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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 J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00383 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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Journal of Agricultural and Food Chemistry

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Comparative proteome approach reveals metabolic changes

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associated with Flammulina velutipes mycelia in response to

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cold and light stress

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Jing-yu Liu,*,†,‡ Ming-chang Chang,*,†,‡ Jun-long Meng,†,‡ Cui-ping Feng,†,‡ Yu Wang†

5

†

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‡

College of Food Science and Engineer, Shanxi Agricultural University, Taigu 030801, China

Shanxi Engineering Research Center of Edible Fungi, Taigu 030801, China

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ABSTRACT: In some industrial processes, cold and light stresses are recognized as two important

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environmental triggers for the transformation of mycelia into fruit-bodies via intermediate primordia in

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Flammulina velutipes cultivation. To gain insights into the regulation mechanism of F. velutipes mycelia

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in response to cold and light stress, proteins expressed abundantly and characteristically at particular

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stress states were investigated by using the isobaric tags for relative and absolute quantitation (iTRAQ)

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labeling technique. Among the 1046 non-redundant proteins identified with high confidence, 264 proteins,

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which were detected as differentially expressed proteins, were associated with 176 specific KEGG

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pathways. In-depth data analysis revealed that the regulatory network underlying the cold and light

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response mechanisms of F. velutipes mycelia were complex and multifaceted, as it included varied

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functions such as rapid energy supply, the biosynthesis of lysine, phenylalanine, tyrosine and γ-

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aminobutyric acid, calcium signal transduction process, dynein-dependent actin and microtubule

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cytoskeleton formation, autolysis, oxidative stress adaption, pigment secretion, tissue and organ

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morphogenesis, and other interesting stress-related processes. Insights about the proteins might shed light

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on an intuitive understanding of the cold and light stress response mechanism underlying the fruiting

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processes of F. velutipes. Furthermore, the data might also provide further insights into the stress

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ACS Paragon Plus Environment


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response mechanism of macro-fungi and valuable information for scientific improvement of some

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mushroom cultivation techniques in practice.

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KEYWORDS: Flammulina velutipes, environmental stress, proteome, iTRAQ, LC-MS/MS

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■ INTRODUCTION

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Due to high nutritional and medicinal value, mushrooms comprise an important macro-fungi class of

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significant commercial relevance.1 However, basic stress physiological studies of mushrooms are given

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less attention than their lower relatives, such as yeasts and filamentous fungi. 2-4 Flammulina velutipes

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(Curt. ex Fr.) Sing, commonly known as golden-needle mushroom, is one of the most popular

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industrialized cultivation mushrooms in the global market. 5 Meanwhile, its culture conditions can be

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conveniently established in a biology laboratory by using some common refrigeration and light apparatus.

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F. velutipes has been recognized as a potentially excellent model fungal species for basic and applied

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studies on macro-fungi.6-9 Preliminary study has indicated that temperature reduction with weak light

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irradiation is an effective regulation method for controlling fruiting process of theses fungi. 9-11 In some

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industrial processes, cold and light stress are environmental triggers that has been routinely used to

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induce transformation of mycelia to primordia and control both yield and quality of F. velutipes fruit-

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bodies. Our previous study also showed that cold stress is one of the pivotal environmental factors that

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influencing mycelium growth and fruit-body formation in F. velutipes, but few primordia came into being

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during F. velutipes mycelium growth under cold stress in complete darkness. 12 This indicates that F.

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velutipes responds to the stimuli of cold and light stress by orchestrating a range of physiological and

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metabolic processes. An understanding of these processes at the molecular level will not only be useful 2


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in the cultivation practice of F. velutipes, but also be valuable in providing basic knowledge on the

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fruiting mechanism of F. velutipes. Unfortunately, the molecular biology of F. velutipes has been studied

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poorly until present. For example, only a handful of genes involved the fruiting process of F. velutipes

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have been isolated and preliminarily characterized.7,8,13 Current knowledge of the cold and light stress

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response mechanism underlying the fruiting processes of F. velutipes remains fragmented. We are still

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far from understanding the mechanism of mycelia in response to cold and light stress in these fungi.

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Proteomics techniques have emerged as powerful tools for the global assessment and measurement

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of systematic flux changes of total cell protein expression at particular biological states. The standard

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gel-based method, one-/two-dimensional (1D/2D) gel electrophoresis followed by mass spectrometry

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(MS), has been chosen and widely practiced for decades.14 Recently, with the advent of chromatography

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and MS techniques, high-throughput shotgun proteomics, liquid chromatograph (LC) followed by MS,

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are being increasingly practiced.15 The isobaric tags for relative and absolute quantitation (iTRAQ)

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labeling coupled to 2D LC-MS/MS technique, one of the LC-based proteome techniques, has been

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developed and proven to be a more suitable tool for analyzing chronological proteomic changes in

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complex developmental processes of fungi.4,9,12,16 In this study, we used the iTRAQ labeling coupled to

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2D LC-MS/MS technique to assess the global chronological changes in protein expression patterns of F.

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velutipes mycelia response to cold and light stress. Furthermore, these protein expression patterns were

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systematically investigated, analyzed and compared to better understand the stress response mechanism

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in F. velutipes mycelia.

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■ MATERIALS AND METHODS

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Strains and Culture Conditions

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Brown fruit-body dikaryon strain Fv13 of F. velutipes were cultured on culture plates (potato 20%,

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glucose 2% and agar powder 2%) at 23-25 °C in darkness. A total of 285 plates were divided randomly

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into three groups: control group, cold stress (CS) group, and cold and light stress (CLS) group. 20 days

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after incubation, the whole plate mycelia of the control group were collected as control samples.

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Subsequently, 95 plates of CS group were transferred and cultured at 12-15 ℃ for 22 days in darkness,

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and the whole plate mycelia were collected as CS samples. 95 plates of CLS group were transferred and

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cultured at 12-15 ℃ in low light (200-300 lx) for 22 days, and the whole plate mycelia were collected as

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CLS samples, not including primordia. All samples were stored at -80 °C until protein extraction.

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Protein Preparation and iTRAQ Labeling

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Protein preparation was performed following published protocols with minor modification. 9 Briefly,

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the control, CS and CLS samples were extracted by STD buffer (SDS 4%, Tris-HCl 150 mM, DTT 1

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mM, pH 8.0) and UT buffer (Urea 8 M, Tris-HCl 150 mM, pH 8.0). Total protein concentration was

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determined by agarose gel and Bradford Assay Kit (Applied Biosystem). Protein digestion was

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performed by trypsin solution (5% trypsin, Promega) overnight at 37 °C. The resulting tryptic peptides

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were collected and incubated with 8-plex iTRAQ reagents application kit (AB Sciex) according to the

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manufacturer’s instructions. CLS samples were labeled with reagent 115; control samples and CS

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samples were labeled with reagent 113 and 114, respectively.

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2D LC-MS/MS Analysis and Data Analysis

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2D LC-MS/MS analysis was performed following published protocols with minor modification. 12

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Briefly, the peptides were fractionated using a C18 column (waters bed 2.1 × 50 mm, 1.7 μm, Applied

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Biosystem). Peptides were eluted with solvent A (20 mM ammonium formate) adjusted to pH 10 and a

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linear gradient solvent B (ACN, 5-35 %) at a flow rate of 600 μL/min. The absorbance was monitored at

89

214 nm. The peptide fractions were collected together to obtain 20 final fractions and subsequently

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separated by nano-HPLC (Eksigent Technologies) on the secondary RP analytical column (Eksigent, 3

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μm, 75 μm × 150 mm) equipped with Triple TOF 5600 MS (Thermo Finnigan). The gradient conditions

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were a linear phase B (98% ACN, 0.1% formic acid) gradient of 5-45 % from 5-100 min at a total

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controlled flow rate of 300 nL/min. Electrospray voltage of the mass spectrometer was 2.5 kV versus the

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inlet. The scanning range of the tandem mass spectrometry was 100 to 1250 m/z. A dynamic exclusion

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time of 25 s per cycle was used to select 25 most intense precursors for fragmentation.

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Tandem mass spectrometry extraction was processed by Proteome Wizard version 3.0.10185 64-bit.

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The MS data of three above mentioned samples were processed using MASCOT (Matrix Science,

98

version 2.3.0) software. Agaricales. Fasta in UniProt (http://www.uniprot.org/) was used as a reference

99

database. Data were normalized by Scaffold Q+ (software version Scaffold-4.4.6). Protein identifications

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and protein probabilities were determined by the Scaffold Local FDR algorithm, a false discovery rate

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(FDR) < 0.05, and the protein prophet algorithm, respectively. 17 In this study, FDR of 2 were chosen as the criteria for protein identification. In all samples, the matrix

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[0.000,0.0188,0.931,0.0490,0.001000];

[0.000,0.0282,0.932,0.0390,0.000700];

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[0.000,0.000,0.929,0.0689,0.00220];

[0.000,0.00940,0.930,0.0590,0.00160];

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[0.000600,0.0377,0.933,0.0288,0.000];

[0.00140,0.0566,0.933,0.00870,0.000];

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[0.000900,0.0471,0.933,0.0188,0.000];

[0.00270,0.0744,0.921,0.00180,0.000]

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[0.000,0.000,0.000,0.000,0.000] were adopted to correct channels according to the published algorithm

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in i-Tracker.18

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Functional Categorization Analysis

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For functional characterization of the differentially expressed proteins (DEPs), the abundance of

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DEPs were detected by p < 0.05 (one-way ANOVA test) and 1.5 fold cut-off. Gene Ontology (GO)

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enrichment of down- or up-regulated DEPs were subsequently conducted according to the information

113

from Swiss-Prot and EBI database (Agaricus). To gain GO mapping and annotation information, data of

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the DEPs was sequentially uploaded to agriGO (http://bioinfo.cau.edu.cn/agriGO/) by using Blast2GO

115

(software version 2.6.6). Enzyme codes of DEPs were sequentially uploaded by our in-house compiled

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PERL program KEGG.pl to KEGG (the Kyoto Encyclopedia of Genes and Genomes) website

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(http://www.genome.jp/kegg/), and mapped to annotated sequences and metabolic pathways in KEGG.

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RNA Extraction, Reverse Transcription and qRT-PCR analysis

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Total RNA of the three samples, control, CS and CLS was extracted according to the manufacturer’s

120

instructions of TRIZOL Reagent (Invitrogen). Genomic DNA was removed using DNase I treatment

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(Promega). Quality of purified RNA was accepted if they had a ratio of 1.8-2.0 at A260/A280. Total RNA

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was reverse transcribed according to the manufacturer’s instructions of Prime-Script® RT reagent Kit

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(Takara). Quantitative real-time Polymerase Chain Reaction (qRT-PCR) was sequentially conducted

124

using SYBR Premix EX TaqTM II (Takara) and a Step One Real-time PCR System (Applied Biosystems).

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To validate our proteome data, the expression patterns of 18 mRNAs, each corresponding to 18

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representative DEPs including nine up-regulated and nine down-regulated proteins, respectively. The

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data were then normalized to the expression levels of the endogenous control gene, GAPDH. Primer pairs

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were designed for qRT-PCR analysis using the Primer 5.0 software. Primer data of the 18 representative

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DEPs are summarized in Table S1. Our data indicated that the amplifications were specific.

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■ RESULTS

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Morphological Changes of Flammulina velutipes Mycelia in Response to Cold and Light Stress

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After being incubated at 23-25°C in darkness for 20 days, the plates of the control group (Fig. 1A)

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were fully colonized with vigorous aerial mycelia (Fig. 1a). After CS treatment, aerial mycelia of CS

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group plates (Fig. 1B) became flattened and secreted some yellow physiological water (Fig. 1b). After

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CLS treatment, a few primordia came into being, but the aerial mycelia of the plates secreted less yellow

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physiological water (Fig. 1C, 1c).

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B

A

C

138 139 140 141 142 143

a

144

c

b

145 146 147

Figure 1. Morphology changes of Flammulina velutipes mycelia in response to cold and light stress. A: control

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sample; B: cold stress sample; C: cold and light stress sample; a: aerial hyphae; b: yellow physiological water; c:

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primordia. All strains were inoculated at the same time.

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Protein Identification and Quantification

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According to the above mentioned criteria for protein identification, 1046 non-redundant proteins

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were obtained from three biological replicates for further comparative analysis. According to a criterion

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of 95% significance and 1.5 fold cutoff, following which a total of 550 non-redundant proteins were

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identified as DEPs. The Venn diagram analysis was subjected to illustrate the 160 up-regulated and 409

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down-regulated DEPs distributions and their overlap between CS and CLS samples (Figure 2).

156 157 158 159 160 161 162 163

A

B

12

CS

1

147

63

CLS

CS

37

309

CLS

Figure 2. Venn diagrams and expression of 551 DEPs identified under cold and light stress in mycelia of Flammulina

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velutipes. The numbers of DEPs with up-regulation (A) and down-regulation (B) under given development stages

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are shown in different segments. CS: cold stress; CLS: cold and light stress.

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Among the 550 DEPs, 286 DEPs, which were characterized as predicted, uncharacterized or putative

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proteins under the reference database, were discarded. In general, two clearly different expression

168

patterns in CS and CLS samples of F. velutipes were generalized among the remainder 264 DEPs. A total

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of 70 DEPs were up-regulated in at least one of two samples (CS or CLS), including one common DEPs

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(acetyltransferase component of pyruvate dehydrogenase complex) in two samples (CS and CLS), while

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two and 66 DEPs were significantly up-regulated in CS and CLS respectively. Among 204 down-

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regulated DEPs in at least one of the two samples, 21 DEPs were common to two samples (CS and CLS),

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while 33 and 149 DEPs were particularly down-regulated in CS and CLS, respectively. The data of up-

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regulated DEPs are summarized in Table 1 and Table 2. The data of down-regulated DEPs are

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summarized in Supplementary Table S2, Table S3 and Table S4.

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Table 1. List of up-regulated proteins under only cold stress in mycelia of Flammulina velutipes. Uniprot ID a

No. 1

D6RQF5_COPC7

Protein name Aminoadipate-semialdehyde

Gene Name

Fold change b CS1/C1

CS2/C2

CS3/C3

CC1G_15694

1.56

1.91

1.91

dehydrogenase 2

V2X4P6_MONRO

Acetyl-hydrolase

Moror_11650

1.71

1.91

1.75

3

D8PKQ0_SCHCM

Acetyltransferase component

SCHCODRAFT_81014

1.67

1.86

1.82

of pyruvate dehydrogenase complex

177 178 179 180 181 No. 1

a

Uniprot: http://www.uniprot.org/.

b

CS: cold stress; C: Control.

Table 2. List of up-regulated proteins under only cold and light stress in mycelia of Flammulina velutipes. Uniprot ID a Q5EGJ1_FLAVE

Protein name Translation elongation factor

Gene Name

Fold change b CLS1/C1

CLS2/C2

CLS3/C3

tef1

1.67

1.57

1.71

EF1-alpha (Fragment) 2

V2WVN8_MONRO

Hsp70 chaperone

Moror_9965

2.50

2.33

2.33

3

D8QLU7_SCHCM

Calmodulin

SCHCODRAFT_80005

1.53

1.67

1.65

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4

D8PR32_SCHCM

40S ribosomal protein S1

RPS1

2.00

1.67

1.84

5

B0DI83_LACBS

Pyruvate carboxylase

PYC

1.60

1.57

1.60

6

B0DHG8_LACBS

Beta-tubulin 1 tubb1

LbTUBb1

2.63

2.30

1.85

7

V2XAN6_MONRO

Calnexin

Moror_7233

1.63

1.59

1.82

8

A0A067P3W4_PLEOS

Histone H4

PLEOSDRAFT_1088653

1.63

1.67

1.60

9

A0A067NVC3_PLEOS

Histone H2B

PLEOSDRAFT_1088190

1.82

1.57

1.56

10

A0A067SYC7_9AGAR

60S ribosomal protein L20

GALMADRAFT_142989

2.33

2.71

2.17

11

V2WVN7_MONRO

Inorganic diphosphatase

Moror_16434

3.00

1.86

1.50

12

A0A067SEN5_9AGAR

Serine hydroxymethyltransferase

GALMADRAFT_257154

1.61

1.67

1.60

13

B0CPE4_LACBS

Cell division control/GTP binding protein

LACBIDRAFT_291428

2.09

2.53

2.31

14

B0DZY3_LACBS

Catalase

LbCAT

4.25

3.75

4.50

15

A8NSX2_COPC7

Ribosomal protein rpl23a

CC1G_12270

2.00

3.33

2.60

16

V2YIE8_MONRO

Alpha-1,4 glucan phosphorylase

Moror_2611

1.53

1.71

1.50

17

D8PXF2_SCHCM

Succinate dehydrogenase

SCHCODRAFT_66862

1.71

1.57

1.80

[ubiquinone] iron-sulfur subunit, mitochondrial 18

R4HKU2_FLAVE

Myosin regulatory light chain

cdc4

1.88

2.00

1.53

19

A0A067NJV8_PLEOS

Autophagy-related protein

PLEOSDRAFT_1097703

1.75

1.20

2.00

20

D8PW86_SCHCM

Phosphoglycerate kinase

SCHCODRAFT_74370

1.63

2.40

2.33

21

V2YDU9_MONRO

Scf complex subunit skp1

Moror_859

2.25

2.11

2.13

22

A8NZV3_COPC7

Superoxide dismutase

CC1G_06963

3.00

1.67

3.00

23

D2JY86_FLAVE

Initiation factor 5a

--

1.60

1.58

1.55

24

V2XI69_MONRO

Proteasome subunit alpha type

Moror_1703

1.50

1.50

1.50

25

V2XUP9_MONRO

2-methylcitrate dehydratase

Moror_6806

1.63

2.17

3.00

26

A0A067N8U4_PLEOS

6-phosphogluconate

PLEOSDRAFT_41725

3.00

3.20

2.71

dehydrogenase, decarboxylating 27

V2XD70_MONRO

60s ribosomal protein l17

Moror_13629

2.33

2.75

2.67

28

A0A067TK21_9AGAR

Proteasome subunit alpha type

GALMADRAFT_235810

1.75

2.00

1.72

29

B0CQH3_LACBS

Acetyltransferase component of

LACBIDRAFT_378984

1.89

1.95

--

pyruvate dehydrogenase complex 30

V2XYL9_MONRO

Cyanide hydratase

Moror_894

1.75

2.00

1.81

31

A8NBN6_COPC7

60S ribosomal protein L27a

CC1G_02496

2.71

2.71

1.78

32

A8N0P0_COPC7

Peptidyl-prolyl cis-trans

CC1G_04343

1.50

2.50

2.17

gpd

1.63

1.57

1.64

isomerase 33

D2SRR0_9AGAR

Glyceraldehyde-3-phosphate

34

V2X183_MONRO

ATP synthase d subunit

Moror_9086

3.50

2.33

2.67

35

B0CU44_LACBS

26S proteasome regulatory

PATPA16201

1.63

1.82

1.71

dehydrogenase (Fragment)

complex, ATPase RPT4 36

A0A067NIX1_PLEOS

Malate dehydrogenase

PLEOSDRAFT_1090652

2.25

2.47

2.32

37

V2WVA6_MONRO

Aryl-alcohol dehydrogenase

Moror_4055

2.83

2.39

2.41

38

A8P7B9_COPC7

Calcium-dependent protein

CC1G_08194

1.75

1.86

1.82

serine/threonine phosphatase 39

V2WZD6_MONRO

Transketolase

Moror_2319

1.62

1.58

1.57

40

V2WRP8_MONRO

60s ribosomal protein l2

Moror_1359

1.63

1.72

1.77

9



41

D8QCI6_SCHCM

Peptidyl-prolyl cis-trans

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SCHCODRAFT_16915

2.25

3.33

2.50

isomerase 42

D8Q077_SCHCM

Isocitrate lyase

SCHCODRAFT_66738

1.55

1.67

1.73

43

V2XH67_MONRO

Elongation factor 2

Moror_4797

2.64

1.75

1.86

44

V2XTA5_MONRO

Proteasome 26s subunit ATPase 3

Moror_12489

4.20

2.43

2.75

45

B0CUA0_LACBS

Actin filament-coating protein

LACBIDRAFT_176790

3.08

3.11

2.78

tropomyosin 46

V2XPR5_MONRO

Dynein light chain cytoplasmic

Moror_14124

1.60

1.50

1.50

47

A0A067SXJ7_9AGAR

Proliferating cell nuclear antigen

GALMADRAFT_102448

1.78

1.50

2.00

48

K5WMW0_AGABU

Transcription elongation factor

AGABI1DRAFT_115721

2.71

3.38

2.60

TEF EF1B 49

V2XT09_MONRO

Fe-containing alcohol

Moror_7341

2.42

2.75

1.90

50

V2X429_MONRO

Glyceraldehyde-3-phosphate

Moror_13000

2.67

3.00

2.00

dehydrogenase 51

A8N1B9_COPC7

Ribosomal protein L4/L1

CC1G_10540

3.67

2.66

--

52

V2XDI0_MONRO

Het-c2 protein

Moror_16482

2.00

1.91

1.67

53

V2X892_MONRO

Sec14 cytosolic factor

Moror_3846

1.81

1.69

1.67

54

B0D3H6_LACBS

Glutamate decarboxylase

LACBIDRAFT_315921

1.95

1.55

1.86

55

A8NHX8_COPC7

Alpha-mannosidase

CC1G_01530

1.65

1.57

1.59

56

A8NN10_COPC7

Class V chitinase ChiB1

CC1G_09921

3.00

3.60

3.22

57

A8NCY6_COPC7

60S ribosomal protein L10

CC1G_08610

2.50

2.00

1.71

58

V2XI54_MONRO

Arg-6 protein

Moror_4407

--

1.83

1.64

59

V2YBK4_MONRO

Microtubule binding protein

Moror_5349

1.80

2.30

2.00

60

B0CVX0_LACBS

Isocitrate lyase

LACBIDRAFT_171643

--

2.67

3.00

61

V2XG31_MONRO

Proteasome subunit beta type

Moror_369

1.60

1.74

1.71

62

V2XWY1_MONRO

Heat shock protein

--

2.07

--

2.13

63

G8A534_FLAVE

Beta-galactosidase

Moror_17812

1.67

1.50

1.56

64

A0A067T146_9AGAR

Eukaryotic translation initiation

HCR1

2.43

2.22

2.06

65

A0A067NJ33_PLEOS

INT6

1.71

1.64

1.58

factor 3 subunit J Eukaryotic translation initiation factor 3 subunit E 66 67

V2XAU1_MONRO

Serine hydroxymethyltransferase

Moror_7089

1.72

1.53

1.52

D8PRY7_SCHCM

Tubulin alpha-1A chain

SCHCODRAFT_9815

5.75

6.67

5.88

182 183

a

Uniprot: http://www.uniprot.org/.

b

CLS: cold and light stress; C: Control.

184

Functional Categorization Analysis

185

Among the 550 DEPs, functional information for the 264 DEPs was characterized by searching for

186

homologous proteins against the genomics information category of Agaricales. Fasta (UniProt) database.

187

The results of enrichment analysis showed that under the category of the cellular component, DEPs were

188

mainly found in cytoplasm, ribosome, membrane, nucleus, and proteasome complex. In the biological

10


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189

process, DEPs were mainly enriched in translation, protein folding, carbohydrate metabolic process, fatty

190

acid biosynthetic process, response to stress and signal transduction. In molecular function, DEPs were

191

mostly related to binding, enzyme activity and structural constituent of ribosome. Unfortunately, 13 of

192

the DEPs had unknown locations. The GO mapping and annotation data of up-regulated and down-

193

regulated DEPs are summarized and shown in Figure 3A and 3B, respectively.

A

Cellular component

B

Cellular component

194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222

Biological process

Molecular function

Biological process

Molecular function

223

Figure 3. Bioinformatics analysis of the above mentioned differentially expressed proteins through Gene Ontology

224

(GO) in three domains: cellular component, biological process and molecular function. The statistics at GO level 2

11



225

is shown in this figure. A: Up-regulated proteins; B: Down-regulated proteins.

226

Metabolic Pathway Analysis

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227

The data of GO analysis demonstrated that the DEPs during the transformation of mycelia into

228

primordia of F. velutipes under cold and light stress were influenced by a crucial and complicated cellular

229

process, particularly the metabolic processes. To gain further insights into pathway information of the

230

metabolic processes, data of the 264 DEPs were sequentially uploaded to KEGG website to annotate

231

sequences and metabolic pathways. The result showed that 139 (52.65 %) of the 264 DEPs were

232

associated with 176 specific KEGG pathways. Further analysis of 70 up-regulated DEPs showed that 42

233

DEPs (60 %) were mainly distributed in 99 specific KEGG pathways. In general, one (aminoadipate-

234

semialdehyde dehydrogenase) of the two up-regulated DEPs at CS samples were associated with

235

biosynthesis of amino acids (path: ko01230), lysine biosynthesis (path: ko00300) and lysine degradation

236

(path: ko00310). 40 of the 67 DEPs that were significantly up-regulated in CLS sample were distributed

237

in 97 specific KEGG pathways and mainly involved in signaling pathway (path: ko04010, path: ko04014,

238

path: ko04020, path: ko04022, path: ko04068, path: ko04152, path: ko04310, path: ko04350,

239

path:ko04910, path: ko04921, path: ko04922), amino acid biosynthesis and metabolism (path: ko00250,

240

path: ko00330, path:ko00460, path: ko00520, path: ko01230), energy metabolism (path: ko00010, path:

241

ko00020, path: 00030, path: ko00190, path: ko00630), glutathione metabolism (path: ko00480) (Figure

242

4), proteasome (path: ko03050), ribosome (path: ko03010) and cell cycle (path: ko04111). Further

243

functions of the DEPs are discussed in the following discussion sections.

244 245

12


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246 247 248 249 250 251 252 253 254 255 256 257 258 259

Figure 4. Representative metabolic pathway maps of differentially expressed proteins involved in glutathione

260

metabolism in KEGG. The colored enzyme codes were noted as follows: 6-phosphogluconate dehydrogenase (EC:

261

1.1.1.44 and 1.1.1.343), ribonucleoside-diphosphate reductase subunit (EC: 1.17.4.1), spermidine synthase (EC:

262

2.5.1.16), gamma-glutamyltranspeptidase / glutathione hydrolase (EC: 2.3.2.2 and 3.4.19.13). Red color: Up-

263

regulated proteins; Green color: Down-regulated proteins; Blue color: other identified enzymes.

264

Transcriptional Expression Pattern Analysis of Representative Differentially Abundant Proteins

265

Due to lack of specific antibodies for most proteins in basidiomycete studies, the western blotting

266

technique was limited to confirm only the most edible mushroom proteome data. In the study, we

267

determined and compared transcriptional expression patterns of 18 mRNAs, each corresponding to 18

268

representative DEPs, by qRT-PCR technique. Our data showed that 13 genes, encoding 60S ribosomal

269

protein L20, Serine hydroxymethyltransferase, malate dehydrogenase, elongation factor 2, proteasome

270

26s subunit ATPase 3, heat shock protein sks2, Tryptophan synthase, 2-cysteine peroxiredoxin, glycoside

271

hydrolase family 13 protein, V-type proton ATPase subunit a, alcohol oxidase-like protein, Glutathione-

13



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272

disulfide reductase and S-adenosylmethionine synthase exhibited similar expression patterns with

273

representative protein expression patterns in the control, CS and CLS samples (Figure 5A). However, the

274

mRNA expression patterns of three genes, encoding inorganic diphosphatase, transketolase and 2-

275

methylcitrate dehydratase, were not consistent with their corresponding up-regulated proteins. Two of

276

the 18 genes, encoding superoxide dismutase and Hsp70 chaperone, showed opposite expression patterns

277

between their mRNAs and proteins (Figure 5B). This result is in accordance with previous reports due

278

to translational or post-translational regulation.9,12,19

279 280 281 282 283 284 285 286 287 288 289

Figure 5. Transcriptional expression analysis of representative proteins as revealed by qRT-PCR. A: Relative mRNA

290

expression patterns of representative up-regulated proteins; B: Relative mRNA expression patterns of representative

291

down-regulated proteins. CS: cold stress; CLS: cold and light stress. The data were then normalized to the expression

292

levels of the endogenous control gene, GAPDH (n = 3, mean ±standard error of the mean, *p-value < 0.05).

293

■ DISCUSSION

294

In all three life super-kingdoms (archaea, bacteria and eukaryotes), the cellular stress response have

295

been linked to essential processing and stability of protein and DNA. 20 Development in proteomic

296

analysis during the last decade provided insights into a systematic understanding of the complex

297

developmental events of filamentous fungi and yeast at the molecular level in response to cell stress

298

exposure.20,21 However, unlike other lower model fungi, the stress proteome of micro-fungi, especially

14


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299

mushrooms, remains largely uncharacterized. In micro-fungi, F. velutipes, a tetrapolar basidiomycete

300

mushroom, has emerged as a potential model system for studying basic mechanisms of genetics,

301

development, and cellular stress response.6,9,11,12 In this study, the stress proteome associated with F.

302

velutipes mycelia in response to cold and light stress were investigated by using iTRAQ labeling coupled

303

to 2D LC-MS/MS technique. Our data showed that some interesting up-regulated DEPs might play a key

304

role in the response mechanism of F. velutipes mycelia. The biological relevance of these up-regulated

305

DEPs is analyzed and discussed under some of the common central metabolism tasks, including carbon

306

and energy metabolism, biosynthesis and metabolism of amino acids, signal transduction pathways, and

307

other interesting stress-related processes.

308

Increased Abundance of Stress-related Proteins of Carbon and Energy Metabolism

309

Our results indicated complex flux changes of cell protein expression systems associated with F.

310

velutipes mycelia in response to cold and light stress in carbon and energy metabolism. The data showed

311

that acetyltransferase component of pyruvate dehydrogenase complex (ACPDC) had higher expression

312

levels under both CS and CLS samples. ACPDC participates in pyruvate metabolism and converts

313

pyruvate to acetyl CoA. Acetyl-CoA is an important molecule that participates in carbohydrate, protein

314

and lipid metabolism. Acetyl-hydrolase, which catalyzes the conversion of acetyl-CoA and H2O into

315

Coenzyme A and acetate, had higher expression levels in mycelia in response to CS, but their expression

316

levels returned to normal under CLS. CoA is the main input for the citric acid cycle (TCA cycle) and

317

fatty acid biosynthesis which are required for fungal mycelia growth and fruit-body formation.22 In fungi,

318

accumulation of acetate also reduced growth by repressing mating and filamentation, and promoted the

319

building block for fatty acid biosynthesis.23 Aldehyde dehydrogenase also serves as the last enzyme in

320

the formation reaction of Neurospora carotenoid.24 This finding revealed that acetyl-hydrolase might play

15



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321

an important role in improving the mycelium adaptability, secreting pigment and reducing damage under

322

CS environment in F. velutipes.

323

We found that some crucial proteins participated in processes like central energy metabolisms for

324

sugar, glycolysis, citric acid cycle, pentose phosphate pathway and glucose release from starch, also had

325

higher expression levels in CLS sample, such as α-1,4 glucan phosphorylase, 6-phosphogluconate

326

dehydrogenase,

327

dehydrogenase, isocitrate lyase, 2-methylcitrate dehydratase, malate dehydrogenase, succinate

328

dehydrogenase [ubiquinone] iron-sulfur subunit and transketolase. The catalysates of these enzymes

329

serve as important intermediates for gluconeogenesis, ATP and fatty acid biosynthesis that play key roles

330

in several processes of fungi, such as the synthesis of biological membranes, cellular structure, protein

331

localization, energy production and storage for fungal growth. 22,25,26 On the basis that low activity of the

332

enzymes in CS sample, high activities of ATP synthase d subunit and ribosomal protein, and

333

morphological characters of F. velutipes mycelia in CLS samples. One plausible explanation for the

334

result is light irradiation stimulation plays a crucial role in the rapid energy supply for the transformation

335

of mycelia into primordia in F. velutipes.

336

Increased Abundance of Stress-related Proteins of Amino Acid Biosynthesis and Metabolism

phosphoglycerate

kinase,

pyruvate

carboxylase,

glyceraldehyde-3-phosphate

337

Amino acids function as building blocks and energy in fungus growth and development. Stress-

338

related metabolites of amino acids also have the potential to integrate environmental stress conditions

339

into physiological outputs and intracellular signals. In this study, our data showed that six proteins

340

associated with amino acid metabolism, including aminoadipate-semialdehyde dehydrogenase

341

(AASDH), aryl-alcohol dehydrogenase (AAD), serine hydroxymethyltransferase (SHMT), glutamate

342

decarboxylase (GD), cyanide hydratase (CHT) and peptidyl-prolyl cis-trans isomerase, increased in

16


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343

abundance during mycelia response to cold and light stress in F. velutipes. Among the six proteins,

344

AASDH had higher expression levels in CS samples. AASDH is an important enzyme for the

345

biosynthesis of lysine in yeasts and higher fungi.27 Accumulation of lysine is beneficial in the convert of

346

pyridoxal phosphate (PLP) from vitamin B6, where PLP plays a key role as a coenzyme in transamination

347

reactions of amino acids and amino sugar.28

348

Our data showed that the activities of AAD, SHMT, GD, and CHT were remarkably increased in

349

CLS samples. In wood-saprophyte fungi and some yeast, AAD serves as an important enzyme for

350

detoxifying aromatic aldehydes released during lignin degradation.29 The catalysate of AAD are involved

351

in the synthesis of phenylalanine and tyrosine that might be advantageous to stress responses in

352

microorganism.30 SHMT, one of PLP-enzymes, catalyzes the inter-conversion of L-Ser and

353

tetrahydrofolate (THF) to Gly and 5,10-CH2-THF. This reaction plays an important role in providing the

354

largest one-carbon fragments available to the cell.31 GD is an enzyme that catalyzes the conversion of

355

glutamate to γ-aminobutyric acid (GABA). GABA levels could serve as the key signal connecting

356

cellular carbon metabolism to developmental or environmental responses of various biotic and abiotic

357

stresses in plants and fungi.32,33 In Stagonospora nodorum, GABA has positive effects on asexual

358

development of mycelia in response to environmental stresses.34 Recent work showed that fruit-bodies

359

of F. velutipes contained the highest amount of GABA among 20 different mushrooms. 35 CHTs that are

360

the key enzymes catalyzing the degradation of cyanide are widely found in filamentous fungi and few

361

other members of Basidiomycota36 and were postulated to serve as nitrogenase.37 In this study, we first

362

found the CHT increased in abundance under light induction in F. velutipes mycelia. This finding

363

revealed that these enzymes mediated regulation of the biosynthesis of lysine, phenylalanine, tyrosine

364

and GABA, the degradation of cyanide, and one-carbon fragments available, which might play important

17



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365

roles in developmental and environmental responses F. velutipes mycelia under CLS environment.

366

Increased Abundance of Stress-related Proteins of Signaling Pathways and Other Biological

367

Processes

368

In fungi, some conserved signaling pathways that control the adaptation to the environmental changes

369

is crucial for cell cycle regulation, reproduction and stress response. Here the result showed a complex

370

picture of signaling pathways associated with mycelia response to light stress in F. velutipes. Our data

371

showed that some interesting stress-related DEPs, such as α-mannosidase, calnexin, calmodulin,

372

calcium-dependent protein serine/threonine phosphatase, cell division control/GTP binding protein, beta-

373

tubulin 1 tubb1, tubulin alpha-1A chain, actin filament-coating protein tropomyosin, microtubule binding

374

protein, dynein light chain cytoplasmic and myosin regulatory light chain, remarkably increased

375

abundance in CLS samples. In the filamentous fungus Aspergillus oryzae, α-mannosidases act on N-

376

linked mannooligosaccharides that are involved in the formation of the membrane. 38 Calnexin is a

377

molecular chaperone that modulates the Ca2+ homeostasis and calcium signal transduction process in

378

endoplasm reticulum. Calnexin is required for mycelium growth and conidiation in the filamentous

379

fungus Aspergillus nidulans under some endoplasmic reticulum stress.39 Calmodulin and calcium-

380

dependent protein serine/threonine phosphatase play critical roles in the regulation of stress responses

381

via the calcium-calcineurin pathway that controls fundamental aspects of survival, morphogenesis and

382

signaling cascades in fungi.40 In the fungus Ustilago, calcium signaling was proved to participate in

383

dynein-dependent microtubule organization that drives tissue morphogenesis.41 GTP-binding proteins

384

coordinate cell division and signaling and participate in forming filaments in eukaryotic cells. In fungi,

385

the proteins function via scaffolding of cytosolic proteins and associate with actin and microtubule

386

cytoskeleton networks to form higher-order structures.42 In eukaryotes, alpha and beta -tubulin are

18


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387

necessary for the assembly of dynamic microtubules.43 Actin filament-coating protein tropomyosin and

388

microtubule binding protein contribute to the binding of the filaments and regulate the diverse range of

389

actin and microtubule cytoskeleton structures, respectively. The interdependent collaboration of actin

390

and microtubule cytoskeletons is crucial for cytokinesis, polarity maintenance, membrane remodeling

391

and tissue morphogenesis during fungal growth.44 Myosin regulatory light chain functions via the action

392

of myosin kinases and reversible phosphorylation to modulate basal actomyosin oscillations that have

393

emerged as key regulators of tissue and organ morphogenesis.45 Cytoplasmic dynein light chain functions

394

in peroxisome biogenesis that attributes to assemble the dynein motor in yeast.46 Cytoplasmic dynein is

395

required for the microtubule-based transport and spatial organization in filamentous fungi.47 Considered

396

together, these findings suggested the coordinated organization of calcium signal transduction process

397

and dynein-dependent actin and microtubule cytoskeletons formation might play a crucial role in the

398

regulation of survival, tissue morphogenesis and signaling cascades during F. velutipes mycelia under

399

CLS environment.

400

In CLS samples, we also found a dramatic increase in the activities of class V chitinase ChiB1, β-

401

galactosidase, autophagy-related protein and Hsp70 chaperone. In synergy with the β-glucanases, ChiB1,

402

a chitinase, may play an important role in the developing fruit-body of Coprinopsis cinerea, especially

403

pileus autolysis.48 In fungi, autophagy-related proteins are essential for triggering autophagy-mediated

404

processes to regulate nuclear dynamics and hyphal differentiation during vegetative growth, hyphal

405

fusion, fruit-body development and sexual reproduction.49,50 Heat-shock protein 70 (Hsp70) chaperones

406

play important roles in stabilizing partially folded protein structures and rescuing the cells from apoptosis

407

during stress that affects a wide range of normal cellular physiology. 51 Our data showed that the activity

408

of superoxide dismutase and catalase were remarkably increased in CLS samples suggesting F. velutipes

19



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409

mycelium adaption to increased oxidative stress induced by light stress. Superoxide dismutase and

410

catalase, located in the peroxisome, are two crucial enzymes for protecting cells and tissues from

411

oxidative damage by reactive oxygen species (ROS) in many fungi. 52 In addition, we observed that

412

expression levels of some initiation and elongation factors associated with the initiation of primordia

413

from mycelia and sexual development in fungi, such as translation elongation factor EF1-alpha53,

414

elongation factors8,54 and transcription factors55, were significantly up-regulated in CLS sample. More

415

broadly, understanding of expression pattern of the DEPs associated with survival, oxidative stress

416

adaption, autolysis and sexual development could be helpful to provide further insights into the response

417

mechanism of F. velutipes mycelia under CLS environment.

418

In conclusion, here, we try to systematically analyze and investigate the stress proteome associated

419

with mycelia in response to cold and light stress in F. velutipes by using iTRAQ labeling coupled to 2D

420

LC-MS/MS technique. Our data showed that three interesting up-regulated DEPs, including acetyl-

421

hydrolase, AASDH and ACPDC, may play an important role in improving the mycelium adaptability

422

and the ability to secrete pigment under CS environment in F. velutipes. The regulatory network

423

underlying the response mechanism of F. velutipes mycelia under CLS environment was complex and

424

multifaceted, as it included varied functions such as the rapid energy supply, the biosynthesis of lysine,

425

phenylalanine, tyrosine and GABA, calcium signal transduction process, dynein-dependent actin and

426

microtubule cytoskeletons formation, autolysis, oxidative stress adaption, tissue morphogenesis, and

427

other interesting stress-related processes. The data might provide further insights into the regulation

428

mechanism of mycelia in response to cold and light stress underlying mycelium growth, development

429

and fruit-body formation in F. velutipes. Furthermore, the data might also shed new light on an intuitive

430

understanding of the stress response mechanism in macro-fungi and provide valuable evidence for

20


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431

scientific improvement of some mushroom cultivation techniques in practice.

432

■ ASSOCIATED CONTENT

433

Table S1: the summarized primer data of 18 representative differentially expressed proteins.

434

Table S2: list of down-regulated proteins under both cold stress and cold and light stress in mycelia of

435

Flammulina velutipes

436

Table S3: list of down-regulated proteins under only cold stress in mycelia of Flammulina velutipes

437

Table S4: list of down-regulated proteins under only cold and light stress in mycelia of Flammulina

438

velutipes

439

■ AUTHOR INFORMATION

440

Corresponding Author

441

*Tel: +86 (0354) 6288325; fax: +86 (0354) 6288325; e-mail: [email protected], [email protected].

442

Author Contributions

443

All authors have given approval to the final version of the manuscript. Notes: The authors declare no

444

competing financial interest.

445

■ ACKNOWLEDGMENTS

446

This study was financially supported by grants from the National Natural Science Foundation of China

447

(no. 31301826), the Key Scientific and Technological Project from Shanxi Province (no. FT2014-03-01),

448

and the National Key Research and Development Program of China (no. 2017YFD0400200). We thank

449

MogoEdit (http://wwq.mogoedit.com) for its linguistic assistance during the preparation of this

450

manuscript.

451

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452

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