Accepted Manuscript Title: Photosynthetic and molecular responses of the marine diatom Thalassiosira pseudonana to triphenyltin exposure Author: Andy Xianliang Yi Priscilla T.Y. Leung Kenneth M.Y. Leung PII: DOI: Reference:

S0166-445X(14)00163-5 http://dx.doi.org/doi:10.1016/j.aquatox.2014.05.004 AQTOX 3841

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

17-2-2014 3-5-2014 5-5-2014

Please cite this article as: Yi, A.X., Leung, P.T.Y., Leung, K.M.Y.,Photosynthetic and molecular responses of the marine diatom Thalassiosira pseudonana to triphenyltin exposure, Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Photosynthetic and molecular responses of the marine diatom Thalassiosira pseudonana to triphenyltin exposure

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Andy Xianliang Yi, Priscilla T.Y. Leung and Kenneth M.Y. Leung*

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The Swire Institute of Marine Science and School of Biological Sciences, The University of

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*Corresponding author

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Hong Kong, Pokfulam, Hong Kong, P.R. China

Corresponding author:

Prof. Kenneth M. Y. Leung

Corresponding address:

School of Biological Sciences, The University of Hong Kong, Pokfulam, Hong Kong, P.R. China

Corresponding email:

[email protected]

Corresponding tel/fax no.:

+852-22990607 (Tel); +852-25176082 (Fax)

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Abstract This study aimed to investigate the responses of the marine diatom Thalassiosira pseudonana

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upon waterborne exposure to triphenyltin chloride (TPTCl) through determining their photosynthetic response, growth performance, and expressions of genes and proteins. Based on

According to photosynthetic

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1.09 μg/L (95% confidence interval (CI): 0.89 - 1.34 μg/L).

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the growth inhibition test, the 96-h IC50 (i.e., median inhibition concentration) was found to be

parameters, the 96-h EC50s (i.e., median effect concentrations) were estimated at 1.54 μg/L

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(95% CI: 1.40 - 1.69 μg/L) and 1.51 μg/L (95% CI: 1.44 - 1.58 μg/L) for the maximum quantum yield of photosystem II (PSII) photochemistry (ΦPo) and the effective quantum yield of

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photochemical energy conversion in PSII (Φ2), respectively. Non-photochemical quenching in

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the algae was increased at low concentrations of TPTCl (0.5-1.0 μg/L) but it decreased gradually Results of gene

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when the TPTCl concentration further increased from 1.0 to 2.5 μg/L.

expressions showed that lipid metabolism related genes were not influenced by TPTCl at 0.5 or

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1.0 μg/L, while silica shell formation genes were down-regulated at 0.5 μg/L. Photosynthesis related genes were up-regulated at 0.5 μg/L TPTCl but were down-regulated at 1.0 μg/L TPTCl. Proteomics analysis revealed that relatively less proteins could be detected after exposure to 1.0 μg/L TPTCl (only about 50-60 spots) compared with that observed in the 0.5 μg/L TPTCl treatment and two control groups (each with about 290-300 protein spots). At 0.5 μg/L TPTCl, five proteins were differentially expressed when compared with the seawater control and solvent control, and most of these proteins are involved in defence function to protect the biological systems from reactive oxygen species that generated by TPTCl. These proteins include oxygenevolving enhancer protein 1 precursor, fucoxanthin chlorophyll a/c protein - LI818 clade, and

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mitochondrial manganese superoxide dismutase, which can function to maintain the capacity of PSII and stabilize the photosynthesis efficiency as reflected by the unchanged ΦPo and Φ2 values at 0.5 μg/L TPTCl.

In contrast, the excess toxicity that caused by TPTCl at the high

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concentration (1.0 μg/L TPTCl) might directly damage the proteins, inhibit their expression, and/or cause the suppression of metabolism as indicated by the down-regulation of most studied

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proteins and genes, which could ultimately inhibit the photosynthesis and growth of the algae.

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Overall, this study comprehensively elucidated the toxicity effects of TPT on T. pseudonana, and partially revealed the molecular toxic mechanisms and corresponding defence responses in this

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model algal species.

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Keywords: growth inhibition; gene expression; proteomics; defence response; TPT

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1. Introduction Diatoms, as unicellular photosynthetic eukaryotes, can be found throughout world's marine

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and freshwater systems (Armbrust et al., 2004). Diatoms are highly productive as primary producers that contribute for about 20% of the total carbon fixation in marine systems and

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comprising the main base of marine food webs (Armbrust, 2009). Because of their important

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marine environments (Davis et al., 2005; Bao et al., 2011).

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ecological role, diatoms are common subjects for elucidating effects of chemical pollution to

Thalassiosira pseudonana, a centric diatom, is widely distributed throughout world's oceans

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(Armbrust et al., 2004). It was the first chosen eukaryotic marine phytoplankton species for whole genome sequencing (Armbrust et al., 2004).

Such genomic information enables

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environmental scientists to investigate the response of T. pseudonana to chemical contaminants such as copper and polycyclic aromatic hydrocarbons (PAHs) at molecular level (Davis et al.,

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2005; Carvalho and Lettieri, 2011). The availability of genomic information, wide distribution and ecological importance of T. pseudonana concomitantly make it an ideal model organism for marine pollution and ecotoxicological studies.

Organotin compounds (OTs) such as tributyltin (TBT) and triphenyltin (TPT) have been widely used since mid-1960s in industrial applications because of their effective biocidal properties (Bennett, 1996). These compounds are extensively applied worldwide as antifouling agents in boat paints to prevent the settlement of fouling organisms on ship hulls and underwater facilities like submerged cages for mariculture (de More, 1996). However, OTs have high but non-specific toxicity, and have been demonstrated to be highly toxic and harmful to many non4

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target marine organisms (Alzieu, 2000). Due to the widespread OT-contamination and adverse impacts to marine organisms, a global ban of OT application in any antifouling system has been enacted since September 2008 (IMO, 2008). However, the applications of these compounds are

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not limited to the anti-fouling paints, but they are also commonly used as biocides in agricultural crops to control fungal diseases and unwanted growth of algae and molluscs (WHO, 1999).

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Although the level of TBT has been declining in many coastal regions worldwide over the past

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decade (e.g. Japan, Harino et al., 1999; the United Kingdom, Oliveira et al., 2009; South Korea, Choi et al., 2009; Iceland, Guðmundsdóttira et al., 2011), there is an increasing trend of TPT

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contamination in Asian coasts such as the marine environment of Hong Kong, Taiwan and Xiamen, China (Meng et al., 2005; Xie et al., 2010; Ho and Leung, in press). Such TPT

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contaminations may be associated with the illegal use of TPT-based paints in antifouling systems,

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(Yi et al., 2012).

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and wastewater and surface run-off contaminated with TPT-based biocides from farming areas

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In general, TPT is as toxic as TBT (Horiguchi et al., 1996). However, there were very limited documented studies on the toxicity of TPT to aquatic species, in particular algal species (Yi et al., 2012). Growth inhibition has been the most studied endpoint for algae, and median inhibition concentrations (IC50) of TPT ranged from 0.59 (Skeletonema costatum; Walsh et al., 1985) to 51.5 μg/L (Monoraphidium minutum; Roessink et al., 2006). Moreover, photosynthesis inhibition, chlorophyll or phycocyanin content, and activity of nitrate reductase have also been applied to study the toxic effect of TPT to algae (Mooney and Patching, 1995; Huang et al., 1996). In recent years, global analyses of genes and proteins have been successfully applied in revealing the effects of chemical contaminants on aquatic organisms (Carvalho and Lettieri,

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2011; Dondero et al., 2011; Leung et al., 2011). A combination of molecular and physiological approaches can enable us to understand the linkage between responses of organisms at different levels and reveal the toxic mechanism of chemical contaminants (Hayes and Bradfield, 2005).

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For example, Chen and Yen (2013) have recently employed this integrative approach to demonstrate that waterborne exposure of nonylphenol can reduce the biomass and root length,

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and lower the chlorophyll content in the Arabidopsis (Arabidopsis thaliana) and such

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physiological changes could be linked to the inhibition of the expression of some essential phosphate proteins. Nevertheless, this integrative approach has yet to be applied to investigate

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the effect of chemical pollutants on marine diatoms such as T. pseudonana.

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This study, therefore, aimed to investigate the effect of TPT on T. pseudonana through

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studying their physiological responses (i.e., photosynthetic response and growth inhibition) and

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molecular responses (i.e., proteome and gene expression) after waterborne exposure to triphenyltin chloride (TPTCl). The results would shed light on the possible toxic mechanisms of

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TPT to marine diatoms.

2. Materials and methods

2.1. Experimental diatom and apparatus Thalassiosira pseudonana (CCMP 1015) were purchased from Provasoli-Guillard National Centre for Culture of Marine Phytoplankton, Bigelow. They were cultured in laboratory under standard test conditions (f/2-Si medium in 32 ppt filtered artificial seawater (FASW); 14 h light:10 h dark cycle; 25 ± 1°C) and applied for toxicity test after at least three sub-cultured

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generations. All of the conical flasks and glass vials that used for the exposure study were cleaned with acid bath and autoclaved before use.

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2.2. Chemical preparation and growth inhibition test

Stock solutions of triphenyltin chloride (TPTCl) were prepared by dissolving TPTCl (> 95%;

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Sigma, USA) in dimethyl sulfoxide (DMSO; ACS reagent, 99.9%; Sigma, USA). Test solutions

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at designated nominal concentrations were made by spiking TPTCl stock solution into filtered

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artificial seawater (FASW; 32 ± 1 ppt, 0.45 μm, Millipore, USA) at appropriate volumes.

An initial cell concentration of 105 cells/mL was exposed to 5 mL test solutions (in a 10 mL

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glass vial) with TPTCl concentrations ranging from 0.1 (0.26 nM) to 2 μg/L (5.2 nM; diluted in

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f/2-Si medium). The algae at the same initial cell concentration were also exposed to the control

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(f/2-Si medium) and solvent control (f/2 medium with 0.01% DMSO) in parallel with the TPTtreated groups. Four to six replicates of each treatment or control group were applied in this

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exposure experiment, which was conducted in an environmental chamber with 14 h light: 10 h dark cycle at 25 ± 1°C for 96 h. The test glass vials were manually shaken every 12 h so as to resuspend the settled cells. The number of cells was estimated using a hemacytometer (Brand GmbH, Wertheim, Germany) after the 96 h exposure, and the relative growth rate was calculated as toxicity endpoint (ASTM, 1993).

2.3. Photosynthesis test Test solutions with TPTCl concentrations ranging from 0.5 (1.3 nM) to 2.5 μg/L (6.5 nM), and without TPTCl addition (i.e., the seawater control and solvent control) were prepared with

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an initial algae cell concentration of 105 cells/mL. For each treatment or control group, there were four replicates. The test glass vials were intensely shaken once every 12 h. After 96 h of exposure at the same conditions as stated above, the glass vials were covered with aluminium

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foil for 2 h and then the fluorescence was monitored by a WATER-PAM fluorometer (Heinz Walz GmbH, Effeltrich, Germany). Briefly, the fluorescence (Fo) was measured using a weak

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measuring light (0.15 µmol photons m-2 s-1) and the fluorescence of the dark-adapted chlorophyll

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was excited to a maximum state (Fm) by a saturating pulse light (> 3,000 μmol photons m-2 s-1). As for light-adapted chlorophyll (LAC), actinic radiation (1,000 µmol photons m-2 s-1) was

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applied continuously and a steady level of the fluorescence (Fs) could be achieved within 5 min. Again, the maximum fluorescence of LAC (Fm') was gained under a saturating pulse of light.

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The photosynthesis parameters which included the maximum quantum yield of photosystem II

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(PSII) photochemistry (ΦPo), the effective quantum yield of photochemical energy conversion

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in PSII (Φ2), and the nonphotochemical parameter (NPQ), were calculated following the

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methods described by Maxwell and Johnson (2000).

2.4. Gene expression analysis

According to the results of the 96-h toxicity tests for growth and photosynthesis inhibition, the diatom was exposed to TPTCl at 0.5 μg/L (1.3 nM; close to IC10 value for growth inhibition) and 1.0 μg/L (2.6 nM; close to IC50 for growth inhibition and EC10 for photosynthesis yield) in parallel with the seawater control and solvent control for genomic and proteomic study.

Test solutions (500 mL) with 0.5 and 1.0 μg/L TPTCl as well as the control and solvent control were prepared in glass conical flasks with initial cell concentration of 105 cells mL-1.

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There were four replicates for each treatment or control group. The test glass conical flasks were intensely shaken once every 12 h. After 96 h of the exposure at the same conditions as stated above, test solutions were filtered by 0.45 μm filter membrane and algae samples were re-

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suspended in a 50 mL falcon tube with 15 mL FASW. The samples were then centrifuged at 4000 rpm, collected in 1.5-mL Eppendorf tubes. Samples were briefly rinsed with autoclaved

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Milli-Q water and stored at -80 °C until proteomic analysis. RNA samples were extracted with

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RNeasy Plant Mini Kit (Qiagen, Germany) according to manufacturer's guidance and RNA concentrations were measured using Nanodrop 2000 (Thermo Scientific, USA), and the integrity

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of RNA samples was verified by 1.5% agarose-formaldehyde gel. As for complimentary DNA (cDNA) synthesis, 1 μg of isolated RNA samples were applied in a total reaction volume of 20

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μL using High Capacity RNA-to-cDNA Kit (Applied Biosystems, USA).

For real-time

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polymerase chain reaction (PCR), 2 μL diluted (x 5 times) reverse-transcripted cDNA samples

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and 250 nM primer sets were applied in each reaction, which was performed on a CFX96 RealTime System (Biorad, CA, USA) with SYBR Green (iQTM SYBR Green Supermix, Biorad, USA)

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as fluorescence dye. Primer sequences of selected genes, including Fucoxanthin-chlorophyll a/c light harvesting proteins (3Hfcp A and 3Hfcp B); Long chain acyl-coA synthetase (lacsA); Silaffin precursor (sil1 and sil3), Silicon transporter 1 (sit1) and glyceraldehyde phosphate dehydrogenase (GAPDH) as the reference gene, are listed in Table 1. Reaction conditions were as follows: 95°C/3 min; 40 cycles of 95°C/15 s, 60°C/30 s, 72°C/30 s. In order to confirm the amplification of specific products, melting curve cycles were continued with the following parameters: 95°C/1 min, and 80 cycles of 65°C/5 s with 0.5°C increase per cycle. Threshold cycle (CT) values were collected to calculate the relative expressions of target genes by 2-∆∆CT (Livak and Schimittgen, 2001).

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2.5. Proteomic analysis 2.5.1. Sample preparation for 2-dimensional gel electrophoresis (2-DE)

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In parallel to the gene expression analysis, algae samples were also collected for proteomic analysis after 96 h exposure using the aforementioned protocol. A total of 16 algae samples (4

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treatment/control groups x 4 replicates) were lysed in 2-DE buffer consisting of 7 M urea, 2 M

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thiourea, 4% CHAPS, 100 mM DTT, 2% ASB (amidosulfobetain) 14 and 2% IPG buffer (BioRad) with the protease inhibitor cocktail (Roche; IPG = immobilized pH gradients). After being

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centrifuged for 20 min at 14000 g, the supernatant was collected and purified with 2-D clean up kit (Bio-Rad, Hercules, USA). The protein concentration was then quantified with the 2D quant

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2.5.2. 2-DE and staining of the gels

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kit (GE Healthcare Life Sciences, Uppsala, Sweden).

Sample buffer (220 μL containing 350 μg protein) was applied to 11 cm IPG strips (BioRad

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Laboratories, USA), pH 4–7 (linear), rehydrated at 50 V for overnight and then subjected to the first dimension separation, i.e., isoelectric focusing (IEF) using a Protean IEF Cell (BioRad Laboratories, USA). Focusing conditions were set as follows: 300 V for 1 h, followed by 600 V for 1 h, 1000 V for 2 h and 8000 V for 20 h, with a total running time of 80 kVhr. After IEF, the strips were equilibrated for 15 min in 10 mL equilibration buffer 1 (6 M urea, 2% SDS, 0.05 M Tris–HCl (pH 8.8), 50% glycerol, and 2% w/v 1,4-DTT) followed by 15 min in 10 mL buffer 2 (same as buffer 1 but containing 2.5% iodoacetamide instead of DTT). Equilibrated IPG strips were then inserted on top of the 12.5% Criterion Tris–HCl gels (BioRad Laboratories, USA) for the second dimension separation. The electrophoresis was conducted under room temperature at

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the current of 15 mA per gel. Protein spots in the gels were stained and detected by a colloidal Coomassie blue G-250 staining method (modified from Candiano et al., 2004).

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2.5.3. Proteome analysis

The stained gels were subsequently scanned using Quantity One (BioRad Laboratories, USA)

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on a GS-800 Calibrated Densitometer (Biorad, USA) and analysed by PDQuest software (ver. Differentially expressed protein spots were classified as

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8.0; BioRad Laboratories, USA).

statistically difference (one-way Analysis of Variance, p < 0.05) with 1.5-times or greater change

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in the mean normalized volume with respect to both control and solvent control (Peart et al. 2005; McCarthy and Smyth, 2009). Together, there must be no significant difference in the protein

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expression profile between the control and solvent control, in terms of differentially expressed

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protein spots. Such comparisons were conducted using appropriate statistics based on four Besides, in order to check the reproducibility, two

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independent experimental replicates.

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technical replicates for each biological sample were conducted throughout the proteomic analysis.

As described previously, differentially expressed spots were excided from the gels and subjected to in-gel trypsin digestion (Leung et al., 2011).

Briefly, in-gel digestion was

performed overnight at 37°C with sequencing grade modified trypsin (Promega, Madison), and peptides were extracted with 5% (v/v) formic acid and 50% (v/v) acetonitrile and further purified using pipette ZipTip® C18 (Millipore, MA, USA). Protein identification was then performed by tandem mass spectrometry analysis using ABI 4800 MALDI TOF/TOF™ MS Analyser (Applied Biosystems, USA). Peptide mass spectra and MS/MS information that acquired were then

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subjected to searching against the NCBI non-redundant database with entities restricted to "other eukaryota" sequences (724,457 entries).

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2.5. Data analysis

To determine IC10 and IC50 (i.e., inhibition concentrations led to 10% and 50% growth

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inhibition, respectively in relation to the control) and EC10 and EC50 (i.e., effect concentrations

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triggered 10% and 50% negative effects on the photosynthesis parameter, respectively relative to the control), experimental data were normalized to the control before being fitted to a sigmoidal

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log(dose)-response (variable slope) using GraphPad Prism version 5.00 (GraphPad software, San Diego). Due to unequal variances among the data, the comparison of NPQ among the four

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treatment/control groups was conducted using Kruskal-Wallis test and followed by Dunn’s post-

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hoc test. To identify the differentially expressed proteins or genes, either one-way analysis of

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variance (ANOVA) with Tukey's post-hoc test (for data with an equal variance) or KruskalWallis test with Dunn's post-hoc test (for data failed the homogeneity requirement for ANOVA)

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was employed.

3. Results

3.1. Effects of TPT on growth and photosynthesis Exposure to TPTCl significantly reduced the relative algal growth rate in a concentrationdependent manner (Fig. 1a). Based on this relationship, IC10 and IC50 was estimated as 0.46 μg/L (95% confidence interval (CI): 0.27-0.69 μg/L) and 1.09 μg/L (95% CI: 0.89-1.34 μg/L), respectively. Similarly, the photochemical quenching, in terms of ΦPo and Φ2, decreased with

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increasing TPTCl concentration (Figs. 1b & c). The sensitivities of T. pseudonana to both photosynthesis endpoints were quite similar, with the EC50 values of ΦPo and Φ2 equal to 1.54 (95% CI: 1.40–1.69 μg/L) and 1.51 μg/L (95% CI: 1.44–1.58 μg/L) and the EC10 values equal to

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1.02 (95% CI: 0.85–1.18 μg/L) and 0.96 μg/L (95% CI: 0.76–1.17 μg/L), respectively. The nonphotochemical parameter NPQ was significantly increased at 0.5 and 1.0 μg/L when compared

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with the solvent control, and it was decreased as the TPTCl concentration increased from 1.0 to

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2.5 μg/L (Fig. 2).

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According to the above results, the exposure TPTCl concentrations of 0.5 μg/L (close to IC10 value for growth inhibition) and 1.0 μg/L (close to IC50 for growth inhibition and EC10 for

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3.2. Gene expression profiles

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photosynthesis test) were selected for the subsequent genomic and proteomic study.

Except LacsA gene, which is involved in the fatty acid biosynthesis, expressions of other

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selected genes in T. pseudonana were all regulated by TPTCl exposure (Fig. 2). For example, 3HfcpA and 3HfcpB genes, which are related with photosynthesis processes, were up-regulated (> 1.5 folds) in T. pseudonana after exposure to 0.5 μg/L TPTCl, while a significant downregulation of 3HfcpA gene was observed in the algae exposed to 1.0 μg/L TPTCl (Fig. 2a). Both silaffin precursor genes (sil1 and sil3) and sit1, which are related to silica shell formation, displayed a significant down-regulation at 1.0 μg/L TPTCl (Fig. 2c).

3.3. Proteome response to TPTCl exposure

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The results of proteome analysis revealed approximated 290–300 total protein spots (mostly fall in pI 4–6) in 2D gels for T. pseudonana after exposure to the seawater control, solvent control and 0.5 μg/L TPTCl treatment, respectively (Fig. 4). In contrast, the total number of

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spots that detected on the 2D gels of 1.0 μg/L TPTCl treated samples was largely reduced to 50– 60 and some of these proteins spots were not present in the 2D gels of the other three groups (i.e.,

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spots 6 to 9; Fig. 4). The four proteins that expressed only at the 1.0 μg/L TPTCl treatment were

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identified as a fucoxanthin chlorophyll a/c protein (FCP), a hypothetical protein, FCP6 and FCP-

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LI818 (Table 2).

Statistical analysis was further performed for detectable differentially expressed protein spots

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among the three groups (i.e., the seawater control, solvent control and 0.5 μg/L TPTCl treatment).

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Compared with the seawater control or solvent control, five proteins (spots 1 to 5) were

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identified to be differentially expressed in the algae exposed to 0.5 μg/L TPTCl, and four of them were up-regulated. The up-regulated proteins were identified as oxygen-evolving enhancer

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protein 1 precursor (OEE1) (26.8-fold), FCP-LI818 (3.2-fold) and mitochondrial manganese superoxide dismutase (Mn-SOD, 3.4-fold; Fig. 5), while the only down-regulated protein was a hypothetical protein.

4. Discussion 4.1. Effects of TPT on growth and photosynthesis Based on growth performance, the 96h-IC50 value of TPTCl on T. pseudonana was found to be 1.09 μg/L (95% CI: 0.89-1.34 μg/L) in the present study, which is comparable to the 72h-

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IC50 value of TPT reported for the same algae species (i.e., 1.34 μg/L; Walsh et al., 1985). The sensitivity of T. pseudonana to TPT is also close to another marine diatom Skeletonema costatum (72h-IC50 = 0.92 μg/L; Walsh et al., 1985). When compared to other algal species such as

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Scenedesmus obliquus (96h-IC50 = 2.62 μg Sn/L; Huang et al., 1996), Porphyra yezoensis (48hIC50 = 3.6 μg/L; Maruyama et al., 1991) and Spirulina subsalsa (8d-IC50 = 15.63 μg/L; Huang

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et al., 2002), T. pseudonana tends to be more sensitive to TPT. Moreover, T. pseudonana is also

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more sensitive to TPT than some freshwater algal species. The reported 72h-IC50 of TPT for four freshwater algae (including Selenastrum capricornutum, Desmodesmus subspicatus,

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Monoraphidium minutum and Scenedesmus quadricauda) ranged from 8.8 to 51.5 μg/L (Roessink et al., 2006).

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In terms of photosynthesis parameters (ΦPo and Φ2), the 96h-EC50 values were found to be

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about 1.5 μg/L for T. pseudonana in the present study. Our results indicated that photosynthesis

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of T. pseudonana was less affected by TPTCl when comparing with its effect on the algal growth. Similar results were also observed for another marine microalga Phaeodactylum tricornutum

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upon exposure to copper (Cid et al., 1995).

The measurement of dark-adapted ΦPo has been used as a sensitive indicator of photosynthesis performance (Maxwell and Johnson, 2000). Decreased ΦPo upon exposure to TPTCl indicated an impairment of PSII photochemical reactions and fewer open reaction centres available in T. pseudonana cells, which could ultimately affect their photosynthesis performance (Kalaji et al., 2011). Φ2 values can give a measure of the rate of linear electron transport (Maxwell and Johnson, 2000). The electron transport chain between plastoquinone A (PQA) and plastoquinone B (PQB) was considered the most susceptible part when exposed to metals (Rutherford and Inoue, 1984; Krupa and Baszyński, 1995). Some herbicides (such as triazines,

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urea and diazine) were able to attach onto the molecule of D1 protein and inhibit the electron transport between PQA and PQB in the PSII, thereby affecting non-cyclic photophosphorylation rate and carbon dioxide assimilation (Rutherford and Inoue, 1984; Genty et al., 1989; Murkowski

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and Sk rska, 2010). TPT has been found to have similar effects on the freshwater green alga Chlorella vulgaris (Murkowski and Sk rska, 2010). In the current study, the decreased Φ2

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values in TPT-treated diatoms suggest that TPTCl might have affected the electron transport

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chain in T. pseudonana as there is a strong linear relationship between Φ2 and the efficiency of

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carbon fixation.

Non-photochemical quenching (NPQ), which occurs in almost all photosynthetic eukaryotes,

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can help to regulate and protect photosynthesis in microalgae (Müller et al., 2001). In the

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present study, increased NPQ values were observed at low concentrations of TPTCl, which is in

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accordance with many other studies which reported an increased value of NPQ related parameters under salt stress (Moradi and Ismail 2007), light stress (Müller et al., 2001) and

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chemical stress (e.g., paraquat, norflurazon, flazasulfuron and atrazine; Frankart et al., 2003). In contrast, our observed decrease in NPQ in T. pseudonana after exposure to high concentrations of TPTCl (≥ 1.5 µg/L) may indicate the possibility of TPTCl binding to the second electron acceptor and hence inhibiting the electron transport process (Frankart et al., 2003).

4.2. Differentially expressed genes Tonon et al. (2005) demonstrated that T. pseudonana contains the necessary fatty acid synthesis, desaturation, elongation, and acylation mechanisms to accumulate polyunsaturated fatty acids, and LacsA is related to biosynthetic pathways of fatty acid derived molecules. In T.

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pseudonana, PAHs could up-regulate the expression of LacsA and therefore affect the lipid metabolism (Bopp and Lettieri, 2007), and a similar result was also found for benzo(a)pyrene (Carvalho et al., 2011). Up to now, there is no information on the effect of organotin compounds

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on the diatom's lipid metabolism. In the green microalga Scenedesmus quadricauda, TPT could trigger an increase of lipid peroxidation (Xu et al., 2011). In the present study, the expression of

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LacsA was not affected by TPTCl even at the concentration as high as 1.0 μg/L (Fig. 2b),

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indicating that the lipid metabolism system is unlikely influenced by TPTCl at 0.5-1.0 μg/L. However, measurement of physiological parameters such as lipid content and lipid peroxidation

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will be required to further confirm the present results.

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A recent review on oxidative stress in microalgae has summarized that the production of

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reactive oxygen species (ROS) in algal species could be caused by different stresses including

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light, metals and other chemicals (Cirulis et al., 2013). TPT is one of the chemicals that have already been reported to increase ROS in another algae species Scenedesmus quadricauda at 4.0 Fucoxanthin-chlorophyll a/c light harvesting protein (FCLHP) is

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μg/L (Xu et al., 2011).

involved in harvesting energy and transferring it to reaction centres, and its encoding genes have been found to respond to different stresses that could cause ROS. For example, FCLHP genes can be up-regulated in T. pseudonana upon Fe-limitation (Thamatrakoln et al., 2012) and in the dinoflagellate Pyrocystis lunula upon exposure to sodium nitrite (Okamoto and Hastings, 2003). In the present study, the encoding genes of FCLHP (3HfcpA and 3HfcpB) showed a nearly identical expression trend that both were significantly up-regulated in T. pseudonana exposed to 0.5 μg/L TPTCl. The enhanced expression of 3HfcpA and 3HfcpB genes at this concentration could contribute to the maintenance of the integrity of the light harvesting complex (Ritter et al.,

17

Page 17 of 40

2010), so that both photosynthesis parameters (ΦPo and Φ2) were not affected at this TPTCl concentration. On the contrary, the down regulation of the two FCLHP encoding genes in T. pseudonana after exposure to 1.0 μg/L TPTCl indicated a reduction of photosynthetic activities.

ip t

Such observations were echoed by the lower values of ΦPo and Φ2 detected at this concentration. Generally, an excess exposure to chemicals (e.g., copper) can cause direct damage to the PSII,

cr

and generate oxidative stress as a result of the PSII dysfunction (Ritter et al., 2010). An

us

increased production of ROS can in turn lead to membrane destabilization and cell death (Srivastava et al., 2005). Accordingly, the population growth of T. pseudonana was inhibited by

an

about 50% at 1.0 μg/L TPTCl as shown in this study. Similar effects on growth, photosynthesis and suppression of expression of FCLHP encoding genes have been found in T. pseudonana

M

after exposure to PAH (Bopp and Lettieri, 2007). In spite of the fact that the expressions of

d

FCLHP-related genes can be influenced by many different stressors, the mechanism of FCLHP

te

upon stress exposure is not yet clearly described which deserves further research.

Ac ce p

Chemical exposure, such as PAHs, can affect the biosilicification process of T. pseudonana through silica uptake or polymerization pathways (Carvalho et al., 2011). In T. pseudonana, silicon transporter protein is involved in silica uptake, and its encoding genes are involved in cell wall formation by directly catalysing silica polymerization (Poulsen and Kroger, 2004). In the current study, the expression of the three associated biosilicification genes (e.g. sit1, sil3 and sit1) were significantly down-regulated at 1.0 μg/L TPTCl, indicating a hinder of the silica uptake process which could result in a reduction in the available intracellular pools of silica and a disturbance of biosilica formation.

Overall, the genes that regulate silica uptake and

18

Page 18 of 40

polymerization in diatom species could be potential biomarkers to monitor TPT pollution in coastal marine environments.

ip t

4.3. Differentially expressed proteins

Proteomic studies on T. pseudonana upon chemical exposures have been carried out in recent

cr

years after the publication of its whole genome (Armbrust et al., 2004). For instance, proteins

us

functioning on photosynthesis, DNA methylation, lipid metabolism and silicon uptake in T. pseudonana were influenced by benzo(a)pyrene (Carvalho and Lettieri, 2011), while their Rieske

an

Fe-S protein and malate dehydrogenase like protein could be affected by exposure to thiobencarb herbicide (Shimasaki et al., 2013). It should be noted that in the present study and these

M

published studies, some proteins are still annotated as predicted or putative proteins, which limit

d

the interpretation of pathways involved in response to TPTCl exposure (Table 2).

te

The FCP-LI818 clade belongs to the light harvesting complex (LHC) superfamily and it has already been discovered in green and brown algae in response to environmental stresses (Dittami

Ac ce p

et al., 2010; Richard et al., 2000). Identification and expression of FCP-LI818 proteins in T. pseudonana have only been conducted upon exposure to high light intensity.

The results

suggested that this group of proteins is likely involved in both photo-protection and lightharvesting in T. pseudonana (Zhu and Green, 2008). Yet, the function of FCP-LI818 proteins against chemical stress has not been uncovered. In general, proteins which belong to LHC family have a key role in controlling the NPQ level, which play a similar role as the PsbS protein of PS-II in plants (Depauw et al., 2012). Thus, in this study, the increased NPQ value in T. pseudonana after exposure to 0.5 µg/L TPTCl might be controlled by the up-regulation of the

19

Page 19 of 40

FCP-LI818 protein. The results also implied that the FCP-LI818 protein might be involved in protection mechanism against TPTCl-induced stress in the diatom.

ip t

The OEE1 protein, which is a highly conserved protein in phototrophic organisms ranging from cyanobacteria and microalgae to higher plant chloroplasts, is an auxiliary component of the In the large-leafed mangrove Bruguiera

cr

PS-II manganese cluster (Heide et al., 2004).

us

gymnorrhiza, an increased expression of this protein was observed to maintain the capacity of PSII complex under NaCl treatment (Sugihara et al., 2000). Besides salinity stress, the OEE1

an

protein was also induced by heat stress in the seaweed Ecklonia cava (Yotsukura et al., 2012). In the present study, the expression of the OEE1 protein was up-regulated by 26.8 folds in T.

M

pseudonana exposed to 0.5 μg/L TPTCl suggesting that this protein could function to maintain

d

the capacity of PSII instead of increasing the number of PSII complexes. It was because the

te

photosynthesis reaction centre D1 protein was not included in the four up-regulated proteins that observed in this study and the photosynthesis parameters were not affected at the 0.5 μg/L

Ac ce p

TPTCl treated diatoms.

The MnSOD protein is a key antioxidant that catalyses the transformation of superoxide to molecular oxygen and hydrogen peroxide so as to protect biological systems (Wolfe-Simon et al., 2006). In T. pseudonana, the MnSOD protein is localized in chloroplasts and can catalyse the destruction of ROS (Wolfe-Simon et al., 2006). When coping with oxidative stress, diatoms substantially rely on the MnSOD than other isoforms of SODs (FeSOD, Cu/ZnSOD and NiSOD) (Peers and Price, 2004). In this study, the up-regulation of the MnSOD in T. pseudonana by

20

Page 20 of 40

TPTCl exposure was possibly associated with remediation of the toxicant-mediated oxidative stress mediated.

ip t

Interestingly, only about 50–60 protein spots were found on the 2D gels of T. pseudonana after exposure to 1.0 μg/L TPTCl and some of these protein spots were not detected in other This may be caused by degradation or damage of the proteins, and

cr

treatments (Fig. 4).

us

disordered metabolism of T. pseudonana after exposure to such a high concentration of TPTCl. A similar observation was reported in the thale cress Arabidopsis thaliana upon exposure to high

an

concentrations of nonylphenol (Chen and Yen, 2013). It could also be explained by the possible variations in biochemical compositions of the diatom under different environmental conditions

M

(Brown et al., 1996). For instance, the diatoms exposed to TPT at 1.0 μg/L (which was close to

d

the IC50 of growth inhibition) likely had a different biochemical composition when compared

Ac ce p

te

with those exposed to the seawater and solvent control groups.

5. Conclusions

The 96h-IC50 of TPTCl for growth inhibition in T. pseudonana was found to be 1.09 μg/L (95% CI: 0.89–1.34 μg/L), while their 96h EC50 values based on the two photosynthesis parameters were estimated at 1.54 μg/L (95% CI: 1.40–1.69 μg/L) and 1.51 μg/L (95%CI: 1.44– 1.58 μg/L) for ΦPo and Φ2, respectively. Non-photochemical quenching (NPQ) was increased in response to lower concentrations of TPTCl (0.5-1 μg/L) but decreased gradually between 1.0 and 2.5 μg/L. The overall molecular responses in T. pseudonana were highly dependent on the exposure concentration (Fig. 6). When the diatom was exposed to 0.5 μg/L TPTCl, defensive mechanisms were induced.

The up-regulation of the MnSOD has offered a significant 21

Page 21 of 40

antioxidant role in the elimination of reactive oxygen species (i.e., dismutation of superoxide radical). In addition, the induction of the OEE1 protein could protect the reaction centre D1 protein from oxidative stress (Heide et al., 2004). On the other hand, the increased expression of

ip t

the FCP-LI818 protein might enhance NPQ, which in turn helped to dissipate the excess energy that generated in this tolerant line under TPTCl-induced stress. Consequently, the elevated NPQ

cr

level would protect PSII and maintain the photosynthesis process. The results are in accordance

us

with the enhanced expression of 3HfcpA and 3HfcpB genes at this concentration, which could contribute to the maintenance of the integrity of the light harvesting complex as well. Overall,

an

all these genes/proteins that up-regulated by TPTCl can function to maintain the capacity of PSII and stabilize the photosynthesis efficiency as revealed by the unchanged ΦPo and Φ2 values. In

M

contrast, when T. pseudonana was exposed to 1.0 μg/L TPTCl, the excess exposure of TPT

d

caused direct damage to the proteins and/or caused the suppression of metabolism as reflected by

te

the down-regulation of most studied proteins/genes, which could ultimately inhibit the growth

Ac ce p

and photosynthesis of the diatom.

Acknowledgements

This work is substantially supported by the Area of Excellence Scheme under the University Grants Committee (Project No. AoE/P-04/2004) of the Hong Kong SAR Government and a Small Project Fund from the University of Hong Kong (HKU) to KMY Leung. The authors also thank HKU for providing a Type-B PhD studentship to AX Yi and a matching fund to support PTY Leung for her postdoctoral fellowship.

22

Page 22 of 40

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ip t

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Table 1. Primer sequences of target genes and inner control Abbreviation

Primer sequences (5'→3')

Reference

Glyceraldehyde-3phosphate dehydrogenase

GAPDH

F: GGAGAAGGCCTCCATGCAT R: TGGAGCCGAGATGACAACCT

Bopp and Lettieri, 2007

Fucoxanthin-chlorophyll a/c light harvesting protein

3HfcpA

F: CTCCCTCCAGGTTCCTGTTG R: AGCGAGCTCAAGGAATCCAA

Bopp and Lettieri, 2007

Fucoxanthin-chlorophyll a/c light harvesting protein

3HfcpB

F:AGTTCGATGAGGAGACCAAGCT R: GGCACGTCCGTTGTTCAAC

Bopp and Lettieri, 2007

Long chain acyl-coA synthetase

lacsA

F: GGCATGTCGTGTGTGGTTTG R: TTGGCCTCGCACAATCG

Bopp and Lettieri, 2007

Silaffin precursor 1

sil1

F: CCGTCACCCTCTCCTGAAAC R: ATGGGAGCAGCGGTAATGG

Bopp and Lettieri, 2007

Silaffin precursor 3

sil3

F: GGTGCAAAGAGTGCCAAGATG R: GCTGCGTCCTCCGACTTTC

Bopp and Lettieri, 2007

Silicon transporter 1

sit1

F: TTGCCGAGGATGCCTAAACTT R: TGACGAGCTACTGCAGGTTCA

Bopp and Lettieri, 2007

Ac ce p

te

d

M

an

us

cr

ip t

Name of the gene

31

Page 31 of 40

4 5 6 7 8

fucoxanthin chlorophyll a/c protein - LI818 clade hypothetical protein THAPSDRAFT_7881 fucoxanthin chlorophyll a/c protein hypothetical protein THAPSDRAFT_7881 fucoxanthin chlorophyll a/c protein 6 fucoxanthin chlorophyll a/c protein -LI818 clade

pI (T/O)

gi|224003107

146

1/18

32/25

5.3/5.4

gi|223997268

141

2/9

27/24

gi|224008172

294

4/22

gi|224011607

485

6/28

gi|224006870

710

7/29

gi|223993505

304

gi|224006870

333

gi|223998112

484

gi|224011607

295

e

ip t

Mr (kDa) (T/O)d

5.4/5.3

Tha lass iosi ra pse udo nan a.

27/24

5.2/5.2

21/22

4.9/5.3

27/31

4.9/4.8

3/27

23/22

4.7/4.8

4/15

22/19

4.9/4.6

8/29

22/19

5.2/4.7

3/18

21/19

4.9/4.9

Ac ce p

te

9

predicted protein

Peptide matched/coverage (%)

cr

3

Protein score (C.I.)c

us

2

oxygen-evolving enhancer protein 1 precursor mitochondrial manganese superoxide dismutase

Accession No.

an

1

Protein

b

M

Spot No.a

d

Table 2. List of identified proteins responsive to triphenyltin chloride (TPTCl) in the diatom

Remarks: a Spot numbers as assigned in Fig. 4. b Proteins identified from database “NCBInr_other Eukaryota". c Mascot score generated by Mascot MS/MS ion search (p < 0.05, ion score > 41 for NCBInr). d Mr and e pI (T, theoretical/O, observed molecular mass and isoelectric point of protein, respectively).

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Figure captions Fig. 1. Triphenyltin chloride (TPTCl) induced inhibition of (a) growth, (b) ΦPo and (c) Φ2 in

ip t

Thalassiosira pseudonana. Data are presented as the percentage that relative to control (n = 4 to 6), and the relative percentage fitted to a log (dose)-normalized response curve (variable slope):

us

R2 = 0.971, Hillslope = -4.67; for Φ2, R2 = 0.986, Hillslope = -5.58.

cr

Y = 1--/(1+10(LogEC50-X)*Hillslope)). For growth inhibition, R2 = 0.940, Hillslope = -2.55; for ΦPo,

an

Fig. 2. Effects of triphenyltin chloride (TPTCl) on nonphotochemical quenching (NPQ) of Thalassiosira pseudonana (bar represents the mean and error bar represents standard deviation; n

M

= 6). Bars with different letters indicate significantly different means of NPQ values (p < 0.05,

d

Kruskal Wallis test and Dunn's post-hoc test). C: seawater control. SC: solvent control.

te

Fig. 3. Relative mRNA expression of (a) 3HfcpA and 3HfcpB genes, (b) LacsA and (c) sil1, sil3

Ac ce p

and sit1 genes upon exposure to different concentrations of triphenyltin chloride (TPTCl). Thalassiosira pseudonana-GAPDH gene was used as a reference housekeeping gene to normalize the expression. C: seawater control. SC: solvent control. Bar represents the mean and error bar represents standard deviation (n = 3). Bars with different letters indicate significantly differential gene expression levels (p < 0.05; one-way ANOVA and Tukey post-hoc test for data passing the Levene’s test for homogeneity of variance; Kruskal-Wallis test and followed by Dunn’s post-hoc test for data failing the Levene’s test).

33

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Fig. 4. Representative 2DE gels of proteomic profiles in Thalassiosira pesudonana in response to (a) seawater control, (b) solvent control, (c) 0.5 µg/L triphenyltin chloride (TPTCl) and (d) 1.0

ip t

µg/L TPTCl. Proteins were separated by 11 cm pH 4-7 IEF strips and followed by separation

cr

using 12% SDS-PAGE. The gels were visualized using Colloidal CBB-G-250.

us

Fig. 5. Statistical representation of the changes in expression levels of the five identified proteins (Spot 1 to 5). C: seawater control. SC: solvent control. Bar represents the mean and error bar

an

represents standard deviation (n = 4). Bars with different letters indicate significantly differential

M

gene expression levels (p < 0.05; one-way ANOVA and Tukey's post-hoc test).

Fig. 6. Summary of possible toxicity pathways of triphenyltin chloride (TPTCl) in Thalassiosira

d

When the diatom was exposed to 0.5 μg/L TPTCl, MnSOD, as the main

te

pseudonana.

antioxidant in T. pseudonana, was up-regulated to catalyse the elimination of active oxygen

Ac ce p

species; the up-regulation of OEE1 could protect the reaction centre D1 protein from oxidative stress; and the increased expression of FCP-LI818 might enhance NPQ, which helped to dissipate the excess energy that generated in this tolerant line under TPTCl-induced stress, and the elevated NPQ level could in turn protect the PSII system and stabilize the photosynthesis efficiency as revealed by unchanged ΦPo and Φ2 values. The enhanced expression of 3HfcpA and 3HfcpB genes at this concentration could contribute to the maintenance of the integrity of the light harvesting complex. In contrast, when T. pseudonana was exposed to 1.0 μg/L TPTCl, the excess toxicity of TPT might directly damage the protein and/or cause the suppression of metabolism as revealed by the down-regulation of most studied proteins/genes, which ultimately inhibited

the

population

growth

and

photosynthesis

in

the

algae.

34

Page 34 of 40

ip t cr us an M d te Ac ce p

Fig. 1

35

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0.6 c bc

ip t

0.5 a

ad

0.3

us

NPQ

0.4

cr

ab

de

an

0.2

M

0.1

e

C

SC

0.5

d

0

1.0

1.5

2.0

2.5

Ac ce p

Fig. 2

te

TPTCl conc. ( g/L)

36

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(a)

2.0

b

1.5

a

a

a

0.5

0.0 C

SC

0.5

1

1.2

an

Lacs A 1.0

0.2 0.0

d

0.6 0.4

(b)

M

0.8

te

Relative mRNA expression

a

c

cr

1.0

a

b

ip t

3Hfcp A 3Hfcp B

us

Relative mRNA expression

2.5

Ac ce p

C

SC

0.5

1

1.6

Relative mRNA expression

1.4 1.2

sil 1 sil 3 sit 1

a a a

(c)

a ab ab a

ab ab

1.0

b b b

0.8 0.6 0.4 0.2 0.0

C

SC

0.5

1

TPTCl ( g/L)

Fig. 3

37

Page 37 of 40

ip t cr us an M Ac ce p

te

d

Fig. 4

38

Page 38 of 40

20000

ip t

a

a

cr

25000

us

b

10000

a a

a a

a a

Spot 2

Spot 3

b a

a

Spot 4

Spot 5

Spot number

Ac ce p

te

Spot 1

M

b

5000

b

an

15000

0

Fig. 5

C SC 0.5 g/L

b

d

Normalized spots volume (arbitrary unit)

30000

39

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ip t cr us an M d te Ac ce p

Fig. 6

40

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