Changes in Relative Thylakoid Protein Abundance Induced by

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Changes in relative thylakoid protein abundance induced by fluctuating light in the diatom Thalassiosira pseudonana Irina Grouneva, Dorota Muth-Pawlak, Natalia Battchikova, and Eva-Mari Aro J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00124 • Publication Date (Web): 30 Mar 2016 Downloaded from http://pubs.acs.org on April 3, 2016

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Journal of Proteome Research

Changes in relative thylakoid protein abundance induced by fluctuating light in the diatom Thalassiosira pseudonana

Irina Grouneva‡, Dorota Muth-Pawlak‡, Natalia Battchikova‡, Eva-Mari Aro‡,*

‡

Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, FI-20520, Finland *

Corresponding author

E-mail addresses: [email protected] [email protected] [email protected]

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Corresponding author: Prof. Dr. Eva-Mari Aro Tykistökatu 6, BioCity A, 6th floor, room 6207, FI-20520 Department of Biochemistry Molecular Plant Biology University of Turku, Finland E-mail: [email protected] Tel. 00358-2-333 5931

Notes The authors declare no competing financial interest.


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Abstract

One of the hallmarks of marine diatom biology is their ability to cope with rapid changes in light availability due to mixing of the water column and the lens effect. We investigated how irradiance fluctuations influence the relative abundance of key photosynthetic proteins in the centric diatom Thalassiosira pseudonana by means of mass spectrometry-based approaches for relative protein quantitation. Most notably, fluctuating light (FL) conditions lead to a substantial overall upregulation of light-harvesting complex proteins as well as several subunits of photosystems II and I. Despite an initial delay in growth under FL, there were no indications of FL-induced photosynthesis limitation, in contrast to other photosynthetic organisms. Our findings further strengthen the notion that diatoms use a qualitatively different mechanism of photosynthetic regulation in which chloroplast-mitochondria interaction has overtaken crucial regulatory processes of photosynthetic light reactions that are typical for survival of land plants, green algae and cyanobacteria.

Keywords: diatoms, fluctuating light, thylakoid proteome, photosynthesis regulation, relative protein quantitation, mass spectrometry



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Introduction

Diatoms are a highly successful group of aquatic photosynthetic eukaryotes responsible for up to 20% of global photosynthesis and therefore playing a significant role in shaping Earth’s climate.1,2,3,4 Due to the turbulent nature of their habitat, marine diatom species have developed numerous strategies for coping with changes in light and nutrient availability. Previous studies have already examined global changes in relative protein abundance in response to iron limitation5,6 and CO2 availability7 by means of datadependent mass spectrometry (MS)-based methods. The full sequencing of two genomes by 20088,9 made diatoms largely accessible to large-scale, high-throughput techniques, including mass spectrometry analysis. In the present study, this potential was utilized in order to investigate the influence of FL intensity on the abundance of photosynthetic proteins. Studies of FL effects on diatoms are highly relevant because of the light properties in aquatic environments. They include a rapid vertical mixing of water layers as well as the lens effect of waves, as a consequence of which phytoplankton can experience high intensity pulses of light on a short timescale.10 Photosynthetic organisms harvest the energy of the sun by means of pigment-protein complexes, also called light-harvesting antennae (Lhcs). Two photosystems (PSII and PSI) further convert this energy into a multi-component electron flow, resulting in the production of reducing equivalents in the form of NADPH and the generation of a proton gradient driving ATP synthesis. Both are required for the subsequent chloroplast-based carbon assimilation. The cell’s capacity for NADPH and ATP production as well as the metabolic demand for these compounds, however, can vary at any given time. On the production side, one reason can be an imbalance between the activation states of PSII and 4 ACS Paragon Plus Environment

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the PSI acceptor side during transitions from dark to light as well as upon rapid changes in light intensity or quality. Consumption of NADPH and ATP depends mainly on the capacity for carbon and nitrogen assimilation. This is why the adjustment of ATP/NADPH ratios in the chloroplast by modulating electron flow is one of the central mechanisms of photosynthesis regulation. In higher plants and green algae, cyclic electron transport (CET11) aids the balance of NADPH production and ATP synthesis. In CET, electrons are diverted towards cycling around PSI rather than reduction of NADP+ while still generating a proton gradient and ATP. Among the most universal and well-studied CET mechanisms are the multi-component protein complex NAD(P)H dehydrogenase (NDH12,13,14) and the PGRL/PGR5 proteins.15 In addition to CET, cyanobacteria, green algae and mosses contain flavodiiron (Flv) proteins16, which are responsible for diverting electrons to alternative acceptors thus preventing oxidative damage to the photosystems. A recent study in the diatom Phaeodactylum tricornutum17, however, showed that chloroplast-mitochondria interaction, and not plastid-localised CET, regulates NADPH/ATP ratios in this organism. It expanded on earlier reports of a close spatial and metabolic interaction between chloroplasts and mitochondria in diatoms.18 In detail, 10% of photosynthetic electron transport is re-routed to the mitochondrion during light phases independent of illumination intensity. In the dark, the process is partially reversed: ATP hydrolysis by the chloroplast ATPase drives a sustained proton motive force across the thylakoid membrane. A mitochondrial alternative oxidase (AOX), responsible for cyanide-insensitive respiration, was shown to be a main component behind this exchange and therefore vital for optimization of photosynthesis. Diatom AOX-driven oxygen consumption was estimated to be responsible for 50% of dark respiration and have a prominent role in the light as well.17 5 ACS Paragon Plus Environment


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An important question remains how diatoms acclimate to strongly fluctuating irradiances and how photosynthesis is regulated under these conditions. The effects of FL on growth, development and photosynthetic capacity in Arabidopsis19,20, cyanobacteria21 and the diatom P. tricornutum22 have already been studied. The latter focused mainly on carbon assimilation capacity of diatoms compared to green algae under two different light regimes, which, however, also differed in overall light irradiance throughout the day. We, on the contrary, made a point of choosing an approach in which the overall photon dosage in both FL and continuous light (CL) remained the same during the course of the day. Our FL regime had a lower background illumination but was interrupted by pulses of high light (HL) every five minutes. Nevertheless, Wagner et al.22 already provided evidence for the superior capacity of FL-grown diatoms for biomass production per absorbed photon. They put this effect down to differences in electron cycling around PSII.22 A previous study on Arabidopsis23 postulated a negative effect of FL on PSI, not PSII, and showed a central role for PGR5 in PSI photoprotection. In cyanobacteria, once again, PSI was discovered to be the main target of photodamage under FL but, unlike in Arabidopsis, it was protected by the activity of the Flv1 and Flv3 proteins21, not PGR5. Diatoms have detectable levels of PGR524 but do not encode Flv proteins.25,16 Neither do diatoms encode any NDH subunits homologues.24 In order to get further insights into the dynamic regulation of light reactions in diatoms, we specifically addressed the difference between fluctuating growth light and the same dosage of constant light in modulation of the proteome of the photosynthetic apparatus in T. pseudonana.

Experimental procedures 6 ACS Paragon Plus Environment

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Cell culture and growth conditions T. pseudonana CCMP1335 (obtained at the Provasoli-Guillard National Centre for Culture of Marine Phytoplankton) was grown as batch cultures at 16°C in a light-dark regime of 14-10 h in f/2 medium supplemented with vitamins.26,27 The growth medium was supplemented with 1.06 x 10-4 M Na2SiO3. Prior to experimental light conditions, cells were maintained at a light intensity of 50 µmol photons m-2 s-1 (growth light, GL). During the experiment, cultures were grown under either a fluctuating light (FL) regime of a background irradiance of 50 µmol photons m-2 s-1 interrupted by one minute of 330 µmol photons m-2 s-1 every five minutes, or under constant intensity of 95 µmol photons m-2 s-1 (CL). In both FL and CL regimes, the sum of total light intensity during the day was equal. This was done in order to avoid any effects induced by a higher overall photon dosage rather than FL. Cultures were only grown under FL or CL for about one week in order to study the immediate effects of light intensity, not long-term adaptation.

Sample preparation Cultures for MS experiments were harvested 2 h after onset of daily illumination. Cultures in the late exponential growth phase (OD750nm = 0.27-0.32) were harvested by centrifugation at 20 000 g for 5 min at 4°C. On average, this corresponded to day 5 for CL cultures and day 7 for FL cultures. The cell pellets were immediately frozen in liquid nitrogen and kept at -80°C until further use. Lysis buffer (50mM Tris-HCl, pH 6.8, 2% SDS) was added to still frozen samples. After mixing, they were incubated at RT for 30 min. Protein content was measured (Direct Detect® Infrared Spectrometer, Merck Millipore, Darmstadt, Germany), and the equivalent of 30 µg was diluted 1:1 with 2x Laemmli buffer28 before 7 ACS Paragon Plus Environment


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being loaded on a 12% (50% acrylamide, 1.3% bis-acrylamide) stacking gel (0.5 M Tris-HCl, pH 6.8) containing 6 M urea and no SDS. In-gel reduction, cysteine modification and overnight trypsin (Gold, MS grade, Promega Madison, USA) digestion was carried out on total cell protein according to Shevchenko et al.29 with slight modifications. Samples were not heated above 33°C due to the presence of urea. A total of four biological replicates and two technical replicates for each biological replicate were subsequently analyzed by MS for every condition (CL and FL).

MS analysis Data-dependent (DDA) analysis was performed on an EASY-nLC 1000 nanoflow liquid chromatograph coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Extracted peptides equivalent to 200 ng were first loaded on a trapping column (0.3 x 5 mm PepMap C18, LC Packings) and subsequently separated inline on a 15 cm C18 column (75 µm x 15 cm, ReproSil 5 µm 200 Å C18, Dr. Maisch GmbH, AmmerbuchEntringen, Germany). The mobile phase consisted of water/acetonitrile (98:2 (v/v)) with 0.1% formic acid (solvent A) or acetonitrile/water (95:5 (v/v)) with 0.1% formic acid (solvent B). A stepped 55 min gradient (35 min 2-20% B, 15 min 20-40% B, 5 min 40-100% B and 5 min 100% B) was used to elute peptides. The Q Exactive instrument was operated with Thermo Xcalibur software (Thermo Fisher Scientific) in positive mode with spray voltage of 2.3 kV. The survey scan (MS) with the detection range of 300-2000 m/z was followed by MS/MS scan of up to ten most intense ions with charge +2 or higher with a resolution of 70000 and 17500 (m/z 200) respectively. The fragmentation was performed in HCD cell with normalized collision energy of 27%. The AGC (automatic gain control) settings were set to a maximum fill time of 120 ms and 250 ms and to obtain maximum number of 1e6 and 2e4 8 ACS Paragon Plus Environment

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ions for MS and MS/MS scans, respectively. The mass window for precursor ion selection was set to 2.0 m/z, the intensity threshold for triggering collection of MS/MS spectra was set to 2.4e2 and the underfill ratio to 0.3%. Dynamic exclusion was 10.0 s. For protein identification purposes, acquired data was searched against a custommade UniProt (down-loaded on 13.11.14) database of two fully sequenced diatom species (P. tricornutum and T. pseudonana, number of sequences: 56819), additional published diatom protein sequences, common contaminants as well as the reversed sequence decoy database using Mascot 2.4 (Matrix Science, London, UK) via Proteome Discoverer 1.4 (Thermo Fisher Scientific). The following settings were used for the database searches: maximum one missed cleavage sites, carboxymethyl (C) as static modification, oxidation (M), deamidated (NQ) and acetyl (N-term) as dynamic modifications. Precursor mass tolerance was set to 5 ppm and the fragment mass tolerance was 0.01 Da. For verification of results, the target decoy PSM validator was included in the workflow. A relaxed false discovery rate of 0.05 was set as target. Label-free quantification was performed using Progenesis QI for proteomics, LC-MS 4.0 (Nonlinear Dynamics, Newcastle upon Tyne, UK). Only proteins with ≥ 3 unique peptides and Anova values ≤ 0.05 were used for quantitation. SRM targeted analysis was performed on a TSQ Vantage (ESI-triple stage quadrupole, Thermo Scientific) instrument operating in the positive ion mode with a capillary temperature of 270°C, spray voltage of +1600 V and collision gas pressure of 1.2 mTorr. Measurements in unscheduled mode were performed with a cycle time of 2.0 s and a dwell time of 25 ms. The Q1 and Q3 peak width was set to 0.7 unit resolution (FWHM) and the isolation width was 0.002 m/z. Indexed retention time (iRT) peptides (Biognosis) were added to the samples. The peptide library was generated by importing DDA results to the Skyline 9 ACS Paragon Plus Environment


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software30 in order to generate SRM transitions. The chromatographic separation of the peptides for SRM was carried out using a similar nanoLC system to the one connected to the Q Exactive instrument (Easy-nLC, Thermo Scientific). Eight proteins were targeted. The total number of transitions was 150. Scheduled runs had a cycle time of 1.8 s, resulting in a minimum dwell time of 45 ms. For detailed information on peptide sequences, specific collision energies and transitions see supplementary table S2.

Variable chlorophyll fluorescence measurements (dualPAM) Fluorescence was measured throughout the morning and cells were taken from growth conditions immediately before measurement. 5 ml of culture corresponding to a cell density of 0.27-0.32 OD were rapidly transferred onto a filter, covered in foil to prevent drying out and dark-adapted for 5 min. A WALZ dualPAM instrument (Walz Mess- und Regeltechnik, Effeltrich, Germany) was used in dual mode (fluorescence and P700 redox kinetics) to record changed in variable chlorophyll fluorescence and the relative absorption changes associated with reduction and oxidation of PSI, measured as absorption changes at 830 nm in transmission mode. After 5 min in the dark Fm was recorded. After darkadaptation cells were exposed to 10 min of HL (650 µmol photons m-2 s-1) and 10 min of subsequent darkness. NPQ was calculated as (Fm-Fm’)/Fm’. On average, PSII yield was 0.650.7 for both CL and FL cultures.

HPLC pigment analysis Photosynthetic pigments were extracted from frozen pellets with 100% acetone. Samples were centrifuged and the supernatant was filtered through a 0.2-μm polytetrafluoroethylene syringe filter. Pigments were analyzed by high-performance liquid 10 ACS Paragon Plus Environment

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chromatography (HPLC, Agilent 1100 Series, Agilent Technologies, Palo Alto, CA) equipped with a diode array detector and a reverse phase C18 column (4 × 125 mm, 5 μm, LiChroCART, Merck KGaA, Darmstadt, Germany). Two buffers (A and B) were used consecutively at a constant flow rate of 0.5 ml min−1. An isocratic run with buffer A, consisted of acetonitrile/methanol/0.1 M Tris-HCl buffer adjusted to pH 8.0 (72:8:3, v/v), for 4 min was followed by a linear gradient of buffer B from 0 to 100% for 15 min. Buffer B consisted of methanol/hexane (4:1, v/v). Pigments were identified based on retention times and absorption spectra compared to an in-house database.

Results and Discussion

In order to eliminate effects related to differences in the amount of available light quanta, the growth conditions of T. pseudonana were designed in a way that the overall photon dosage in both CL and FL remained the same during the course of the day, similarly to the study performed on Arabidopsis.23 Compared to CL, the FL regime had a lower background illumination but was interrupted by pulses of HL every five minutes (see Experimental Procedures for details).

Physiological characterisation Initially, T. pseudonana demonstrated slower growth under FL compared to CL but after a lag phase of around two days, FL cultures were able to reach the same cell densities as the ones grown under CL (figure 1). However, a clear difference was also observed in the colouration of the cultures. FL-grown cultures were dark brown in colour while CL cultures were pale brown. Absorption spectra of intact cells revealed that FL-grown cells contained 11 ACS Paragon Plus Environment


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more overall pigment than CL cultures at the same cell density (OD750nm = 0.3, figure 2). Thus, FL caused significant changes in cellular metabolism manifested in retardation of initial growth and later on in increased amounts of pigments. The photosynthetic performance of CL- and FL-grown cells was assessed by measuring specific physiological parameters. We monitored PSII quantum yield, non-photochemical quenching (NPQ) and PSI (P700) photooxidation capacity. PSII yield, a parameter correlating with photosynthesis activity and integrity of PSII, was comparable between CL and FL cultures at 0.65-0.7. This lead to the conclusion that FL did not cause any limitation upon nor net damage to the PSII pool. Non-photochemical quenching comprises a class of photoprotective mechanisms widely distributed among photosynthetic organisms and can be measured by monitoring changes in variable fluorescence. Compared to higher plants, diatoms are known to show high induction rates and levels of NPQ, making it one of their predominant photoprotective mechanisms.31 One major mechanism of NPQ is triggered by low lumenal pH and the presence of specific de-epoxidized carotenoids (zeaxanthin in higher plants and predominantly diatoxanthin in diatoms).32,33 We found no significant differences in the kinetics or intensity of NPQ between FL and CL cultures (figure 3). This was an indication that FL did not induce an upregulation of photoprotective mechanisms and probably did not lead to a higher acidification of the lumen. It was also tested whether the FL regime used in this study had any adverse effects on the functionality of PSI. In diatoms, the capacity for P700 oxidation can be measured after application of a saturating light pulse. A P700 oxidation curve presents a relative estimate of the amount of photooxidizable, and therefore active, PSI units as well as the redox state of the PSI acceptor side. FL cultures seemed to have a higher amount of active PSI units (figure 12 ACS Paragon Plus Environment

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4). Apart from that, no differences in the kinetics of oxidation, initial re-reduction by electrons from PSII and subsequent steady-state oxidation was observed between CL and FL cultures (figure 4). This lead to the conclusion that PSI was not a target for damage under FL.

Proteomics We proceeded to investigate the overall effect of FL on the proteome of T. pseudonana, with a special focus on photosynthetic proteins. Due to sample complexity, a two-step isolation protocol was used, involving cell lysis and in-gel protein clean-up followed by trypsin digestion.29 With this method, we were able to obtain reliable data with good sequence coverage for proteins of interest. MS analysis of non-fractionated, total protein samples yielded around 1380 protein identifications based on at least two peptide of middle and high confidence, with FDR ≤ 0.05. This represented ca. 11.7% of the total predicted proteome of T. pseudonana. The obtained proteome coverage was comparable to previous studies on unfractionated samples.5,6 In the present study, 562 proteins were quantified based on two or more unique peptides and Anova values ≤ 0.05 (p-value). The remaining proteins had either only one unique, good-quality peptide or the overall differences in peptide abundance between CL and FL was not statistically significant (high p-values). Out of the 562 quantified proteins, 162 displayed a differential regulation of ≥ 0.58-fold (supplementary table S1). Relative abundance of photosynthesis-related proteins in FL cultures in comparison to CL conditions is summarised in table 1. In some ambiguous cases, additional information on subcellular localisation is also provided. The plastid proteome prediction by Gruber et al.34 was used to verify that proteins were indeed targeted to the chloroplast. In addition to photosynthesis-related proteins, we were further interested in possible changes in 13 ACS Paragon Plus Environment


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mitochondrial proteins because of the recently shown close metabolic exchange between the two organelles in adjusting photosynthetic ATP/NADPH ratios.17 Relative abundances of mitochondrial proteins are therefore included in table 1 as well. In order to confirm the results obtained with the method described above, the quantitation for several selected proteins was verified using a targeted approach called selected reaction monitoring (SRM).35,36,37 The results of the targeted approach, summarised in table 2, correlated well with data obtained by global quantitation, confirming the overall tendency and the extent of up- or downregulation of proteins presented in table 1. In particular, SRM verified FL-induced downregulation of the Lhcx6 protein even though only one peptide could be targeted. Our simplified approach proved to be suitable for large scale, rapid screening of overall photosynthetic protein changes occurring in different growth conditions and thus for identifying targets for more detailed studies.

Lhc and Lhc-like proteins The most pronounced differences in the proteome between FL- and CL-grown diatoms were observed in the abundance of Lhc and Lhc-like proteins, also called fucoxanthinchlorophyll a-binding proteins (FCPs). They are integral thylakoid membrane proteins containing three transmembrane helices and binding chlorophylls and carotenoids.38 Functionally, these proteins are separated into three main groups in diatoms: i) Lhcf, forming the main, detachable antennae, which can serve both photosystems, ii) Lhcr, mainly comprising the PSI-specific antennae, and iii) Lhcx, proteins involved in photoprotection and structural integrity. For a recent review on diatom Lhcs see Gundermann and Büchel.38 In total, 29 Lhcs and Lhc-like proteins were quantified in the present study. Lhcfs and Lhcrs, the 14 ACS Paragon Plus Environment

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most abundant and thus extensively studied FCPs, were substantially upregulated under FL (1.89-0.68-fold). Likewise, two representatives of the Lhcx group, Lhcx5 and Lhcx6_1, were significantly upregulated in FL. Intriguingly, two Lhc proteins, Lhcr5 and Lhcx6, displayed a distinct difference in expression compared to all other Lhcs. Lhcx6 showed 1.49-fold downregulation in FL compared to CL. A study by Zhu and Green39 on mRNA level revealed a possible involvement of Lhcx6 in photoprotection in HL. Their study showed upregulation of Lhcx6 transcripts in response to a shift to HL. In the present study, on the contrary, the Lhcx6 protein was downregulated in FL as compared to CL and the results of the global analysis on this protein were confirmed by SRM (table 2). This provided evidence that the background growth light, rather than the HL pulse in the FL illumination condition, determines the acclimation strategy of diatoms to FL, as also observed for cyanobacteria.21 Thus, the strong downregulation of the Lhcx6 protein might indicate less need for photoprotection under FL. The other differently behaving protein, Lhcr5, is likely to be a PSI antenna protein.38 It was 1.81-fold downregulated in FL compared to CL, in variance with the general increase of many other antenna proteins under FL. Thus, the specific role of Lhcr5 in the complexity of diatom Lhc proteins remains to be elucidated. Additionally, ten further Lhc-like proteins of unknown function and lower homology were distinctively upregulated in FL compared to CL (table 1). Since our knowledge on acclimation strategies of diatoms to varying light environments is only emerging, it remains to be confirmed whether diatoms rely on yet different mechanisms than so far revealed for cyanobacteria, algae and plants.16 Lhc proteins contain pigment-binding sites, and the overall increase in their abundance under FL was accompanied by an increase in the entire pigment pool (figure 2). 15 ACS Paragon Plus Environment


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This was in agreement with an observed upregulation of enzymes involved in pigment biosynthesis (table 1). Qualitatively, fucoxanthin to chlorophyll a ratios were found to remain the same between CL and FL cultures (0.879 ± 0.22 in CL and 0.850 ± 0.09 in FL). The only qualitative difference was observed in a lower level of diadinoxanthin (Ddx) in FL-grown cells (Ddx/Chl a = 0.677 ± 0.2 compared to 0.365 ± 0.02 in FL). This further corroborates the conclusion that diatoms grown in FL experienced a lesser need for photoprotection. However, the higher level of Ddx observed in CL-grown cells seems not to be involved in non-photochemical energy dissipation, since NPQ levels were similar in CL and FL cultures (figure 3). Our knowledge on the organization of Lhc proteins in the thylakoid membrane of diatoms is far from complete, and more data is needed to present appropriate models of their localisation and function. Nonetheless, our data provide compelling evidence of the plasticity of diatom acclimation to changing light environments, which largely relies on dynamics in the expression of various Lhc proteins.

PSII/PSI The multi-protein PSII complex is the site of water oxidation, resulting in oxygen evolution and supply of electrons for the photosynthetic electron transport chain (reviewed by McEvoy and Brudvig40). Our results demonstrated that in T. pseudonana, PSII subunits, and especially the proteins of the oxygen evolving complex (OEC), were upregulated under FL (table 1). This suggested a higher photosynthetic capacity of FL-grown cultures. The abundance of two proteases involved in PSII repair (FtsH A0T0S3 and B8BVM2), however, was unchanged in FL, indicating that short HL pulses did not induce higher rates of PSII turnover.


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The PSI complex supplies electrons to ferredoxin, which in turn serves as a substrate for the ferredoxin-NADP+-reductase (FNR). FNR is an enzyme responsible for the reduction of NADP+ to NADPH. Under FL, PSI was modified in a slightly different way compared to PSII. Amounts of PsaA and PsaB, the two proteins forming the core of PSI, were not changed substantially in global analysis (although SRM analysis on PsaA showed upregulation of 0.77fold, see table 2). Yet the peripheral subunits of the acceptor side of PSI, PsaL, PsaD and PsaE, were significantly upregulated in FL growth conditions (see table 1). Two further proteins, B8BUW3 and B8BRQ8, that might be associated with PSI24 were likewise upregulated under FL. These proteins display a low homology to any proteins outside the diatom clade and their function remains to be resolved. Moreover, B8BUW3 appears to be specific to centric diatoms and has no homologue in P. tricornutum. Nevertheless, both proteins have been identified in native gels as components of the diatom thylakoid membrane.24 While there are detailed studies on PSII repair and turnover in diatoms,41 little is known about the dynamics of PSI, apart from the fact that its subunit composition differs from higher plants.42 Diatoms lack subunits PsaG, H, K, N, O and P but encode an additional subunit, PsaM. It therefore remains conceivable that PSI employs unknown auxiliary proteins and is less susceptible to damage compared to PSI of higher plants and green algae. Unfortunately, the quantitation of the plastid-targeted isoform of FNR (B8CGL9), which operates as electron acceptor from ferredoxin, was not statistically significant in our dataset. Nevertheless, two other FNR isoforms, B8C0N7 and B8C4K5, were identified and quantified in the present study. Neither of them was differentially regulated in FL. The closest annotated homologues for the two proteins were the root-type isoforms in higher plants. In higher plants, root-type FNRs are localised within non-photosynthetic plastids.43 It is of note that the above two proteins lack a clear plastid targeting signal sequence and 17 ACS Paragon Plus Environment


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were classified as non-plastid by Gruber et al.34 Therefore, the function of these two FNRlike proteins remains to be clarified. The dynamics and flexibility of both PSII and PSI subunit composition appears to play an intriguing role in acclimation of diatoms to changing growth environments. However, further efforts are necessary to investigate the functional organisation, and possible formation of super- and megacomplexes, in the thylakoid membrane of diatoms.

Cyt b6f The cytochrome b6f complex is the third major component of the electron transport chain. Three known subunits of the complex, PetA (cytochrome f), PetB (cytochrome b6) and PetC (the Rieske protein), were successfully quantified. No differential regulation was observed for these proteins (table 1). Thus, unlike PSI and PSII, the Cytb6f complex maintained similar levels in FL and CL growth conditions. A second PetC isoform encoded in the T. pseudonana genome (designated as PetC2), which lacks a targeting signal sequence, was not identified on the protein level in the present study. Our dataset contained a further 2Fe-2S Rieske-type protein (B8CBE4) with possible relevance for the Cytb6f complex, however. On the one hand, this protein contains a plastid targeting signal sequence and showed upregulation under FL in our study. Therefore, it could be speculated that this additional protein might be part of a sub-population of Cytb6f complexes with a separate, regulatory role in photosynthetic electron flow. On the other hand, it also matches nitrite reductase domain features and its localisation within the chloroplast is unknown. In Synechocystis, for example, two out of three present PetC isoforms were found to be localised in the thylakoid membrane while a third one was found


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in the cytoplasmic membrane.44 Therefore, it is also possible that protein B8CBE4 might not be part of the thylakoid membrane.

Photosynthesis regulation. CET Recent findings suggested that CET (especially PSI-mediated CET) does not play any significant role in photosynthesis regulation in diatoms.17 Therefore, two proteins known to be involved in CET in plants,15 PGR5 and PGRL, were of particular interest in our study. Both proteins, which are also present in the thylakoid membrane of diatoms,24 were identified and quantified. Our global study revealed that both proteins were slightly upregulated in FL; however, the fold change was lower than the accepted threshold of 0.58. Therefore, we quantified them also using the SRM approach. While PGR5 showed no significant changes with either method (0.49 in global analysis and 0.38 in SRM), the PGRL protein showed a 0.68-fold upregulation in SRM. Based on these results, we cannot entirely exclude the possibility of PGRL playing a role in acclimation of T. pseudonana to FL conditions. Consequently, even though PGR5/PGRL was shown not to be involved in CET,17 these proteins might still play a protective or structural role in diatoms and their function in photosynthesis remains to be elucidated.

Photosynthesis regulation. Mitochondria In a recent study, Bailleul et al.17 described that in P. tricornutum the NADPH/ATP ratio is regulated by chloroplast-mitochondria interactions. A mitochondrial alternative oxidase (AOX) was further shown to be vital for optimisation of photosynthesis. In T. pseudonana, the closest match to the mitochondrial AOX protein characterised by Bailleul et al.17 is the AOX2 (B8CF38). This protein showed 0.49-fold downregulation in FL, which was not 19 ACS Paragon Plus Environment


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substantial according to selected criteria. The lack of changes in relative abundance of AOX2 in our experiment points towards constitutive levels of AOX irrespective of light conditions. This matches findings by Bailleul et al.17 who demonstrated that AOX activity did not vary with light intensity, further stressing its crucial role under all light conditions. The second AOX isoform present in T. pseudonana (AOX1_2) was not detected in our dataset. Further, the mitochondrial Rieske protein B8BX01 as well as two cytochrome c oxidase subunits (Cox), all representatives of the respiratory electron transport chain, were identified and quantified in our study. They too remained unchanged in FL. Three soluble mitochondrial proteins significantly changed their abundance under FL. Two enzymes involved in phosphoenolpyruvate (PEP) conversion demonstrated reverse regulation. PEP carboxykinase, responsible for converting oxaloacetate into PEP and CO2, showed upregulation (0.58-fold). In contrast, PEP carboxylase, responsible for oxaloacetate synthesis, showed downregulation (0.93-fold). Taken together and assuming a correlation between protein abundance and activity, the above observations indicate a depletion of oxaloacetate and an increased demand for PEP in the mitochondria under FL. Further, a malate dehydrogenase (MDH1), converting malate into oxaloacetate, was found to be upregulated in our dataset (0.05-fold). According to Kustka et al.7 this enzyme could be localised either in the chloroplastic endoplasmic reticulum (CER), also called periplastidic space (PPS), or the mitochondrion. While no conclusions on metabolic exchange can be drawn from enzyme abundance alone, our results allow to envisage a pathway where malate, generated in the chloroplast, is converted to oxaloacetate and further to PEP in the mitochondria, effectively functioning as a “malate valve”.45 Such a pathway might indeed be part of the machinery for exchanging reducing equivalents between chloroplast and mitochondria. This possibility was already discussed previously.18,17 However, a possible 20 ACS Paragon Plus Environment

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overlap with another metabolic pathway is worth mentioning briefly at this point. The enzymes described above might also participate in a C4 photosynthesis-like carbon concentrating mechanism.7 Ever since an initial report by Reinfelder et al. in 200046 on the diatom Thalassiosira weissflogii, there has been a discussion if C4 photosynthesis operates in diatoms. A body of more recent evidence refuted such a possibility, at least in T. pseudonana.47,48,49,50 However, this is to highlight that enzymes involved in C4 interconversion in diatoms might be situated at the cross-section of competing processes, including amino acid metabolism in the mitochondria.47 Taken together with the uncertainty of their subcellular localisation, the above results should be interpreted with caution. An intriguing line of future investigation could be a closer look into the protein composition of diatoms under prolonged dark periods during which the mitochondria-chloroplast exchange might be reversed.17

Acknowledgements Research was financially supported by the Academy of Finland Centre of Excellence Project number 271832 (E.M.A.), the Academy of Finland Project number 273870 (E.M.A.) as well as a personal Academy of Finland grant 250216 (I.G.). We are grateful to Biocenter Finland and the Proteomics facility of the Turku Center for Biotechnology (BTK) for the possibility to run our MS measurements and technical support.

Supporting information Table S1. Full list of quantified proteins, global analysis, ratios between FL and CL, Progenesis QI output



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Table S2. List of peptide sequences, transitions and collision energies used in SRM experiment

References

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(21) Allahverdiyeva, Y.; Mustila, H.; Ermakova, M.; Bersanini, L.; Richard, P.; Ajlani, G.; Batchikova, N.; Cournac, L.; Aro, E.-M. Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light. Proc. Natl. Acad. Sci. USA 2013, 110, 4111-4116. (22) Wagner, H.; Jakob, T.; Wilhelm, C. Balancing the energy flow from captured light to biomass under fluctuating light conditions. New Phytol. 2006, 169, 95-108. (23) Suorsa, M.; Järvi, S.; Grieco, M.; Nurmi, M.; Pietrzykowska, M.; Rantala, M.; Kangasjärvi, S.; Paakkarinen, V.; Tikkanen, M.; Jansson, S.; Aro, E.-M. PROTON GRADIENT REGULATION5 is essential for proper acclimation of Arabidopsis photosystem I to naturally and artificially fluctuating light conditions. Plant Cell 2012, 24, 2934-2948. (24) Grouneva, I.; Rokka, A.; Aro, E.-M. (2011). The thylakoid membrane proteome of two marine diatoms outlines both diatom-specific and species-specific features of the photosynthetic machinery. J. Proteome Res. 2011, 10, 5338-5353. (25) Zhang, P.; Allahverdiyeva, Y.; Eisenhut, M.; Aro, E.-M. Flavodiiron proteins in oxygenic photosynthetic organisms: Photoprotection of photosystem II by Flv2 and Flv4 in Synechocystis sp. PCC 6803. PLoS One 2009, 4, e5331. (26) Guillard, R. R. L.; Ryther, J. H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 1962, 8, 229-239. (27) Guillard, R. R. L. Culture of phytoplankton for feeding marine invertebrates. In Culture of Marine Invertebrate Animals, Smith W.L. and Chanley M.H., Eds; Plenum Press: New York, 1975; pp 26-60. (28) Fling, S. P.; Gregerson, D. S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris buffer system without urea. Anal. Biochem. 1985, 155, 83-88. 25 ACS Paragon Plus Environment


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Advances in Photosynthesis and Respiration, Hohmann-Marriott, M. F., Ed.; Dordrecht: Springer Science+Business Media: Dordrecht, 2014; 39, pp 21-37. (39) Zhu, S.-H.; Green, B. R. Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress. Biochim. Biophys. Acta 2010, 1797, 1449-1457. (40) McEvoy, J. P.; Brudvig, G. W. Water-splitting chemistry of photosystem II. Chem. Rev. 2006, 106, 4455-4483. (41) Wu, H.; Roy, S.; Alami, M.; Green, B. R.; Campbell, D. A. Photosystem II photoinactivation, repair, and protection in marine centric diatoms. Plant Physiol. 2012, 160, 464-476. (42) Jensen, P. E.; Bassi, R.; Boekema, E. J.; Dekker, J. P.; Jansson, S.; Leister, D.; Robinson, C.; Scheller, H. V. Structure, function and regulation of plant photosystem I. Biochim. Biophys. Acta 2007, 1767, 335-352. (43) Hanke, G. T.; Kurisu, G.; Kusunoki, M.; Hase, T. Fd:FNR electron transfer complexes: evolutionary refinement of structural interactions. Photosynth. Res. 2004, 81, 317-327. (44) Schultze, M.; Forberich, B.; Rexroth, S.; Dyczmons, N. G.; Roegner, M.; Appel, J. Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechcystis sp. PCC 6803. Biochim. Biophys. Acta 2009, 1787, 1479-1485. (45) Scheibe, R. Malate valves to balance cellular energy supply. Physiol. plantarum 2004, 120, 21-26. (46) Reinfelder, J. R.; Kraepiel, A. M. L.; Morel, F. M. M. Unicellular C4 photosynthesis in a marine diatom. Nature 2000, 407, 996-999.



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(47) Tanaka, R.; Kikutani, S.; Mahardika, A.; Matsuda, Y. Localization of enzymes relating to C4 organic acid metabolisms in the marine diatom, Thalassiosira pseudonana. Photosynth Res. 2014, 121, 251-263. (48) Roberts, K.; Granum, E.; Leegood, R. C.; Raven, J. A. C3 and C4 pathways of photosynthetic carbon assimilation in marine diatoms are under genetic, not environmental, control. Plant Physiol. 2007, 145, 230-235. (49) Clement, R.; Dimnet, L.; Maberly, S C.; Gontero, B. The nature of the CO2concentrating mechanisms in a marine diatom, Thalassiosira pseudonana. New Phytol. 2016, 209, 1417-1427. (50) Kroth, P. G.; Chiovitti, A.; Gruber, A.; Martin-Jezequel, V.; Mock, T.; Schnitzler Parker, M.; Stanley, M. S.; Kaplan, A.; Caron, L.; Weber, T.; Maheswari, U.; Armbrust, E. V.; Bowler, C. A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis. PLoS One 2008, 1, e1426.

Table 1. Relative changes in abundance of photosynthetic proteins in T. pseudonana grown under continuous light (CL) or fluctuating light (FL). Ratios between FL and CL (given in bold) are expressed as log2(FL/CL) and based on label-free quantitation of nonfractionated samples (Progenesis QI, ≥ 2 peptides, Anova (p-value) < 0.05). Upregulation of ≥ 0.58 is marked in red; downregulation of ≤ -0.58 is marked in green. LHC: light-harvesting complex; PS: photosystem; FNR: Ferredoxin-NAD(P)+-oxidoreductase; CET: cyclic electron transport. Description/putative function

UniProt ID T. pseudonana

Fold change log2(FL/CL)

LHC 28 ACS Paragon Plus Environment

Spectral Anova (pcount/unique value) peptides

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Lhcf9 Lhcf4 Lhcf1 Lhcf11 Lhcf5 Lhcf10 Lhcf6 Lhcr12 Lhcr4 Lhcr1 Lhcr3 Lhcr7 Lhcr14 Lhcr10 Lhcr5 Lhcx5 Lhcx6_1 Lhcx6 Lhc-like FCP2 Lhc-like LhcA (PSI) Lhc-like FCP3 Lhc-like Lhc-like, fragment FCP-like Lhca2 Lhca6

B8BS67 B8CFG5 B8CFW3 B8BVI1 B8CEV5 B5YM25 B8BX92 B8BU32 B8C0K3 B8C8Q0 B8C2K6 B8BYV4 B8C0K4 B8C2Y4 B5YM80 B8BSG2 B5YLU3 B8CGG1 B8BX75 B8C0G1 B8C5N8 B8LE61 B8CFG4 B8CEQ3 B8CDK8 B8LE60 B8C770 B8BUU4 B8BYV3

1.89 1.89 1.85 1.34 1.07 1.07 0.77 1.2 1.14 1 1 0.85 0.68 0.68 -1.79 1 0.77 -1.43 1.81 1.32 1.32 1.2 1.07 1.07 1.07 1 0.93 0.93 0.49

15/5 12/3 14/4 4/4 15/5 3/3 12/9 4/2 2/2 4/4 3/3 4/4 6/6 2/2 2/2 2/2 4/4 4/4 2/2 2/2 2/2 3/3 13/4 2/2 5/3 3/3 2/2 2/2 4/4

0.0049 1.1x10-6 0.0003 0.0028 0.0014 3.1x10-9 9.2x10-7 0.0003 2.3x10-5 1x10-7 6.5x10-7 7.1x10-5 0.0041 0.0001 5.4x10-9 0.0011 4.3x10-7 5.6x10-6 0.0098 4.3x10-7 1.7x10-7 7.3x10-5 7.8x10-7 0.0001 1.5x10-5 2.4x10-8 0.0003 2.3x10-5 6.6x10-5

B8CC14 B8BVI4 B8C4I5 B8BSY9 A0T0T0 A0T0W2 A0T0T1 A0T0N2 B8BX97

0.93 0.93 0.85 0.85 0.68 0.68 0.58 0.49 0.38

8/8 7/6 13/12 7/7 8/6 8/8 4/3 3/3 5/5

6.6x10-8 6.3x10-6 6.7x10-6 6.1x10-7 5.2x10-7 5x10-5 4.1x10-6 0.0005 0.0003

B8LEQ8 A0T0T5 A0T0U5 A0T0U3 A0T0M8 A0T0M9 B8BUW3

1.26 0.77 0.77 0.58 0.49 0.38 1.07

3/3 6/6 4/4 4/4 11/11 7/7 2/2

5.6x10-5 5.8x10-6 4.1x10-6 0.0046 1.9x10-8 6.2x10-5 6.2x10-6

PSII Psb31 (OEC) PsbU (OEC) PsbO (OEC) PsbQ’ OEE3 (OEC) PsbD (D2) PsbA (D1) PsbC (CP43) PsbV (OEC) Psb27, PSII assembly

PSI PsaE, fragment PsaD PsaL PSI assembly Ycf4 PsaA PsaB putative PSI-associated



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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B8BRQ8

0.93

3/3

1x10-5

FNR, not plastid FNR-like, not plastid

B8C0N7 B8C4K5

0.26 -0.49

10/4 6/4

0.0377 0.0006

Cytb6/f Rieske, plastid Apocytochrome f (petA) Cytochrome b6 (petB) Rieske, 2Fe-2S (petC)

B8CBE4 A0T0R9 A0T0T6 B8BVG6

0.68 0.38 0.38 0.38

3/3 9/8 3/2 5/5

1.6x10-5 0.001 0.0067 0.0094

PGR5 PGRL

B8C035 B8BZQ3

0.49 0.38

2/2 8/8

0.0006 8.4x10-5

ATP synthetase, chloroplastic AtpF AtpD AtpA AtpB

A0T0P2 A0T0P3 A0T0P4 A0T0R6

0.49 0.49 0.26 0.26

8/6 11/9 16/10 16/15

0.0007 0.0007 0.0014 0.0061

Assembly/repair ycf90 FtsH, plastid FtsH, plastid

A0T0T8 A0T0S3 B8BVM2

0.26 -0.14 -0.26

3/3 19/17 21/21

0.031 0.0085 0.0065

Thioredoxin Thioredoxin f

B8BVB7

0.68

2/2

1.3x10-6

A0T0U6 B8BWB1

0.58 0.49

9/8 5/3

0.0005 0.0002

A0T0N5 B8BXS1

0.38 0.38

3/2 7/4

0.0184 0.0003

B8C163 B8BQU2 A0T0N6 B5YN92

0.38 0.26 0.14 0.14

7/7 16/15 26/24 25/17

9.1x10-5 0.0069 0.0486 0.0453

B8CDB2 B8BW15 B8C1W6 B8C448

0.85 0.58 0.58 0.58

5/5 4/4 5/5 5/4

0.0009 1.1x10-5 0.001 0.0002

putative PSI-associated

FNR

CET

Carbon assimilation CbbX, Rubisco expression Ribulose-phosphate 3epimerase RubisCO small, rbcS Fructose-bisphosphate aldolase CbbX, “red” RubisCO activase GAPDH, plastid RubisCO large, rbcL Phosphoglycerate kinase Pigment biosynthesis/xanthophyll cycle Carotene desaturase Chlorophyll synthesis Protochlorophyllide reductase Zeaxanthin epoxidase


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Zeaxanthin epoxidase Geranyl-geranyl reductase Mg-protoporphyrin IX chelatase Chlorophyll synthesis Carotene 7,8-desaturase Diadinoxanthin de-epoxidase Mitochondria Malate dehydrogenase, mitochondria or CER Phosphoenolpyruvate carboxykinase Cyt b-c1 complex, Rieske Cytochrome c oxidase su1, Cox1 Cox2 Elongation factor G PPC2 Alternative oxidase, AOX2 Mitochondrial chaperonin Phosphoenolpyruvate carboxylase, PPC1

B8BUH8 B8CDB3 B8BVG3

0.38 0.38 0.26

6/6 14/13 11/11

0.0077 0.0003 0.0066

B8C067 B8CCC6 B8C775

0.26 0.14 -0.26

6/6 8/7 3/3

0.0039 0.0148 0.0038

B8BQC2

0.85

4/4

3x10-6

B8C274

0.58

10/9

0.0002

B8BX01 Q3S276

0.38 0.26

4/4 3/3

0.0009 0.0069

Q3S2A6 B8CET1 B8C1R7 B8CF38 B5YLQ5 B8BYW8

0.26 -0.26 -0.38 -0.49 -0.49 -0.53

2/2 22/22 20/18 2/2 14/13 20/17

0.0007 0.0059 0.0417 5.2x10-5 1.1x10-8 0.0061

Table 2. Relative fold change in protein abundance between T. pseudonana cultures grown under continuous light (CL) or fluctuating light (FL) based on SRM analysis. Ratios are expressed as log2(FL/CL). Four biological replicates were used for CL conditions and two biological replicates for FL conditions. For each peptide, four transitions were targeted. Lhcx6 was an exception, with only one detected peptide for which six transitions were targeted, including b-ions. Upregulation of ≥ 0.58 is marked in red; downregulation of ≤ 0.58 is marked in green. Protein_ID Lhcf4_B8CFG5 Psb31_B8CC14 PsaL_A0T0U5 PsaA_A0T0M8 PGRL_B8BZQ3

Number of peptides (transitions) targeted 3 (12) 5 (20) 2 (8) 5 (20) 5 (20)

Fold change log2(FL/CL) 1.63 1.2 1 0.77 0.68


p-value 0.0268 0.0159 0.018 0.0085 0.0455


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PGR5_B8C035 Lhcx6_B8CGG1

0.38 -1.32

3 (12) 1 (6)

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0.042 0.021

Figure legends Figure 1. Growth curve of T. pseudonana cultures grown under continuous light (CL) or fluctuating light (FL) based on optical density (OD) measured at 750 nm, logarithmic scale Figure 2. Absorption spectra ranging from 300 to 750 nm of T. pseudonana intact cells grown under continuous light (CL) or fluctuating light (FL). Cultures were measured at an optical density of 0.27-0.32. Spectra were recorded at room temperature and normalized to 750 nm. Figure 3. NPQ of T. pseudonana cultures grown under continuous light (CL) or fluctuating light (FL). Cells were dark-adapted for 5 min prior to measurement. Cultures were measured at an optical density of 0.27-0.32. NPQ was recorded during 10 min of high light illumination (650 µE m-2 s-1) and a consequent dark phase (10 min). NPQ was calculated as (Fm-Fm’)/Fm’. Figure 4. PSI photooxidation kinetics, measured as P700+ absorbance change at 830 nm, of T. pseudonana cultures grown under continuous light (CL) or fluctuating light (FL). Cultures were measured at an optical density of 0.27-0.32. Average of nine measurements of three biological replicates.


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For TOC only



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Figure 1 growth curve 82x63mm (300 x 300 DPI)

ACS Paragon Plus Environment

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Figure 2 absorption 272x208mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3 NPQ 82x57mm (300 x 300 DPI)


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Figure 4 P700 272x208mm (300 x 300 DPI)


Changes in Relative Thylakoid Protein Abundance Induced by

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