Article pubs.acs.org/JAFC
Comparative Proteome Reveals Metabolic Changes during the Fruiting Process in Flammulina velutipes Jing-yu Liu,*,†,‡ Ming-chang Chang,*,†,‡ Jun-long Meng,†,‡ Cui-ping Feng,†,‡ Hui Zhao,† and Ming-liang Zhang† †
College of Food Science and Engineering, Shanxi Agricultural University, Taigu 030801, China Shanxi Engineering Research Center of Edible Fungi, Taigu 030801, China
‡
S Supporting Information *
ABSTRACT: Understanding the molecular mechanisms regulating the fruiting process in macro-fungi, especially industrially cultivated mushrooms, has long been a goal in mycological research. To gain insights into the events accompanying the transformation of mycelia into fruit-bodies in Flammulina velutipes, proteins expressed characteristically and abundantly at primordium and fruit-body stages were investigated by using the iTRAQ labeling technique. Among the 171 differentially expressed proteins, a total of 68 displayed up-regulated expression levels that were associated with 84 specific KEGG pathways. Some up-regulated proteins, such as pyruvate carboxylase, aldehyde dehydrogenase, fatty acid synthase, aspartate aminotransferase, 2-cysteine peroxiredoxin, FDS protein, translation elongation factor 1-alpha, mitogen-activated protein kinases (MAPKs), and heat-shock protein 70 that are involved in carbohydrate metabolism, carotenoid formation, the TCA cycle, MAPK signaling pathway, and the biosynthesis of fatty acids and branched-chain amino acids, could serve as potential stagespecific biomarkers to study the fruiting process in F. velutipes. Knowledge of the proteins might provide valuable evidence to better understand the molecular mechanisms of fruit-body initiation and development in basidiomycete fungi. Furthermore, this study also offers valuable evidence for yield improvement and quality control of super golden-needle mushroom in practice. KEYWORDS: Flammulina velutipes, fruiting process, proteome, iTRAQ, LC−MS/MS
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INTRODUCTION Understanding the molecular mechanisms regulating the fruiting process in macro-fungi, especially industrially cultivated mushrooms, has long been a goal in mycological research.1,2 During the development of mushrooms, the mycelia transform into fruit-bodies via intermediate primordia, which is a critical and complicated process.3,4 As a white rot fungus, Flammulina velutipes is widely cultivated worldwide for its high nutritional and medicinal value. In F. velutipes cultivation, both yield and quality of F. velutipes differ largely depending on induction of primordia and control of fruit-body development. Meanwhile, F. velutipes is a low-temperature fungus whose mycelia can grow vegetatively at 20−24 °C and transform into fruit-bodies at 12− 15 °C in low light.5 These favorable culture conditions can be conveniently built using industrial or laboratory refrigeration equipment. F. velutipes has been recognized as a potential useful model fungal species to study fruit-body formation in mushrooms.6−8 Several genes involved in fruit-body formation of F. velutipes have been investigated at the molecular level.9−12 However, current knowledge of F. velutipes fruit-body formation remains fragmented. Unlike other model organisms, its proteome remains largely uncharacterized. Furthermore, proteomics analysis could provide insight related to systematic flux changes of F. velutipes fruit-body formation. Proteomics techniques are powerful tools for the identification of quantitative changes in protein expression in response to stress exposure in filamentous fungi.13 In the past decade, proteomics has become an indispensable complement to genome and transcriptome approaches in fungal biology.14 © 2017 American Chemical Society
The standard procedures of proteomics are 2-dimensional gel electrophoresis (2-DE) or liquid chromatography coupled with mass spectrometry (LC−MS).15 Protein expression patterns during the fruit body induction of F. velutipes were investigated by 2-DE, and 22 differentially expressed protein spots were found and 4 of the spots had identified by N-terminal sequencing.7 With the recent advent of chromatography, mass spectrometry, and bioinformatics, gel-based techniques have been complemented and challenged by LC-based techniques, particularly high-throughput shotgun proteomics.16 Coupled with two-dimensional liquid chromatography tandem mass spectrometry (2D LC−MS/MS), the iTRAQ labeling technique has proven to be a powerful tool for analyzing chronological changes of proteomic profiles and investigating candidate genes and signaling pathways associated with complex developmental processes of filamentous fungi.13 The technique is proved to be more suitable to gain insights into metabolic changes during different development stages in F. velutipes.5,17 In this study, iTRAQ coupled with 2D LC−MS/ MS was used to assess proteome changes associated with fruitbody formation in F. velutipes. Furthermore, chronological changes in protein expression patterns at primordia and fruitbody stages were investigated, compared, and examined. Received: Revised: Accepted: Published: 5091
March 10, 2017 May 27, 2017 June 1, 2017 June 1, 2017 DOI: 10.1021/acs.jafc.7b01120 J. Agric. Food Chem. 2017, 65, 5091−5100
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Journal of Agricultural and Food Chemistry
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version, 138 749 entries) assuming the digestion enzyme trypsin. Scaffold Q+ (version Scaffold_4.4.6, Proteome Software Inc.) was used to quantitate label based quantitation peptide and protein identifications. Protein identifications were accepted if they were at a 99% (95%) confidence level for protein identification as determined by a false discovery rate (FDR) of ≤1% (5%) by the Scaffold Local FDR algorithm. Protein probabilities were assigned by the Protein Prophet algorithm.18 Channels were corrected by the matrix [0.000,0.000,0.929,0.0689,0.00220]; [0.000,0.00940,0.930,0.0590,0.00160]; [0.000,0.0188,0.931,0.0490,0.001000]; [0.000,0.0282,0.932,0.0390,0.000700]; [0.000600,0.0377,0.933,0.0288,0.000]; [0.000900,0.0471,0.933,0.0188,0.000]; [0.00140,0.0566,0.933,0.00870,0.000]; [0.000,0.000,0.000,0.000,0.000]; [0.00270,0.0744,0.921,0.00180,0.000] in all samples according to the algorithm described in i-Tracker.19 All normalization calculations were performed using medians to multiplicatively normalize data. Function Analysis. GO enrichment and KGG pathway analysis of differentially expressed proteins (DEPs) were conducted according to the information from the GO databases and KEGG Pathway, respectively. The GO project described the roles of proteins in three domains: cellular component, molecular function, and biological process. A GO term or pathway was considered a significant enrichment of differential proteins if the p-value was 0.05 (ANOVA test) were discarded. Following this criterion, a total of 171 DEPs were identified. In general, three clearly different expression patterns during fruit-body formation in F. velutipes were generalized among the 171 DEPs. A total of 68 DEPs (Figure 2A) were up-regulated under at least 1 of the 3 F. velutipes stages, including 41 DEPs at 2 stages (primordia and young fruit-bodies), while 21 and 6 DEPs were particularly up-regulated at primordia and young fruit-bodies, respectively. Among 103 down-regulated DEPs (Figure 2B) in at least one development stage, 63 DEPs were shared by 2 stages (primordia and young fruit-bodies), while 27 and 13 DEPs were particularly down-regulated at primordia and young fruit-bodies, respectively. The summarized data of up-regulated proteins are shown in Table 1 and Table 2. The summarized data of down-regulated proteins are shown in Tables S1 and S2. 5092
DOI: 10.1021/acs.jafc.7b01120 J. Agric. Food Chem. 2017, 65, 5091−5100
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Functional Categorization Analysis. Functional information for the 171 DEPs were founded using BLASTP (http:// www.ncbi.nlm.nih.gov/BLAST/). Enrichment analysis against agriGO (http://bioinfo.cau.edu.cn/agriGO/) showed that, in the cellular component, DEPs were mainly enriched in the GO terms of ribosome, cytoplasm, nucleus, and component of the cellular membrane. In biological processes, DEPs were mainly found in translation, carbohydrate metabolic process, metabolism, and transport. While under the category of molecular function, DEPs were mostly related to enzyme regulator activity, antioxidants, and binding. Unfortunately, 11 DEPs had unknown locations. The summarized GO mapping and annotation data of up-regulated proteins and down-regulated proteins are shown in parts A and B of Figure 3, respectively.
Figure 2. Venn diagrams and expression of 171 DEPs identified by iTRAQ during fruit-body initiation and development in Flammulina velutipes. The numbers of DEPs with down-regulation (A) and upregulation (B) under given development stages are shown in different segments.
Table 1. List of up-Regulated Proteins in Primordia and Young Fruit-Bodies of Flammulina velutipes average foldchangeb
a
Uniprot IDa
protein name
gene name
P/C
Y/C
K5W8H5_AGABU B0D9J1_LACBS D0VB17_9AGAR V2XTQ9_MONRO V2XPL1_MONRO K9HWC2_AGABB K5Y3R7_AGABU D6RM71_COPC7 V2XLD8_MONRO D7GKX4_SCHCO V5NDN0_LEUGO A0A067NZB1_PLEOS K9HHZ0_AGABB B0E052_LACBS V2XET7_MONRO V2X746_MONRO A8N7Q5_COPC7 D2Y6R3_FLAVE V2X447_MONRO A8N3 V6_COPC7 V2WTP2_MONRO V2X1J9_MONRO V2XZ71_MONRO V2YXI4_MONRO B0E2L4_LACBS A0A067P9S7_PLEOS A0A067NHG4_PLEOS Q12097_FLAVE A0A067NAR2_PLEOS D2JY94_FLAVE A8N640_COPC7 V2X1R9_MONRO A0A067NRD1_PLEOS B0D6H6_LACBS O74163_FLAVE V2XPY0_MONRO V2XEA8_MONRO D8PQW8_SCHCM V2YJT1_MONRO V2YEI1_MONRO V2WU06_MONRO
V-type proton ATPase subunit a urease translation elongation factor 1-alpha (fragment) transcription factor threonine synthase T-complex protein 1 subunit gamma T-complex protein 1 subunit delta T-complex protein 1 epsilon subunit T-complex protein 1 spermidine synthase-saccharopine dehydrogenase (fragment) serine protease 1 S-adenosylmethionine synthase pyruvate carboxylase plasma membrane H+-transporting ATPase phosphoenolpyruvate carboxykinase phospho-2-dehydro-3-deoxyheptonate aldolase lysine-tRNA ligase immunomodulatory protein (fragment) Hsp70 family chaperone lhs1 histone acetyltransferase type B subunit 2 heterogeneous nuclear ribonucleoprotein hrp1 heat shock protein sks2 heat shock protein guanylate kinase guanine nucleotide-binding protein alpha-4 subunit glycosyltransferase family 4 protein glycoside hydrolase family 51 protein FDS protein fatty acid synthase complex protein expansin family protein delta-aminolevulinic acid dehydratase D-arabinitol dehydrogenase cytochrome b-c1 complex subunit Rieske, mitochondrial CipC1 protein, concanamycin induced protein C (fragment) C1 protein bifunctional purine biosynthesis protein ade17 Atp-citrate synthase aspartate aminotransferase (fragment) alcohol oxidase-like protein 5-methyltetrahydropteroyltriglutamate- homocysteine S-methyltransferase 2-cysteine peroxiredoxin
AGABI1DRAFT_111634 LACBIDRAFT_291133 tef Moror_6368 Moror_14189 AGABI2DRAFT_189151 AGABI1DRAFT_111201 CC1G_14362 Moror_8024 spe-Sdh
2.19 1.91 1.94 3.18 2.03 2.20 2.28 2.93 2.14 2.73 1.85 2.85 1.53 1.58 1.84 1.51 2.77 6.14 1.95 2.03 1.74 3.65 2.08 1.66 3.30 2.96 3.12 6.99 2.42 2.75 2.14 1.93 1.94 1.98 4.83 1.68 1.62 1.75 3.13 3.86 3.72
1.60 1.89 1.62 2.36 2.33 1.74 2.23 2.12 2.03 1.65 1.57 2.15 1.69 1.50 1.77 1.69 2.03 4.06 1.89 1.86 1.55 3.14 1.79 1.51 3.13 3.06 4.03 5.91 1.81 2.67 1.57 1.60 1.58 1.91 3.83 1.78 1.57 1.58 2.90 2.94 3.64
PLEOSDRAFT_1052256 AGABI2DRAFT_193409 LACBIDRAFT_315237 Moror_16330 Moror_13998 CC1G_02312 Moror_12985 CC1G_00568 Moror_7732 Moror_8916 Moror_660 Moror_6932 LACBIDRAFT_318523 PLEOSDRAFT_1060842 PLEOSDRAFT_1043210 PLEOSDRAFT_33075 CC1G_01960 Moror_11289 PLEOSDRAFT_1088400 LbCipC1 C1 Moror_14083 Moror_16546 SCHCODRAFT_42521 Moror_10380 Moror_7843 Moror_15689
Uniprot: http://www.uniprot.org/. bP, primordia; Y, young fruit-bodies; C, control (mycelia). 5093
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Journal of Agricultural and Food Chemistry Table 2. List of Up-Regulated Proteins at Only Primordia or Young Fruit-Body Stages of Flammulina velutipes
average fold-changeb Uniprot ID
a
protein name
Up-Regulated Proteins at Only Primordium Stages V2Y0D9_MONRO tyrosine-tRNA ligase A0A067U0J3_9AGAR triosephosphate isomerase A8NV31_COPC7 transketolase V2XKQ8_MONRO Rps25ap A0A067NYX2_PLEOS pyruvate dehydrogenase E1 component subunit alpha V2YTF9_MONRO Premrna splicing factor D8QEV1_SCHCM Peptidyl-prolyl cis−trans isomerase B0CY35_LACBS NAD-aldehyde dehydrogenase A0A097ZNK8_FLAVE methyltransferase V2WZS1_MONRO ketol-acid reductoisomerase V2WVN8_MONRO Hsp70 chaperone D8PX23_SCHCM heat shock protein HSS1 A0A067P0S7_PLEOS glycoside hydrolase family 31 protein B0D3B6_LACBS glycoside hydrolase family 3 protein V2XLZ6_MONRO glycoside hydrolase family 13 protein V2WK12_MONRO dihydroxy-acid dehydratase V2XZE6_MONRO D-3-phosphoglycerate dehydrogenase K5W9Z2_AGABU chorismate synthase V2X2P0_MONRO aldehyde dehydrogenase B0CQR5_LACBS 6-phosphogluconolactonase K5W4 V4_AGABU mitogen-activated protein kinase Up-Regulated Proteins at Only Young Fruit-Body Stage V2XEN3_MONRO T-complex protein eta subunit (Tcp-1-eta) V2X5W5_MONRO sulfur metabolism regulator MTAP_COPC7 S-methyl-5′-thioadenosine phosphorylase C7E3U3_HYPMA heat shock protein 70 B0D9Q1_LACBS fatty acid synthase A9Q1C5_CLINE elongation factor 1-alpha (fragment) a
gene name
P/C
Y/C
Moror_262 GALMADRAFT_233292 CC1G_06175 Moror_8875 PLEOSDRAFT_1091334 Moror_13969 SCHCODRAFT_78835 LACBIDRAFT_188764 Moror_4591 Moror_9965 SCHCODRAFT_74624 PLEOSDRAFT_1061529 LACBIDRAFT_189477 Moror_1652 Moror_11846 Moror_17187 AGABI1DRAFT_81437 Moror_1794 LACBIDRAFT_291579 AGABI1DRAFT_54872
1.62 1.73 1.51 1.54 1.80 1.50 1.91 1.58 1.67 1.82 1.58 1.90 1.65 2.00 1.57 1.58 1.50 2.23 1.95 1.97 1.55
1.11 1.06 1.33 1.20 1.40 1.32 1.44 1.30 1.28 1.26 1.10 1.33 1.39 1.11 1.21 1.09 1.29 1.31 1.35 1.15 1.07
Moror_4808 Moror_5545 CC1G_06667 HSP70 FAS-2 tef-1
0.95 1.14 1.11 1.33 1.17 1.34
2.37 1.82 1.80 1.60 1.58 1.57
Uniprot: http://www.uniprot.org/. bP, primordia; Y, young fruit-bodies; C, control (mycelia).
Metabolism Pathway Analysis. The result of GO analysis showed that proteins differentially expressed during fruit-body formation of F. velutipes were influenced by a variety of cellular processes, particularly metabolic processes. Among the 171 DEPs, 105 (61.4%) were associated with 159 specific KEGG pathways. Further analysis of 68 up-regulated DEPs showed that 48 DEPs (70.6%) were mainly distributed in 84 specific KEGG pathways. Among the 48 DEPs, 29 DEPs (60.4%) that were up-regulated at two stages (primordia and young fruitbodies) were associated with biosynthesis of amino acids (path, ko01230), citrate cycle (TCA cycle) (path, ko00020), purine metabolism (path, ko00230), MAPK signaling pathway (path, ko04010), protein processing in endoplasmic reticulum (path, ko04141) and alanine, aspartate, and glutamate metabolism (path, ko00250). Fifteen DEPs (31.3%) that were up-regulated at only primordium stage were mainly involved in biosynthesis of amino acids (path, 01230), cysteine and methionine metabolism (path, ko00270), spliceosome (path, ko03040), and MAPK signaling pathway (path, ko04010). Four DEPs (8.3%) that were up-regulated at only the young fruit-body stage involved in purine metabolism (path, ko00230), alanine, aspartate, and glutamate metabolism (path, ko00250) (Figure 4) and starch and sucrose metabolism (path, ko00250). Further functions of these differentially expressed proteins are discussed in the following section. Transcriptional Expression Analysis of Selected Proteins as Revealed by qRT-PCR. The Western blotting technique is the best method to confirm the proteome data.
However, this is limited by the unavailability of specific antibodies for most edible mushroom proteins. Therefore, we determined the expression patterns of 15 mRNAs, each corresponding to the distinct 15 DEPs mentioned above, by qRT-PCR to validate our proteome data. The results showed that 9 genes, encoding 5-methyltetrahydropteroyltriglutamatehomocysteine S-methyltransferase, FDS protein, V-type proton ATPase subunit a, S-adenosylmethionine synthase, glycoside hydrolase family 3 protein, heat shock protein sks2, transketolase, class V Chitinase ChiB1, and T-complex protein 1 epsilon subunit showed similar expression patterns at all three developmental stages with their protein expression patterns (Figure 5A). However, the mRNAs of four genes, encoding lysine-tRNA ligase, glucose-6-phosphate isomerase, fatty acid synthase complex protein, and catalase, exhibited expression patterns that are different with their corresponding proteins. Another two genes, encoding ATP synthase subunit beta and beta-glucosidase, showed opposite expression patterns between their mRNAs and proteins (Figure 5B). The summarized primer data of 15 representative DEPs are shown in Table S3.
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DISCUSSION From a cultivation perspective, processes at the fruit-body formation stage are the primary relevant factors in the industrialized production of commercial mushrooms. However, we are still far from understanding the molecular and physiological mechanisms of fruit-body formation. In the cultivated fungi, F. velutipes has emerged as a potentially 5094
DOI: 10.1021/acs.jafc.7b01120 J. Agric. Food Chem. 2017, 65, 5091−5100
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Figure 3. Bioinformatics analysis of the above-mentioned differentially abundant proteins through gene ontology (GO) in three domains: biological process, molecular function, and cellular component. The statistics at GO level 2 is shown in this figure: (A) up-regulated proteins and (B) downregulated proteins.
excellent model system for studies of fungal genetics, development, biochemistry, and environmental stress.6,11 In the present study, the experiments were undertaken to investigate chronological changes of protein expression associated with primordia induction and fruit-body development in F. velutipes by using an iTRAQ-coupled 2D LC−MS/ MS technique. The proteomics data revealed that some interesting up-regulated proteins may play an important role during primordia induction and fruit-body development in F. velutipes. More than four functional categories were selected to analyze the data sets. The biological relevance of these differentially expressed proteins is discussed below. Increased Abundance of Energy Metabolism Related Proteins. The enzymatic mechanisms of microbial plant cell wall degradation are complex, and require an enormous diversity of glycoside hydrolases and carbohydrate lyases.20
The results showed complex protein abundance change patterns associated with fruit-body formation of F. velutipes at the molecular level. In the present study, we found that globally and differentially expressed proteins participated in energy metabolism pathways including oxidative phosphorylation, fatty acid biosynthesis and metabolism, tricarboxylic acid cycle, pentose phosphate pathway, oxidative phosphorylation, pentose and glucuronate interconversions, starch and sucrose metabolism, and glycolysis/gluconeogenesis. Our data showed that some interesting proteins that had higher expression levels at two stages (primordia and young fruit-bodies) are involved in energy metabolism, such as pyruvate carboxylase, phosphoenolpyruvate carboxykinase, phospho-2-dehydro-3-deoxyheptonate aldolase, Atp-citrate synthase, V-type proton ATPase subunit a, and plasma membrane H+-transporting ATPase. Pyruvate carboxylase (PC) is a 5095
DOI: 10.1021/acs.jafc.7b01120 J. Agric. Food Chem. 2017, 65, 5091−5100
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Figure 4. Representative metabolic pathway maps of differentially abundant enzymes involved in alanine, aspartate, and glutamate metabolism in KEGG. The colored enzyme codes were noted as follows: glutamine synthetase (EC, 6.3.1.2); carbamoyl-phosphate synthase (aspartate carbamoyltransferase/dihydroorotase) (EC, 6.3.5.5); glutamate decarboxylase (EC, 4.1.1.15); 1-pyrroline-5-carboxylate dehydrogenase (EC, 1.2.1.88).
Figure 5. Transcriptional expression analysis of representative proteins as revealed by qRT-PCR. (A) Relative mRNA expression patterns of selected proteins are similar to their protein expression patterns. (B) Relative mRNA expression patterns of selected proteins are not consistent with their protein expression patterns.
mitochondrial enzyme that plays a crucial role in gluconeogenesis during starvation or fasting growth.21 PC also serves an important anaplerotic role for amino acid catabolism and tricarboxylic acid (TCA) cycle, when intermediates are removed for different biosynthetic purposes. Increase flux of fatty acid synthesis and the TCA cycle are required for fungal growth.22 In the present study, high levels of PC activity, together with high activities of phosphoenolpyruvate carboxykinase, phospho-2-dehydro-3-deoxyheptonate aldolase, Atpcitrate synthase, V-type proton ATPase subunit a, and plasma membrane H+-transporting ATPase at the primordium and young fruit-body stages demonstrate that these enzymes may play an important role in energy metabolism during F. velutipes
mycelia to fruit-bodies transformation. One plausible explanation for this is that regulating fungal fruit-body formation and the sexual cycle are required for rapid energy requirements. Transketolase and 6-phosphogluconolactonase play a crucial role in connecting the pentose phosphate pathway to glycolysis for the synthesis of biologically important molecules in all organisms.23 In the nonoxidative phase of the pentose phosphate pathway, glucose is converted into ribulose 5phosphate, which is further processed to synthesize biomolecules including coenzyme A, ATP, and nucleotides.24 Coenzyme A is the main input for a series of reactions known as the TCA cycle and in the biosynthesis of fatty acids which are required for fungal growth and fruit-body 5096
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Journal of Agricultural and Food Chemistry formation.22,25 Aldehyde dehydrogenases, which are polymorphic enzymes, are widespread among white-rot basidiomycete fungi, where they participate in the detoxification of aromatic aldehydes to their corresponding carboxylic acids.26 Aldehyde dehydrogenase is also responsible for the last reaction in the formation of Neurospora carotenoid.27 The pigment also has a more general function as physiologically important compounds found in the key regulator of differentiation of vertebrate embryogenesis retinoic acid and fungal pheromone trisporic acid.28,29 Glycoside hydrolases (GHs) are important enzymes for carbohydrate metabolism in archaea, bacteria, fungi, plants, and mammals.30 Family GH 13, also known as the alphaamylase family, contains oligo-1,6-glucosidase, α-glucosidase, and dextran glucosidase.31 Glycoside hydrolase family 3 (GH3) from filamentous fungi have been widely used for the supplementation of cellulases. β-glucosidases from Aspergillus aculeatus (AaBGL1) belongs to the GH3 and shows high activity toward cellulosan.32 Glycoside hydrolase family 31 αglucosidase was found to be involved in starch utilization and to convert soluble starch to nigerose and maltose in fungi.33 Our data showed that the above-mentioned six proteins were significantly up-regulated at the primordium stage, but its expression levels returned to normal at the young fruit-body stage. These findings could have important implications for these differentially expressed proteins might play key roles in energy metabolism during F. velutipes mycelia to primordia transformation. In this study, we found fatty acid synthase expression dramatically increased in young fruit-bodies. In eukaryotes, fatty acid synthases (type I FASs) have evolved two strikingly distinct types, the metazoan and the fungal FAS (fFAS) that catalyzes the de novo synthesis of fatty acids that are essentially the same in all organisms.34 Fatty acids are central components of biological membranes, serve as the cellular structure, energy production and storage, and act as second messengers or as covalent modifiers governing protein localization and the biosynthesis of other biologically important molecules.35 Knowledge of the proteins associated with energy metabolism pathways is helpful to better understand the molecular mechanisms underlying fruit-body initiation and development in F. velutipes. Increased Abundance of Amino Acid Biosynthesis and Metabolism Related Proteins. In the fungi, amino acids serve multiple purposes, such as proteins building blocks, nitrogen storage, stress response, and signaling transport. In this study, our results showed that seven proteins related to amino acid metabolism, including aspartate aminotransferase, urease, 5-methyltetrahydropteroyltriglutamate-homocysteine Smethyltransferase, threonine synthase, S-adenosylmethionine synthase, and lysine-tRNA ligase, had higher expression levels at two stages (primordia and young fruit-bodies). Aspartate aminotransferase (AST) catalyzes the interconversion of aspartate or glutamate to the corresponding ketoacid that plays a crucial role for both amino acid biosynthesis and degradation. Glutamine synthesis, or accumulation, provides a richer nutritional environment leading to increased TORC1 (Target of Rapamycin complex 1) signaling and faster growth of yeast.36 In amino acid degradation, high activities of AST can produce glutamate from α-ketoglutarate that forms ammonium ions by subsequent oxidative deamination, which are excreted as urea. The hydrolysis of urea catalyzed by urease to produce carbamate plays an important role in the biosynthesis of inosine, which is an intermediate in a chain of purine
nucleotides reactions.37 In some medically important fungi, higher activity of urease may play an important role in pathogenesis of these fungi.38 5-Methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MetE) and threonine synthase (TS) are essential enzymes for methionine metabolism. In plants and yeasts, MetE catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in formation of L-methionine (L-Thr).39 TS catalyzes the hydrolysis of O-phospho-L-homoserine to synthesize L-Thr de novo.40 In the following steps, L-Thr is further catalyzed into S-adenosylmethionine (SAM-e) by S-adenosylmethionine synthetase (SAM). Together with high activities of methyltransferase, SAM catalyzes the synthesis of SAM-e that serves as a methyl group donor in transmethylation of proteins and nucleic acids and also a precursor in the biosynthesis of phytohormone ethylene, which takes part in many important biological processes in plants such as cell differentiation, cell expansion, the synthesis of polyamines, biotin, and nicotianamine.41 In addition, we observed that the expression level of four proteins, including NAD-aldehyde dehydrogenas, ketol-acid reductoisomerase, and chorismate synthase were significantly up-regulated at the primordium stage, but its expression levels dropped to basal levels at the young fruit-body stage. NAD +-aldehyde dehydrogenase catalyzes the oxidation of ethanol to acetyl-CoA that is advantageous to the cell for biosynthetic purposes in yeast. Higher activity of NAD+-aldehyde dehydrogenases serve as candidates for 4-aminobutyrate (GABA) and β-alanine production.42 GABA is a ubiquitous 4-carbon amino acid that plays an important role in metabolism and cell signaling during subjection to various biotic and abiotic stresses.43 Ketol-acid reductoisomerase (KARI) and dihydroxyacid dehydratase are essential enzymes for the biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine), pantothenate and coenzyme A (CoA) in bacteria, fungi, and plants. Amino acids, particularly the branched-chain amino acid leucine, have been shown to stimulate mTOR complex 1 (mTORC1) activation and to control TORC1 signaling in Saccharomyces cerevisiae.44 The mechanistic target of mTORC1 integrates environmental and intracellular signals to regulate cell growth and inhibit catabolic processes such as autophagy.45 Chorismate synthase is the enzyme involved in the last step of the shikimate pathway that plays a central role in the biosynthesis of three aromatic amino acids (L-tryptophan, Lphenylalanine, and L-tyrosine) and numerous secondary metabolites in bacteria, fungi, and plants. In plants, three aromatic amino acids are used for the synthesis of proteins and numerous natural products, such as hormones, cell wall components, pigments, and alkaloids.46 Chorismate synthase also serves as a potential target of antifungal chemotherapy for human pathogenic yeast and filamentous fungi.47 These findings could indicate that branched-chain amino acids and aromatic amino acids might play an important role in F. velutipes mycelia to fruit-bodies transformation. Increased Abundance of Signaling Pathways and Other Processes Related Proteins. In this study, the expressions of 2-cysteine peroxiredoxin, FDS protein, translation elongation factor 1-alpha, and C1 protein dramatically increased at two stages (primordia and young fruit-bodies). Peroxiredoxins (Prxs) are ubiquitous and abundant antioxidant enzymes that help to control intracellular peroxide levels and regulate cell proliferation. A growing body of evidence implicates hydrogen peroxide as an important cellular signaling 5097
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molecule in eukaryotes.48 In the model organisms Schizosaccharomyces pombe and S. cerevisiae, 2-cysteine peroxiredoxins (2-Cys Prxs) have been shown to act as molecular chaperones and regulate signal transduction in cell cycle and DNA damage responses.49 In plants, this protein plays a role in seedling development and light stress response.50 FDS protein is specifically expressed during fruit-body differentiation in F. velutipes.8 Our data showed that mRNA levels and protein abundances of the protein were consistent. Translation elongation factor 1-alpha (TEF α1) is thought to play a major role in regulating cell wall morphogenesis. It is thought to play an important and major role in the transition from mycelia to primordia of Pleurotus tuberregium and Termitomyces heimii.4,51 Unfortunately, the function of C1 protein is unknown. In addition, we observed that the expression level of mitogen-activated protein kinase were significantly up-regulated at the primordium stage, and expression levels of heat-shock protein 70 and elongation factor 1-alpha were significantly upregulated at the young fruit-body stage. Mitogen-activated protein kinases (MAPKs) serve as an essential trigger of MAPK signaling pathway in fungi. In yeast, MAPK pathways are responsible for cell cycle arrest and mating in response to pheromone stimulation.52 Heat-shock protein 70 (Hsp70) is a central component of the cellular network of molecular chaperones, ribosomes, and folding catalysts to intracellular organelles.53 Elongation factor 1-alpha (eEF1a), a GTPase, and an actin bundling protein participates in cell growth and proliferation processes, including cytoskeleton organization, mitotic apparatus formation, and signal transduction.54 In the study, higher activity of these differentially expressed proteins suggests that the enzymes might play an important role in the initiation of primordia and differentiation of young fruit-bodies. In conclusion, here, we try to use a new proteomic profiling technology, an iTRAQ-coupled 2D LC−MS/MS technique, to investigate differences in protein expression patterns during fruit-body formation in F. velutipes. We found some upregulated proteins, which were involved in carbohydrate metabolism, carotenoid formation, the TCA cycle, MAPK signaling pathway, and biosynthesis of fatty acids and branchedchain amino acids, could serve as potential stage-specific biomarkers to provide insights into the transformation of mycelia to fruit-bodies in F. velutipes. The results of this study may be helpful in better understanding the molecular mechanisms underlying fruit-body formation in basidiomycete fungi. Furthermore, the data of metabolic changes during the fruiting process in F. velutipes also provide valuable evidence for better quality control and scientific improvement of some mushroom cultivation processes in practice.
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Article
AUTHOR INFORMATION
Corresponding Authors
*Phone: +86 (0354) 6288325. Fax: +86 (0354) 6288325. Email:
[email protected]. *Phone: +86 (0354) 6288088. Fax: +86 (0354) 6288788. Email:
[email protected]. ORCID
Jing-yu Liu: 0000-0001-8888-4231 Author Contributions
All authors have given approval to the final version of the manuscript. Funding
This study was supported by grants from the National Natural Science Foundation of China (Grant No. 31301826) and the Key Scientific and Technological Project from Shanxi Province (Grant No. FT2014-03-01). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01120. List of down-regulated proteins in primordium and young fruit-bodies of Flammulina velutipes (Table S1); list of down-regulated proteins at only primordium or young fruit-body stages of Flammulina velutipes (Table S2); and summarized primer data of 15 representative differentially expressed proteins (Table S3) (PDF) 5098
DOI: 10.1021/acs.jafc.7b01120 J. Agric. Food Chem. 2017, 65, 5091−5100
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