophotometer. For measuring the 522 nm absorption change (Δ 522 nm) which reflects the 'activation of DT' (see [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]), spectra were performed on dark-acclimated cells (for 20 min) and cells exposed to light (5 min, 2000 µmol photons m -2 s -1 ) as previously described [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]; the half-bandwidth was 0.5 nm. For partial inhibition of NPQ, cells were incubated prior to illumination (20 min, dark) with dithiothreitol (DTT) or NH 4 Cl with a final concentration of 25 µM and 2 mM, respectively (see [START_REF] Lavaud | In diatoms, a transthylakoidal proton gradient alone is not sufficient for non-photochemical fluorescence quenching[END_REF]). 77K fluorescence emission spectra were measured with an F-3000 and F-4500 Hitachi spectrophotometer. Halfbandwidth for emission was 2.5 (F-4500) and 3 (F-3000) nm, respectively. Samples were dark-acclimated (20 min) and then concentrated at 1 g Chl a on a Millipore AP-20 prefilter that was immediately frozen in liquid nitrogen before measurement. The same procedure was used for illuminated cells (2000 µmol m -2 s -1 for 5 min) albeit a Chl a amount of 3 µg was immediately frozen after illumination. The angle between the filter and the light source/detector was 45°. For complete inhibition of NPQ, cells were incubated prior to illumination (20 min, dark) with 0.2 mM DTT or 5 mM NH 4 Cl [START_REF] Lavaud | In diatoms, a transthylakoidal proton gradient alone is not sufficient for non-photochemical fluorescence quenching[END_REF].

Chlorophyll fluorescence yield and the non-photochemical fluorescence quenching (NPQ).

Chlorophyll fluorescence yield was monitored with a modified PAM-101 fluorometer (Walz, Effeltrich, Germany) as described previously [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF] (see Figure 1A for a typical recording). Fluorescence was excited by a very weak (non-actinic) modulated 650 nm light beam. After 20 min of dark-acclimation, continuous actinic light of adjustable intensity was applied. 600 ms pulses of white light (4000 µmol photons m -2 s -1 ) were admitted by an electronic shutter (Uniblitz, Vincent, USA, opening time 2 ms) placed in front of a KL-1500 quartz iodine lamp continuously switched on in order to monitor the evolution of NPQ. The average fluorescence (acquisition time 33 µs) measured during the last 400 ms of the pulse was taken as F m or F m '. For each experiment, 2 mL of cell suspension were used. Sodium bicarbonate was added at a concentration of 4 mM from a freshly prepared 0.2 M stock solution in distilled water to prevent any limitation of the photosynthetic rate by carbon supply. Standard fluorescence nomenclature was used [START_REF] Van Kooten | The use of chlorophyll fluorescence nomenclature in plant stress physiology[END_REF]. F o and F m are defined as the minimum PS II fluorescence yield of dark-acclimated cells and the maximum PS II fluorescence yield reached in such cells during a saturating pulse of white light, respectively. The maximum photosynthetic efficiency of PS II is the ratio F v /F m where F v is the variable part of the fluorescence emission and is equal to F m -F o . NPQ (also known as SV m ) was quantified by the 'Stern-Volmer' expression F m /F m '-1, where F m ' is the maximum PS II fluorescence yield of light-acclimated cells [START_REF] Krause | Chlorophyll fluorescence and photosynthesis: the basics[END_REF].

Results

The general photosynthetic properties of the strains of Phaeodactylum tricornutum (P.t.2 strain) and Skeletonema costatum used here have been described earlier [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]. In our growth conditions, both species have comparable light harvesting efficiency and photosynthetic capacity. As previously shown, the extent of NPQ in P. tricornutum was about twice the one in S. costatum; it holds true under saturating and over-saturating irradiances [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 under prolonged exposure to over-saturating intensities [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF].

Differences in the relationship between the XC and NPQ between Phaeodactylum tricornutum and Skeletonema costatum cells

The de-epoxidation of DD into DT was measured for the two species under the same light conditions as for NPQ (Fig. 1 and Fig. S1). While in P. tricornutum (P.t.2 strain) the deepoxidation started for a light intensity of 150 µmol photons m -2 s -1 , it was already active at 50 µmol photons m -2 s -1 in S. costatum. For the two species, the de-epoxidation state (DES) reached a near maximum around 750 µmol photons m -2 s -1 . As the size of the total pool of DD+DT remained stable along the range of irradiances, no de novo DT synthesis (i.e. DT synthesis independent from the xanthophyll cycle, see [START_REF] Olaizola | Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom[END_REF][START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]) occurred. In these conditions, the maximal synthesis of DT was lower in P. tricornutum (2.5 ± 0.01 mol. 100 mol Chl a -1 ) than in S. costatum (6.8 ± 0.54 mol. 100 mol Chl a -1 ) corresponding to a maximal DES of 27% and 40%, respectively (Fig. 1A, Table 1). For comparison, P. tricornutum 'high NPQ' cells (see Table 1 for their characteristics) showed a maximal DES of 46% (Fig. 1A, open circles). Under these light conditions, there was a tight correlation between the amount of DT and NPQ as previously reported for several diatom species [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF][START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF] (Fig. 1B). However, in contrast to P. tricornutum, for S. costatum the relationship depended on the illumination pattern. When it was fixed to 5 min and the irradiance increased from 0 to 2000 µmol photons m -2 s -1 (Fig. S1), the NPQ vs. DT relationship showed an exponential feature (Fig. 1B). NPQ barely developed (0.2) until the DT amount reached a value close to 2 mol DT. 100 mol Chl a -1 . Interestingly, the inflexion point of NPQ development corresponded to an amount of DT synthesized for a light intensity of about 400 µmol photons m -2 s -1 (Fig. S1), which was just below the light intensity for reaching the maximum photosynthetic capacity (Fig. S2) [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]. When the irradiance was fixed to a maximum of 2000 µmol photons m -2 s -1 and the illumination time increased from 0 to 30 min, the NPQ vs. DT relationship was linear with a slope twice lower than the one observed in P. tricornutum (as previously reported in [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]).

By illuminating the cells with harsher conditions (2000 µmol m -2 s -1 for over 30 min and up to 1 h), the NPQ vs. DT relationship remained true only for P. tricornutum (continuous line). Indeed, for illumination times longer than 30-45 min, the slope of the relationship NPQ/DT decreased to 0.09 in S. costatum (Fig. 1B, closed triangles, dashed line). During such high and prolonged light exposure de novo synthesis of DT occurs with a similar rate in P. tricornutum and S. costatum [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]. It can be therefore concluded that in S. costatum part of the newly synthesized molecules of DT were less or not involved in the development of subsequent NPQ. This feature was confirmed by the recording of the epoxidation kinetics of DT back to DD after short (5-15 min) and long (1 h) exposures to 2000 µmol m -2 s -1 (Fig. 2). While after 5 and 15 min exposure the epoxidation kinetics were similar in both species and well in concordance with the relaxation of NPQ, it was drastically different after 1 h exposure (Fig. S3). In S. costatum, the epoxidation kinetics and NPQ relaxation showed disconnected patterns (Fig. 2B, Fig. S3). During the first 30 min of recovery, there was a fast DT epoxidation similar as in P. tricornutum (Fig. 2A). The amount of DT involved (2.4 ± 1.2 mol. 100 mol Chl a -1 ) was similar to the amount of DT synthesized de novo (2.4 ± 1.6 mol. 100 mol Chl a -1 ). In contrast to P. tricornutum, it was accompanied by only a small change in NPQ (13%).

After 1 h of recovery, nearly all DT molecules were converted back to DD in P. tricornutum while a large DT pool (3.2 ± 0.9 mol. 100 mol Chl a -1 ) persisted in S. costatum. NPQ remained high too (2/3s of the value after the light extinction). Both sustained DT and NPQ could not be recovered by the addition of the uncoupler NH 4 Cl after 15 or 25 min of recovery (data not shown) [START_REF] Lavaud | In diatoms, a transthylakoidal proton gradient alone is not sufficient for non-photochemical fluorescence quenching[END_REF], hence the ΔpH was most probably already broken. Additionally, when the light (same intensity) was again switched on after 30 min of recovery, the development of NPQ was faster but not higher (x 2, data not shown) as previously observed for P. tricornutum [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]. These features fit well with the proposed regulatory features of NPQ in diatoms (see [START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[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]). Lastly, the dark recovery of the F v /F m was higher in P. tricornutum whatever the illumination time (15 or 60 min) (Fig. S4).

Light-dependent absorption changes related to the XC and NPQ

It has been shown earlier that the absorption increase at 522 nm (522 nm) accounts for the so-called 'activation' of the DT molecules, an essential step in the switching of the PS II LHC antenna to the quenching state (see [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][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]). Fig. 3A shows the 522 nm in S. costatum and in P. tricornutum cells with different extents of NPQ (see Table 1 for their characteristics) which were obtained by growing the cells under different conditions: 1) a classical low light regime (16h L:8h D) with a cell harvest during the exponential phase of growth ('CL exp.'), 2) the same light regime with a cell harvest during the stationary phase of growth ('CL stat.'), 3) an intermittent light of the same intensity (5 min light: 55 min dark) ('IL exp.'). In order to complete the range of NPQ, we included a second strain of P. tricornutum (P.t.4), which is known for its lower NPQ than the strain P.t.2 [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF] and C. meneghiniana whose NPQ mechanism is more complex than the one of P. tricornutum [START_REF] Grouneva | The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana[END_REF][START_REF] Grouneva | A new multi-component NPQ mechanism in the diatom Cyclotella meneghiniana[END_REF]. In these conditions, the four strains reacted rather similarly. The maximum photosynthetic efficiency of PS II (F v /F m , Table 1) of 'CL exp.' cells was very similar (Table 1 [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[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] Materna | First induced plastid genome mutations in an alga with secondary plastids: psbA mutations in the diatom Phaeodactylum tricornutum (Bacillariophyceae) reveal consequences on the regulation of photosynthesis[END_REF]).

Although the growth rate was not measured for all species, it decreases 10-fold in P. tricornutum P.t.2 'IL exp.' cells due to the 8-fold lower daily amount of light supplied by the IL light regime [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF]. Under a fluctuating light regime not as harsh as the IL regime used here, the growth rate of P. tricornutum P.t.4, S. costatum and C. meneghiniana was decreased by a factor of 2 to 4.5 (data compiled from [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[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] Su | The impact of nonphotochemical quenching of fluorescence on the photon balance in diatoms under dynamic light conditions[END_REF]).

In contrast, the effect of the light regimes on the pool size of DD+DT, the deepoxidation of DD to DT (DES and DT amount) and on NPQ in the four species/strains was different. In general, 'CL stat.' cells showed a higher NPQ than 'CL exp.' cells due to the larger DD pool and the higher subsequent DT synthesis [START_REF] Arsalane | Influence of the pool size of the xanthophyll cycle on the effects of light stress in a diatom: Competition between photoprotection and photoinhibition[END_REF]. While P. tricornutum P.t.2 'IL exp.' cells exhibited a higher NPQ [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF][START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF], the IL treatment had an adverse effect on S. costatum xanthophyll synthesis and NPQ (Table 1). Consequently, the 522 nm band was lower in S. costatum compared to P. tricornutum (P.t.2 strain) whatever the types of cells considered (see Table 1). NPQ in P.t.4 reacted the same way as in P.t.2 to growth conditions while for C. meneghiniana it was closer to S. costatum (Table 1). The extent of NPQ exponentially correlated with the amplitude of the 522 nm band (Fig. 3B). In contrast, and as expected, NPQ linearly correlated with the total amount of synthesized DT with a decreasing slope in the order P. tricornutum > S. costatum > C. meneghiniana (Fig. S5). The NPQ/522 nm relationship also held true for cells of P. tricornutum (P.t.2, 'low NPQ') in which NPQ was more than half abolished (-60%) by the use of moderate concentrations of an uncoupler (NH 4 Cl) of the ΔpH and of an inhibitor of the DD de-epoxidation (DTT, dithiotreitol) [START_REF] Lavaud | In diatoms, a transthylakoidal proton gradient alone is not sufficient for non-photochemical fluorescence quenching[END_REF]. Although S. costatum and C. meneghiniana showed a larger xanthophyll pool size and a higher DT synthesis than P. tricornutum (see also Fig. 1A), NPQ was lower (see also Fig. 1B) in direct correlation with the lower capacity for DT 'activation' in the NPQ process (i.e. lower 522 nm) (Table 1). This situation was especially extreme with 'high NPQ' cells: the huge amount of synthesized DT (about 16 and 37 mol. 100 DT Chl a -1 for S. costatum and C. meneghiniana, respectively) did not lead to the expected large increase of NPQ which instead followed the slight increase of the 522 nm band.

NPQ capacity and chlorophyll fluorescence emission properties

In order to gain more insight into the fluorescence emission properties of the species examined, we performed 77K fluorescence emission spectra on dark-acclimated cells (Fig. 4). The majority of the Chl a fluorescence arose from the 687 nm peak while a second emission band peaking at 711 nm in P. tricornutum was showing a slight shoulder in S. costatum and C. meneghiniana (not shown). In P. tricornutum (P.t.2 and P.t.4 strains) the amplitude of the 711 nm band increased with the NPQ capacity of the cells, following a linear relationship whose slope was dependent on the strain (Fig. 5, Table 1).

Probably the most illustrative situation concerns the P.t.2 'medium and high NPQ' cells: the synthesis of 1.7 fold more DT molecules led to a much higher NPQ (2.7 fold) than expected regarding the moderate increase of DT activation (522 nm); instead the 2.7 fold NPQ increase correlated well with the one of the fluorescence emission at 711 nm.

Although slight variations in the 711/687 nm fluorescence ratio (F711/F687) were observed in S. costatum (Fig. 4B, Table 1), no clear relationship could be established (Fig. 5). No changes were observed in C. meneghiniana.

It has been described earlier in P. tricornutum how the 711 nm fluorescence band can arise from the PS II at both room temperature and 77K [START_REF] Fujita | On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature[END_REF], and how it could be related to the capacity for excess energy dissipation [START_REF] Miloslavina | Ultrafast fluorescence study on the location and mechanism of nonphotochemical quenching in diatoms[END_REF][START_REF] Fujita | On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature[END_REF]. When measured after illumination (5 min, 2000 µmol m -2 s -1 ) in P. tricornutum (P.t.2 strain), the 77K fluorescence signal was decreased by a factor of about 3 and F711/F687 increased by 31% (Table 2) (Fig. S6). When NPQ was inhibited by the addition of NH 4 Cl or DTT, the change of F711/F687 was abolished (Table 2, Fig. S6). By illuminating the cells with farred light (200 µmol photons m -2 s -1 ), we did not observe any change of F711/F687 (data not shown).

Discussion

The inter-species differences of the involvement of DT in NPQ

The DT molecules are distributed among several pools which show a spatial and functional heterogeneity [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 is believed that DT can be : 1) free in the lipid matrix [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], 2) in the lipid matrix in the close vicinity to the PS II LHC antenna [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], 3) bound to the LHC antenna system for the harvesting of light [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] Gildenhoff | Oligomerization and pigmentation dependent excitation energy transfer in fucoxanthinchlorophyll proteins[END_REF]; 4) bound to specific FCP trimers where they participate to NPQ [START_REF] Gundermann | Factors determining the fluorescence yield of fucoxanthin-chlorophyll complexes (FCP) involved in non-photochemical quenching in diatoms[END_REF]. A change of balance between these pools can occur during light acclimation [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][START_REF] Grouneva | A new multi-component NPQ mechanism in the diatom Cyclotella meneghiniana[END_REF]. We show that it is also true between species.

Obviously the addressing of DT to the four pools is different in S. costatum (and C. meneghiniana) compared to P. tricornutum. First, the fact that the slope of the NPQ vs.

DT relationship is twice lower in S. costatum indicates that twice less DT molecules are involved in NPQ, i.e. there are 50% less DT in pool 4. Second, while DT de novo synthesis significantly occurs in both species, it is mainly inefficient for NPQ in S. costatum (and C. meneghiniana, [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]) in contrast to P. tricornutum [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]. Hence, in the latter the de novo synthesized DT appears to be mainly part of the pool 4. Interestingly, de novo synthesized DT was recently proposed to be bound by Lhcx proteins and to participate to the sustained part of NPQ (i.e. qI) [START_REF] Wu | Photosystem II photoinactivation, repair, and protection in marine centric diatoms[END_REF][START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF], thus enhancing the dissipative capacity of the PS II LHC antenna under prolonged and harsh illumination conditions. In contrast, it is probable that DT is addressed to the pools 1 and/or 2 in S. costatum as also shown for C. meneghiniana [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]. This is supported by their fast dark epoxidation illustrating their easy access to the DT epoxidase in the lipid matrix of the thylakoid membrane [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] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. Here, they likely participate to the prevention of lipid peroxidation [START_REF] Olaizola | Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom[END_REF][START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[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]. Hence, in S. costatum and P. tricornutum, DT would have different main physiological roles with a higher involvement in NPQ in P. tricornutum. Consequently, from a species to another, NPQ often does not correlate with the amount of DT and with the DES [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF][START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. This discrepancy between the two species was especially true when the light exposure was short and of moderate intensity. In these conditions, S. costatum synthesizes significant amounts of DT with only low or even no development of NPQ. A similar relationship was documented before in cells where the ΔpH build-up was chemically manipulated, the lowest ΔpH generating a significant amount of DT with barely any NPQ [START_REF] Lavaud | In diatoms, a transthylakoidal proton gradient alone is not sufficient for non-photochemical fluorescence quenching[END_REF]. Such discrepancy was explained before thanks to P. tricornutum mutants showing a DT synthesis divided by half by genetic means [START_REF] Lavaud | Silencing of the violaxanthin de-epoxidase gene in the diatom Phaeodactylum tricornutum reduces diatoxanthin synthesis and non-photochemical quenching[END_REF]. It implies the socalled 'activation' of DT which necessitates the protonation of some FCP binding sites during the ΔpH build-up [START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][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] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. The entering of DT into an 'activated' state triggers the switch of the PS II LHC antenna to the quenching state. We propose that the discrepancy between DT synthesis and NPQ in S. costatum is due to a weak/slow 'activation' of DT in conditions of low ΔpH due to a limited protonation of the PS II LHC antenna. It is supported by the fact that the NPQ vs. DT vs. light intensity relationship followed two clear phases in S. costatum with a breakpoint corresponding to the intensity for saturating photosynthesis; this is in total contrast with P. tricornutum.

The efficiency of DT in the NPQ process: the Δ522 nm absorption change

The above mentioned 'activation' of DT (section 4.1.) can be followed by an absorption change at 522 nm (Δ522 nm) similar to the qE-related Δ525/535 nm in higher plants [START_REF] Ruban | The photoprotective molecular switch in the photosystem II antenna[END_REF][START_REF] Ruban | The super-excess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF]. It is supposed to illustrate the effective involvement of DT as an allosteric regulator of a conformational change in the PS II LHC antenna, a crucial step in the NPQ process [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF][START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. The conformational change is believed to be an aggregation of part of the PS II LHC antenna generated by the protonation of FCP binding sites [START_REF] Goss | Regulation and function of xanthophyll cycle-dependent photoprotection in algae[END_REF]. The inhibition of Δ522 nm by NH 4 Cl and DTT confirms this view. The effect of DTT most likely excludes a relationship to an electrochromic shift signal [START_REF] Bailleul | Electrochromism: a useful probe to study algal photosynthesis[END_REF]. Δ522 nm thus provides a very useful tool to measure the efficiency of DT in the NPQ process. Indeed, the difference between the relationships NPQ vs. DT (Fig. S5) and NPQ vs. Δ522 nm (Fig. 3B) demonstrates that the 522 nm fingerprint is different from the total DT amount in the way it correlates with NPQ for all the species, i.e. the total amount of DT was far from accounting for the DT which was effectively involved in NPQ ('activated' DT) especially in S. costatum and C. meneghiniana. This discrepancy explains the above mentioned lack of correlation between the DT amount and the DES, and NPQ (section 4.1). We could additionally broaden the validity of the Δ522 nm fingerprint to the centric diatom representatives S. costatum and C. meneghiniana. The correlation of Δ522 nm with NPQ validates the crucial importance of the amount of 'activated' DT to determine the extent of NPQ in that order: P. tricornutum > C. meneghiniana  S. costatum. We could partially manipulate this general trend by modifying the cellular amount of DD+DT thanks to different growth conditions.

Apart from the amount of DT and its binding to the PS II LHC antenna (section 4.1.), two reasons could explain the Δ522 nm inter-species differences: 1) a fewer number of proton binding sites in the FCP proteins (Lhcf and Lhcx) involved in NPQ, 2) a lower protonation of these sites, their number being stable, as illustrated by the DT-independent modification of NPQ by the extent of the ΔpH/lumen acidification [START_REF] Grouneva | A new multi-component NPQ mechanism in the diatom Cyclotella meneghiniana[END_REF][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]. Nevertheless and although we did not measure the ΔpH, it is reasonable to propose that at least in P. tricornutum and S. costatum the extent of FCP protonation was similar since they showed a similar electron transport rate (see [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]). The first reason, although more plausible, is at this stage impossible to verify as we do not know which of the LHC proteins might bind protons in diatoms although both Lhcf and Lhcx proteins have been proposed [START_REF] Gundermann | Factors determining the fluorescence yield of fucoxanthin-chlorophyll complexes (FCP) involved in non-photochemical quenching in diatoms[END_REF]. The exponential pattern of the NPQ vs. Δ522 nm relationship illustrates an essential feature of the diatom NPQ regulation: above a certain value (NPQ of about 1.5), NPQ increases much faster than the Δ522 nm fingerprint, i.e. the increase of DT 'activation' cannot fully account for the huge increase of NPQ, especially in 'high NPQ' cells. Hence, there is another partner amplifying the dissipative process in the LHC antenna system of PS II.

Amplification of the NPQ process: the chlorophyll 711 nm fluorescence emission band (Chl a 711 )

The presence of a Chl a 711 emission band at 77K (or in the range 710-717 nm depending on the studies and the species) has been reported before in diatoms. It is species-dependent [START_REF] Fujita | On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature[END_REF][START_REF] Goedheer | Chlorophyll a forms in Phaeodactylum tricornutum: Comparison with other diatoms and brown algae[END_REF][START_REF] Goedheer | Comparison of the long-wave chlorophyll fluorescence in various green and blue-green algae and diatoms[END_REF][START_REF] Shimura | Some properties of the chlorophyll fluorescence of the diatom Phaeodactylum tricornutum[END_REF]. Similarly, we showed that while Chl a 711 can be high in P. tricornutum, its amplitude is strongly limited in S. costatum and C. meneghiniana whatever the growth conditions. The present work additionally depicts the following characteristics for Chl a 711 : 1) it is positively correlated to the inter-species (P. tricornutum > S. costatum  C. meneghiniana) and intra-species (P.t.2 strain > P.t.4 strain) differences of the NPQ capacity; 2) in P. tricornutum and to a much lower extent in S. costatum (but not in C. meneghiniana), its amplitude is increased together with the pool size of DD/DT by changing the growth conditions (see also [START_REF] Fujita | On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature[END_REF][START_REF] Goedheer | Chlorophyll a forms in Phaeodactylum tricornutum: Comparison with other diatoms and brown algae[END_REF][START_REF] Goedheer | Comparison of the long-wave chlorophyll fluorescence in various green and blue-green algae and diatoms[END_REF]); 3) it is quenched under high light exposure but not as much as PS II Chl a 687 , and this change is abolished by NH 4 Cl and DTT; 4) it is insensitive to red light ruling out any involvement of a state-transitions (i.e. qT) in the quenching process. Some studies, using 77K spectroscopy, classically linked Chl a 711 to the core of PS I and its specific FCP antenna (see [START_REF] Ikeda | Photosystem I associated with fucoxanthin-cholorophyll-binding proteins from a marine centric diatom, Chaetoceros fragile[END_REF][START_REF] Juhas | Properties of photosystem I antenna protein complexes of the diatom Cyclotella meneghiniana[END_REF]) while others, using both room temperature and 77K spectroscopy, demonstrated, it can also originate from PS II by using DCMU or a preferential excitation of fucoxanthin pigments [START_REF] Miloslavina | Ultrafast fluorescence study on the location and mechanism of nonphotochemical quenching in diatoms[END_REF][START_REF] Fujita | On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature[END_REF][START_REF] Chukhutsina | Variations in the first steps of photosynthesis for the diatom Cyclotella meneghiniana grown under different light conditions[END_REF]. In that framework, and although we did not measure it for all strains/species under all growth conditions, it is noteworthy that the stoichiometry PS II:PS I was similar in P. tricornutum P.t.2 'low' and 'high' NPQ cells [START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF]. The PS II:PS I difference was higher (+23%) between P. tricornutum P.t.2 and S. costatum [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] with more PS I vs. PS II in P. tricornutum. In diatoms, a large change of PS II:PS I stoichiometry is usually generated by an increase in the light intensity and/or by nutrient deficiency [START_REF] Juhas | Properties of photosystem I antenna protein complexes of the diatom Cyclotella meneghiniana[END_REF][START_REF] Allen | Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation[END_REF]. Hence, the Chl a 711 differences observed here between at least these three types of cells cannot be fully explained by a difference in PS II:PS I stoichiometry. Although it amounts to few percents of total chlorophyll, Chl a 711 was shown to drain an appreciable fraction of excitation energy from the PS II [START_REF] Goedheer | Chlorophyll a forms in Phaeodactylum tricornutum: Comparison with other diatoms and brown algae[END_REF][START_REF] Goedheer | Comparison of the long-wave chlorophyll fluorescence in various green and blue-green algae and diatoms[END_REF][START_REF] Shimura | Some properties of the chlorophyll fluorescence of the diatom Phaeodactylum tricornutum[END_REF] and was proposed to be responsible for dissipating excess energy [START_REF] Fujita | On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature[END_REF]. The Chl a 711 emission band was earlier suggested to arise from aggregated proteinpigment complexes close but spatially separated from the PS II [START_REF] Goedheer | Chlorophyll a forms in Phaeodactylum tricornutum: Comparison with other diatoms and brown algae[END_REF][START_REF] Goedheer | Comparison of the long-wave chlorophyll fluorescence in various green and blue-green algae and diatoms[END_REF][START_REF] Owens | Photosystem II heterogeneity in the marine diatom Phaeodactylum tricornutum[END_REF]. Owens [START_REF] Owens | Photosystem II heterogeneity in the marine diatom Phaeodactylum tricornutum[END_REF] further demonstrated that Chl a 711 is sensitive to the energization of the thylakoid membrane which explains its NH 4 Cl sensitivity. Recently, a time-resolved fluorescence analysis of the diatom LHC system brought evidence for a 710 nm fluorescence emission band which resembles our Chl a 711 [START_REF] Miloslavina | Ultrafast fluorescence study on the location and mechanism of nonphotochemical quenching in diatoms[END_REF]. It is present in P. tricornutum but much less in C. meneghiniana and it exists in both unquenched (darkness) and quenched (high light) states. The 710 nm band was assigned to a FCP complex functionally disconnected from both PS I and PS II upon high light exposure [START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF][START_REF] Miloslavina | Ultrafast fluorescence study on the location and mechanism of nonphotochemical quenching in diatoms[END_REF]. It was additionally proposed that this part of the LHC antenna is in an aggregated state similar to the oligomerized LHCII involved in NPQ in higher plants [START_REF] Miloslavina | Far-red fluorescence: a direct spectroscopic marker for the LHCII oligomer formation in non-photochemical quenching[END_REF]. Interestingly, the Chl a 710 associated to PS I was shown to arise from FCP complexes weakly bound to the PS I core where they act as a 'shallow sink' of excitation energy [START_REF] Yamagishi | Shallow sink in an antenna pigment system of photosystem I of a marine centric diatom, Chaetoceros gracilis, revealed by ultrafast fluocrecence spectroscopy at 17 K[END_REF]. These observations indicate that Chl a 711 (or Chl a 710 ) probably plays a role in NPQ. Because the quenching was more important on Chl a 687 than on Chl a 711 , we propose that Chl a 711 serves at amplifying the part of the quenching which directly relies on DT (i.e. related to Chl a 687 ). The amplitude of Chl a 711 (or F711/F687 nm) measured in dark-acclimated cells is a good fingerprint of the level of NPQ amplification at light. It explains in part the inter-species differences in NPQ; i.e. in contrast to P. tricornutum, Chl a 711 had only a minor role in the extent of NPQ in S. costatum and C. meneghiniana; it was also reported before that Chl a 710-711 is lower in C. meneghiniana [START_REF] Miloslavina | Ultrafast fluorescence study on the location and mechanism of nonphotochemical quenching in diatoms[END_REF].

The consequence of NPQ inter-species/strains differences on the ecophysiology of diatoms

All and all, the differences in the NPQ extent observed between species and strains of the same species (P. tricornutum) can be explained by the extent of Δ522 nm and of Chl a 711 together with the distribution of DT within the photosynthetic machinery, but independently of the total amount of DD+DT and of the DES. We propose that a high NPQ ability (as observed in the P. tricornutum P.t.2 strain) would rely on 1) a high amount of DT which effectively participate to NPQ (Δ522 nm fingerprint); 2) the capacity to form many/large functionally disconnected oligomeric FCP complexes (Chl a 711 fingerprint) in order to amplify the DT-dependent quenching process. These features are in dramatic contrast with the ones measured for S. costatum. The most relevant specific features of NPQ regulation in S. costatum are ( [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[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] and this study) the coupling between i) the early onset of NPQ, i.e. already for a light intensity corresponding to the one the cells are acclimated to, ii) the slow development of NPQ vs.

DT vs. light for non-saturating irradiances, iii) the apparent slow recovery of NPQ related to a limited epoxidation of DT (also reported for C. meneghiniana and P. tricornutum [START_REF] Goss | The importance of a highly active and delta-pH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]), iv) the low investment of de novo synthesized DT in the enhancement of NPQ. These features, together with the absence of a PS II CET [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] (which can explain in part the points i) and ii)) cannot promote a similarly strong and fast irradiance-tuning of NPQ as in P. tricornutum. Ultimately, as shown here and before [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], these differences lead to a higher susceptibility of S. costatum to photoinhibition during exposure to excess light and/or to a fluctuating light climate negatively inferring on its photosynthetic productivity and growth. Such record brings a molecular basis for explaining the adaptation of the regulatory photosynthetic machinery to the aquatic light environment as proposed before [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF][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 | 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 | Photoprotective capacity as functional trait in planktonic algae: relationship between xanthophyll cycle and ecological characteristics in three diatoms[END_REF]. Indeed, the general characteristics for high NPQ in the P. tricornutum P.t.2 strain, which originates from an estuarine habitat, fit well with an adaptation to fast and strong light fluctuations while the ones for low NPQ fit better with the slow and moderate light changes that characterize the habitat the strain of S. costatum used here originates from (a Mediterranean semi-enclosed bay). The recent report that S. costatum and C. meneghiniana behave similar under fluctuating light conditions [START_REF] Su | The impact of nonphotochemical quenching of fluorescence on the photon balance in diatoms under dynamic light conditions[END_REF] further supports the same impact of their seemingly close regulatory NPQ machinery on their photophysiology. Also supporting and extending conclusions to strains/ecotypes of the same species, is the fact that the low NPQ P. tricornutum P.t.4 strain shows an NPQ vs. DT relationship which slope is similar to the one of S. costatum [START_REF] Lavaud | Silencing of the violaxanthin de-epoxidase gene in the diatom Phaeodactylum tricornutum reduces diatoxanthin synthesis and non-photochemical quenching[END_REF]. Different from the P.t.2 strain, P.t.4 originates from a high latitude habitat characterized by a low light environment [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF]. As observed here and before [START_REF] Bailleul | An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light[END_REF] in P. tricornutum strains/ecotypes, the amounts of DD/DT, of Chl a 711 FCP oligomers and of Lhcx1 are concomitantly influenced by the light environment the cells are adapted to. The slope of the NPQ vs. DT relationship [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF][START_REF] Dimier | Photoprotective capacity as functional trait in planktonic algae: relationship between xanthophyll cycle and ecological characteristics in three diatoms[END_REF] therefore could be a good indicator of the adaptation of a species/strain to an aquatic niche as a function of the light climate. The present work further confirms and extends the potential role for the light-dependent fast regulation of photosynthesis, and especially NPQ, in the ecophysiology of diatom phytoplanktonic and microphytobenthic communities [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: Mechanisms, evolution and ecophysiology[END_REF][START_REF] Perkins | The application of variable chlorophyll fluorescence to microphytobenthic biofilms[END_REF][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] Cartaxana | Physiological versus behavioral photoprotection in intertidal epipelic and epipsammic benthic diatom communities[END_REF]. It could explain in part the ecological diversity of diatoms [START_REF] Kooistra | The origin and the evolution of the diatoms: Their adaptation to a planktonic existence[END_REF] which has been participated to their ecological success in contemporary oceans [START_REF] Kooistra | The origin and the evolution of the diatoms: Their adaptation to a planktonic existence[END_REF][START_REF] Armbrust | The life of diatoms in the world's oceans[END_REF][START_REF] Falkowski | The evolution of modern phytoplankton[END_REF]. cells is shown by a continuous line (circles, see also [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF][START_REF] Lavaud | Influence of the diadinoxanthin pool size on photoprotection in the marine planktonic diatom Phaeodactylum tricornutum[END_REF]). tricornutum (P.t.2 strain) and S. costatum. For each of the species, the three NPQ types of cells ('low', 'medium' and 'high', see Table 1) were used. The absorption band at 522 nm (Δ522 nm) is highlighted. B-Relationship between NPQ and Δ522 nm. NPQ was measured after 5 min illumination at 2000 µmol photons m -2 s -1 . For each of the species, P. tricornutum (P.t.2 strain, circles), S. costatum (triangles) and Cyclotella meneghiniana (squares), the three NPQ types of cells ('low', 'medium' and 'high', see Table 1) were used. The exponential relationship was NPQ = 0.35 x e (1.24 Δ522 nm) (R 2 = 0.93). For P. tricornutum, the same measurements were performed on the 'low NPQ' cells in presence of NH 4 Cl (2 mM) and dithiothreitol (DTT, 25µM) during the illumination (open circles).

P.t.4,
See the text for further details. Data (±SD) are the average of three independent measurements. 1). The spectra were normalized at their 687 nm maximum. and C. meneghiniana (squares), the three NPQ types of cells ('low', 'medium' and 'high', see Table 1) were used. The linear relationship was NPQ = 8.10 x F711/687 nm (R 2 = 0.99) and NPQ = 4.62 x F711/687 nm (R 2 = 0.98) for the P.t.2 and P.t.4 strains, respectively. Data (±SD) are the average of three independent measurements.

Figure 1 .

 1 Figure 1. A-The de-epoxidation state (calculated as DT/(DD+DT) x 100) in S. costatum,

Figure 2 .

 2 Figure 2. Dark relaxation of NPQ (closed symbols) and DT epoxidation back into DD

Figure 3 .

 3 Figure 3. A-Light-treated minus dark-acclimated difference absorption spectra in P.

Figure 4 .

 4 Figure 4. 77K Chl a fluorescence emission spectra excited at 440 nm of P. tricornutum

Figure 5 .

 5 Figure 5. Relationship between NPQ and the ratio of Chl a fluorescence emission at 711

  8 h Light:Dark cycle; 'IL', 'intermittent light': 5 min/55 min L:D cycle (see the Material and methods section for details); exp. growth, exponential phase of growth; stat. growth, stationary phase of growth; the fluorescence ratios F v /F m (the maximum photosynthetic efficiency of PS II) and F711/F687 were measured on dark-acclimated cells (see also Fig.4); Δ 522 nm X 10 2 (the absorption change at 522 nm), NPQ and DES (de-epoxidation state of diadinoxanthin (DD) into diatoxanthin (DT) in %: (DT/(DD+DT)x100), DD and DT in mol. 100 mol Chl a -1 ) were measured after 5 min at 2000 µmol s -1 m -2 (see also Fig.1and Fig.3).

*

  There was no significant difference between the dark samples whatever the conditions (student test, p>0.05). Treatments: dark-acclimation was for 20 min, light exposure was 2000 µmol photons m -2 s -1 during 5 min, DTT (0.2 mM) and NH 4 Cl (5 mM) were applied 15 min in the dark before subsequent measurement either in the dark or at light. Values are average of three measurements ± SD. Full spectra are shown as Supplemental Material (Fig.S6).

Figure 1 -

 1 Figure 1-Lavaud and Lepetit

Figure 2 -

 2 Figure 2-Lavaud and Lepetit

Figure 3 -PFigure 5

 35 Figure 3-Lavaud and Lepetit

  ). F v /F m slightly decreased in 'CL stat.' cells while it was maintained in 'IL exp.' cells. The growth rate of 'CL exp.' cells was similar (P. tricornutum P.t.2, 2.08 ± 0.08 d -1 ; P.t.4, 2.54 ± 0.2 d -1 ; S. costatum, 2.01 ± 0.09 d -1 ; C. meneghiniana, 2.11 ± 0.10 d -1 ) while it dramatically decreased to about 0 in 'CL stat'

	conditions (P. tricornutum P.t.2, 0.05 ± 0.02 d -1 ; P.t.4, 0.07 ± 0.03 d -1 ; S. costatum, 0.02 ±
	0.01 d -1 ; C. meneghiniana, 0.03 ± 0.02 d -1 ) (part of the data compiled from

Table 1 741 Photophysiological properties of the species/strains and of the NPQ type of cells 742 used in this study 743

 1

	Species	Growth	F v /F m DD+DT F711/F687 DES	DT	Δ 522 nm	NPQ
	and	status						X 10 2
	type of cells
	P. tricornutum (P.t.2)	'CL',	0.65 ±	8.8 ± 0.4	0.35 ±	27	2.38 ±	1.3 ± 0.10 2.38 ±
	'low NPQ'	exp.	0.02		0.06		0.1		0.24
	P. tricornutum (P.t.2)	'CL',	0.61 ±	13.6 ±	0.50 ±	36	4.9 ±	2.1 ± 0.10 3.50 ±
	'medium NPQ'	stat.	0.03	0.9	0.07		0.3		0.25
	P.t.2,	'IL',	0.68 ±	17.4 ±	1.22 ±	46	8.0 ±	2.5 ± 0.20 9.40 ±
	'high NPQ'	exp.	0.02	2.3	0.26		1.1		0.61
	P. tricornutum (P.t.4)	'CL',	0.67 ±	12.7 ±	0.48 ±	38	4.8 ±	n.d.	1.58 ±
	'low NPQ'	exp.	0.02	0.2	0.01		0.1		0.11
	P.t.4,	'CL',	0.60 ±	23.8 ±	0.62 ±	43	10.2 ±	n.d.	2.63 ±
	'medium NPQ'	stat.	0.03	0.2	0.09		0.1		0.19
	P.t.4,	'IL',	0.64 ±	30.9 ±	1.00 ±	61	18.9 ±	n.d.	4.08 ±
	'high NPQ'	exp.	0.02	0.7	0.19		0.4		0.21
	S. costatum (S.c.)	'IL',	0.64 ±	8.3 ± 2.1	0.19 ±	33	2.7 ±	0.33 ±	0.51 ±
	'low NPQ'	exp.	0.01		0.02		0.7	0.15	0.07
	S.c.	'CL',	0.59 ±	18.1 ±	0.29 ±	40	7.2 ±	1.00 ±	1.35 ±
	'medium NPQ'	exp.	0.07	0.1	0.01		0.05	0.15	0.29
	S.c.	'CL', 0.69 ±	26.9 ±	0.32 ±	59	15.9 ±	1.73 ±	2.5 ±

'CL', 'continuous light': 16 h:

Table 2 Effect of light and inhibitor treatments on the pattern of 77K chlorophyll fluorescence emission as measured by the ratio F711/F687 nm in Phaeodactylum tricornutum (P.t.2 strain) 'medium NPQ' cells (see Table 1)

 2

		Dark-acclimated cells	Illuminated cells
	No inhibitors	0.78 ± 0.05 *	1.02 ± 0.09
	+DTT	0.81 ± 0.06 *	0.77 ± 0.07

Acknowledgements

The authors thank Prof. P. Kroth (University of Konstanz, Germany) and Prof. C. Wilhelm (University of Leipzig, Germany) for the access to some of the instruments used here, and B. Rousseau and M. Desnos for their technical assistance. This work was supported by the DFG-German Research Council (grant LA2368/2-1 to JL), the DAAD-German Academic Exchange Agency (grant to BL), the CNRS-Centre National de la Recherche Scientifique (for JL) and the French consortium 'CPER Littoral' (for JL and BL).

Appendice A. Supplementary data

Supplementary data can be found under the numbering Figure S1 to Figure S6.