iTRAQ-Based Quantitative Proteomic Analysis Reveals Proteomic Changes in Mycelium of Pleurotus ostreatus in Response to Heat Stress and Subsequent Recovery

Abstract

High temperature is a key limiting factor for mycelium growth and development in Pleurotus ostreatus. Thermotolerance includes the direct response to heat stress and the ability to recover from heat stress. To better understand the mechanism of thermotolerance in P. ostreatus, we used morphological and physiological analysis combined with an iTRAQ-based proteomics analysis of P. ostreatus subjected to 40°C for 48 h followed by recovery at 25°C for 3 days. High temperature increased the concentrations of thiobarbituric acid reactive substances (TBARS) indicating that the mycelium of P. ostreatus were damaged by heat stress. However, these physiological changes rapidly returned to control levels during the subsequent recovery phase from heat stress. In comparison to unstressed controls, a total of 204 proteins were changed during heat stress and/or the recovery phase. Wherein, there were 47 proteins that responded to both stress and recovery conditions, whereas 84 and 73 proteins were responsive to only heat stress or recovery conditions, respectively. Furthermore, quantitative real-time PCR (qRT-PCR) confirmed differential expression of nine candidate genes revealed that some of the proteins, such as a mitogen-activated protein kinase (MAPK), phenylalanine ammonia-lyase (PAL), and heat shock protein (HSP), were also regulated by heat stress at the level of transcription. These differentially expressed proteins (DEPs) in mycelium of P. ostreatus under heat stress were from 13 biological processes. Moreover, protein-protein interaction analysis revealed that proteins involved in carbohydrate and energy metabolism, signal transduction, and proteins metabolism could be assigned to three heat stress response networks. On the basis of these findings, we proposed that effective regulatory protein expression related to MAPK-pathway, antioxidant enzymes, HSPs, and other stress response proteins, and glycolysis play important roles in enhancing P. ostreatus adaptation to and recovery from heat stress. Of note, this study provides useful information for understanding the thermotolerance mechanism for basidiomycetes.

Keywords: Pleurotus ostreatus; TBARS; heat stress; proteomics; recovery.

FIGURE 1

Different colony shapes and mycelial morphology of P. ostreatus in response to heat stress and recovery. (A) CK1; (B) incubated for 5 days at 28°C; (C) HS sample; (D) CK2; (E) RC sample; and (F) incubated for 3 days at 40°C following heat stress.

FIGURE 2

The TBARS concentration in mycelium after heat stress and recovery for P. ostreatus. HS group was cultivated for 5 days and subjected to heat stress for 2 days. RC group was allowed to recover for 3 days after exposure to heat stress. Data were analyzed by Duncan’s ANOVA test. Error bars represent the standard deviation of three replicates. The asterisks indicate the significance of differences between treatments and their corresponding controls (^∗P < 0.05).

FIGURE 3

Venn diagram of differentially expressed proteins that were up- or down-regulated (A) by heat stress or recovery and total number (B,C) of identified DEPs from heat stress or recovery. The “+” and “-” indicate up- and down-regulated proteins, respectively. The “++” and “–” indicate up- and down-regulated under both heat stress and recovery, respectively. The “+-” indicates up-regulated under heat stress and down-regulated during recovery and the “-+” indicates down-regulated under heat stress but up-regulated during recovery.

FIGURE 4

Bioinformatics analysis of DEPs responsive to heat stress (A) and subsequent recovery (B) in P. ostreatus mycelia compared to the control group through gene ontology (GO) in biological process (BP).

FIGURE 5

Bioinformatics analysis of DEPs responsive to heat stress (A) and subsequent recovery (B) in P. ostreatus mycelia compared to the control group through gene ontology (GO) in cell component (CC).

FIGURE 6

Bioinformatics analysis of DEPs responsive to heat stress (A) and subsequent recovery (B) in P. ostreatus mycelia compared to the control group through gene ontology (GO) in molecular function (MF).

FIGURE 7

The KEGG pathway enrichment analysis of the DEPs in P. ostreatus mycelium under heat stress (A) and subsequent recovery (B). Top 10 enrichment in KEGG pathway maps of the DEPs. P-value was calculated using Fisher’s exact test.

FIGURE 8

Protein–protein interaction network analysis among the significantly expressed proteins in P. ostreatus mycelium under heat stress (A) and subsequent recovery (B) using String software.

FIGURE 9

Transcriptional expression analysis of representative proteins as revealed by qRT-PCR. The relative mRNA expression levels of matched differentially abundant proteins including mitogen-activated protein kinase HOG1 (A), beta-glucan synthesis-associated protein (B), phenylalanine ammonia-lyase (C), malate synthase (D), methylthioribulose-1-phosphate dehydratase (E), 78 kDa glucose-regulated protein homolog (F), heat shock protein 60 (G), heat shock protein 90 (H), and heat shock protein 104 (I). Gapdh was used as the reference gene. Mean values and standard deviations of three biological replicates are shown. The asterisks indicate the significance of differences between treatments and their corresponding controls (^∗∗P < 0.01, ^∗P < 0.05).