Article Cite This: J. Agric. Food Chem. 2018, 66, 3716−3725
pubs.acs.org/JAFC
A Comparative Proteome Approach Reveals Metabolic Changes Associated with Flammulina velutipes Mycelia in Response to Cold and Light Stress Jing-yu Liu,*,†,‡ Ming-chang Chang,*,†,‡ Jun-long Meng,†,‡ Cui-ping Feng,†,‡ and Yu Wang† †
College of Food Engineering, Shanxi Agricultural University, Taigu 030801, China Shanxi Engineering Research Center of Edible Fungi, Taigu 030801, China
‡
S Supporting Information *
ABSTRACT: In some industrial processes, cold and light stresses are recognized as two important environmental triggers for the transformation of mycelia into fruit-bodies via intermediate primordia in Flammulina velutipes cultivation. To gain insights into the mechanism of regulation of F. velutipes mycelia in response to cold and light stress, proteins expressed abundantly and characteristically at particular stress states were investigated by using the isobaric tags for the relative and absolute quantitation labeling technique. Among the 1046 nonredundant proteins identified with a high degree of confidence, 264 proteins, which were detected as differentially expressed proteins, were associated with 176 specific KEGG pathways. In-depth data analysis revealed that the regulatory network underlying the cold and light response mechanisms of F. velutipes mycelia was complex and multifaceted, as it included varied functions such as rapid energy supply, the biosynthesis of lysine, phenylalanine, tyrosine, and γaminobutyric acid, the calcium signal transduction process, dynein-dependent actin and microtubule cytoskeleton formation, autolysis, oxidative stress adaptation, pigment secretion, tissue and organ morphogenesis, and other interesting stress-related processes. Insights into the proteins might shed light on an intuitive understanding of the cold and light stress response mechanism underlying the fruiting processes of F. velutipes. Furthermore, the data might also provide further insights into the stress response mechanism of macro-fungi and valuable information for scientific improvement of some mushroom cultivation techniques in practice. KEYWORDS: Flammulina velutipes, environmental stress, proteome, iTRAQ, LC−MS/MS
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INTRODUCTION Because of their high nutritional and medicinal value, mushrooms comprise an important macro-fungi class of significant commercial relevance.1 However, basic stress physiological studies of mushrooms are given less attention than their lower relatives, such as yeasts and filamentous fungi.2−4 Flammulina velutipes (Curt. ex Fr.) Sing, commonly known as the golden-needle mushroom, is one of the most popular industrialized cultivation mushrooms in the global market.5 Meanwhile, its culture conditions can be conveniently established in a biology laboratory by using some common refrigeration and light apparatus. F. velutipes has been recognized as a potentially excellent model fungal species for basic and applied studies on macro-fungi.6−9 Preliminary study has indicated that decreasing the temperature with weak light irradiation is an effective method for controlling the fruiting process of these fungi.9−11 In some industrial processes, cold and light stress are environmental triggers that have been routinely used to induce the transformation of mycelia to primordia and control both the yield and the quality of F. velutipes fruit-bodies. Our previous study also showed that cold stress is one of the pivotal environmental factors that influences mycelium growth and fruit-body formation in F. velutipes, but few primordia came into being during F. velutipes mycelium growth under cold stress in complete darkness.12 This indicates that F. velutipes responds to the stimuli of cold and light stress by orchestrating a range of physiological and metabolic © 2018 American Chemical Society
processes. An understanding of these processes at the molecular level will be not only useful in the cultivation of F. velutipes but also valuable in providing basic knowledge about the fruiting mechanism of F. velutipes. Unfortunately, the molecular biology of F. velutipes has been studied poorly until now. For example, only a handful of genes involved in the fruiting process of F. velutipes have been isolated and preliminarily characterized.7,8,13 Current knowledge of the cold and light stress response mechanism underlying the fruiting processes of F. velutipes remains fragmented. We are still far from understanding the mechanism of mycelia in response to cold and light stress in these fungi. Proteomics techniques have emerged as powerful tools for the global assessment and measurement of systematic flux changes of total cell protein expression in particular biological states. The standard gel-based method, one-dimensional (1D) and two-dimensional (2D) gel electrophoresis followed by mass spectrometry (MS), has been chosen and widely used for decades.14 Recently, with the advent of chromatography and MS techniques, high-throughput shotgun proteomics, liquid chromatography (LC) followed by MS, are being increasingly practiced.15 The isobaric tags for relative and absolute Received: Revised: Accepted: Published: 3716
January 22, 2018 March 7, 2018 March 27, 2018 March 27, 2018 DOI: 10.1021/acs.jafc.8b00383 J. Agric. Food Chem. 2018, 66, 3716−3725
Article
Journal of Agricultural and Food Chemistry
0.0188, 0.931, 0.0490, 0.001000], [0.000, 0.0282, 0.932, 0.0390, 0.000700], [0.000, 0.000, 0.929, 0.0689, 0.00220], [0.000, 0.00940, 0.930, 0.0590, 0.00160], [0.000600, 0.0377, 0.933, 0.0288, 0.000], [0.00140, 0.0566, 0.933, 0.00870, 0.000], [0.000900, 0.0471, 0.933, 0.0188, 0.000], [0.00270, 0.0744, 0.921, 0.00180, 0.000], and [0.000, 0.000, 0.000, 0.000, 0.000] were adopted to correct channels according to the published algorithm in i-Tracker.18 Functional Categorization Analysis. For functional characterization of the differentially expressed proteins (DEPs), the abundances of DEPs were detected by p < 0.05 (one-way analysis of variance test) and a 1.5-fold cutoff. Gene Ontology (GO) enrichment of down- or upregulated DEPs was subsequently conducted according to the information from the Swiss-Prot and EBI database (Agaricus). To gain GO mapping and annotation information, data of the DEPs were sequentially uploaded to agriGO (http://bioinfo.cau.edu.cn/agriGO/) by using Blast2GO (version 2.6.6). Enzyme codes of DEPs were sequentially uploaded by our in-house compiled PERL program KEGG.pl to KEGG (the Kyoto Encyclopedia of Genes and Genomes) Web site (http://www.genome.jp/kegg/) and mapped to annotated sequences and metabolic pathways in KEGG. RNA Extraction, Reverse Transcription, and Quantitative Real-Time Polymerase Chain Reaction Analysis. Total RNA of the three samples, control, CS, and CLS, was extracted according to the manufacturer’s instructions of TRIZOL Reagent (Invitrogen). Genomic DNA was removed using DNase I treatment (Promega). The quality of purified RNA was accepted if they had a ratio of 1.8− 2.0 at A260/A280. Total RNA was reverse transcribed according to the manufacturer’s instructions that accompanied the Prime-Script RT Reagent Kit (Takara). Quantitative real-time polymerase chain reaction (qRT-PCR) was sequentially conducted using SYBR Premix EX TaqII (Takara) and a Step One Real-time PCR System (Applied Biosystems). To validate our proteome data, the expression patterns of 18 mRNAs, each corresponding to 18 representative DEPs, including nine upregulated and nine downregulated proteins, were examined. The data were then normalized to the expression levels of the endogenous control gene, GAPDH. Primer pairs were designed for qRT-PCR analysis using Primer version 5.0. Primer data of the 18 representative DEPs are summarized in Table S1. Our data indicated that the amplifications were specific.
quantitation (iTRAQ) labeling coupled to the 2D LC−MS/MS technique, one of the LC-based proteome techniques, has been developed and proven to be a more suitable tool for analyzing chronological proteomic changes in complex developmental processes of fungi.4,9,12,16 In this study, we used iTRAQ labeling coupled to the 2D LC−MS/MS technique to assess the global chronological changes in protein expression patterns in the response of F. velutipes mycelia to cold and light stress. Furthermore, these protein expression patterns were systematically investigated, analyzed, and compared to better understand the stress response mechanism in F. velutipes mycelia.
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MATERIALS AND METHODS
Strains and Culture Conditions. Brown fruit-body dikaryon strain Fv13 of F. velutipes was cultured on culture plates (20% potato, 2% glucose, and 2% agar powder) at 23−25 °C in darkness. A total of 285 plates were divided randomly into three groups: control group, cold stress (CS) group, and cold and light stress (CLS) group. Twenty days after incubation, the whole plate mycelia of the control group were collected as control samples. Subsequently, 95 plates of the CS group were transferred and cultured at 12−15 °C for 22 days in darkness, and the whole plate mycelia were collected as CS samples; 95 plates of the CLS group were transferred and cultured at 12−15 °C in low light (200−300 lx) for 22 days, and the whole plate mycelia were collected as CLS samples, not including primordia. All samples were stored at −80 °C until protein was extracted. Protein Preparation and iTRAQ Labeling. Protein preparation was performed following published protocols with minor modification.9 Briefly, the control, CS, and CLS samples were extracted with STD buffer [4% sodium dodecyl sulfate (SDS), 150 mM Tris-HCl, and 1 mM dithiothreitol (DTT) (pH 8.0)] and UT buffer [8 M urea and 150 mM Tris-HCl (pH 8.0)]. The total protein concentration was determined with an agarose gel and a Bradford assay kit (Applied Biosystems). Protein digestion was performed with a trypsin solution (5% trypsin, Promega) overnight at 37 °C. The resulting tryptic peptides were collected and incubated with an 8-plex iTRAQ reagents application kit (AB Sciex) according to the manufacturer’s instructions. CLS samples were labeled with reagent 115; control samples and CS samples were labeled with reagents 113 and 114, respectively. 2D LC−MS/MS Analysis and Data Analysis. 2D LC−MS/MS analysis was performed following published protocols with minor modification.12 Briefly, the peptides were fractionated using a C18 column (Waters bed, 2.1 mm × 50 mm, 1.7 μm, Applied Biosystems). Peptides were eluted with solvent A (20 mM ammonium formate) adjusted to pH 10 and a linear gradient solvent of B (ACN, from 5 to 35%) at a flow rate of 600 μL/min. The absorbance was monitored at 214 nm. The peptide fractions were collected together to obtain 20 final fractions and subsequently separated by nano-high-performance liquid chromatography (Eksigent Technologies) on the secondary RP analytical column (Eksigent, 3 μm, 75 μm × 150 mm) equipped with a Triple TOF 5600 MS instrument (Thermo Finnigan). The gradient conditions were a linear phase B (98% ACN and 0.1% formic acid) gradient from 5 to 45% from 5 to 100 min at a total controlled flow rate of 300 nL/min. The electrospray voltage of the mass spectrometer was 2.5 kV versus the inlet. The scanning range of tandem mass spectrometry was m/z 100−1250. A dynamic exclusion time of 25 s per cycle was used to select the 25 most intense precursors for fragmentation. Tandem mass spectrometry extraction was processed by Proteome Wizard version 3.0.10185 64-bit. The MS data of three samples mentioned above were processed using MASCOT (Matrix Science, version 2.3.0). Agaricales. Fasta in UniProt (http://www.uniprot.org/) was used as a reference database. Data were normalized by Scaffold Q+ (version 4.4.6). Protein identifications and protein probabilities were determined by the Scaffold Local FDR algorithm, a false discovery rate (FDR) of
