Light harvesting proteins regulate non-photochemical fluorescence quenching in the marine diatom Thalassiosira pseudonana

Algal Research 12 (2015) 300–307

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Light harvesting proteins regulate non-photochemical fluorescence quenching in the marine diatom Thalassiosira pseudonana Yue-Lei Dong, Tao Jiang, Wei Xia, Hong-Po Dong ⁎, Song-Hui Lu ⁎⁎, Lei Cui Research Center for Harmful Algae and Marine Biology, Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China

a r t i c l e

i n f o

Article history: Received 11 May 2015 Received in revised form 18 September 2015 Accepted 23 September 2015 Available online xxxx Keywords: T. pseudonana NPQ Xanthophyll cycle Lhcx Proteomics

a b s t r a c t Diatoms utilize various mechanisms to enhance heat dissipation when they are subjected to drastic fluctuations in the light intensity. The activation of the xanthophyll cycle (XC) leading to non-photochemical fluorescence quenching (NPQ) is one of the important mechanisms. We used the model diatom Thalassiosira pseudonana to investigate the factors controlling the kinetics of NPQ and XC. By adding chemicals to cells exposed to excess light, we found that the increase in NPQ during high light (HL) exposure could not be inhibited by NH4Cl and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), while the increase in diatoxanthin (Dt) could be prevented by DCMU and not by NH4Cl, suggesting that the characteristics of NPQ do not solely depend on the presence of the XC. During HL exposure, the up-regulation of the light harvesting complex protein ×6 (Lhcx6) and Lhcx genes suggests that they are involved in photoprotection. The addition of DCMU and NH4Cl significantly elevated the transcript levels of three of the Lhcx genes during HL treatment, especially Lhcx6. The results suggest that reactive oxygen species (ROS) generated by DCMU and transthylakoid ΔpH changes elicited by NH4Cl may contribute to the development of NPQ during HL exposure by inducing the gene expression of Lhcx instead of controlling the XC. Among these, Lhcx6 protein may play a key role in managing light responses in T. pseudonana. Proteomic data demonstrated that the elevated Calvin cycle and increased synthesis of antioxidants, pseudouridines and plastoglobulins may raise the capability of diatoms to cope with light stress. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Diatoms, eukaryotic unicellular microalgae, are widespread in oceans and rivers and are estimated to account for 20% of the primary productivity in the world [1,2]. Why are diatoms so successful? One important reason is that diatoms possess an excellent capability to cope with rapid changes in light intensity resulting from fast agitation of surface waters in the ocean. NPQ is a vital photoprotective process in plants and algae that dissipates surplus light energy in the form of heat. The mechanism of NPQ is well characterized in higher plants and is associated with the XC. The XC in higher plants embraces the conversion from the violaxanthin to zeaxanthin in high light and the counter reaction in low light [3]. However, The XC in diatoms is different from that in higher plants. It involves the de-epoxidation of diadinoxanthin (Dd) to Dt in high light treatment and the back reaction in low light irradiance or in darkness [4,5]. Although

⁎ Correspondence to: H.-P. Dong, Research Center for Harmful Algae and Marine Biology, Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (H.-P. Dong), [email protected] (S.-H. Lu).

http://dx.doi.org/10.1016/j.algal.2015.09.016 2211-9264/© 2015 Elsevier B.V. All rights reserved.

the XC is essential for thermal dissipation, the formation of transthylakoid ΔpH [6] and PsbS protein in the PSII antenna are also necessary for NPQ induction [7]. In Phaeodactylum tricornutum, the application of certain uncouplers such as NH4Cl, the ADRY reagent and the protonophore 2,4-dinitrophenol, which can effectively dissipate the transthylakoid ΔpH, inhibited NPQ and the conversion of Dd to Dt [8]. This suggests that NPQ in P. tricornutum relies on the transthylakoid ΔpH. DCMU, which restrains photosynthetic electron transfer, was used to study relationship between photosynthetic electron transport and NPQ in P. tricornutum. The addition of DCMU to P. tricornutum cells exposed to surplus light hardly affected NPQ and the functioning of the XC [8]. The authors proposed that the XC may be activated by cyclic electron transport in PSI in the presence of DCMU. T. pseudonana is a species of marine centric diatom and is the first eukaryotic marine phytoplankton used for whole genome sequencing. The relationship between NPQ and the XC in T. pseudonana has not been reported to date. For higher plants under high light stress, conformational changes of light harvesting complexes (Lhc) promote the thermal dissipation of surplus light energy, which is triggered by protonation of PsbS protein [9]. In T. pseudonana, the Lhcx6 protein in the Lhc was found to participate in dissipation of surplus light energy during high light stress [10]. This suggests that the Lhcx6 protein in diatoms has a similar function to PsbS in higher plants. Phylogenetic analysis found five LI818-like

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genes (Lhcx) in the genome of T. pseudonana, including Lhcx1, Lhcx2, Lhcx4, Lhcx5 and Lhcx6 [11]. To date, roles of Lhcx genes in the regulation of NPQ remain largely unknown for T. pseudonana. In this study, the marine model diatom T. pseudonana was used to examine the responses of NPQ, XC and Lhcx genes to excess light treatment in the presence of various chemicals. Several responses to inhibitors in T. pseudonana were found which were different from those in P. tricornutum. Furthermore, a comparative proteomics method was employed to examine the proteomic changes in T. pseudonana under excess light stress. The purpose of this proteomic study was to figure out which metabolic pathways in diatom cells are involved in the response to excess light stress and attempt to provide evidence for changes in the XC and NPQ at the protein level. These results will provide new insights into functioning of the XC and NPQ formation in marine diatoms during excess light acclimation. 2. Materials and methods 2.1. Growth conditions Axenic T. pseudonana was obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota and was grown in artificial seawater supplemented with f/2 [12]. Cultures were incubated at 19 °C under a 12:12 light:dark regime with a light intensity of 30 μmol photons m−2 s−1 (LL). Light intensity was measured using a quantum scalar laboratory radiometer (QSL-2100, Biospherical Instruments Inc.). To ensure full acclimation of cells, the cultures were grown under the LL condition for at least 4 weeks. Periodic dilution was used to maintain the exponential growth phase of the cultures. In vivo fluorescence of the cultures was monitored using a Turner Designs Model 10 Fluorometer (Turner Designs, CA, USA). In determining NPQ and the relative electron transfer rate through PSII (rETR), LL-acclimated cells were subjected to 800 μmol photons m−2 s−1 (HL) treatment for 1 h, followed by 1 h of LL recovery and subsequently 1 h of HL exposure again. In other experiments, the cells under LL conditions were divided into two treatments. One treatment was divided into several 1-L Erlenmeyer flasks and exposed to 200 μmol photons m−2 s−1 (ML) for 48 h for proteomic analysis or to HL and ML for quantitative real-time PCR. The other treatment was kept under LL as a control. Three biological replicates for all experiments were used to assure statistical significance. 2.2. Inhibitor treatments For the effect of inhibitors on NPQ, Dithiothreitol (DTT, 1 mM, in water), NH4Cl (10 mM, in water) and DCMU (10 μM, in ethanol; final ethanol concentration, 2‰) were supplemented 15 min before HL treatment. For the effect of inhibitors on pigment composition, DTT, NH4Cl and DCMU were supplemented 30 min before HL treatment following 30 and 60 min of HL treatment.

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HPLC system (Agilent Technologies, CA, USA) with a Symmetry C8 column (4.6 × 150 mm). 2.5. Two-dimensional electrophoresis (2-DE) and gel analysis Protein extraction was implemented according to a previous procedure [14]. In the last step of extraction, dry protein powder was obtained and then dissolved in a rehydration buffer (containing 7 M urea, 2 M thiourea, and 4% (w/v) CHAPS). The protein concentration was measured with a 2-D Quant Kit (GE Healthcare). The procedure of 2-DE followed the method proposed by Lee and Lo (2008) [14] with minor modification. Briefly, 300 μg of protein samples were incubated with a 17-cm IPG strip (pH 4–7, Bio-Rad). Isoelectric focusing (IEF) was carried out using a Protean IEF Cell (Bio-Rad). The procedure of voltage/time was described as follows: 12 h at 50 V, 2 h at 200 V, 1 h at 500 V, 2 h at 1000 V, 4 h at 10,000 V and 5 h at 10,000 V for 50,000 V h. After IEF, the gel strip was incubated with the first equilibration buffer for 15 min, followed by the second equilibration buffer containing 1% iodoacetamine (instead of DTT) for another 15 min. The seconddimensional electrophoresis was performed in a PROTEAN II Cell (BioRad). The gels were stained using a Blue Silver method as described as in Candlano et al. [15] when electrophoresis was completed. Gel images were captured using an image scanner at 300 dpi resolution. Gel comparison was carried out using PDQuest 2-D™ Analysis software (Bio-Rad, USA). Three gels from biological replicates were analyzed for control and treatment groups. The protein spots that had a ratio N 2.0-fold and significant variations (P b 0.05) were considered differentially expressed. Among these, the spots with clear background were selected for mass spectrometric analyses. 2.6. Mass spectrometry (MS) analyses and protein identification Digestion of protein spots followed a previous method [16]. Briefly, after gel pieces were destained and dried, they were incubated with trypsin solution at 37 °C overnight. On the next morning, 1 μL of the slurry supernatant was spotted on a target plate and allowed to dry completely. And then, 0.1 μL of matrix solution was added to the target and mixed with the digested peptides. MS analysis was performed with an ultrafleXtreme spectrometer (Bruker) according to the instruction manual of the spectrometer. All spectra were acquired in default mode. For MS/MS analysis of each protein spot, the 10 strongest precursor ions were elected. All acquired sample spectra were processed using FlexAnalysis 3.3 software (Bruker). The MS and MS/MS spectra were combined using the BioTools 3.0 software and searched against the protein database of T. pseudonana from NCBI using the MASCOT software (version 2.3.02). The parameters of the database searches were as follows: trypsin as digesting enzyme; peptide mass tolerance, 100 ppm; fragment mass tolerance, 0.6 Da; miss cleavage, 1. Protein identification was considered as confident when the total Mascot score and the ion score were greater than the minimum significant score (56 and 44 for the Mascot score and ion score, respectively) with E-value = 0.

2.3. Chlorophyll fluorescence measurements

2.7. Quantitative real-time PCR (qRT-PCR)

rETR and NPQ were measured at room temperature using a PhytoPAM Phytoplankton Analyzer (Walz, Germany). NPQ was counted using the Stern–Volmer parameter as NPQ = Fm / fm′ − 1.

The transcript levels of diadinoxanthin deepoxidase (DDE) and the Lhcx genes under HL treatment without or with inhibitors were analyzed. In addition, to validate the results from proteomes, timeresolved expression profiles of genes for five important differentially expressed proteins were studied. Total RNA was obtained with TRIzol according to the instruction manual of Invitrogen. The first strand of cDNA was synthesized with the AMV First Strand cDNA Synthesis Kit using random primers. The resulting cDNA was diluted 8-fold as the qRT-PCR template. Amplicons were quantified in qRT-PCR reactions with ABI SybrGreen PCR Master Mix (Applied Biosystems). Additionally, qRT-PCR was performed in a LightCycler 480 instrument according to

2.4. Pigment analysis Fifteen-milliliter culture was filtered on a GF/F glass fiber filter (Whatman), immediately frozen in liquid nitrogen, and stored at − 80 °C. Extraction of pigments and HPLC procedure were performed as described as in Jakob and colleagues [13]. The pigment extract was filtered and then separated by an Agilent 1200

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standard methods (Roche) as previously described [17]. The 2ΔCT method was used to evaluate the relative gene expression normalized to the endogenous control gene actin. ΔCT values were obtained by subtracting the mean values of the experimental genes from a mean of the control genes for each sample. Triplicate biological replicates were performed. The qRT-PCR primers are listed in Supplementary Table A1. 3. Results and discussion 3.1. Changes in NPQ and the XC during HL exposure NPQ is like a ruler to gauge the extent of the surplus light energy dissipation under HL stress. For higher plants and algae, it is one of the foremost photoprotective mechanisms [18]. During 1 h of HL treatment, a strong and continuous increase in NPQ was observed, followed by a sharp decrease during 1 h of LL recovery and a rapid increase once again during a subsequent HL treatment of 1 h (Fig. 1A). After 0.5 h and 1 h of HL exposure, the percentage of Dt increased while the percentage of Dd decreased; the Dd + Dt concentration did not change (Table 1). The results indicated that short-term HL exposure activates

Table 1 The content of Dt and Dd+Dt in T. pseudonana cells when the different treatments were applied. Treatment 0.5 h LL HL HL+NH4Cl HL+DCMU HL+DTT 1h LL HL HL+NH4Cl HL+DCMU HL+DTT

Dt μg/100 μg Chl a

Dd+Dt μg/100 μg Chl a

Dd/(Dd+Dt) Dt/(Dd+Dt) (%) (%)

0.86 ± 0.06 1.68 ± 0.36* 1.92 ± 0.13 0.49 ± 0.10* 0.65 ± 0.01* 1.47 ± 0.12 2.14 ± 0.33* 1.92 ± 0.04 0.38 ± 0.05** 0.55 ± 0.13**

10.75 ± 0.94 10.63 ± 0.83 10.34 ± 1.19 8.57 ± 0.81* 9.45 ± 0.68 16.49 ± 0.89 13.07 ± 1.59 10.34 ± 0.44 7.72 ± 0.17* 8.89 ± 2.20*

92 84 81 94 93 91 84 81 95 94

8 16 19 6 7 9 16 19 5 6

Data represent the means of three independent samples ± standard deviations. Asterisks represent significant differences (P b 0.05). HL (800 μmol photons m−2 s−1)-treated cells were compared with LL (30 μmol photons m−2 s−1)-acclimated cells. The HL plus inhibitor-treated cells were compared with HL-treated cells.

the conversion from Dd to Dt but does not affect the size of Dd + Dt store. A significant amount of Dt could be detected under LL conditions, which is contrary to the result obtained by Zhu et al. [10]. These data show that the increase of Dt content may be required for the development of NPQ in T. pseudonana. 3.2. Influence of inhibitors on NPQ and the XC

Fig. 1. NPQ (A) and rETR (B) during 1 h of HL (800 μmol photons m−2 s−1) treatment, 1 h of LL (30 μmol photons m−2 s−1) recovery, and subsequently, 1 h of HL treatment. Control, cultures without inhibitors; +DCMU, cultures were incubated with DCMU (10 μM) for 15 min before starting the experiment; +DTT, cultures were incubated with DTT (1 mM) for 15 min before starting the experiment; +NH4Cl, cultures were incubated with NH4Cl (10 mM) for 15 min before starting the experiment. Data represent the average and standard deviation for n = 3.

The addition of DTT partially inhibited the increase in NPQ during HL treatment and resulted in a decline in rETR (Fig. 1A and B). In addition, DTT reduced the Dd+Dt pool and thus likely blocked the de novo synthesis of Dd+Dt. Dt content and percentage of Dt in HL were reduced significantly by DTT (Table 1). These data indicated that as an inhibitor of the violaxanthin de-epoxidase enzyme (VDE) in higher plants [19, 20], DTT blocked Dd de-epoxidation and Dt synthesis in T. pseudonana to a great extent. It is noteworthy that the NPQ value with DTT treatment was higher than that without DTT during the LL recovery period. These observations are different from the results obtained in the diatom P. tricornutum, where DTT treatment strongly inhibited the induction of NPQ during HL treatment and did not prevent relaxation of NPQ during the LL recovery period [21]. T. pseudonana possesses one DDE and two violaxanthin deepoxidase-like enzymes (VDL) [11]. It is likely that DDE in T. pseudonana might be less sensitive to DTT than DDE in P. tricornutum. For a slight decrease in NPQ during LL recovery, one may raise the possibility that the partial NPQ induced during DTT inhibition depends on other unknown mechanisms that are independent of Dt. Another possibility is that DTT may also prevent the fast epoxidation of Dt in LL conditions. Further study is needed to uncover the exact mechanism. The addition of NH4Cl did not inhibit the increase in NPQ and Dt and did not block the conversion of Dd to Dt but did lead to a consecutive decrease in rETR during HL treatment (Fig. 1A and B, Table 1). NH4Cl is an uncoupler that can decrease the formed transthylakoid proton gradient (ΔpH) during illumination [22]. It has been shown that a weak proton gradient would suffice to induce a slow conversion of Dd to Dt [23]. Our data suggest that the addition of NH4Cl may not dissipate the ΔpH completely while other effectors may contribute to the increase in NPQ. It has been reported that in uncoupled chloroplasts (pHin = pHout), rETR slows down with a decline in the lumen pH [24]. It is likely that the presence of NH4Cl results in the decrease in the lumen pH. However, the exact mechanism is unclear. On the other hand, HL plus the NH4Cl treatment did not affect the Dd+Dt pool size. Notably, NPQ increased continuously in the presence of DCMU during HL treatment (Fig. 1A). This is consistent with the results obtained in P. tricornutum [21]. Moreover, the addition of DCMU significantly reduced the amount and percentage of Dt in HL (Table 1). These data suggest that the increase in NPQ is independent of Dt in the presence of

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Fig. 2. Relative gene expression (ΔCT) of DDE (A), Lhcx1 (B), Lhcx2 (C), and Lhcx6 (D) during HL treatment. DCMU and NH4Cl were applied 15 min before the light treatment. Data represent means of three independent samples ± standard deviation. Asterisks represent significant differences (P b 0.05). The HL-treated samples were compared with the respective control samples. The HL+DCMU-treated or HL+NH4Cl-treated samples were compared with the respective HL-treated samples. DDE, diadinoxanthin de-epoxidase; Lhcx, light harvesting complex protein x.

DCMU. Interestingly, in P. tricornutum, DCMU induced a pronounced deepoxidation, suggesting that the NPQ increase is derived from the increase in Dt [21]. Obviously, in T. pseudonana, a different mechanism

led to the increase in NPQ in HL plus DCMU. It has been suggested that chlororespiratory and cyclic electron flow in PSI may induce the transthylakoid ΔpH in DCMU-treated P. tricornutum cells [8]. However,

Fig. 3. Two-dimensional electrophoretograms of T. pseudonana in response to ML (200 μmol photons m−2 s−1) exposure. Proteins were extracted from T. pseudonana and separated using 2-DE. LL and ML are gels for the LL-acclimated samples and 48 h ML-treated samples, respectively. Red circles on the gels indicate the differentially expressed spots. Numbers 1 to 31 on the LL and ML gels is in accordance with the spots numbered in Table 2.

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it is likely that the ΔpH in T. pseudonana may not be enough to activate Dd de-epoxidase. It is deduced that the pH change may induce the increase in NPQ by other triggers. In addition, the DCMU plus HL treatment may produce large amounts of ROS [25]. This may lead to conformational changes of the PSII complex, which induces the development of NPQ. We also observed that the Dd + Dt pool size was reduced in the HL plus DCMU treatment relative to the HL treatment (Table 1), suggesting that as in P. tricornutum, de novo synthesis of Dd + Dt might be activated by the redox state of the PQ pool. It is noteworthy that a rapid decrease in NPQ was observed during the shift from HL to LL (Fig. 1A). This suggests that the epoxidation of Dt to Dd is very fast in T. pseudonana in LL treatment. On the other hand, the relaxation of NPQ was inhibited in the presence of DCMU during the LL recovery conditions. Similar results were also found in P. tricornutum [26]. NADPH is an essential cofactor for Dt epoxidase. DCMU completely inhibited rETR during HL treatment (Fig. 1B) and blocked the reduction of NADP + to NADPH [27], which could inhibit the decrease in NPQ and Dt epoxidation during the LL phase. This suggests that efficient Dt epoxidation may be prevented by the NADPH shortage. It is likely that active NADPH consumption leads to the shortage in the chloroplast after HL exposure. We also observed that the addition of NH 4 Cl inhibited the decrease in NPQ during the LL recovery. It is assumed that NH 4 Cl may prevent Dt epoxidation. This observation in T. pseudonana is contrary to the result obtained in P. tricornutum, i.e., that the addition of NH 4 Cl can suppress partly NPQ in the presence of DCMU [26]. Taken together, our data demonstrated that the responses of

T. pseudonana in NPQ and the XC to inhibitors are different from those of P. tricornutum; other than the XC, other effectors support the development of NPQ. 3.3. Effect of inhibitors on DDE and LHCX transcripts DDE transcript dropped by 50% after 1 h of exposure to HL and remained unchanged after 2 and 3 h of HL exposure (Fig. 2). This suggests that Dd de-epoxidation may mainly take place during 1 h of HL treatment. The addition of DCMU caused a decrease in the DDE transcript level after 1 and 2 h of HL exposure, indicating that DCMU can inhibit de-epoxidation of Dd to Dt. This observation is consistent with a significant reduction of Dt in the presence of DCMU (Table 1). Notably, the addition of NH4Cl did not elicit a change in the DDE transcript after 1 h of HL exposure, which is in agreement with changes of Dt in the presence of NH4Cl (Table 1). HL treatment induced remarkable transcript changes in Lhcx. After 1 h of exposure to HL, Lhcx1 was upregulated some 600-fold compared to LL, Lhcx2 increased approximately 344-fold, and Lhcx6 increased 26fold (Fig. 2B, C, and D). As the time of HL exposure increased, the levels of Lhcx1, Lhcx2 and Lhcx6 declined significantly after 2 h, but were still much higher than in LL levels. The huge increase in Lhcx6 transcript was reflected by the respective protein levels in 2-DE (see below) and supported previous findings [10], suggesting that Lhcx1, Lhcx2 and Lhcx6 participate in the photoprotective response of diatoms. Given that during HL treatment both NPQ and Dt increased continuously (Fig. 1A, Table 1), we hypothesized that Dt molecules synthesized de

Table 2 The differentially expressed proteins identified in Thalassiosira pseudonana after ML treatment. Ratios represent fold changes in relative abundance. A ratio greater than 2.0-fold indicates that a protein is significantly upregulated (P b 0.05). A ratio less than −2.0-fold indicates that a protein is decreased (P b 0.05). Three biological replicates are performed. Spot no.

Protein name

Protein mass (Da)

Protein score

Isoelectric point

Ratio, ML/LL

Photosynthesis and carbon fixation 16 Rubisco expression protein 28 Rubisco expression protein 18 Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) 24 Rubisco expression protein 25 Fucoxanthin chlorophyll a/c protein-LI818 clade (Lhcx6) 10 Fucoxanthin chl a/c light-harvesting protein (Lhcr3) 3 Fucoxanthin chl a/c light-harvesting protein (Lhcr3) 5 Photosystem II stability/assembly factor HCF136 4 Ferredoxin-NADP reductase

32,360.02 32,360.02 13,830.82 32,360.02 21,306.12 21,519.75 21,519.75 40,301.84 38,193.77

131 305 76.9 150 86.1 165 101 93.8 121

5.8 5.8 5.13 5.8 4.58 5.56 5.56 4.89 5.77

23.82 41.57 6.65 5.57 293.32 5.15 −2.21 −2.05 8.29

Energy production 1 F-type H+-transporting ATPase subunit gamma 8 Putative v-type h-ATPase subunit 20 F-type H +-transporting ATPase subunit gamma 11 F-type ATPase beta subunit

30,821.36 24,246.51 30,821.36 53,410.57

220 305 81.9 67.6

4.83 5.26 4.83 4.86

3.36 12.02 52.11 −4.49

Protein metabolism 12 Cyclophilin-type peptidyl-prolyl cis-trans isomerase 31 Pseudouridine synthase 7 tRNA-2′-O-ribose methyltransferase 27 40S ribosomal protein-like protein

18,740.08 39,104.21 59,901.94 27,300.96

164 54.4 51.6 160

4.8 9.81 6.1 5.97

3.29 3.96 3.38 3.34

Signaling and glycolysis 29 Signal peptide protein 6 Signal peptide protein 13 Signal transduction protein with cbs domains 17 Signal peptide protein 9 Phosphoglycerate mutase 15 Phosphoglycerate mutase

13,830.82 13,830.82 20,962.73 13,830.82 32,558.73 32,558.73

120 62 104 76.9 79.6 255

5.13 5.13 6.51 5.13 6.32 6.32

5.88 2.10 3.45 7.91 −2.54 −4.62

Antioxidant defense 19 Chaperone, heat shock protein 70 30 Plastoglobulin 21 Cytochrome C peroxidase 23 HEAT repeat domain containing protein 26 Thioredoxin/protein disulfide isomerase 22 Hsp70-type chaperone

72,389.85 58,938.41 29,101.55 150,609 18,535.17 65,640.02

144 339 74.7 69.1 113 91.3

4.67 4.47 5.38 5.09 4.31 4.52

2.19 35.52 2.23 5.36 2.49 −2.90

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novo are combined with the increased Lhcx proteins, which facilitates the increase of NPQ. In P. tricornutum, Lhcx1, Lhcx2 and Lhcx3 play pivotal roles in excess light energy dissipation [21,28,29], while in the diatom Chaetoceros neogracile, the Lhcx5 and Lhcx6 homologs are upregulated markedly during 6 h of HL exposure [30]. These results demonstrate that Lhcx genes respond in different ways depending on the diatom species. Interestingly, the addition of DCMU significantly elevated the gene expression of Lhcx1, Lhcx2 and Lhcx6 during 1 and 3 h of HL treatment (Fig. 2B, C, and D). DCMU changes the PQ pool redox state by binding to the secondary electron-accepting plastoquinone of PSII (Q B). In P. tricornutum, it has been demonstrated that the redox state of the PQ pool can regulate gene expression of Lhc proteins and play an important role in the light acclimation of diatoms [21]. In HL conditions, the PQ pool should be reduced while QA is oxidized. DCMU might prevent this possible PQ regulation by holding the oxidized status of the PQ pool. However, DCMU did not block the gene expression of Lhcx, showing that another mechanism is acting in T. pseudonana. As mentioned above, DCMU creates high amounts of ROS, which can elicit gene expression [31]. It is likely that the joint impact of HL and DCMU on Lhcx is greater than the impact of the DCMU on the PQ pool. Therefore, it is speculated that ROS generated by excess light exposure is also a signal regulating the cellular light response. Surprisingly, the addition of NH4Cl caused a marked increase in the Lhcx1, Lhcx2 and Lhcx6 transcript levels in HL treatment (Fig. 2B, C, and D). The strongest induction occurred in the Lhcx6 transcript. Compared to HL, Lhcx6 increased 21-

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fold, 70-fold and 175-fold after 1, 2 and 3 h of HL treatment, respectively. This suggests that the Lhcx6 plays a vital role in acclimation responses of diatoms to HL. If we hypothesize that NH4Cl partially dissipates the ΔpH across the thylakoid membrane, the consecutive increase in NPQ in the presence of NH4Cl may be mainly triggered by the marked increase in the Lhcx6 protein. The change in ΔpH may induce the gene expression of Lhcx. In T. pseudonana, the function of Lhcx6 may be similar to Lhcx1 in P. tricornutum, where Lhcx1 is like a molecular calculator that quantitatively controls NPQ production in diatoms [29]. 3.4. Proteomic responses to excess light stress To understand the molecular mechanisms of the rise in NPQ and the functioning of the XC under excess light stress, we used 2-DE to compare the protein expression between LL-acclimated cells and MLtreated cells. After getting rid of the smeared background, approximately 1200 clear spots were found for each sample. By using gel analysis software, 56 proteins with significant difference were picked out and analyzed by mass spectrometry (Fig. 3). Of these, 47 proteins were identified by mass spectrometry. These proteins with functional annotation are listed in Table 2. One Lhcx6 protein was upregulated 293-fold after ML exposure (Table 2). In T. pseudonana, the transcripts of Lhcx6 increased consecutively during 10 h of HL treatment [10]. The Lhcx6 protein has been suggested to be combined with Dt and to participate directly in

Fig. 4. Relative expression (ΔCT) pattern of five important genes corresponding to differentially expressed proteins during ML exposure. LL represents LL-acclimated cultures, and ML represents cultures after exposure to ML for 3, 6, 12, 24, and 48 h. Error bars represent the standard deviations of the means generated from triplicates. * indicates significant differences (P b 0.05) relative to LL-acclimated cultures. RuBisCO, Ribulose-1,5-bisphosphate carboxylase/oxygenase.

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responses of diatoms to HL [10]. Our proteomic result confirmed the key photoprotective role of Lhcx6 in T. pseudonana. Two Lhcr3 proteins were identified; one was upregulated 5.2-fold after ML treatment and the other downregulated 2.2-fold. These data suggest that the Lhcr3 protein may also participate in photoprotection. Lhcr proteins have been shown to combine Dd or Dt [32]. In P. tricornutum, the transcript levels of Lhcr6, Lhcr8, and Lhcr10 are upregulated significantly after HL treatment [28]. The photosystem II stability/assembly factor HCF136 was downregulated 2.3-fold after ML exposure. In Arabidopsis thaliana, HCF136 is indispensable for the assembly and stability of the photosystem II [33]. Downregulation of HCF136 suggests changes in the photosystem II reaction center under ML treatment. A ferredoxin-NADP reductase (FNR) increased 8.3-fold after ML exposure (Table 2). In the chloroplast, FNR is responsible for the transfer of electrons from ferredoxin to NADPH in the photosynthetic electron transfer chain. The transcript of FNR was markedly upregulated when LL-acclimated cells were exposed to ML for 6, 24 and 48 h (Fig. 4A). Upregulation of FNR under ML stress suggests more efficient NADP+ photoreduction. Two F-type H+-transporting ATPases localized to the chloroplast thylakoid membrane were increased 3.4- and 52-fold, respectively, after ML exposure. This suggests that excess light energy is used to produce more ATP by photophosphorylation after ML exposure. In addition, upregulation of ATPase increased acidity in the thylakoid lumen that facilitates the activation of de-epoxidase. One ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) increased 6.7-fold, and three Rubisco expression proteins that regulate the activities of RuBisCO increased 5.6- to 41.6-fold after ML exposure. The transcript of one RuBisCO increased significantly when cells were exposed to ML for 12 and 48 h (Fig. 4C). The data suggest that the Calvin cycle in ML-treated cells was elevated to fix more carbon relative to LLacclimated cells. Several antioxidants increased significantly after ML exposure, including cytochrome C peroxidase, thioredoxin/protein disulfide isomerase, and the HEAT repeat domain-containing protein. In addition, a pseudouridine synthase showed a 4.0-fold upregulation after ML exposure (Table 2). The transcript of pseudouridine synthase was significantly upregulated after 12 and 48 h of ML exposure (Fig. 4B). It was shown that pseudouridine, which is the nucleoside present in tRNA, can reduce radiation-induced chromosome aberrations in human lymphocytes [34]. Therefore, following ML exposure, upregulation of pseudouridine synthase resulted in the synthesis of more pseudouridine, which may promote the capability to resist excess light stress. Interestingly, a plastoglobulin (PGL) was found to be upregulated 35.5-fold after ML exposure. Putative PGL genes are found in cyanobacteria and eukaryotic algae, and plastoglobulins (PGLs) have been shown to play roles in the protection of the thylakoid membrane in cyanobacteria [35]. Our data confirmed the presence of PGL in T. pseudonana and suggested that PGLs may be involved in the repair of photooxidative damage under excess light exposure. A signal transduction protein with cbs increased 3.5-fold after ML exposure, and its transcript was upregulated markedly after 48 h of ML exposure (Table 2 and Fig. 4D). This protein may be used to transmit excess light signals to downstream pathways. Our proteomic data demonstrated that other mechanisms in diatoms participated in photoprotection besides the XC and NPQ. The elevated synthesis of ATP and NADPH may direct excess light energy to the XC and the Calvin cycle. The marked upregulation of Lhcx6, Lhcr3 and ATPases contributes to the activation of the XC and NPQ induction. The increased synthesis in antioxidants, pseudouridines and PGLs elevates the capability to resist excess light in diatoms. 4. Conclusions In this study, T. pseudonana showed response patterns to inhibitors under HL and LL conditions that differ from those of P. tricornutum. This may be due to the pronounced difference in the genome structures between the two diatoms. The link between the development of NPQ

and Dt accumulation is not mandatory, suggesting that NPQ do not solely rely on the presence of the XC. In P. tricornutum, it has been shown that ΔpH changes regulate the level of NPQ only by controlling expression of epoxidase and deepoxidase of the XC [36]. Our data demonstrated that besides the redox state of the PQ pool, ΔpH changes also contribute to the development of NPQ by inducing the expression of Lhcx genes, although we do not know how these signals are transmitted to the nucleus. Among these Lhcx genes, Lhcx6 plays a key role in responses of T. pseudonana to HL stress. The Lhcx6 protein may be similar to PsbS in higher plants, where it induces the initiation of NPQ through perceiving pH changes in thylakoid lumen and controls the level of NPQ. In addition, our proteomic data showed that the elevated Calvin cycle and increased synthesis of antioxidants, pseudouridines and PGLs may raise the capability of diatoms to cope with light stress. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.algal.2015.09.016.

Acknowledgements This study was funded by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11020304), the Guangdong Natural Science Foundation through grant S2013010013406, the National Natural Science Foundation of China through grant 41206126, and the China Postdoctoral Science Foundation through grant 2014M562251. The authors would like to thank Feiyu Chen for the assistance with pigment analysis.

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