Responses of the Mushroom Pleurotus ostreatus under Different CO2 Concentration by Comparative Proteomic Analyses

Background: Pleurotus ostreatus is a popular edible mushroom in East Asian markets. Research on the responses of P. ostreatus under different carbon dioxide concentrations is limited. Methods: Label-free LC-MS/MS quantitative proteomics analysis technique was adopted to obtain the protein expression profiles of P. ostreatus fruiting body pileus collected under different carbon dioxide concentrations. The Pearson correlation coefficient analysis and principal component analysis were performed to reveal the correlation among samples. The differentially expressed proteins (DEPs) were organized. Gene ontology analysis was performed to divide the DEPs into different metabolic processes and pathways. Results: The expansion of stipes was inhibited in the high CO2 group compared with that in the low CO2 group. There were 415 DEPs (131 up- and 284 down-regulated) in P. ostreatus PH11 treated with 1% CO2 concentration compared with P. ostreatus under atmospheric conditions. Proteins related to hydrolase activity, including several amidohydrolases and cell wall synthesis proteins, were highly expressed under high CO2 concentration. Most of the kinases and elongation factors were significantly down-regulated under high CO2 concentration. The results suggest that the metabolic regulation and development processes were inhibited under high CO2 concentrations. In addition, the sexual differentiation process protein Isp4 was inhibited under high CO2 concentrations, indicating that the sexual reproductive process was also inhibited under high CO2 concentrations, which is inconsistent with the small fruiting body pileus under high CO2 concentrations. Conclusions: This research reports the proteome analysis of commercially relevant edible fungi P. ostreatus under different carbon dioxide concentrations. This study deepens our understanding of the mechanism for CO2-induced morphological change in the P. ostreatus fruiting body, which will facilitate the artificial cultivation of edible mushrooms.


Introduction
Mushrooms are macrofungi with epigeous or hypogeous distinct fruiting bodies [1][2][3]. The aggregation of hyphae occurs in the early stage of fruiting body development [4], and fruiting body shape can be immediately affected by environmental factors, including nutrients, humidity, temperature, carbon dioxide concentration, gravity, and light [5]. Mushrooms sense light, gravity, and carbon dioxide concentration to develop a proper pileus to enable the effective diffusion of spores. Environmental factors for fruiting body induction (light, temperature, and nutrients as three examples), development (light, gravity, and carbon dioxide concentration as three examples), and maturation or senescence (during/after sporulation and after artificial harvesting) of mushroom-forming basidiomycetes were researched in previous studies, wherein carbon dioxide affects the development of mushrooms [6][7][8]. Carbon dioxide is an important trace gas in the Earth's atmosphere. Variation in its concentration can affect the growth and survival of living organisms on the Earth.
Previous studies demonstrated that CO 2 is transported through membranes, is sensed by organisms, and acts as a key signaling molecule to control growth, differentiation, virulence, biotic interactions, etc. [9][10][11][12]. In yeast, CO 2 can be transported into the cells mainly by simple diffusion, is then converted to HCO 3− , and maintains CO 2 /HCO 3− homeostasis by carbonic anhydrase [13]. CO 2 can also contribute to morphology, mating, sporulation, phenotypic switching, and virulence processes of fungi via the adenylyl cyclase/cAMP pathway [14]. Lu et al. revealed a new regulatory mechanism of CO 2 signaling in fungi hyphal development by reducing Ume6 phosphorylation and degradation [15]. CO 2 concentration could affect the development of fungal fruiting bodies. In particular, the differentiation of pileus would be inhibited under high CO 2 concentration. Edible fungi need to consume a large amount of oxygen and metabolize CO 2 in the growth process [16][17][18][19]. The excessive CO 2 in the mushroom shed can inhibit the growth of mushrooms, and even lead to CO 2 poisoning, manifested as the premature aging of mushrooms, formation of mushroom shape, etc., which may be caused by CO 2 accumulation generated in the growth process of mushrooms due to poor ventilation [20,21]. Excessive CO 2 in mushroom shed poses a threat to the health of mushroom cultivation management personnel, and thus deserves close attention. The appropriate CO 2 concentration could stimulate the differentiation of fruiting bodies, and excessive CO 2 concentration could inhibit the growth of hypha [22]. In the fruiting body stage, edible fungi are more sensitive to CO 2 concentration. When the sporocarp is formed, it has strong respiratory function and the demand for oxygen increases sharply. When the CO 2 concentration reaches more than 0.1%, it produces a toxic effect on the sporocarp. When the concentration of CO 2 reaches 5%, it inhibits the differentiation of the pileus and even affects the formation of fruiting bodies. Therefore, the ventilation time should be reasonably determined according to the varieties of edible fungi and the growth period of various agents.
The Influence of the gaseous condition on fruiting body shape is relevant in commercial mushroom production. Respiration and the concentration of carbon dioxide during fruiting body formation have been investigated [6]. Respiration activity increases during primordia formation in the development of fruiting bodies, and a high concentration of carbon dioxide affects fruiting body morphology. Sensitivity to carbon dioxide has been investigated in the commercially cultivated mushroom varieties of Flammulina velutipes, Pleurotus ostreatus, Pholiota nameko (microspora), and Lentinula edodes; of these, P. ostreatus is one of the most sensitive to carbon dioxide [6,23]. In many mushroom species, the pileus is not fully developed and the stipe is spindly and elongated at a high carbon dioxide concentration. The morphology is similar to that produced in the absence of light. In the early stage of fruiting body development, sensitivity to carbon dioxide is more pronounced [6]. Elevated carbon dioxide affects the synthesis of the cell wall component R-glucan [7] and fruiting body cell morphology. The regulation of CO 2 concentration in the cultivation environment is mainly achieved by setting the sealing time and ventilation time. However, the knowledge of the CO 2 regulation mechanism in higher fungi is still unknown [5].
In the present study, we cultivated P. ostreatus at different concentrations of CO 2 and the proteomes of the fruiting bodies were analyzed, which may help us to understand the mechanism underlining the fruiting body development of this mushroom.

Materials and Methods
2.1. Culture Conditions and Acquisition of the P. ostreatus Samples P. ostreatus strain PH11 mycelia were cultured and subcultured in PDA media. For fruiting body production, the strain PH11 was inoculated into solid media in polypropylene bags (5 cm × 30 cm, 50 µm thickness). The formula of solid media was as follows: 55% cottonseed hull, 30% sawdust, 10% bran, 3% gypsum, 0.5% potassium dihydrogen phosphate, 0.5% urea, and 1% glucose, and the ratio of substrates to water was 1:1.5; the pH was natural. The cultivation bags with solid media were sterilized at 121 • C for 2 h, incubated at root temperature for 72 h, and sterilized at 120 • C for 2 h for the second time. Vegetative growth of P. ostreatus mycelia was performed at 25 • C with a humidity of 70% in darkness. After 40 days cultivation, the primordium was stimulated by water injection, low temperature, and light in a high-humidity environment. After the primordium formed, the high CO 2 group bags were transferred into a CO 2 chamber with 90-95% humidity, 12 h light/12 h dark, at 20 • C. Fruiting production at high CO 2 concentration was carried out in a ZCLY-180ES CO 2 chamber (Zhichu, Shanghai) with 90-95% humidity. The humidity and light were controlled by wireless humidifier and light. The control group bags were incubated in atmosphere with 90-95% humidity, 12 h light/12 h dark, and at 20 • C. The fruiting bodies were grown under different CO 2 concentrations (1%, 0%) for 72 h, and the fruiting bodies were collected and stored at −80 • C for further analysis.

Protein Extraction and Peptide Digestion
Total proteins were extracted from the frozen P. ostreatus samples according to the following protocol: 100 mg frozen sample was taken into the centrifuge tubes and then 1 mL UT buffer (8 M urea, 0.1 M Tris-HCl pH 8.5) containing Thermo HALT protease and phosphatase inhibitor cocktail was added. The tissueLyser II was used to break the sample at 150 Hz for 60 s. The cell extract was treated by ultrasonication for 24 s (on for 6 s, off for 15 s). Tissue debris was removed by centrifugation (12,000× g for 10 min at 4 • C), and the supernatant was transferred into a new tube. Protein concentration was determined using the Micro BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). After adding 15 mg dithiothreitol (DTT), the sample was incubated at 37 • C for 1 h [24].
Afterwards, enzymolysis was performed according to the FASP method created by Wiśniewski et al. [25]. The extracted proteins (100 µg) dissolved in 300 µL UA buffer were taken into Pierce Protein Concentrators PES (10 K MWCO, 0.5 mL) (Thermo Fisher) to remove the low-molecular-weight impurities by centrifuging at 10,000× g for 30 min. A total of 50 mM of iodoacetamide was added to alkylate the proteins for 30 min at room temperature in the dark. The proteins were washed with 200 µL UA and 300 µL of 50 mM NH 4 HCO 3 after removing the buffer by centrifugation. A total of 2 µg modified trypsin (Promega) in 100 µL of 50 mM NH 4 HCO 3 was added into the ultrafiltration tube in a mass proportion of 1:50 (enzyme/protein). Enzymolysis was performed with gentle shaking at 37 • C for 12 h. After that, peptides were collected by centrifugation at 10,000× g for 15 min, and the residue peptides in the ultrafiltration tube were washed with 50 µL of 50 mM NH 4 HCO 3 one more time. The salt in the pooled elutes was removed by using Merck Millipore ZipTip C18 resin (Darmstadt). Additionally, peptide concentration was measured by utilizing Pierce Quantitative Colorimetric Peptide Assay. The peptide sample was lyophilized on an RVC 2-25 CD plus vacuum concentrator (Christ), and stored at −80 • C for further analysis.

Label-Free LC-MS/MS Quantitative Proteomics Analysis
This analysis was performed with desalted peptides, which were reconstituted in 10 µL 0.1% formic acid, and was carried out using a Nano-LC system coupled with Orbitrap Fusion TM Tribrid TM (Thermo Fisher Scientific). The peptide sample (1 µL) was injected onto the Acclaim PepMap 100 nano trap column (75 µm × 2 cm, nanoViper 2PK C18, 3 µm, 100 Å) and then separated on an Acclaim PepMap TM 100 analytical column (75 µm × 15 cm, nanoViper C18, 3 µm, 100 Å), and eluted in a 75 min nonlinear gradient program (mobile phase A: 0.1% (w/w) formic acid in water, mobile phase B: 0.1% (w/w) formic acid in 80% acetonitrile; 0-5 min, 4% to 8% B; 5-50 min, 8% to 20% B; 50-60 min, 20% to 30% B; 60-73 min, 30% to 90% B, 73-75 min, 95% B). The Orbitrap Fusion was operated in positive ion mode with spray voltage set at 2.2 KV and source temperature at 275 • C. The MS instrument was operated in data-dependent acquisition mode (DDA), with full MS scans over a mass range of m/z 350-1500 with detection in the Orbitrap (120 K resolution) and with auto gain control (AGC) set to 100,000 or a maximum ion injection time of 50 ms. For the survey scan, ten of the most abundance precursor ions with a charge state of 2+ to 6+ were selected for higher-energy collisional-dissociation (HCD) fragment analysis. The dynamic exclusion parameter was set at 60 s. For each group, four biological repeats were performed [24].

Peptides and Proteins Identification
The raw data were analyzed using Proteome Discoverer software suite Version 2.0 (Thermo Fisher Scientific) against the P. ostreatus PC15 proteome database from Uniprot (Proteome ID: UP000027073, accessed on January 2021) [26]. Protein identification was supported by at least two unique peptides with a false discovery rate lower than 0.05.

Bioinformatics Analyses
Raw data obtained from Proteome Discovery software were normalized. The P. ostreatus proteome was annotated and functionally enriched using the Gene Ontology tool (http://geneontology.org/, accessed on 26 April 2022) according to cellular component (CC), molecular function (MF), and biological process (BP). Gene Set Enrichment Analysis (GSEA) was used for interpreting gene expression data [27]. GO analysis was performed by using the R-ggcyto 1.24.0 tool [28].

Phenotype of P. ostreatus under Different CO 2 Concentrations
To investigate the effects of CO 2 variations on the development of P. ostreatus PH11, young fruiting bodies of P. ostreatus PH11 were kept under a low concentration (atmospheric conditions) or high concentration (1%) of CO 2 . The fruiting body development was observed at both low and high CO 2 conditions ( Figure 1A-D); however, the stipes were much shorter in length (3.9 cm, 9.5 cm) and much thinner in the 1% carbon dioxide concentration (HCO) group as compared with the atmospheric condition (LCO) group ( Figure 1B,E). A significant, quick pileus expansion at 48 h was recorded in the LCO group, whereas the pileus growth was significantly inhibited with no basidiospore shedding in the HCO group ( Figure 1C,F). A difference in the pileus diameter was observed under low CO 2 conditions compared with high CO 2 conditions, suggesting a significant change in the function of the pileus due to the variation in CO 2 conditions.

Proteomic Analysis of P. ostreatus Grown under Different CO 2 Conditions
There are 56 unique proteins in HCO and 140 unique proteins in LCO. There were 1137 shared proteins in two different CO 2 treatments according to the Venn diagram ( Figure 2D). Principal component analysis shows that the four replicates of LCO (green) clustered more closely than HCO (red). Compared with the LCO group, the HCO group demonstrated a more divergent expression level. The second principal component varied greatly in HCO, especially HCO-4 and HCO-1. Pearson correlation shows that the correlation among different treatments was high with the lowest correlation coefficient of 0.93 (between HCO-4 and LCO-3), even in different carbon dioxide concentrations (Figure 2A,C, Table S1). The protein expression profiles of the LCO group and HCO group displayed a higher correlation (>0.93) with each other. Furthermore, the principal component analysis showed similar results ( Figure 2B). Additionally, Pearson correlation coefficient analysis and principal component analysis results reveal that the two groups of samples were reasonable with good correlations among biological repeats.  Compared with P. ostreatus under normal conditions, there were 131 differentially expressed proteins in P. ostreatus treated with 1% CO 2 concentration (Figures 3 and 4). Of 131 DEPs, 48 were uncharacterized proteins, which need to be annotated. A wide variety of DEPs were annotated with different biological functions. For instance, some DEPs were hydrolases (such as A0A067NG45 and A0A067NTT4), which catalyze the hydrolysis of a chemical bond. Ribosomal proteins, such as A0A067NG60 and A0A067N2P4, play roles in the formation and functioning of the ribosome. Chitinases, including A0A067N5C9 and A0A067NX05, play significant roles in stipe cell wall extension in mushrooms. Some DEPs are histones (including A0A067NL48 and A0A067P7W0), which provide structural support for a chromosome.
Carbohydrate, organic substance, peptide, and other metabolic processes were active in the 1% carbon dioxide concentration group (HCO), which indicates that the cellular metabolic process is active in high-carbon-dioxide-concentration conditions. From the molecular function aspect, hydrolase activity, catalytic activity, peptidase activity, the structural constituent of ribosome, and flavin-adenine-dinucleotide binding also confirm that material transformation and metabolism occur frequently in P. ostreatus.   Table S3).  The eigengene adjacency heatmap shows that MEred and MEgreen clustered with MEturquoise and MEyellow. MEblue clustered with MEbrown. There were 20 hub genes in six modules, wherein 43 proteins were uncharacterized proteins (blue, brown, green, red, turquoise, yellow). Many modules showed more or less intersection in gene function, especially between MEbrown and MEblue. Ribosomal protein was shared in MEbrown, MEblue, and MEyellow modules. Alkaline phosphatase was shared in MEbrown and MEblue modules. Autophagy-related protein was shared in MEblue and MEred modules. Extracellular metalloproteinase was shared in MEblue and MEbrown modules. Glycoside hydrolase was shared in MEgreen, MEbrown, and MEred modules. Glycosyltransferase was shared in MEgreen and MEturquoise modules (Figures 8 and 9, Tables S4 and S5).

Discussion
CO 2 is an end product of cellular respiration [14]. CO 2 is a critical cellular signaling molecule in all organisms. In most basic aspects of life, the transport of CO 2 through membranes has fundamental roles. High CO 2 concentrations are sensed by cells independent of O 2 of pH via specific signaling pathways, causing distinct effects (phenotypes) [15]. The CO 2 -induced enhancement of plant growth indicates that rising atmospheric CO 2 has contributed to the shrubland expansions of the past 200 years [10]. CO 2 plays a key role in respiration in mammals, microbial photosynthesis in plants and algae, and chemoreception in insects. During sexual reproduction, CO 2 inhibits cell-cell fusion but not filamentation [18]. Elevated CO 2 directly or indirectly influences plant-biotic interactions. For instance, elevated CO 2 alters reactive oxygen signaling, phytohormone secondary metabolism, and defense-associated development. Elevated CO 2 also directly or indirectly influences herbivory-or pathogenesis-related traits in pest and pathogen populations and alters predator-prey interactions by interfering with chemical communications and indirect defenses in pests [20]. In a previous study, carbon dioxide was shown to influence the initiation of the fruiting body development of mushrooms such as Schizophyllum commune, F. filiformis, and Agaricus bisporus [29,30]. However, research on the responses of P. ostreatus under different carbon dioxide concentrations is limited. The inhibition of pileus expansion by high CO 2 levels was especially conspicuous in P. ostreatus, causing a trumpet-shaped deformation of the pileus, occasional swelling of the stipe accompanied by sponge-like tissue, and, occasionally, malformed small pileus surfaces.
The expansion of the pileus, the basidiospore-forming area, is inhibited at high CO 2 concentrations. As we can see from the results, the expression of the sexual differentiation process protein Isp4 (A0A067N4P4) was significantly down-regulated in the HCO group. Sexual differentiation process proteins have been studied in Schizosaccharomyces pombe and some other fungi [31,32], and isp4 has been found to be up-regulated by nitrogen-starvationinduced meiosis. A0A067N4P4, which might be involved in meiosis and reproduction in P. ostreatus, has 53.0% amino-acid sequence identity with Isp4 in S. pombe (NP_595653). The unmatured pileus at high CO 2 concentrations also inhibits the sexual production process. In the natural environment, a high CO 2 concentration indicates poor ventilation, which is not good for basidiospore dispersal. Therefore, the mushroom might control the sexual process by controlling the fruiting body morphology.
GO enrichment indicated that enzymes related to hydrolase activity [GO:0016810] were significantly enriched (with the lowest p-value) at a high CO 2 concentration. Several amidohydrolases were significantly highly expressed in the HCO group, and no amidohydrolase was significantly down-regulated in the LCO group. Alfonso et al. reported that amidohydrolase might be related to autolysis in penicillin, and Donovan et al. reported that amidohydrolase might be related to cell wall lysis [33,34]. Therefore, the high expression of amidohydrolase in the HCO group indicates that cell wall biosynthesis might be influenced by a high CO 2 concentration. Indeed, some cell-wall-synthesis-related proteins were also significantly highly expressed at a high CO 2 concentration (A0A067NEG7), such as chitooligosaccharide oxidase (A0A067NL12), chitin deacetylase, and chitinase-3-like protein 2 (A0A067NHR3). The results indicate that the morphological change in P. ostreatus at a high CO 2 concentration was regulated by the differential expression of cell wall biosynthesis.
Kinase-mediated protein phosphorylations play pivotal roles in the regulation of cellular processes. Physiological CO 2 concentrations induce filamentation by the direct stimulation of cyclase activity. Filamentation is mediated by second messengers, such as cyclic adenosine 3 ,5 -monophosphate (cAMP) synthesized by adenylyl cyclase. The link between cAMP signaling and CO 2 sensing is conserved in fungi, and the cAMP signaling pathway in turn controls the growth, differentiation, and virulence factors of fungi [35,36]. Different kinases may be involved in the subsequent regulation of cellular processes. In high CO 2 concentrations, the expression of many kinases was significantly down-regulated, such as A0A067NLY8, A0A067NNC2, A0A067NJ97, A0A067NHQ7, A0A067N5R0, A0A067NR52, A0A067NIN8, A0A067PB97, A0A067N9N0, A0A067NAM8, and A0A067NE46. However, no predicted kinase was highly expressed under high CO 2 concentrations. In this study, the sample for proteomic analysis is the pileus part. The small pileus combined with the proteomic analysis results suggests that the metabolic activity is lower in high CO 2 conditions. High CO 2 concentrations might inhibit the expression of kinases, which further inhibit the metabolism and development of the fruiting body pileus.
Seven elongation factors were identified in proteomic analysis (Table S1): the expression of five elongation factors was significantly down-regulated in high CO 2 concentrations, the expression of one elongation factor was insignificantly down-regulated in high CO 2 concentrations, and one was only expressed in low CO 2 concentration. Translation elongation factor complexes play a central role in protein synthesis, delivering aminoacyl-tRNAs to the elongating ribosome, and the expression of elongation factor is indispensable in eukaryotes [37][38][39]. The down-regulation of elongation factors at high CO 2 concentrations might inhibit the protein synthesis, which is inconsistent with the down-regulation of kinases. Therefore, the elongation factors might be involved in CO 2 -mediated morphological change in P. ostreatus.

Conclusions
This research investigates the proteome analysis of commercially relevant edible fungi P. ostreatus under different carbon dioxide concentrations. The stipes were shorter and thinner in the HCO group compared with the LCO group. A larger pileus diameter was observed in the LCO group compared with the HCO group. The differentially expressed proteins under higher and lower CO 2 concentrations were presented and discussed in the present study. Based on our results, the expression of kinases and elongation factors are regulated under different CO 2 concentrations, and, as a result, the expressions of proteins related to cell wall synthesis and sexual differentiation process proteins are significantly changed. Proteins in these processes can serve as a target for selective molecular breeding. Further studies are still needed to analyze the function of other proteins, such as proteins related to cAMP signal processing or bicarbonate metabolism, in CO 2 response in P. ostreatus.

Conflicts of Interest:
The authors declare no conflict of interest.