Dr Bernard Lepetit
email: bernard.lepetit@univ-lr.fr

' Lienss'

Sabine Sturm

Alessandra Rogato

Ansgar Gruber

Matthias Sachse

Angela Falciatore

Peter G Kroth

Johann Lavaud

Plastoquinone pool redox regulation in diatoms

Abstract

In diatoms the process of energy-dependent chlorophyll fluorescence quenching (qE) has an important role in photoprotection. Three components are essential for qE: 1) the lightdependent generation of a transthylakoidal proton gradient, 2) the de-epoxidation of the xanthophyll diadinoxanthin (Dd) into diatoxanthin (Dt), and 3) specific nuclear-encoded antenna proteins, called LHCX. We used the model diatom Phaeodactylum tricornutum to investigate the concerted light acclimation response of the qE key components LHCX, proton gradient and xanthophyll cycle pigments (Dd+Dt), and to identify the intracellular light responsive trigger. At high light exposure, the up-regulation of three of the LHCX genes and the de novo synthesis of Dd+Dt led to a pronounced rise of qE. By inhibiting either the conversion of Dd to Dt or the translation of LHCX genes, the qE amplification was abolished and the diatom cells suffered from stronger photoinhibition. Artificial modification of the redox state of the plastoquinone (PQ) pool via DCMU and DBMIB resulted in a disturbance of the Dd+Dt synthesis in an opposite way. Moreover, we could increase the transcription of two of the four LHCX genes under low light conditions by reducing the PQ pool using DBMIB. Altogether, our results underline the central role of the redox state of the PQ pool in the light acclimation of diatoms. Additionally, they emphasize strong evidence for the existence of a plastid-to-nucleus retrograde signaling mechanism in an organism with plastids that derived from secondary endosymbiosis.

List of abbreviations

β-car, β-carotene; Chl a, chlorophyll a; Chl c, chlorophyll c; CHX, cycloheximide; Cyt, cytochrome; DBMIB, 2,5-Dibromo-6-isopropyl-3-methyl-1,4-benzoquinone; DCMU, 3-(3,4dichlorophenyl)-1,1-dimethylurea; Dd, diadinoxanthin; Dd+Dt, diadinoxanthin+diatoxanthin; ΔpH, transthylakoidal proton gradient; Dt, diatoxanthin; DTT, dithiothreitol; Fx, fucoxanthin; HL, high light; LHC, light harvesting complex; LL, low light; mHL, medium high light; NPQ, non-photochemical fluorescence quenching; 1 O 2, singlet oxygen; PQ, plastoquinone; qE, energy-dependent chlorophyll fluorescence quenching; ROS, reactive oxygen species; VDE, violaxanthin de-epoxidase; VDL, violaxanthin de-epoxidase like; VDR, violaxanthin de-epoxidase related

Introduction

Diatoms are eukaryotic microalgae found in any aquatic habitat. They contribute to the global primary production to a similar extent as terrestrial tropical rain forests and grasslands (Geider et al. 2001). As such, they are at the onset of most marine and freshwater food-webs (Armbrust 2009). Because of their ecological importance, the genomes of several diatom species have meanwhile been sequenced and are publically available (see [START_REF] Tirichine | Decoding algal genomes: tracing back the history of photosynthetic life on Earth[END_REF]. Especially the physiological studies on the molecular level of the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum revealed a plethora of unique features [START_REF] Wilhelm | The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae[END_REF]Armbrust 2009;Bowler et al. 2010). Some of these features arise from the chimerical origin of the genome which is due to horizontal gene transfer from bacteria and to the evolution of the diatom chloroplasts via secondary endosymbiosis (Bowler et al. 2010). After each endosymbiotic event, gene transfer from the endosymbiont to the nucleus occurred, permanently modifying the genome composition of the host cell. Still, the diatom chloroplast genome retained a small amount of approximately 130 protein encoding genes (Oudot-Le [START_REF] Secq | Chloroplast genomes of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana: comparison with other plastid genomes of the red lineage[END_REF]). As a further consequence of secondary endosymbiosis, diatom plastids are surrounded by an envelope of four membranes reflecting the endosymbiotic origin of these organelles (Green 2011).

Gene transfer from the chloroplast into the nucleus is common to all eukaryotic phototrophs. Because many of the plastid localized proteins are encoded by the nuclear genome and because most of the plastid protein complexes are chimeras of nuclear and plastid encoded proteins, both the plastidic and the nuclear gene expression as well as the respective protein import into the plastids require a fine-tuned regulation (Allen et al. 2011). Thus, anterograde (nucleus to plastid) and retrograde (plastid to nucleus) signaling pathways are widespread processes to coordinate gene expression in green algae and land plants [START_REF] Kleine | Plastid signalling to the nucleus: messengers still lost in the mists?[END_REF].

Control of photosynthetic gene expression and enzyme activity is coordinated by a number of molecular triggers including photosynthetic metabolites, hormones, chlorophyll and carotenoid biosynthesis precursors, photoreceptors, stress-generated reactive oxygen species (ROS), and soluble redox active compounds [START_REF] Kleine | Plastid signalling to the nucleus: messengers still lost in the mists?[END_REF][START_REF] Pfannschmidt | Plastidial retrograde signalling -a true plastid factoror just metabolite signatures?[END_REF]Foyer et al. 2012). A further important signaling trigger is the redox state of the plastoquinone (PQ) pool of the plastid electron transport chain. Besides its well-known involvement in the irradiance-dependent regulatory process of state-transitions in green microalgae and plants, it has a key role for the regulation of both chloroplast and nuclear gene expression under light changing conditions [START_REF] Pfannschmidt | Chloroplast redox signals: how photosynthesis controls its own genes[END_REF]. In diatoms, the involvement of photoreceptors in photosynthetic regulatory processes has been revealed recently (Depauw et al. 2012), while redox regulation via thioredoxins seem to be of minor importance for the regulation of the Calvin cycle [START_REF] Kroth | A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis[END_REF], even if thioredoxins may control the activity of carboanhydrases in the pyrenoid [START_REF] Kikutani | Redox regulation of carbonic anhydrases via thioredoxin in chloroplast of the marine diatom Phaeodactylum tricornutum[END_REF]. The redox state of the PQ pool is of crucial importance in several photosynthetic regulatory processes such as the PSII electron cycle [START_REF] Feikema | Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum[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]), the non-photochemical fluorescence quenching (NPQ) [START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: mechanisms, evolution and ecopysiology[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 the chlororespiration (Caron et al. 1987;Dijkman and Kroon 2002;Grouneva et al. 2009). The dynamics of the PQ pool redox state as a function of light intensity differ between diatom species and between diatoms and land plants [START_REF] Ruban | The superexcess energy dissipation in diatom algae: comparative analysis with higher plants[END_REF][START_REF] Lavaud | Fast regulation of photosynthesis in diatoms: mechanisms, evolution and ecopysiology[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]. So far, no evidence for an involvement of the PQ pool in redox signaling in diatoms has been published.

Diatoms possess an outstanding capacity to tolerate light fluctuations regularly punctuated by excess light exposure, which might be one of the reasons for their ecological success (Brunet and Lavaud 2010). The central mechanism for the fast regulation of photochemistry under changing light conditions is the NPQ. It takes places in the light-harvesting complex antennae (LHC) of PSII where excessively absorbed light energy is dissipated as heat [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, the most important part of NPQ is the energy dependent fluorescence quenching, qE, while the photoinhibitory quenching, qI, is low, and the state-transition quenching, qT, does not exist at all (Goss and Jakob 2010). qE requires the irradiance-dependent establishment of a transthylakoidal proton gradient (ΔpH) and the conversion of the xanthophyll diadinoxanthin (Dd) into diatoxanthin (Dt) in the presence of a ΔpH (Goss and Jakob 2010). Together with the reverse reaction in the absence of the ΔpH this process is called 'xanthophyll cycle' and is equivalent to the viola-, anthera-and zeaxanthin xanthophyll cycle in land plants [START_REF] Jahns | The role of the xanthophyll cycle and of lutein in photoprotection of photosystem II[END_REF]. Furthermore the presence of LHCX proteins is essential (Bailleul et al. 2010). In addition to LHCX, diatoms contain other members of the LHC protein superfamily like LHCF which constitute the peripheral light harvesting complex, and LHCR which are tightly linked with PSI and whose function remains still to be elucidated [START_REF] Lepetit | Molecular dynamics of the diatom thylakoid membrane under different light conditions[END_REF]. Located in the plastids, both the enzymes for the interconversion of the xanthophyll cycle pigments diadino-and diatoxanthin (Dd+Dt), the Dd de-epoxidases and the Dt epoxidases (Coesel et al. 2008;[START_REF] Lohr | Carotenoid metabolism in phytoplankton[END_REF], as well as the LHCX proteins [START_REF] Kroth | A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis[END_REF][START_REF] Zhu | Light harvesting and photoprotection in diatoms: Identification and expression of LI818-like proteins[END_REF]Engelken et al. 2012) are encoded in the nucleus. Dd+Dt and LHCX have been shown to be synthesized de novo during prolonged high light stress [START_REF] Olaizola | Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom[END_REF]Bailleul et al. 2010;[START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF]. De novo synthesis of Dd+Dt does not necessarily lead to higher NPQ [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[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]), but can enhance the antioxidant activity within the thylakoid membranes [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]. Nevertheless, reduced expression of the gene encoding the Dd de-epoxidase clearly leads to both lower Dt synthesis and qE [START_REF] Lavaud | Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching[END_REF]. Overor under-expression of LHCX1 promotes higher or lower qE, respectively, and the upregulation of LHCX2 and LHCX3 might confer more efficient photoprotection (Bailleul et al. 2010). In the present study we monitored the changes of Dd+Dt and LHCX during prolonged high light exposure and the subsequent low light exposure. Because light stress creates an increased reduction of the electron transport chain, we tested the possible involvement of the redox state of the PQ pool in the qE acclimation response using different light conditions and inhibitor treatments. By identifying a link between the redox state of the PQ pool and the regulation of the amount of the qE key players LHCX and Dd+Dt pigments, we deliver a so far missing evidence for plastid-to-nucleus retrograde signaling in diatoms.

Results

Expression of LHCX genes under different light conditions

LHCX gene expression was studies in P. tricornutum cells that had been cultured for 33 h in darkness, darkness/ low light (LL, 40 µmol photons m -2 s -1 ) or darkness/ medium low light (mHL, 750 µmol photons m -2 s -1 ) conditions. Except for the cells kept in the dark, the standard 16/8 h day/ night rhythm was maintained. In prolonged dark conditions, LHCX4 gene expression showed a significant increase during daytime while gene expression of LHCX2 and LHCX3 decreased (Fig. 1A). At 9:00 in the morning, after light had normally been switched on, the transcript level of LHCX1 reached its maximum, after which it declined to increase again at the same time the next day. This rhythmic diurnal expression suggests the presence of a circadian regulation. LL conditions had a pronounced effect on the expression of LHCX1, LHCX2 and LHCX3. The two latter genes were slightly but significantly induced until noon while LHCX1 was highly up regulated. mHL conditions strongly increased the expression of LHCX2 and LHCX3. During the following night, the respective amounts of mRNA almost reached their initial level indicating a fast regulation of transcript increase and decrease. The extra light intensity at mHL conditions compared to LL also resulted in an increase of LHCX1 transcription, albeit to a lesser extent. LHCX4 did not show any differences regarding transcription at both light treatments compared to cells kept in the dark.

In order to evaluate the effect of a high light exposure equivalent to full sunlight in nature, the cells were exposed to 2000 µmol photons m -2 s -1 (HL) [START_REF] Long | Photoinhibition of Photosynthesis in Nature[END_REF] for 2 h, and their recovery from HL stress was recorded during a subsequent LL exposure of 4 h. In this case the transcription of LHCX1, LHCX2 and LHCX3 was up-regulated while LHCX4 appeared to be light insensitive (Fig. 1B). In contrast to cells grown at mHL conditions, LHCX3 was the most strongly up-regulated gene (more than 100 fold), followed by LHCX2 and LHCX1. After 4 h of recovery at LL, LHCX3 reached its initial transcript level, indicating an extremely rapid up-and down-regulation. The reduction of transcripts was slower for LHCX2, while LHCX1 transcripts even decreased below its initial level because of the daily regulation pattern.

Changes of pigments and NPQ during mHL and HL treatments

During the mHL exposure (16 h) no alterations were observed regarding the amounts of chlorophyll c (Chl c) and β-carotene (β-car) per chlorophyll a (Chl a) (Fig. 2A). The fucoxanthin (Fx) content per Chl a increased just after the onset of light and then slightly decreased. Pigments of the violaxanthin cycle, which were observed by [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF], could not be recorded under our experimental conditions. The Dd+Dt concentration increased almost fourfold (Fig. 2A) and the Dt amount became higher than Dd. Remarkably, the Dd+Dt pool size kept scaling up during the first 3 h of darkness and did not decrease during the resting night period. After HL treatment (2 h) we detected an equally strong increase of Dd+Dt (Fig. 2B and Fig. 3A). The de novo synthesis of Dd+Dt started approximately 30 min after the light onset as already reported before [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[END_REF]) and rapidly declined within the 4 h of LL recovery. In contrast, during dark recovery the amount of Dd+Dt remained stable (Fig. 3A). Also, we did not observe major differences in the amount of all other pigments indicating a rather independent synthesis and degradation of Dd+Dt from Fx and β-Car. Interestingly, the epoxidation of Dt to Dd occurred rapidly and almost completely during the recovery period, no matter whether LL or darkness was employed (Fig. 3B).

An inverse relationship was observed between NPQ and F v /F m during the mHL exposure. The NPQ increased, albeit with a much lower slope during the second half of the light treatment, while F v /F m concomitantly decreased (Suppl. Fig. 3). The NPQ did not relax completely during the dark recovery period. A strong increase of NPQ was also observed during the HL experiments (Fig. 3C). Its maximal value was lower than during the mHL treatment (1.65 to 2.75) which can be explained by the shorter light treatment (2 h vs. 16 h). NPQ completely relaxed during the LL recovery period but not in darkness (Fig. 3C). The addition of NH 4 Cl, which usually relaxes NPQ efficiently in P. tricornutum [START_REF] Wilhelm | The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae[END_REF], could not reduce the NPQ persisting in the dark (Fig. 3C).

The NPQ/Dt relationship reflects the Dt quenching efficiency (Lavaud et al. 2002a;Lavaud et al. 2004), which in the mHL experiments showed a highly linear correlation (R 2 =0.98) up to a threshold value of 20 mol Dt (100 mol Chl a) -1 at the 15:00 time point (Suppl. Fig. 4A).

Beginning at 18:00, the slope of the relationship strongly decreased. The respective values during the HL treatment yielded a strong linear correlation (R 2 =0.96) up to a timespan of 60 min and a threshold concentration of 7 mol Dt (100 mol Chl a) -1 , while the slope of the relationship during the second hour of illumination dramatically dropped (Suppl. Fig. 4A andB). Moreover, the values for the steep slopes of the NPQ/Dt relationships in mHL and HL were different (0.125 vs. 0.218) (Suppl. Fig. 4A).

Influence of DTT and CHX on NPQ, Dd+Dt and LHCX synthesis

Because the photoacclimative responses were already fully activated during the two hours of HL exposure, the inhibitor treatments were performed at these conditions. DTT, an inhibitor of the de-epoxidation reaction [START_REF] Yamamoto | The effects of dithiothreitol on violaxanthin deepoxidation and absorbance changes in the 500-nm region[END_REF], had no effect on the increase of the Dd+Dt pool size (Fig. 3A, see 'DTT+'), but blocked the Dd de-epoxidation during the first 30 min of illumination (Fig. 3B). Afterwards, when the Dd+Dt pool size increased, a considerable amount of Dt was produced even in the presence of DTT. To exclude the possibility that the inhibitor effect is reduced after 30 min of HL, DTT was alternatively added every 20 min (Fig. 3B, 'DTT+'). The supplementary DTT dose slowed down the Dt synthesis while not completely inhibiting it. As expected, both DTT treatments (DTT and DTT+) strongly inhibited the induction of NPQ (Fig. 3C,D) and resulted in a dramatic decrease of the functional fraction of PSII (Fig. 4).

CHX blocks the translation of nuclear encoded genes on cytosolic 80s ribosomes and therefore does not influence gene translation in the organelles [START_REF] Obrig | The Mechanism by which Cycloheximide and Related Glutarimide Antibiotics Inhibit Peptide Synthesis on Reticulocyte Ribosomes[END_REF][START_REF] Setkov | Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: a radioautographic and cell fusion study[END_REF]). As a matter of fact, while HL treatment induced the synthesis of nuclear encoded LHCX2 and LHCX3 proteins (Fig. 5) as expected from the transcript level measurements (Fig. 1), the addition of CHX prevented it. In contrast, CHX had no inhibiting effect on the plastid encoded PsbB protein, instead even a slightly increased amount of PsbB was detected as observed before in other diatoms [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF]). We did not notice any differences in the amounts of LHCX1 after HL treatment. This might either be due to an oversaturation of the Western blot because of the already rather high initial LHCX1 amount or to a different regulation on the posttranslational level. Addition of CHX did not alter the de novo synthesis of Dd+Dt and the amount of Dt produced during HL exposure (Fig. 3A,B), but it completely prevented the decrease of Dd+Dt during the LL recovery period, similarly to the dark recovery experiments in the absence of the inhibitor. However, after 30 min of HL CHX retarded a further increase of NPQ almost completely (Fig. 3C) and increased the degree of PSII photoinactivation, although not as pronounced as DTT (Fig. 4). As expected, we obtained the highest degree of photoinactivation by adding both CHX and DTT.

Only during the first 30 min of HL and CHX treatment we found a highly linear correlation of Dt versus NPQ (R 2 =1), thereafter the slope almost dropped close to zero. The threshold concentration for Dt participating in NPQ decreased to a value of around 5 mol Dt (100 mol Chl a) -1 (Suppl. Fig. 4B).

Effect of DCMU and DBMIB on the Dd+Dt pool size

In diatoms a chlororespiration process exists which influences the redox state of the PQ pool in the dark [START_REF] Jakob | Activation of diadinoxanthin de-epoxidase due to a chlororespiratory proton gradient in the dark in the diatom Phaeodactylum tricornutum[END_REF]Dijkman and Kroon 2002;[START_REF] Wilhelm | The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae[END_REF]Grouneva et al. 2009). As we saw a clear difference in the Dd+Dt pool size during dark or LL recovery, DCMU was utilized to modify the PQ pool redox state. By binding to Q B , DCMU generates a reduced Q A -and an oxidized PQ pool [START_REF] Trebst | Inhibitors in the functional dissection of the photosynthetic electron transport system[END_REF]. Applied during the LL recovery conditions, DCMU prevented the decrease of the Dd+Dt pool (Fig. 3A), comparably to the dark recovery in the absence of the inhibitor. On the other hand, DCMU induced a pronounced de-epoxidation, probably due to the onset of cyclic electron transport around PSI (Fig. 3B). By applying DCMU directly at the beginning of the HL exposure, i.e. by mimicking LL conditions regarding the redox state of the PQ pool, no Dd+Dt pool size increase was observed, but again a strong de-epoxidation occurred (Fig. 3A). In order to verify these results, we used DBMIB which usually shows the opposite effect compared to DCMU regarding the redox state of the PQ pool. DBMIB binds close to the plastoquinol oxidizing Qo-site of the cytochrome (Cyt) b 6 f complex and consequently induces a reduced PQ pool under LL which hence mimics HL conditions [START_REF] Kurisu | Structure of the Cytochrome b6f Complex of Oxygenic Photosynthesis: Tuning the Cavity[END_REF][START_REF] Trebst | Inhibitors in the functional dissection of the photosynthetic electron transport system[END_REF].

Because this effect is known to depend on the concentration of DBMIB, we tested different amounts in order to define a suitable DBMIB concentration necessary to induce a reduced PQ pool following two main criteria: 1) a high F v /F m served as a proof that DBMIB does not bind to Q B which can occur at too high concentrations [START_REF] Vener | Plastoquinol at the quinol oxidation site of reduced cytochrome bf mediates signal transduction between light and protein phosphorylation: Thylakoid protein kinase deactivation by a single-turnoverÇëflash[END_REF], and 2) we checked for a reduced NPQ compared to the control because of the limited proton translocation at the Cyt b 6 f complex [START_REF] Strzepek | Photosynthetic architecture differs in coastal and oceanic diatoms[END_REF]. The suitable DBMIB concentration was defined to be 1 µM (Suppl. Fig. 5) which is consistent with concentrations used in green microalgae [START_REF] Steinbrenner | Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: Regulation by photosynthetic redox control[END_REF] and in land plants [START_REF] Tullberg | Photosynthetic electron flow regulates transcription of the psaB gene in pea (Pisum sativum L.) chloroplasts through the redox state of the plastoquinone pool[END_REF]. 1 µM DBMIB at a light intensity of 100 µmol photons m -2 s -1 (LL 100 ) was sufficient to support a considerable electron flow from PSII to PSI while neither inducing any Dd de-epoxidation nor any decrease of F v /F m (data not shown). DBMIB + LL 100 led to a 20 % increase of Dd+Dt in contrast to untreated cells (Fig. 6). A reduced PQ pool could therefore induce an HL response under LL conditions.

Effect of DCMU and DBMIB on the LHCX transcript levels

The described DBMIB + LL 100 HL mimicking system was then applied for further analysis of LHCX transcript levels. While the LL 100 treatment alone did not induce remarkable transcript changes of any of the LHCX, the addition of DBMIB caused a slight rise of the LHCX1 transcript level (Fig. 7A) and a pronounced increase of LHCX2 (20 fold after 60 min) (Fig. 7B). In contrast, the amount of LHCX3 transcripts did not change (Fig. 7C). Hence, for two of the three light induced LHCX, gene expression was specifically stimulated by an increased reduction state of the PQ pool. The addition of DCMU during HL treatment did not produce the opposite effect of DBMIB, even when the light intensity was decreased to 500 µmol photons m -2 s -1 (Suppl. Fig. 6), most probably because the combined effect of HL and DCMU on photosynthesis was higher than the DCMU generated artificial LL effect. Instead, we used DCMU during the DBMIB + LL 100 treatment in order to stop the electron flow from PSII to the Cyt b 6 f, relaxing the reduced PQ pool redox state. This abolished the effect of DBMIB on the transcript levels of LHCX1 and LHCX2 (Fig. 7A andB) and demonstrated that DBMIB did not simply induce transcription of these genes due to its presence per se but to its effect on the redox state of the PQ pool. The light insensitive LHCX4 gene responded differently. Its transcription was slightly induced by the DBMIB + LL 100 treatment (Fig. 7D) but it was neither abolished by the addition of DCMU nor increased during the HL + DCMU treatment (Suppl. Fig. 6). LHCX4 apparently reacted specifically on the presence of DBMIB rather than on the changes of the redox state of the PQ pool itself.

Discussion

The role of Dt and LHCX in NPQ

The presented results strengthen previous findings whereupon LHCX1, LHCX2 and LHCX3 but not LHCX4 are involved in the photoacclimative/-protective response of diatoms [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF]Bailleul et al. 2010). The Pt4 strain studied here shows a clearer HL response regarding LHCX1 gene transcription (but not resolved on the protein level) than the Pt1 strain used in the experiments performed by Nymark and co-workers and Bailleul and coworkers. Albeit in both strains LHCX1 is the predominant LHCX protein under LL conditions, Pt1 contains higher levels of LHCX1 mRNA and protein (Bailleul et al. 2010), which might limit its ability to increase their amount under HL.

The increase of gene transcripts is often well paralleled by the respective protein changes [START_REF] Lee | A dynamic model of proteome changes reveals new roles for transcript alteration in yeast[END_REF]Dyhrman et al. 2012). After 2 h of HL the huge increase of LHCX2 and LHCX3 transcripts was reflected by the respective protein levels. This coincided with a higher Dt concentration, resulting from the de-epoxidation of mostly de novo synthesized Dd. As a consequence of the increase of both, Dt and LHCX, a higher NPQ was established. Similar results were also obtained recently in T. pseudonana [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF]. An important question is how these two enhancers of qE may interact. One hint comes from the finding that the amount of Dt alone is only a weak indicator for the quenching capacity [START_REF] Lavaud | General features of photoprotection by energy dissipation in planktonic diatoms (Bacillariophyceae)[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][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]). If suitable protein binding sites are missing, a larger number of Dt molecules can be dissolved in the thylakoid lipids and then does not participate in NPQ [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]. Indeed, the ratio of NPQ to Dt was considerably different in mHL and HL cultures. Moreover, addition of CHX blocked the increase of NPQ beginning after 30 min of HL treatment. This was also the time when de novo synthesis of Dd+Dt started. It therefore can be assumed that, in order to participate in NPQ, the de novo synthesized Dt molecules need the equivalent amounts of newly synthesized nuclear encoded pigment binding proteins. Besides LHCX1, LHCX2 and LHCX3, only four LHCR genes are stronger expressed at HL compared to LL conditions [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF]. LHCR proteins constitute the PSI antenna in diatoms [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]. There are indications that LHCX proteins (except LHCX1/FCP6, see Beer et al. 2006;[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] appear to be rather weakly associated to the thylakoid membrane or to the classical light harvesting complexes [START_REF] Zhu | Characterization of LI818-like genes under various stress conditions in the marine diatom Thalassiosira pseudonana[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]), similar to their LHCSR/LI818 relatives in C. reinhardtii (Richard et al. 2000). As the amount of Dd+Dt bound to LHCR in PSI remains constant at changing 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] Another peculiarity of our data is the strong difference of NPQ during dark or LL recovery conditions after HL treatment. This difference obviously was not due to photoinhibition, as for both recoveries the light treatment before was identical. It is known that full NPQ recovery in diatoms occurs only at LL conditions, but not in darkness [START_REF] Wilhelm | The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae[END_REF]Grouneva et al. 2009). So far this effect was related to the Dt epoxidation reaction, which might be limited in darkness due to the chlororespiration driving the buildup of the ΔpH, and due to a shortage of NADPH [START_REF] Jakob | Activation of diadinoxanthin de-epoxidase due to a chlororespiratory proton gradient in the dark in the diatom Phaeodactylum tricornutum[END_REF][START_REF] Wilhelm | The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae[END_REF]Grouneva et al. 2009). In our experiments the epoxidation was not different in LL or darkness. After 4 h of recovery only a minor amount of Dt was present in the cells (1.1 and 0.5 mol Dt (100 mol Chl a) -1 in samples from LL and darkness, respectively, Fig. 3A). Moreover, the addition of the uncoupler NH 4 Cl did not relax the NPQ persisting in the dark. Different to the experiments by Jakob, Goss and Grouneva and co-workers mentioned above, our cultures were longer exposed to HL. Obviously, some part of the NPQ created here relies on other processes, which are independent of Dt. Dt-independent NPQ already has been demonstrated in diatoms and was interpreted to be related to a PSII cycle and/or to a rearrangement of the PSII core (Lavaud et al. 2002b;Eisenstadt et al. 2008).

The regulation of xanthophyll cycle pigment synthesis

Our data strongly support the assumption that the redox state of the PQ pool might be the trigger for the HL induced de novo synthesis of Dd+Dt. The HL+DCMU treatment, which generates massive amounts of singlet oxygen ( 1 O 2 ) (Flors et al. 2006), prevented the increase of Dd+Dt thus excluding a possible triggering role of this ROS, in contrast to its influence on the LHCX gene expression (see below). However, as by binding to Q B DCMU blocks the photochemical linear electron flow towards NADP+ [START_REF] Trebst | Inhibitors in the functional dissection of the photosynthetic electron transport system[END_REF], the amount of NADPH, which is required in the early steps of carotenoid biosynthesis [START_REF] Lohr | Isoprenoid biosynthesis in eukaryotic phototrophs: A spotlight on algae[END_REF], might indirectly limit the de novo synthesis of Dd+Dt. In contrast, DBMIB equally reduces linear electron flow by preventing electron flow towards the Cyt b6f complex [START_REF] Trebst | Inhibitors in the functional dissection of the photosynthetic electron transport system[END_REF]), but induced Dd+Dt de novo synthesis. Hence, an NADPH shortage effect as the actual reason for the differences in Dd+Dt de novo synthesis seems rather unlikely compared to the triggering by the redox state of the PQ pool.

All known enzymes involved in carotenoid synthesis are nuclear encoded, but the carotenoid synthesis itself takes place in the chloroplast (Coesel et al. 2008;Frommolt et al. 2008;[START_REF] Lohr | Carotenoid metabolism in phytoplankton[END_REF]). The Dd+Dt synthesis was unaffected by the nuclear translation inhibitor CHX, hence its regulation must occur at the post-translational stage and most probably in the chloroplast.

Additionally, inhibition of plastid translation does also not reduce Dd+Dt de novo synthesis in HL [START_REF] Olaizola | Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom[END_REF][START_REF] Wu | Photosystem II Photoinactivation, Repair and Protection in Marine Centric Diatoms[END_REF]. While genes encoding Dd synthesizing enzymes are up-regulated in diatoms that are shifted from darkness to light with moderate intensity (Coesel et al. 2008), their expression is not further induced in LL to HL shift experiments similarly to our experimental conditions [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF]). Thus, the post-translational regulation of Dd+Dt de novo synthesis seems not to require any newly expressed nuclear or plastid encoded factor, which could activate the carotenoid synthesizing enzyme activity. All these features strongly resemble the situation in the green microalga Haematococcus pluvialis, where the HL induced de novo synthesis of the xanthophyll astaxanthin is triggered by the redox state of the PQ pool via post-translational regulation [START_REF] Steinbrenner | Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: Regulation by photosynthetic redox control[END_REF]. In contrast to the Dd+Dt synthesis, the effect of CHX illustrates the need for the expression of nuclear encoded gene(s) during the LL decrease of the Dd+Dt pool size. Unlike in what was proposed by [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF], this degradation of Dd+Dt does not result in an increase of the amount of Fx under our experimental conditions but instead Dd+Dt are probably recycled.

This fits with recent results pointing out that Fx is not synthesized from Dd but that both xanthophylls share the same precursor pathway (Dambek et al. 2012).

Another interesting observation was that DTT blocked the de-epoxidation effectively only during the first 30 min of HL treatment. Even the addition of supplemental DTT, in order to rule out a reduced efficiency during the treatment, did not restore the full inhibitory effect, although it clearly retarded the synthesis of Dt. So far, no Dt biosynthesis pathway excluding the de-epoxidation from Dd has been identified [START_REF] Lohr | Carotenoid metabolism in phytoplankton[END_REF]. P. tricornutum possesses one violaxanthin de-epoxidase (VDE), two VDE like enzymes (VDL) and one VDE related enzyme (VDR) (Coesel et al. 2008;Bertrand 2010). While the role of VDE is clear, the function of VDL/VDR remains speculative. Studies on P. tricornutum mutants containing a reduced amount of VDE indicate that these alternative de-epoxidases are involved in Dd to Dt conversion [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 is possible that during DTT inhibition VDR and VDL perform the Dd de-epoxidation reaction, as they might be less sensitive to DTT than VDE.

Corresponding to the xanthophyll cycle pigments, their synthesis might be induced in HL, explaining the absence of Dd de-epoxidation in the first 30 min of HL. Indeed, in contrast to the VDE, the transcription of these alternative de-epoxidases is up-regulated during a short term light stress [START_REF] Nymark | An integrated analysis of molecular acclimation to high light in the marine diatom Phaeodactylum tricornutum[END_REF].

The regulation of LHCX gene expression

The transcript level of LHCX1, LHCX2 and LHCX3 responded to light in a dose-dependent manner. As conclusively demonstrated, the transcription could be induced in LL conditions via a reduced PQ pool for LHCX1 and LHCX2 but not for LHCX3. Blocking the gene expression of LHCX1 and LHCX2 with DCMU in HL was not possible. As mentioned above, besides keeping the PQ pool oxidized, DCMU creates high amounts of 1 O 2 (Flors et al. 2006), which in plants is the most harmful ROS [START_REF] Triantaphylidès | Singlet oxygen is the major reactive oxygen species involved in photooxidative damage to plants[END_REF][START_REF] Triantaphylidès | Singlet oxygen in plants: production, detoxification and signaling[END_REF] and which is able to induce nuclear gene expression (op den [START_REF] Op Den Camp | Rapid Induction of Distinct Stress Responses after the Release of Singlet Oxygen in Arabidopsis[END_REF]Fischer et al. 2004). We assume that LHCX1 and LHCX2 are also induced by 1 O 2 , which superimposes on the effect of the PQ pool redox state. Most likely LHCX3 is also strongly induced by ROS, as it exhibited a higher relative induction upon addition of DCMU than in HL without DCMU (Suppl. Fig. 6). This suggests a photoprotective role of LHCX3 at conditions where the light stress is harsh and is in line with our results, where LHCX3 was the relatively strongest transcribed LHCX gene only at 2000 µmol photons m -2 s -1 light exposure.

LHCX1, LHCX2 and LHCX3 are furthermore up-regulated in response to blue light via a cryptochrome mediated pathway (Coesel et al. 2009). Similarly, promoters in Arabidopsis react to various stimuli [START_REF] Staneloni | Abscisic Acid, High-Light, and Oxidative Stress Down-Regulate a Photosynthetic Gene via a Promoter Motif Not Involved in Phytochrome-Mediated Transcriptional Regulation[END_REF]). Hence, LHCX gene expression is apparently fine-tuned by the light quality (cryptochrome photoreceptor) as well as the light intensity either in situations that generate an imbalance between the photosynthetic light reactions and the Calvin cycle (redox state PQ pool) or during prolonged excess light exposure (i.e. 'light stress'; ROS). Importantly, all of these triggers might participate altogether in the cellular light response, albeit to different extents, as a function of the light conditions.

Which part of the electron transport chain exactly generates the redox signal?

By applying DCMU and DBMIB we were able to mimic a LL or HL phenotype, respectively, similarly like it has been performed in the green alga Dunaliella tertiolecta in the pioneer work by Escoubas et al. (1995). Besides green algae, comparable experiments succeeded in cyanobacteria [START_REF] Li | A Redox-Responsive Regulator of Photosynthesis Gene Expression in the Cyanobacterium Synechocystis sp. Strain PCC 6803[END_REF] and land plants [START_REF] Karpinski | Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress[END_REF][START_REF] Pfannschmidt | Photosynthetic control of chloroplast gene expression[END_REF][START_REF] Tullberg | Photosynthetic electron flow regulates transcription of the psaB gene in pea (Pisum sativum L.) chloroplasts through the redox state of the plastoquinone pool[END_REF]. While, regarding the effects of DBMIB and DCMU, it is clear that the generated signal is related to the redox state of the PQ pool, its exact localization in the plastidic electron transport chain remains ambiguous [START_REF] Pfannschmidt | Chloroplast redox signals: how photosynthesis controls its own genes[END_REF]. The Qo-site of the Cyt b 6 f complex, which binds plastoquinol, activates state-transitions and probably other redox controlled processes via kinase activation [START_REF] Zito | The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase[END_REF]), but it does not account for all PQ pool redox dependent processes (Baier and Dietz 2005). Because in our experiments DCMU inhibited the DBMIB effect in LL, we proved that the binding of DBMIB to the Cyt b 6 f complex alone is not responsible for the redox mediated response. Moreover, in addition to prevent an increase of Dd+Dt in HL due to the oxidized PQ pool, DCMU also inhibited a decrease in LL. In all light conditions DCMU fixed the Dd+Dt pool size to its actual status.

As at these distinct conditions different degrees of de-epoxidation are induced (Fig. 3B) which directly depend on the generated ΔpH [START_REF] Jakob | Unusual pH-dependence of diadinoxanthin deepoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum[END_REF], one can assume either a chlororespiration or a PSI cyclic electron transfer activity. Both processes would change the redox state of the PQ pool. It is therefore tempting to speculate that the PQ pool mediated signal would need not one but two triggers: one is the redox state of the PQ pool itself and the second one might be the redox state of Q A which works as an on/off switch for the downstream PQ pool redox regulation. Alternatively Q B could be involved because binding of DCMU here might inactivate this possible regulatory site preventing any regulation by the PQ pool. The rate-limiting step in the photosynthetic electron transport chain is the diffusion and the reoxidation of the PQ pool [START_REF] Haehnel | Photosynthetic Electron Transport in Higher Plants[END_REF][START_REF] Rochaix | Regulation of photosynthetic electron transport[END_REF]. In HL conditions Q A should be partially oxidized, while the pool itself is reduced, as long as the light reactions are faster than the enzymatic reactions of the Calvin cycle. As a consequence, the synthesis of Dd+Dt is initiated. In LL conditions, Q A and the PQ pool are oxidized and the Dd+Dt pool size decreases. As long as Q A is reduced, which in our experiments was induced by adding DCMU or by the sudden transfer of the cells from HL to darkness (as demonstrated by Dijkman and Kroon 2002; Grouneva et al. 2009), there is neither an increase nor a decrease of the Dd+Dt pool size independent of the oxidation state of the PQ pool. Why should diatoms have an additional on/off switch within the PSII core? One explanation might be based on the very unusual electron fluxes in diatoms which are still far from being understood. These include: 1) a vivid exchange of electrons between the electron transport chains of chloroplasts and mitochondria [START_REF] Prihoda | Chloroplast-mitochondria cross-talk in diatoms[END_REF], 2) a cyclic electron transfer in PSII (Lavaud et al. 2002b;[START_REF] Feikema | Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum[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], 3) a chlororespiration process which can modify the redox balance of the quinones and of PQ [START_REF] Jakob | Activation of diadinoxanthin de-epoxidase due to a chlororespiratory proton gradient in the dark in the diatom Phaeodactylum tricornutum[END_REF]Dijkman and Kroon 2002;Grouneva et al. 2009;Cruz et al. 2011), and 4) a recombination of Q A -and Q Bwith the oxidized states of the Mn Cluster in the PSII core induced by conformational changes (Eisenstadt et al. 2008). Therefore, an additional switch provided by the acceptor side of PSII (Q A and Q B ) might help, in combination with the redox state of the PQ pool, to sort the different electron flows and to fine-tune the synthesis of xanthophylls and the transcription of light-responsive genes like LHCX, depending on the source of electrons.

Conclusion

The redox state of the PQ pool is used as a widespread physiological mechanism in green algae and land plants for signaling within the plastid as well as for retrograde signaling from the plastid to the nucleus [START_REF] Pfannschmidt | Chloroplast redox signals: how photosynthesis controls its own genes[END_REF]Baier and Dietz 2005;Fey et al. 2005;[START_REF] Kleine | Plastid signalling to the nucleus: messengers still lost in the mists?[END_REF]). To our knowledge, no indications for such signaling processes have been reported before in photosynthetic organisms with secondary plastids. In diatoms, a PQ pool redox regulation and retrograde signaling was so far regarded as unlikely [START_REF] Wilhelm | The regulation of carbon and nutrient assimilation in diatoms is significantly different from green algae[END_REF], especially when taking into account their complex evolutionary history (Bowler et al. 2010) and the abundance of several light sensors (Depauw et al. 2012), which might partially replace this function. We now have demonstrated that diatoms also use the redox state of the PQ pool for controlling the light acclimation of the plastid which includes retrograde signaling to the nucleus to regulate photosynthetic gene expression. As the PQ pool oxidation is the slowest step within the electron transport chain, it is the ideal sensor for redox imbalances. This raises the exciting question how the redox signal can cross the four membranes of the plastid envelope, which is even not answered yet in land plants and green algae, although their plastids possess only two membranes [START_REF] Kleine | Plastid signalling to the nucleus: messengers still lost in the mists?[END_REF]. Moreover, as diatoms are evolutionary and physiologically closely related to other members of the Heterokontophyta like Phaeophyceae, Chrysophyceae, Xanthophyceae or Pelagophyceae (Adl et al. 2005), it is tempting to assume that such mechanism also exists in these groups and might even be a universal feature of photosynthetic organisms.

Materials and Methods

Cell culturing and light treatments

Phaeodactylum tricornutum (University of Texas Culture Collection, strain 646, so-called 'Pt4') was grown in airlift tubes in a day/night rhythm of 16/8 h with a light intensity of 40 µmol photons m -2 s -1 (low light, LL) at 20°C. Cells were cultured in Provasoli's enriched f/2 seawater medium [START_REF] Guillard | Culture of phytoplankton for feeding marine invertebrates[END_REF] using Tropic Marin artificial sea salts (Dr. Biener GmbH, Germany) at a final concentration of 50 % compared to natural seawater. Three days after inoculation the cultures in their exponential phase of growth were concentrated to 10 mg L -1 Chl a. They were again acclimated to LL for 1 h before the different experiments were started, and sodium carbonate (4 mM) was added to prevent carbon limitation. Light exposure was provided by four 65 W white light energy saving lamps (Lexman, France) adjusted at distinct distances, in a custom made glass cylinder which was continuously cooled by a water bath at 20°C. Cell dispersal was secured by a magnetic stirrer. A long term experiment started at 24:00 o'clock in darkness. At 8:00 cells were either kept in darkness, exposed to LL or exposed to 750 µmol photons m -2 s -1 (medium high light, mHL) until 24:00, followed by a further dark cycle of 8 h and a subsequent 1 h darkness or LL/ mHL exposure. In a short term experiment, cells were exposed to 2 h of high light (HL, 2000 µmol photons m -2 s -1 starting at 12:00), followed by 4 h of recovery using either LL or darkness with a subsequent 1 h HL treatment. In a third experiment cells were exposed for 2 h to 100 µmol photons m -2 s -1 (LL 100 ) only, starting at 12:00.

Sampling

Samples were taken as indicated in the Results section. For HPLC analysis, 400 µL of cell suspension were filtered on a Membrane Isopore Polycarbonate filter 1.2 µm (Millipore, USA) and immediately frozen in liquid nitrogen. Pigments were isolated and analyzed the same day. For RNA and protein analyses, 4 mL of cell suspension were centrifuged 4 min at 4°C and 4000 g. The supernatant was discarded, and the 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.

Chlorophyll determination

Chl a was extracted using successively 10 % Methanol and 90 % Acetone. Its concentration was determined in a spectrophotometer (Hitachi U-5100, Japan) according to Jeffrey and Humphrey (1975).

Inhibitor treatments

Dithiothreitol (DTT, 500 µM, in H 2 O) was added 15 min before HL exposure. In an alternative experiment, DTT in the same concentration was additionally supplied every 20 min during the HL treatment. Cycloheximide (CHX, 2 µg mL -1 , in H 2 O) was added 45 min before HL treatment. Ammonium chloride (NH 4 Cl, 5 mM, in H 2 O) was supplemented after 2 h of LL recovery following 2 h of HL treatment. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 10 µM, in Ethanol, final concentration of Ethanol 2 ‰) was added either 10 min before the HL treatment or prior to the recovery period. 2,5-Dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB, 1 µM, in Ethanol, final concentration of Ethanol 0.5 ‰) was supplied 10 min before starting the LL 100 exposure.

Chlorophyll fluorescence measurements

Chlorophyll fluorescence was measured with a Diving-PAM (Walz, Germany), whose optic guide was directly immersed into the algal suspension. Saturating flashes were sent at different time points (see results). As the surrounding light interfered with the measurements, immediately before starting the flash the light was switched off, which was assumed to be fast enough to avoid maximal fluorescence relaxation, in line with previous findings [START_REF] Havaux | A theoretical and experimental analysis of the qP and qN coefficients of chlorophyll fluorescence quenching and their relation to photochemical and nonphotochemical events[END_REF]. The maximum photosynthetic efficiency of PSII was determined as (F m -F o )/F m =F v /F m and the effective quantum yield of PSII as F v' /F m' . Non-photochemical fluorescence quenching (NPQ) was calculated using the Stern-Volmer parameter as NPQ = F m /F m' -1.

Photoinactivation was determined as 1/F o -1/F m according to [START_REF] Park | Light inactivation of functional photosystem II in leaves of peas grown in moderate light depends on photon exposure[END_REF] and [START_REF] He | The rate coefficient of repair of photosystem II after photoinactivation[END_REF]. It has been shown to well describe the photoinactivation also in diatoms [START_REF] Wu | Distinctive photosystem II photoinactivation and protein dynamics in marine diatoms[END_REF]Campbell and Tyystjärvi 2012). Here, the value of LL acclimated cells before starting the HL treatment was taken as 100 %. For investigating the optimal concentration of DBMIB, fluorescence kinetics were measured during 5 min of illumination with 2000 µmol photons m -2 s -1 with a 2500 Schott white light lamp (Germany).

Pigment analysis

Pigment extraction and HPLC analysis followed the protocol established by [START_REF] Jakob | Activation of diadinoxanthin de-epoxidase due to a chlororespiratory proton gradient in the dark in the diatom Phaeodactylum tricornutum[END_REF]. Samples were analyzed with a Hitachi LaChrom Elite HPLC system (Japan) equipped with a 10°C cooled autosampler and a Nucleosil 120-5 C18 column (Maccherey-Nagel, Germany).

RNA extraction and LHCX transcript level analysis by Real-time PCR

RNA was extracted using Trizol (Invitrogen, USA) and the RNeasy Mini kit (Qiagen, Germany). Contaminations of DNA were removed with TurboDNAse (Ambion, USA). An equal amount of RNA was used for all samples to be transcribed into cDNA using the QuantiTect reverse transcription kit (Qiagen, Germany). Resulting cDNA was diluted 2-fold in Nuclease-free water. For Real-time PCR analysis, 1 µL of cDNA was used in a 20 µL qPCR assay containing also the primer pairs and the GoTaq Mastermix (Promega, USA). A geNorm v.3.5 analysis [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF] was performed according to the authors instructions, showing that 18S and RPS were both well suited as endogenous reference genes under conditions used for growing of the cells (Suppl. Fig. 1). Disadvantageous for 18S as reference gene were the lower Ct-values compared to LHCX genes due to its high abundance (around 14 compared to 20-30 for the LHCX genes), leading to a decreased sensitivity for weaker expressed genes. However, when DBMIB and DCMU were supplied, 18S expression was clearly more stable than RPS expression (Suppl. Fig. 2). Therefore, the gene encoding 18S was chosen as the endogenous reference gene. Primer sequences can be found in Suppl.

Table 1. Real-Time PCR was performed on a Stratagene Mx3005P system (Agilent, USA) using a two-step protocol. Samples were measured in triplicates. At the end of each run melting curve analysis was applied to confirm the specific amplification of the template and to exclude false positive fluorescence due to side reactions. C T -values were obtained by utilizing PCR Miner 3.0 [START_REF] Zhao | Comprehensive algorithm for quantitative real-time polymerase chain reaction[END_REF]. Relative transcript levels were calculated according to [START_REF] Pfaffl | Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in realtime PCR[END_REF] and using the REST© software tool.

LHCX protein analysis

Protein extraction, Western blot and ECL detection followed the protocol described in Coesel with 100 µmol photons m -2 s -1 (LL 100 ). In these conditions, the diatoxanthin amount was less than 2 % of the total diadinoxanthin+diatoxanthin pool size in both experimental conditions.

In case of the inhibitor treatment, 1 µM DBMIB was added 10 min before the start of the experiment. Depicted are the mean values of four independent measurements (±SD). with 100 µmol photons m -2 s -1 (LL 100 ). In these conditions, the diatoxanthin amount was less than 2 % of the total diadinoxanthin+diatoxanthin pool size in both experimental conditions.

In case of the inhibitor treatment, 1 µM DBMIB was added 10 min before the start of the experiment. Depicted are the mean values of four independent measurements (±SD).

Figure legends Fig. 1 :

 legends1 Figure legends

Fig. 2 :

 2 Fig.2: Pigment evolution during the mHL (A) and HL (B) experiment. In A), the recovery period was performed at darkness, while in B) recovery was monitored at LL conditions.After 4 h of recovery from HL, cells were again exposed to 1 h of HL treatment. Values are the mean of three (A) and five (B) independent experiments (±SD).

Fig. 3 :

 3 Fig. 3: Diadinoxanthin+diatoxanthin (Dd+Dt) (A) and diatoxanthin (Dt) (B) content and NPQ (C,D) during 2 h of HL treatment, 4 h of LL recovery and subsequently 1 h of HL treatment.Control: cultures without inhibitors; dark recovery: after 2 h of HL exposure 4 h of darkness followed instead of 4 h of LL; CHX: cultures were incubated with cycloheximide (CHX, 2 µg mL -1 ) 45 min before starting the experiment; DTT: cultures were incubated with DTT (500 µM) 15 min before starting the experiment; DTT+: DTT (500 µM) was added additionally every 20 min during the HL treatment and 20 min before the second HL treatment; LL rec+DCMU: DCMU (10 µM) was added before starting the LL recovery period; DCMU+dark rec: DCMU (10 µM) was added 10 min before light exposure; recovery was performed in darkness; dark rec+NH 4 CL: recovery was performed in darkness; after 2 h of darkness, NH 4 Cl (5 mM) was added (indicated with an arrow). Samples represent the mean of two to five independent experiments (±SD), except for the NPQ values of DCMU treated samples during the recovery time, where only one experiment was performed.

Fig. 4 :Fig. 6 :

 46 Fig. 4: Functional fraction of PSII (in %) during 2 h of HL treatment as an indicator of PSII photoinactivation calculated by 1/F o -1/F m . The ratio 1/F o -1/F m obtained before the start of the light treatment was taken as 100%. For explanation of the legend, see Fig. 3. DTT+CHX:DTT (500 µM) and cycloheximide (CHX, 2 µg mL -1 ) were added 15 and 45 min before light exposure, respectively. ±SD

Fig. 7 :Fig. 1 :

 71 Fig. 7: Relative gene expression of LHCX1 (A), LHCX2 (B), LHCX3 (C), and LHCX4 (D) during illumination with 100 µmol photons m -2 s -1 (LL 100 ). Relative LHCX transcription levels referring to 18S gene expression were normalized to the initial amount. White: no chemical treatment; dark grey: 1 µM DBMIB was applied 10 min before the light treatment; light grey: in addition to DBMIB 10 µM DCMU was applied 10 min before the light treatment. Analyses were performed at the indicated time points and values represent the mean of three independent samples (±SD). Asterisks represent significant changes (p-value <0.05, Pair Wise Fixed Reallocation Randomization Test© performed by REST©) of the DBMIB treated samples compared to the respective control samples and, when available, also compared to the respective DBMIB+DCMU treated samples. LHCX4 was significantly up-regulated in the presence of DBMIB compared to the control conditions at each time point. As this

Fig. 2 :Fig. 3 :Fig. 4 :Fig. 6 :

 2346 Fig.2: Pigment evolution during the mHL (A) and HL (B) experiment. In A), the recovery period was performed at darkness, while in B) recovery was monitored at LL conditions.After 4 h of recovery from HL, cells were again exposed to 1 h of HL treatment. Values are the mean of three (A) and five (B) independent experiments (±SD).

Fig. 7 :

 7 Fig. 7: Relative gene expression of LHCX1 (A), LHCX2 (B), LHCX3 (C), and LHCX4 (D) during illumination with 100 µmol photons m -2 s -1 (LL 100 ). Relative LHCX transcription levels referring to 18S gene expression were normalized to the initial amount. White: no chemical treatment; dark grey: 1 µM DBMIB was applied 10 min before the light treatment; light grey: in addition to DBMIB 10 µM DCMU was applied 10 min before the light treatment. Analyses were performed at the indicated time points and values represent the mean of three independent samples (±SD). Asterisks represent significant changes (p-value <0.05, Pair Wise Fixed Reallocation Randomization Test© performed by REST©) of the DBMIB treated samples compared to the respective control samples and, when available, also compared to the respective DBMIB+DCMU treated samples. LHCX4 was significantly up-regulated in the

  Relative gene transcript levels of LHCX1, LHCX2, LHCX3 and LHCX4 during exposure at 2000 or 500 µmol photons m -2 s -1 . Relative LHCX transcription levels referring to 18S gene expression were normalized to the initial amount at time point 0, which was accordingly 1 (not indicated). As the relative gene expression of LHCX3 was much higher than the relative gene expression of all other three LHCX genes, an extra y-scale on the right was introduced. When indicated (+DCMU), DCMU (10 µM) was applied to the samples 10 min before starting the light exposure. Regarding the DCMU 500 µmol photons m -2 s -1 conditions, only time points 0 and 30 min of only one sample were analyzed. Values of the DCMU 2000 µmol photons m -2 s -1 dataset represent the mean of two independent samples (±SD). Regarding control conditions, only time points 0 and 60 min of two independent samples were analyzed, as a more detailed analysis was already performed under these conditions presented in Fig.1.

  ;Juhas and Büchel 2012), we hypothesize that de novo synthesized Dd+Dt molecules bind to the newly synthesized LHCX proteins conferring a higher NPQ. Interestingly, PsbS, which is responsible for NPQ in land plants, binds no xanthophylls (Dominici et al. 2002;Bonente et al. 2008), while the LHCX related LHCSR3 in Chlamydomonas does (Bonente et al. 2011).

Acknowledgments

We thank Christian Wilhelm and Lars Dietzel for valuable discussions and Graham Peers for providing the LHCSR antibody.

The authors thank the German Academic Exchange Service (DAAD) for a postdoctoral grant to BL, the CNRS and the French consortium CPER 'Littoral' (to JL and BL), the DFG (grant LA-2368/2-1 to JL, grant KR-1661/7-1 to PGK), the ANR (grant DiaDomOil ANR-12-BIME-0005-03 to AF) and the Universität Konstanz for their financial support.

0 : 0 0 3 : 0 0 6 : 0 0 9 : 0 0 1 2 : 0 0 1 5 : 0 0 1 8 : 0 0 2 1 : 0 0 0 : 0 0 3 : 0 0 6 : 0 0 9 : 0 0 0 : 0 0 3 : 0 0 6 : 0 0 9 : 0 0 1 2 : 0 0 1 5 : 0 0 1 8 : 0 0 2 1 : 0 0 0 : 0 0 3 : 0 0 6 : 0 0 9 : 0 0 0 : 0 0 3 : 0 0 6 : 0 0 9 : 0 0 1 2 : 0 0 1 5 : 0 0 1 8 : 0 0 2 1 : 0 0 0 : 0 0 3 : 0 0 6 : 0 0 9 : 0 0 Supplemental Table 1: Primer sequences used for the gene expression analysis.