Article pubs.acs.org/jpr
SWATH-MS Quantitative Proteomic Investigation Reveals a Role of Jasmonic Acid during Lead Response in Arabidopsis Fu-Yuan Zhu,†,‡,§ Wai-Lung Chan,† Mo-Xian Chen,‡ Ricky P. W. Kong,∥ Congxi Cai,⊥ Qiaomei Wang,⊥ Jian-Hua Zhang,*,‡ and Clive Lo*,† †
School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong, China School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China § Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China ∥ SCIEX, Hong Kong, China ⊥ Department of Horticulture, Zhejiang University, Hangzhou 310058, China ‡
S Supporting Information *
ABSTRACT: Lead (Pb) pollution is a growing environment problem that continuously threatens the productivity of crops. To understand the molecular mechanisms of plant adaptation to Pb toxicity, we examined proteome changes in Arabidopsis seedlings following Pb treatment by SWATH-MS, a label-free quantitative proteomic platform. We identified and quantified the expression of 1719 proteins in water- and Pb-treated plants. Among them, 231 proteins showed significant abundance changes (151 elevated and 80 reduced) upon Pb exposure. Functional categorization revealed that most of the Pb-responsive proteins are involved in different metabolic processes. For example, down-regulation of photosynthesis and biosynthesis of isoprenoids and tetrapyrroles in chloroplasts were observed. On the contrary, pathways leading to glutathione, jasmonic acid (JA), glucosinolate (GSL), and phenylpropanoid production are up-regulated. Experimental characterizations demonstrated a rapid elevation of endogenic JA production in Pb-treated Arabidopsis seedlings, while a JA-deficient mutant and a JA-insensitive mutant showed hypersensitivity to root inhibition by Pb, implicating an essential role of JA during Pb responses. Consistently, methyl jasmonate supplementation alleviated Pb toxicity in the wildtype and JA-deficient mutant. Furthermore, GSL levels were substantially enhanced following Pb treatment, while such induction was not detected in the JA mutant, suggesting that the Pb-induced GSL accumulation is JA-dependent. Overall, our work represents the first SWATH-MS analysis in Arabidopsis and highlights a potential mediating role of JA during Pb stress. KEYWORDS: SWATH, Arabidopsis, lead response, jasmonic acid, glucosinolates
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INTRODUCTION Heavy-metal pollution is a growing global threat in the environment, and it continuously threatens human health and crop productivity.1 Among those pollutants, the abundant toxic metal lead (Pb) has become a great concern due to the amplification of its toxic effects through the food chain and its persistence in the environment.2 Pb is also recognized as the second most hazardous substance next to arsenic by the Agency for Toxic Substances and Disease Registry (ATSDR 2003). There are various toxic responses and effects on living organisms upon Pb exposure. In human, Pb adversely affects the functions of most organs and the development of brain and nervous system. In plants, the potential Pb-induced toxic effects include restriction of growth, influence of nutrient uptake, disruption of water status, and inhibition of photosynthesis,3 thus seriously affecting crop supply and agriculture.1 To cope with different heavy metal stresses in the environment, plants have developed a range of potential cellular mechanisms © XXXX American Chemical Society
involved in the detoxification process such as metal binding to the cell wall, efflux pumping of metals at the plasma membrane, chelation of metal in the cytosol, compartmentation of metals in the vacuole, and the induction of antioxidants.4 In recent years, a number of functional genes implicated in Pb tolerance in plants have been identified, including Arabidopsis PLEIOTROPIC DRUG RESISTANCE 8 and 12 (AtPDR8 and AtPDR12), rice HEAVY-METAL ATPase 9 (OsHMA9), Arabidopsis ACYL-COA BINDING PROTEIN 1 (AtACBP1), and sorghum extracellular leucine-rich repeat protein 2 (SbLRR2), providing different new insights into plant responses to Pb stress. AtPDR8 and AtPDR12 are ABC transporter proteins,5,6 while OsHMA9 is a P(1B)-type metal transporter.7 AtACBP1 is a Pb-binding protein, and its overexpression enhances Pb tolerance in Arabidopsis.8 Transgenic expression of SbLRR2 in Arabidopsis also enhances Pb tolerance, Received: March 24, 2016
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DOI: 10.1021/acs.jproteome.6b00258 J. Proteome Res. XXXX, XXX, XXX−XXX
Article
Journal of Proteome Research
pH 8.0), incubated at 95 °C for 5−10 min, and briefly cooled in an ice bath. The samples were centrifuged at 16 000g at 4 °C twice. Clear supernatants were collected and mixed with 4× volume of chilled 80% acetone for overnight protein precipitation at −20 °C. Proteins were then pelleted at 1600g centrifugation for 15 min at 4 °C and pellets were washed with 10× volume of 80% acetone. Protein pellets were air-dried for 5−10 min at room temperature before dissolving in 50 μL of urea buffer (6 M urea in 200 mM MOPS-Cl/4 mM CaCl2, pH 8.0). Protein concentrations were determined by the Bradford method (Bio-Rad). Protein samples (100 μg each) were diluted to 1 μg/μL with 100 mM ammonium bicarbonate (ABC) and reduced by 10 mM DTT at 50 °C for 40 min with gentle shaking. Reduced samples were cooled to room temperature and alkylated by 40 mM iodoacetamide for 30 min in darkness. They were then further diluted to 0.5 μg/μL with 100 mM ABC, and the urea concentration was kept below 2 M. Trypsin digestion was performed (enzyme/protein 1:50 w/w) overnight at 37 °C. Subsequently, trypsinized samples were acidified with 10% trifluoroacetic acid, desalted in SepPak C18 cartridges (Waters), according to the manufacturer’s instructions, and dried in speed vacuum concentrator. Peptide samples were stored at −80 °C if not analyzed immediately or dissolved in 0.1% formic acid for LC−MS/MS analysis.
possibly through the upregulation of AtPDR12 expression.9 Meanwhile, transcriptome analyses in plants upon exposure to heavy metals including Pb have been performed by several groups,10−12 improving our understanding of Pb stress responses in plants at mRNA level. However, protein abundances are not always reflected by the corresponding mRNA levels but may also depend on post-transcriptional and post-translational regulations.13 Previously, there were two proteomics investigations of Pb responses in plants (Catharanthus roseus and Typha angustifolia) using the 2D gel-based approach.14,15 However, such approach is not satisfactory for accurate quantification of differentially expressed proteins and large-scale identification of proteins.16,17 Furthermore, no comparative analysis of proteome changes during Pb stress in the model plant Arabidopsis has been reported. On the contrary, proteomics investigations on the responses to cadmium, phytohormones (e.g., abscisic acid), pathogen challenge, and mineral nutrient deficiency in Arabidopsis are available.18−21 In the present work, proteome changes in Arabidopsis seedlings upon Pb exposure are determined by SWATH-MS (Sequential Windowed Acquisition of All Theoretical Mass Spectra), which is a label-free quantitative proteomics approach recently developed. SWATH-MS is based on a data-independent acquisition (DIA) strategy for consistent and reproducible quantification of peptides with high complexity.22,23 Using a fast and highresolution Q-TOF mass spectrometer, data are acquired by repeated cycling via given sequential windows over the whole chromatographic elution range to generate a complete record of all analytes in a sample. SWATH-MS has been employed for unbiased and untargeted quantitation of proteome changes in microbial, human, and animal systems.24−29 Here we report a total of 1719 proteins, which were quantitatively identified by SWATH-MS in Arabidopsis seedlings, 231 of which were found to be differentially expressed during Pb stress (at a fold change of >1.2 or
