Effects of Refrigerated Storage on Restarted Morphological Development of Dictyophora indusiata Fruiting Bodies

Abstract

Mature Dictyophora indusiata fruiting bodies are brittle and broken easily during storage. Peach-shaped Dictyophora indusiata before maturity respond well to refrigerated maintenance, but some cannot resume their development after storage. This study analyzed the effects of refrigerated storage and transportation on the restarted development of Dictyophora indusiata fruiting bodies using quantitative transcriptome analysis. The refrigerated (4 °C, 3 d) peach-shaped Dictyophora indusiata (CK) was used as the control. After induction treatment for 3 d (26 ± 2 °C, 95 ± 3% RH), 81.25% of CK could achieve restarted development and mature (D-M), whereas 18.75% failed (D-P) to restart. Quantitative transcriptome analysis revealed that 1389 and 4451 differentially expressed genes (DEGs) were identified in the D-P and D-M groups when compared with the results for the CK group, respectively. DEG annotation and functional analysis revealed that D-P did not initiate energy and nutrient metabolism. Most DEGs involving the phosphatidylinositol signaling pathway and the MAPK signaling pathway were significantly downregulated or unchanged in the D-P and significantly upregulated in the D-M groups. These results suggested that the phosphatidylinositol signaling pathway may play a crucial role in transmitting environmental signals and initiating the morphogenesis of CK, and that the downstream MAPK signaling pathway may be responsible for signal transmission, thereby regulating cellular activities. This study provides a theoretical basis for regulating the growth and development of postharvest Dictyophora indusiata fruiting bodies.

1. Introduction

Dictyophora indusiata (D. indusiata) is an edible mushroom with numerous bioactive compounds that enhance immune, anti-tumor, and anti-aging properties [1,2]. D. indusiata fruiting bodies are highly valued for their nutritional and medicinal benefits and are commonly consumed as a nutritious food in Asian countries, particularly in China [3]. D. indusiata undergo four major growth phases: the primordia stage, the ball-shaped stage, the peach-shaped stage, and the mature stage, among which the mature D. indusiata is susceptible to aging and autolysis [4]. Furthermore, the D. indusiata fruiting bodies are prone to being broken during transportation. D. indusiata fruiting bodies, harvested at the peach-shaped stage, are suitable for to transportation due to their small size, regular shape, and resistance to crushing. The peach-shaped D. indusiata can develop and mature within a few hours after harvest [5]. Currently, a substantial number of peach-shaped D. indusiata are harvested and sold. Nevertheless, low-temperature storage can disrupt or prevent the development of peach-shaped D. indusiata, even causing some samples to fail to mature. Therefore, it is crucial to clarify the potential mechanism for inhibiting the development and maturity of peach-shaped D. indusiata after refrigerated storage and transportation.

Stipe elongation, primarily driven by the lateral elongation of stipe cells, is a remarkable feature in developing edible mushroom fruiting bodies [6]. Studies have confirmed that cell wall-degrading enzymes are essential in stipe elongation [7]. For example, the apical stipes wall of Coprinopsis cinerea undergoes continuous and stable cell wall elongation for over 2 h due to chitinases ChiE1 and ChiIII [8]. The combination of endo-β-1,3-glucanase ENG and extracellular β-1,6-glucanase BGL2 induced apical stipe wall elongation in an acid-dependent manner [8,9,10,11]. Therefore, the combined action of hydrolytic and synthetic enzymes on the cell wall components during the postharvest stipe elongation of edible mushroom fruiting bodies promotes cell wall remodeling. Notably, the hydrolytic and synthetic enzymes that regulate cell wall remodeling are the “executors” of stipe elongation, and the upstream signaling processes and key genes are the “controllers” of cell wall remodeling and stipe elongation. In yeast, SLT2 kinase activated the transcription factor RLM1, thereby regulating the expression of downstream cell wall integrity-related genes, such as chitin synthase gene CHS3 and β-1,3-glucan synthase gene FKS1 [12,13]. Furthermore, the regulatory activity of RLM1 is mainly activated by the upstream MAPK pathway [14]. Therefore, cell signals may activate transcription factors through signaling cascades, thereby regulating enzymes related to cell wall remodeling and ultimately leading to the development and maturity of edible mushroom fruiting bodies.

The natural maturation process of D. indusiata fruiting bodies has been systematically studied. By comparing the cell morphology and metabolites of peach-shaped and mature D. indusiata, the previous study found that the D. indusiata cell membrane and cell wall undergo significant hydrolysis to adapt to the rapidly expanding cell structure as the D. indusiata stipe elongates [4]. Moreover, the transcriptomic and proteomic analyses found that the regulatory pathway of the stipe cell wall remodeling of D. indusiata during the morphological development consists of three key components: environmental response and signal transduction, signaling and transcriptional regulation, and signal execution to remodel the cell wall [5,15]. Therefore, we speculated that refrigerated storage and transportation may impact the key signaling pathways of peach-shaped D. indusiata, thereby hindering its maturation. However, there has been no report on the mechanism by which refrigerated storage inhibits the development and maturity of peach-shaped D. indusiata.

In this study, the maturation rate of the refrigerated (4 °C, 3 d) peach-shaped D. indusiata stored for 3 d under controlled temperature and humidity conditions (26 ± 2 °C, 95 ± 3% RH) was determined. Additionally, the regulatory mechanisms prompting the failure to initiate the morphological development of the refrigerated peach-shaped D. indusiata were systematically investigated through quantitative transcriptome analysis. This research provides valuable information for regulating the postharvest morphological development of refrigerated peach-shaped D. indusiata.

2. Materials and Methods

2.1. Materials

Fresh D. indusiata were hand-harvested at the peach-shaped stage from a cultivated field in Kunming (Yunnan, China) and transported immediately, within 24 h, to the laboratory at low temperatures (4–10 °C). Peach-shaped D. indusiata with a length and width of about 3 cm × 5 cm were selected for the experiments. The TRIzol^® reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA). TruseqTM RNA Sample Prep Kits, HiSeq X Reagent Kits, and NovaSeq Reagent Kits were purchased from Illumina Inc. (San Diego, CA, USA). Agencourt AMPure XP was obtained from Beckman Coulter (Brea, CA, USA).

2.2. Sample Preparation

Each group consisted of six biological replicates, and each replicate consisted of eight peach-shaped D. indusiata. The peach-shaped D. indusiata were placed on the water-soaked gauze, sprayed with water every 8 h, and stored in a constant temperature and humidity chamber (BPS-50CL, Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China) for 3 d under controlled temperature and humidity conditions (26 ± 2 °C, 95 ± 3% RH) and photographed daily to document their morphological development. The peach-shaped D. indusiata at day 0 was used as the control group (CK group), and the immature peach-shaped D. indusiata (D-P group) and mature D. indusiata (D-M group) at day 3 after induction treatment were used as the experimental groups.

2.3. Calculation of Maturation Rate

Stipe elongation and skirt opening indicate the normal development and maturation of D. indusiata. The maturation rate on day 3 was used to analyze the development of D. indusiata. After induction treatment, the number of mature D. indusiata was recorded daily, and the maturation rate was calculated using the following formula:

$$\mathrm{M}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\mathrm{\%})=\frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{m}\mathrm{a}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e} Dictyophora indusiata}{\mathrm{T}\mathrm{h}\mathrm{e} \mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} Dictyophora indusiata}\times 100$$

2.4. RNA Extraction

Six samples were randomly collected from the CK, D-P, and D-M groups, respectively. The samples were washed and dried, and their stipe and umbrella parts were collected. Samples were ground into powder in liquid nitrogen using a cryo-mill (Wonbio-96, Shanghai Wanbo Biotechnology Co., Ltd., Shanghai, China), with three replicates per group. Total RNA was extracted using TRI-zol reagent, following the manufacturer’s instructions [16]. The RNA concentration and purity, the RNA integrity, and the RIN value were assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), agarose gel electrophoresis, and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively.

2.5. RNA-Seq Library Construction, Assembly, and Sequencing

The TruseqTM RNA sample prep Kit was used for library construction. The requirements for single library construction were a total RNA quantity ≥ 1 μg, an RNA concentration ≥ 35 ng/μL, OD260/280 ≥ 1.8, and OD260/230 ≥ 1.0. The mRNA was isolated from the total RNA by the A-T base pairing of polyadenosinic acid (Poly A) and magnetic beads with oligo (dT). The mRNA is randomly fractured into small fragments of approximately 300 bp using a fragmentation buffer, and random hexamers are added to synthesize cDNA from mRNA under reverse transcriptase. The sequencing was performed on the Illumina NovaSeq 6000 (Illumina Inc., San Diego, CA, USA) platform [17], producing 150 bp pair-end reads. The raw data was filtered using fastp software (Version 0.19.5, https://github.com/OpenGene/fastp) to remove low-quality reads and obtain clean data. The clean data was assembled using Trinity software (Version v2.8.5, https://github.com/trinityrnaseq/trinityrnaseq/wiki). The assembly results were evaluated using TransRate software (Version v1.0.3, http://hibberdlab.com/transrate/) and BUSCO software (Version 3.0.2, https://busco.ezlab.org/) to assess quality.

2.6. Functional Annotation

The unigenes obtained from transcriptome sequencing were compared using six databases to obtain functional annotated information, including NCBI_NR (NCBI Non-Redundant Protein Repository, http://ftp.ncbi.nlm.nih.gov/blast/db/), Swiss-Prot (http://web.expasy.org/docs/swiss-prot_guideline.html), Pfam (http://pfam.xfam.org/), COG (Clusters of Orthologous Groups of proteins, http://www.ncbi.nlm.nih.gov/COG/), GO (Gene Ontology, http://www.geneontology.org/), and KEGG (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/). The overall comparation of transcriptomic data among CK, D-P, and D-M groups were analyzed on the free online Majorbio Cloud Platform (http://www.majorbio.com), and the above-mentioned software and databases were accessed on 10 December 2021.

2.7. Statistical Analysis and Bioinformatics Analysis

The statistical analyses were performed using ANOVA and Duncan’s multiple range tests, employing 21.0 SPSS software (IBM Corp., Armonk, NY, USA), and a value of p < 0.05 was regarded as statistically significant. DEGs were screened according to the |log2FC| ≥ 1 and p-adjust < 0.05. The KEGG database was used for pathway enrichment analyses. The KEGG pathway was considered significantly enriched using Fisher’s exact test, with a corrected p value < 0.1.

3. Results and Discussion

3.1. Morphological Development and Maturation Rate of Refrigerated Peach-Shaped D. indusiata

To visually analyze the development of refrigerated peach-shaped D. indusiata, this study conducted induction treatment (26 ± 2 °C, 95 ± 3% RH) for 3 d on peach-shaped D. indusiata (CK group) that had undergone refrigerated storage and transportation at 4–10 °C for 3 d. The maturation rate of peach-shaped D. indusiata increased significantly (p < 0.05) with the extension of induction time (Figure 1A,B). After 3 d of induction treatment, 81.25% of the peach-shaped D. indusiata developed into mature D. indusiata (D-M group), while 18.75% remained immature and peach-shaped, labeled the immature peach-shaped D. indusiata (D-P group) (Figure 1C). As the induction time increased, the quality of peach-shaped D. indusiata decreased. Thus, the D-P group could not complete morphological development.

3.2. Annotation of D. indusiata Unigenes

To explore the mechanism by which refrigerated storage and transportation inhibited the restart of morphological development of peach-shaped D. indusiata, quantitative transcriptome analysis was performed on three groups (CK, D-P, and D-M) of D. indusiata fruiting bodies. On average, each sample obtained 7430791666 bp of effective bases, with a GC percentage ranging from 47.02% to 47.48%. The Q20 percentage was over 97%, and the base error rate averaged less than 0.027%, indicating that the sequencing results were reliable [18]. After removing the redundant sequences, 13,078 individual genes (unigenes) were obtained, and most unigene lengths were between 200 and 2000 bp.

The obtained unigene sequences were compared using BLAST (Basic Local Alignment Search Tool, Version 2.9.0), and 10,254 unigenes were annotated, accounting for 78.41% of the total unigenes. Among them, the NR database had the highest number of annotated unigenes (9948), accounting for 76.07% of the total unigenes, followed by the GO database (8791, 67.22%), the Pfam database (8084, 61.81%), the Swiss-Prot database (8068, 61.69%), and the KEGG database (6398, 48.92%); the COG database exhibited the lowest number of annotated unigenes (3011, 23.02%).

3.3. DEGs Analysis

Compared with the CK group, the D-P group had 563 upregulated and 826 downregulated DEGs, whereas the D-M group had 3185 upregulated and 1266 downregulated DEGs (Figure 2A), suggesting that the development of the refrigerated peach-shaped D. indusiata into the mature D. indusiata involved substantial DEGs. Notably, there were 3097 upregulated and 1090 downregulated DEGs in the D-M group compared with the D-P group, and these DEGs may be involved in regulating the development and maturation of refrigerated peach-shaped D. indusiata. Additionally, there were 3394 identical DEGs between the CK vs. the D-M group and the D-P vs. the D-M group (Figure 2B), and these were mainly associated with variations in the morphology of the D. indusiata fruiting bodies, specifically in regards to the differences between the peach-shaped stages and the mature stages. Only 706 identical DEGs were found between the CK vs. the D-P group and the CK vs. the D-M group (Figure 2C), with the differences mainly related to induction treatments. The results suggested that the morphological development of the D. indusiata fruiting bodies had a more significant impact on gene expression than did the induction treatments.

3.4. KEGG Functional Enrichment Analysis of DEGs

A KEGG enrichment analysis was performed based on DEGs to elucidate the effect of refrigerated storage and transportation on the failure of peach-shaped D. indusiata to resume development and maturation.

The upregulated DEGs in the CK vs. D-P comparison group were attributed to significantly enriched steroid biosynthesis, hippo signaling pathway, and homologous recombination; the upregulated DEGs in the CK vs. D-M comparison group were attributed to significantly enriched ribosomes; the upregulated DEGs in the D-P vs. D-M comparison group were significantly enriched in regards to the citrate cycle (TCA cycle) and ribosomes. In comparison to the CK group, the downregulated DEGs in the D-P group were not attributed to the enriched pathway; in contrast, the downregulated DEGs in the D-M group were attributed to 13 significantly enriched pathways related to substance catabolism (such as mismatch repair, sesquiterpenoid and triterpenoid biosynthesis, tryptophan metabolism, linoleic acid metabolism, autophagy-yeast, peroxisome, and starch and sucrose metabolism). Compared to the D-P group, the downregulated DEGs in the D-M group were attributed to 15 significantly enriched pathways related to metabolism (such as meiosis-yeast, ascorbate and aldarate metabolism, phenylalanine metabolism, homologous recombination and fatty acid degradation). These results indicated that the failure of D-P to resume morphological development may be attributed to the impact of refrigerated storage and transportation on the material and energy metabolism of peach-shaped D. indusiata.

3.5. The Phosphatidylinositol Signaling System May Serve as the Signaling Switch to Prevent the Maturation of Refrigerated Peach-Shaped D. indusiata

The phosphatidylinositol signaling system can regulate cell proliferation, differentiation, apoptosis, and membrane transport [19]. The phosphatidylinositol signaling system is activated by phosphatidic acid (PA), which is produced and accumulated through the specific hydrolysis of phosphatidylethanolamine (PE) by phospholipase D (PLD). The accumulated PA is catalyzed by phosphatidate cytidylyltransferase (EC: 2.7.7.41) and CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT, EC: 2.7.8.11) to produce phosphatidylinositol (PI). PI is then further catalyzed by phosphatidylinositol 4-kinase B (PI4K) and 1-phosphatidylinositol-4-phosphate 5-kinase (PIP5K) to obtain phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), respectively. Ultimately, PI and its series of products can be hydrolyzed by phosphatidylinositol phospholipase C (PLC) to generate the signaling molecules diacylglycerol (DG) and inositol 1,4,5-trisphosphates (I(1,4,5)P3), which activate protein kinase C (PKC), thereby inducing the mitogen-activated protein kinase (MAPK) cascade reaction [20,21].

This study identified 6 DEGs involved in the phosphatidylinositol signaling system, including PLD, CDIPT, myotubularin-related protein 6/7/8 (MTMR6/7/8, EC: 3.1.3.64, EC: 3.1.3.95), phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), PI4K, and PLC (Figure 3). In the D-P group, PTEN and MTMR6/7/8 were upregulated, while PLD and PI4K were downregulated compared to the results for the CK group. It can be inferred that the downregulated PLD in the D-P group inhibited the hydrolysis activity of PE, leading to a reduction in PA production. Additionally, the downregulated PI4K reduced the conversion of PI to PI(4)P. Therefore, in the D-P group, the accumulation level of PA was insufficient to activate the downstream kinases in the phosphatidylinositol signaling system and activate signaling molecules such as DG and I(1,4,5)P3 due to the downregulated PLD. Notably, compared with CK group, PLD, CDIPT, PI4K, PLC, and MTMR6/7/8 were significantly upregulated in the D-M group. Among them, the upregulation of PLD promoted PE hydrolysis and increased PA accumulation levels, and the upregulation of CDIPT accelerated PI generation. The upregulation of PI4K and PLC was related to the accelerated production rate of final products (DG and I(1,4,5)P3). These results suggested that key enzymes in the phosphatidylinositol signaling system of the D-M group were activated, resulting in the accelerated production of signal molecule DG. The changing trend of DEGs in the D-P vs. D-M comparison group was similar to that in the CK vs. D-M comparison group, except for the MTMR6/7/8 upregulation and PLD and PI4K downregulation in the former. Additionally, studies have shown that PTEN can dephosphorylate phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) to phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) and hydrolyze PI to phosphatidylinositol 3-phosphate (PI(3)P) [22,23]. Compared to the results for the CK group, PTEN was upregulated in the D-P group and downregulated in D-M group, suggesting that PTEN expression might exert an inverse regulatory influence on the activation of the phosphatidylinositol signaling system.

In contrast to the results for the D-M group, most of the key kinases in the phosphatidylinositol signaling system were downregulated or unchanged in the D-P group, suggesting that in the D-P group, the phosphatidylinositol signaling system was not activated in response to environmental stimuli, thereby achieving environmental signal transduction. Therefore, it was likely that the phosphatidylinositol signaling system acts as a signal switch that inhibits the maturation of peach-shaped D. indusiate after refrigerated storage and transportation.

3.6. MAPK Signaling Pathway Mediates Signaling of the Maturation of Refrigerated Peach-Shaped D. indusiata

The MAPK signaling pathway is a crucial cascade pathway in eukaryotic cells that converts external stimuli into cellular responses, regulating cell growth, proliferation, differentiation, and apoptosis [24]. The cell wall integrity (CWI) MAPK sub-pathway, a downstream signaling cascade of the phosphatidylinositol signaling system, is vital for maintaining cellular homeostasis [25]. In this sub-pathway, specific kinases, including Ras homolog gene family member A (Rho1), RHO1 GDP-GTP exchange protein 1/2 (Rom1,2), and classical protein kinase C alpha type (Pkc1), can be activated by PI in the phosphatidylinositol signaling system [5,26]. Furthermore, Pkc1 can also be activated by PLC products, such as DG and I(1,4,5)P3.

The CWI MAPK sub-pathway included nine DEGs, including cell wall integrity and stress response components (Wsc1,2,3), Rom1,2, Rho1, Pkc1, GTPase-activating protein SAC7 (Sac7), tyrosine-protein phosphatase 2/3 (Ptp2,3), cyclin-dependent kinase (Cdc28), G2/mitotic-specific cyclin 2 (Clb1/2), and 1,3-beta-glucan synthase (Fks2) (Figure 4). Compared with the results for the CK group, Cdc28 was upregulated, and Sac7 was downregulated in the D-P group, whereas the D-M group expressed eight DEGs (WSC, Rom1,2, Rho1, Pkc1, Sac7, Ptp2,3, Cdc28, Clb2, Fks2) upregulated and one DEG (Cdc28) downregulated. Rho1, a regulatory subunit of β-1,3-glucan synthase, activates the MAPK cascade through binding and activates PKC1. However, Sac7, a GTPase-activating protein for Rho1, inhibited Rho1 overexpression and preferentially antagonized the Rho1-Pkc1 stress response pathway, without affecting glucan synthesis [27,28]. The downregulated expression of Sac7 in the D-P group indicated that the Rho1-Pkc1 stress response was not activated or did not reach the level of negative feedback regulation. Ptp2,3 is a key negative regulator in the CWI MAPK sub-pathway that binds to Mpk1 (Slt2) and dephosphorylates Mpk1, preventing Mpk1 overexpression and leading to growth defects [24,29]. Although Slt2 was not identified at the transcriptional level in this study, the high expression of Ptp2,3 in the D-M group may indicate the activation of Slt2, which is the binding substrate of Ptp2,3. Therefore, the kinase cascade in the CWI MAPK sub-pathway of the D-M group was upregulated and activated.

As in the CK group, the CWI MAPK sub-pathway was not activated in the D-P group that failed to resume morphological development after induction treatment. However, in the D-M group that resumed morphological development after induction treatment, the CWI MAPK sub-pathway was activated, and its cascade reaction completed signal amplification and conduction. Notably, the upregulated Fks2 will respond to signaling and co-regulate the expression of β-1,3-glucan synthase with the Rho-type GTPases at the transcriptional level, which in turn enhances the synthesis of the cell wall components (β-1,3-glucan) [30,31]. Therefore, refrigerated storage and transportation interrupted the activation of the CWI MAPK sub-pathway of peach-shaped D. indusiata, thereby affecting the synthesis of the cell wall components.

3.7. Cell Wall Remodeling Is Crucial for Inhibiting Stipe Elongation and the Morphogenesis of Refrigerated Peach-Shaped D. indusiata

Chitin and glucan are the primary cell wall components of most edible fungi [32,33]. This study identified seven chitin-degrading enzymes, nine chitin synthases, nine β-l,3-glucan-degrading enzymes, and two β-l,3-glucan synthases (Figure 5).

Compared to the levels for the D-P and CK groups, the expression levels of most chitinases were lower in the D-M group. Among them, the expression level of chitin-degrading enzyme TRINITY_DN2410_c0_g1 remained no significant difference between the D-P and CK groups (p > 0.05), while its gene expression was significantly lower in the D-M group (p < 0.05), i.e., 78% and 75%, compared with the levels in the D-P and CK groups, respectively. Furthermore, the expression of endochitinase B1 (TRINITY_DN3122_c0_g1) in the D-M group was 5.43-fold and 2.28-fold higher than that in the D-P and CK groups, respectively, which suggested that chitin hydrolysis was inhibited in immature peach-shaped D. indusiata, but remained active after D. indusiata completed stipe elongation. For chitin synthase, compared with the CK group, the expression of TRINITY_DN244_c0_g1 significantly decreased in the D-P group and significantly increased in the D-M group (p < 0.05); the expression of TRINITY_DN7438_c0_g6, TRINITY_DN4936_c0_g1, and TRINITY_DN569_c0_g1 significantly increased in the D-P group (p < 0.05) and decreased in the D-M group. These results indicated that chitin synthesis significantly differed in D-P and D-M groups. However, the gene expression of most of the chitin synthases in the D-M group was lower than that in the D-P and CK groups, indicating that chitin synthesis decreased during the mature stage. Interestingly, compared to CK group, the expression of most genes of the β-1,3-glucan-degrading enzymes and synthases was significantly decreased in the experimental group (p < 0.05), especially in the D-M group. These results suggested that the degradation and synthesis of β-1,3-glucan continued, despite inhibiting the stipe extension process.

In short, the degree of degradation and synthesis of chitin and β-1,3-glucanase decreased in the D-P and D-M groups, especially in the latter. Zhou et al. [8] observed that the double knockdown of two chitinases (ChiE1 and ChiIII) resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall elongation in the native stipes, suggesting that chitinases played a critical role in extending the stipe cell walls of Coprinopsis cinerea. These results suggested that extensive degradation and synthesis of stipe cell wall components during stipe elongation was critical for the morphological remodeling in D. indusiata, thus explaining why peach-shaped D. indusiata in the D-P group failed to resume morphological development.

3.8. Restricted Energy Metabolism Inhibits the Energy Supply for the Maturation of Refrigerated Peach-Shaped D. indusiata

During the morphological development process of refrigerated peach-shaped D. indusiata after induction treatment, in addition to signal transduction in response to environmental stimuli and signaling to initiate gene regulation, the execution process (such as stipe cell wall expansion, stipe elongation, and morphological development) requires a large energy supply. Therefore, the relevant DEGs of the main energy metabolism pathways were identified and sorted.

Hexokinase (HK), phosphofructokinase (PFK), phosphoglycerate kinase (PGK), and pyruvate kinase (PK) are key enzymes in the glycolysis pathway [34]. HK and PFK are involved in the preparatory phase of glycolysis (consuming two ATP), while PGK and PK are involved in the release phase (generating ten ATP) [35]. Among these, HK and PFK are involved in the preparatory phase of glycolysis (consuming two ATP molecules), while PGK and PK are involved in the subsequent release phase of glycolysis (generating ten ATP molecules). In comparison to the CK group, four unigenes of HK and three unigenes of PGK were not significantly different in the D-P group, but they were significantly upregulated in the D-M group; for two unigenes of PK, one was insignificantly and one significantly downregulated in the D-P group, while one was significantly upregulated and one was significantly downregulated in D-M group (Figure 6A). This might indicate that unlike in the D-P group, the glycolysis pathway was activated in mature D. indusiata, resulting in the generation of substantial energy for execution process. The tricarboxylic acid cycle represents a crucial pathway of aerobic respiration, which provides the energy necessary for life activities by completely oxidizing and decomposing the substrates. Pyruvate dehydrogenase E1 component (PDHA, PDHB), citrate synthase (CS), and isocitrate dehydrogenase (IDH1, IDH3) are key enzymes in the tricarboxylic acid cycle [36]. Compared to the CK groups, most of the unigenes identified as the above-mentioned rate-limiting enzymes showed no significant difference in D-P group, whereas all of them were significantly upregulated in the D-M group, indicating that the latter exhibited vigorous aerobic respiration (Figure 6A). Succinate dehydrogenase (SDH) is recognized as a pivotal hub in the tricarboxylic acid cycle and respiratory chain [37]. Seven SDH unigenes were all significantly downregulated in D-P group and significantly upregulated in D-M group, which indicated that respiration was restricted in the D-P group.

The respiratory chain consists of five protein complexes, designated as complexes I to V, which have catalytic functions and are integral to mitochondrial energy metabolism [38,39]. The 112 DEGs were annotated in the five protein complexes. Of these, 28, 7, 17, 19, and 41 DEGs were identified in complexes I, II, III, VI, and V, respectively (Figure 6B). Most of the respiratory chain-related DEGs were not significantly different in the D-P group and were significantly upregulated in the D-M group compared to the CK group, suggesting that refrigerated storage inhibited the energy metabolism of peach-shaped D. indusiata, causing their failure to resume morphological development, even after the induction treatment. Consequently, impaired energy metabolism was a crucial factor in preventing the restart of morphogenesis in refrigerated peach-shaped D. indusiata.

4. Conclusions

This study analyzed the morphological development of refrigerated peach-shaped D. indusiata after induction treatment and explored the mechanism of failure to restart morphological development. The results found that 18.75% of the refrigerated peach-shaped D. indusiata failed to respond to environmental stimuli and initiate signaling (lacked activation of the phosphatidylinositol signaling system and the CWI MAPK sub-pathway), and thus failed to initiate cell wall remodeling. Meanwhile, respiration intensity (glycolysis, tricarboxylic acid cycle, and respiratory chain) could not meet the energy requirements for stipe elongation and morphological development due to the inhibitory effect of low-temperature refrigeration on respiration. Conversely, 81.25% of the refrigerated peach-shaped D. indusiata achieved morphological development by activating the phosphatidylinositol signaling system and CWI MAPK sub-pathway after induction treatment. Consequently, the maturation of peach-shaped D. indusiata can be inhibited by blocking the accumulation of signaling molecules through phospholipase inhibitors or by blocking the signaling cascade through phosphorylation inhibitors. This study offers crucial insights into the mechanisms by which refrigerated storage impedes the morphological development of peach-shaped D. indusiata and provides a research direction for developing postharvest preservation technology for D. indusiata.