Article pubs.acs.org/est
Analysis of the Proteome of the Marine Diatom Phaeodactylum tricornutum Exposed to Aluminum Providing Insights into Aluminum Toxicity Mechanisms Jun Xie,†,¶ Xiaocui Bai,‡,¶ Michel Lavoie,§ Haiping Lu,⊥ Xiaoji Fan,† Xiangliang Pan,# Zhengwei Fu,† and Haifeng Qian*,†,‡,#
Downloaded by RUTGERS UNIV on September 4, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.est.5b03272
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College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, P. R. of China ‡ Department of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang 310032, P. R. of China § Quebec-Ocean and Takuvik Joint International Research Unit, Université Laval, Québec City, Québec G1V 0A6 Canada ⊥ College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. of China # Xinjiang Key Laboratory of Environmental Pollution and Bioremediation, Chinese Academy of Sciences, Urumqi, Xinjiang 830011, P. R. of China S Supporting Information *
ABSTRACT: Trace aluminum (Al) concentrations can be toxic to marine phytoplankton, the basis of the marine food web, but the fundamental Al toxicity and detoxification mechanisms at the molecular levels are poorly understood. Using an array of proteomic, transcriptomic, and biochemical techniques, we describe in detail the cellular response of the model marine diatom Phaeodactylum tricornutum to a short-term sublethal Al stress (4 h of exposure to 200 μM total initial Al). A total of 2204 proteins were identified and quantified by isobaric tags for relative and absolute quantification (iTRAQ) in response to the Al stress. Among them, 87 and 78 proteins performing various cell functions were up- and down-regulated after Al treatment, respectively. We found that photosynthesis was a key Al toxicity target. The Al-induced decrease in electron transport rates in thylakoid membranes lead to an increase in reactive oxygen species (ROS) production, which cause increased lipid peroxidation. Several ROS-detoxifying proteins were induced to help decrease Al-induced oxidative stress. In parallel, glycolysis and pentose phosphate pathway were upregulated in order to produce cell energy (NADPH, ATP) and carbon skeleton for cell growth, partially circumventing the Alinduced toxicity effects on photosynthesis. These cellular responses to Al stress were coordinated by the activation of various signal transduction pathways. The identification of Al-responsive proteins in the model marine phytoplankton P. tricornutum provides new insights on Al stress responses as well as a good start for further exploring Al detoxification mechanisms.
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INTRODUCTION Aluminum (Al) is the third most abundant element in the Earth’s crust.1 This element enters freshwater systems mainly through weathering and erosion and then reaches the marine environment.2 Although anthropogenic Al sources to aquatic systems are thought to be smaller than natural sources at the global scale,2 anthropogenic inputs of aluminum in aquatic systems as for other metals are likely increasing in Asian countries3 resulting in the contamination of some marine environments, in which Al concentrations can reach levels of concerns. Even though the total dissolved Al concentrations in the open ocean is in the low nanomolar (to subnanomolar) range,4,5 total dissolved Al concentrations of 85−389 nM (with a peak of 1130 nM) have been reported in surface coastal waters affected by aluminum-rich freshwater inputs.6 Such high © XXXX American Chemical Society
concentrations are close to or higher than the total Al concentrations known to inhibit the growth rate of (at least) one marine diatom (Nitzschia closterium, EC10 ± IC95% = 519 ± 111 to 927 nM),7,8 raising concerns for the potential toxicity of aluminum in sensitive organisms such as phytoplankton in the marine environment. New technologies now enable the rapid study of a wide array of metal toxicity targets (at the genetic, transcriptomic, and proteomic levels) in a given organism. However, the use of these new tools has been restricted mostly to higher plants. One recent and promising high-throughput analytical technique Received: July 6, 2015 Revised: August 23, 2015 Accepted: August 26, 2015
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DOI: 10.1021/acs.est.5b03272 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Downloaded by RUTGERS UNIV on September 4, 2015 | http://pubs.acs.org Publication Date (Web): September 1, 2015 | doi: 10.1021/acs.est.5b03272
Environmental Science & Technology
analyzer MINI-PAM-II (Walz, Germany). The maximum quantum yield of photosystem II (Fv/Fm), the effective quantum yield of PSII (ΦII), and the relative electron transport rate of PSII (rETR) were calculated. Reactive Oxygen Species and Malondialdehyde Assays. Reactive oxygen species (ROS) concentrations were measured using the fluorescent probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) following the instructions supplied with the ROS Assay Kit (Beyotime Institute of Biotechnology, Haimen, China). The lipid peroxidation level was determined by measuring malondialdehyde (MDA) according to the method provided in the MDA kit (Beyotime Institute of Biotechnology, China). Gene Transcription Analysis. Total RNA was extracted from algal cells using RNAiso (Takara Company, Dalian, China) according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA using a reverse transcriptase kit (Toyobo, Tokyo, Japan). After mixing each cDNA samples with the SYBR Green PCR reagents (Toyobo, Tokyo, Japan), the amount of cDNA was measured by real-time quantitative PCR (qRT-PCR) using an Eppendorf Master Cycler ep RealPlex4 (Wesseling-Berzdorf, Germany). Actin was used as a housekeeping gene to standardize the results by eliminating variations in the quality of the cDNA. The relative quantification of gene transcription among the treatment groups was achieved using the 2−ΔΔCt method.18 The sequence of the primer pairs designed to amplify the selected genes are listed in Table S2. Protein Extraction, iTRAQ Labeling, and LC-MS/MS Analysis. After 4 h of Al treatment, total proteins from P. tricornutum (approximately 0.3 g of algal fresh weight) were extracted and digested. After iTRAQ (AB Science) labeling, equal amounts of labeled peptides from each group were mixed and resolved into 15 fractions by HPLC, followed by Q Exactive mass spectrometry (Thermo Fisher Scientific). The resulting MS/MS data were searched against a Uniprot P. tricornutum protein database with MaxQuant (v1.0.13.13) with 1% false detection rate. Evaluation of the function of differentially expressed proteins was performed using Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, functional enrichment analysis, and secondary structure analysis. The GO classification was done using UniProt-GOA database and InterProScan soft. We used wolfpsort to predict subcellular localization of the Alresponsive proteins. Data Analysis. The iTRAQ results for protein identification and quantification were selectively filtered before exportation according to several criteria. An ion score or expected cutoff less than 0.05 (with 95% confidence) was required for protein identification. A threshold of 1.3-fold up- or down-regulation was chosen to identify significant over- or under-expression of proteins, respectively, in addition to p-value
