The Molecular Mechanism of Mycelial Incubation Time Effects on Primordium Formation of Pleurotus tuoliensis Through Transcriptome and Lipidomic Analyses

Abstract

Pleurotus tuoliensis is a precious edible mushroom with a long cultivation cycle. Despite being cultivated in China for nearly 30 years, research on the molecular mechanisms underlying its primordium formation remains limited. In this study, the molecular mechanisms by which incubation time affects the primordium formation of P. tuoliensis were investigated using RNA-seq technology and lipid content detection. Our research revealed that the transcription of genes involved in lipid metabolism and lipid levels changed significantly during different incubation periods. Distinct differences were observed in gene transcription associated with signaling pathways, sphingolipid metabolism, fatty acid metabolism, and steroid biosynthesis in mycelia cultured for varying days and then stimulated by low temperature and light. These findings indicate that lipid accumulation and alterations in mycelial cell membrane components during incubation may affect the mycelial response to environmental signals, subsequently regulating primordium formation. This study revealed the crucial role of lipid metabolism during incubation in the primordium formation of P. tuoliensis, providing a novel perspective for investigating the molecular mechanism underlying fruiting body development.

1. Introduction

Pleurotus tuoliensis, which used to be identified as Pleurotus nebrodensis, is a species of precious edible mushroom with a long cultivation cycle. The fruiting body is rich in lysine, arginine, vitamin D, mineral elements, and trace elements, which have high nutritional value [1]. The fruiting bodies contain numerous polysaccharides with health benefits, playing roles in protecting cardiovascular health, regulating immune activity, regulating blood lipids, and protecting the liver [2,3,4,5]. In view of their high economic value, many scholars have studied the breeding, cultivation technology, physiology, postharvest storage, and deep processing of P. tuoliensis [6,7,8,9].

Pleurotus tuoliensis is a medium-to-low-temperature fructification edible mushroom, and its optimum temperature for mycelial growth is generally 25 °C. Light and low-temperature are necessary to form primordia, and many studies have been conducted on the influence of environmental factors on primordium formation, such as blue light being favorable for the induction of primordium formation [10]. After 10 days of induction at 14 °C and 300–1000 lx with blue light, mycelia kink and finally form primordia [11]. Blue light affects the growth of primordia and fruiting bodies through glycolysis and pentose phosphate pathways [12]. In addition to environmental factors, the physiological state of mycelia can also affect the yield and quality of P. tuoliensis. The reproductive growth of edible mushrooms requires the consumption of many nutrients [13]. After spawn running is completed, mycelia can fully absorb and store nutrients from the substrate. Under appropriate environmental stimulation, mycelia undergo kinking and eventually form primordia. After 37 days of spawning in the dark, P. tuoliensis mycelia form primordia after 7 days of stimulation with low temperature at 17 °C and light [14].

In recent years, transcriptomics has been widely used to research the analysis of key genes and pathways involved in growth and development stages, pigment formation [15], and autolysis [16]. Transcriptome analysis of the mycelia and primordia of Pleurotus ostreatus revealed that 69 genes, which are involved mainly in energy metabolism, signal transduction, membrane proteins, and other pathways, were upregulated in the primordium stage [17]. Members of the Zn₂Cys₆-type zinc finger transcription factor family in P. ostreatus were identified via transcriptome analysis, and 13 candidate genes that play a role in fruiting body formation and the heat stress response were identified [18]. Transcriptome analysis searched for Lentinula edodes fruiting body-specific genes, such as aspartic protease, gamma-glutamyl transpeptidase, and cyclohexanone monooxygenase, which are involved in fruiting body maturation and the isolation of functional substances [19]. Transcriptome analyses revealed that blue light promoted the growth of L. edodes primordia and fruiting bodies, and DDR-48 heat shock protein genes involved in primordia morphogenesis were screened [20]. The maturity and development of transcriptomic technology further promoted the basic research of edible mushrooms.

Lipidomics has been applied in the research on edible mushrooms recently and can reveal complete molecular information about lipids in biological samples [21]. A lipidomics analysis of eight wild edible mushrooms identified lysophosphatidylethanolamine (16:1) and ceramide non-hydroxy fatty acid-dihydrosphingosine (d23:0–10:0) as potential biomarkers to distinguish different mushroom species [22]. Untargeted lipidomics analysis combined with chemometrics was applied to distinguish the edible mushrooms from poisonous mushrooms [23]. Lipidomic profiles and lipid dynamic changes during the growth of Morchella sextelata were analyzed, which revealed that glycerophospholipid metabolism was the major pathway involved in the growth of M. sextelata [24].

In this study, the molecular mechanisms underlying primordium formation in P. tuoliensis were investigated by assessing the effects of various mycelial incubation periods, sequencing the transcriptomes at different developmental stages, screening key genes and validating via RT-qPCR, and detecting the presence of related substances by lipidomics analysis. The present study further contributes to understanding the mechanism of primordium formation in P. tuoliensis.

2. Materials and Methods

2.1. Sample Collection

The P. tuoliensis CCMSSC 02607 strain was obtained from the Center for Mushroom Spawn Standards and Control of China (CCMSSC). The CCMSSC02607 strain was punched with a hole punch (φ7 mm), inoculated in PDA medium (15.6 g of potato dextrose agar powder dissolved in 400 mL of distilled water, sterilized at 121 °C for 20 min), and cultured at 25 °C in the dark for 4 days (T1) and 6 days (T3), respectively. Then, two different treatment groups were cultured at 15 °C with alternate light (12 h light, 12 h dark) for 13 days (T2 and T4) (

Table 1. Samples and treatment conditions.

Sample	Culture Condition
T1	Culture at 25 °C for 4 days in the dark
T2	Cultured at 25 °C for 4 days in the dark, then cultured at 15 °C with alternate light for 13 days
T3	Culture at 25 °C for 6 days in the dark
T4	Cultured at 25 °C for 6 days in the dark, then cultured at 15 °C with alternate light for 13 days

). Mycelia and primordia were collected and stored at −80 °C after being snap-frozen in liquid nitrogen for subsequent experiments.

2.2. Library Preparation and RNA-Seq

Four treatments were set up with three replicates in each treatment, with a total of 12 samples in transcriptome sequencing. Transcriptome sequencing was carried out at Beijing Biomarker Technologies Co., Ltd. Total RNA was extracted from the mycelia and primordia of P. tuoliensis. Purity, concentration, and integrity of the RNA samples were tested to guarantee the use of qualified samples for transcriptome sequencing. mRNAs were enriched with magnetic beads with oligo (dT), which were randomly disrupted with fragmentation buffer. The first cDNA strand was then synthesized with six-base random hexamers, and the second cDNA strand was synthesized by adding buffer, dNTPs, RNase H, and DNA polymerase I. The resulting cDNA fragments were purified using AMPure XP beads and then subjected to end repair through the addition of a single “A” base and the ligation of Illumina multiplex barcode adapters, followed by the separation of the fragments via gel purification using AMPure XP beads. Finally, the cDNA libraries were constructed via PCR to create a total of 12 libraries.

After library construction, the effective library concentration (library effective concentration > 2 nmol/L) was accurately quantified via q-PCR to ensure library quality. After library inspection, the different libraries were pooled according to the target downstream data volume and sequenced via the Illumina platform.

Before performing data analysis, we ensured that reads were of high enough quality to ensure the accuracy of subsequent analyses. Reads containing joints and filtered low-quality reads were removed to obtain high-quality clean data. HISAT2 (v 2.0.4) software was used to obtain the RNA sequencing experimental reads for an efficient comparison system. The reads on the pair were assembled via StringTie to construct more complete transcripts and better assess expression.

2.3. Identification of Differentially Expressed Genes (DEGs)

To identify DEGs, the expression levels of transcripts were calculated via the fragments per kilobase of transcript per million fragments mapped (FPKM) method [25]. The fold change indicates the ratio of expression between two samples (groups). The false discovery rate (FDR) was obtained by correcting for the differential significance of the p-value. The differential expression gene was selected according to DESeq2_edgeR, and fold change ≥ 2.0 and an FDR < 0.01 were used as screening criteria.

The functional annotation of the DEGs was performed via the non-redundant protein sequence database (NR) (https://www.ncbi.nlm.nih.gov/ (accessed on 25 December 2020)), gene ontology (GO) (https://www.geneontology.org/ (accessed on 25 December 2020)), cluster of orthologous groups (COG) (https://www.ncbi.nlm.nih.gov/research/cog-project/ (accessed on 25 December 2020)), eukaryotic orthologous groups (KOG) (https://ftp.ncbi.nlm.nih.gov/pub/COG/KOG/ (accessed on 25 December 2020)), the protein family database (Pfam) (http://pfam.xfam.org/ (accessed on 25 December 2020)), Kyoto encyclopedia of genes and genomes (KEGG) (https://www.kegg.jp/kegg/kegg1.html (accessed on 25 December 2020)), Swiss-Prot (https://www.expasy.org/resources/uniprotkb-swiss-prot (accessed on 25 December 2020)), and other databases to obtain annotation information for the genes.

2.4. Reverse Transcription-Quantitative PCR (RT-qPCR) Analysis

Twenty-five DEGs identified by RNA-seq were selected for RT-qPCR validation. RNA extraction was performed using the E.Z.N.A^® Plant RNA Kit from Omega Bio-Tek, Inc., Norcross, GA, USA. The RNA concentrations were measured via an Agilent 2100 Bioanalyzer. The RNA was used as a template to obtain cDNA using the HiScript^® II 1st Strand cDNA Synthesis Kit from Nanjing Vazyme Biotech Co., Ltd., Nanjing, China. The qPCR primers for the target genes were designed via the online website IDT (https://sg.idtdna.com/Scitools/Applications/RealTimePCR/Default.aspx (accessed on 13 April 2024)), as shown in Table S1. The expression of the target gene was examined via RT-qPCR. RT-qPCR amplification was performed as follows: 95 °C for 30 s, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s, followed by a 72 °C extension for 30 s. The relative gene expression was calculated via the 2^−ΔΔCT method, with β-actin used as an internal reference gene [26].

2.5. Lipid Metabolomic Analysis

To analyze lipid differences after different mycelial incubation periods, mycelia were collected after culture in the dark from 4 d to 6 d. For lipid analysis, the lipid extracts from three biological replicates were separated via an Orbitrap Exploris 120 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at Biomarker Technologies Co., Ltd., Beijing, China. Finally, based on the mass spectrometry analysis results, a comparison of the mycelium lipid composition and quantification was conducted using ultra-high-performance liquid chromatography quadruple time-of-flight mass spectrometry (UHPLC-QTOF/MS) [27].

2.6. Statistical Analysis

In this study, each experiment was performed three times. Statistical analyses were performed using SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA) with the data generated above. Multiple comparisons were conducted via one-way analysis of variance (ANOVA). When the p-value was < 0.05, results were considered statistically significant. Figures were plotted with GraphPad Prism version 8 (GraphPad Software Inc., San Diego, CA, USA).

3. Results

3.1. Incubation Time Affects Primordium Formation

After 4 days of mycelial incubation at 25 °C in the dark (T1) and then stimulation by light and cold subsequently for 13 days (T2), the mycelia kinked but failed to form primordia. However, when the mycelia were incubated for a duration of 6 days (T3) and subjected to the same stimulation for 13 days (T4), they were able to form primordia after hyphal knot formation. The results indicated that incubation time is one of the important factors affecting the formation of primordia in P. tuoliensis (Figure 1).

3.2. Transcription of Genes Involved in Lipid Metabolism Significantly Changed During the Incubation Period

To investigate the mechanism by which primordium formation is affected by various incubation periods (T1 vs. T3), genes whose expressions changed during incubation were screened (Table S2). There were 242 DEGs affected by incubation time, of which 188 were upregulated, and 54 were downregulated (Figure S1). KEGG analyses showed that the upregulated DEGs were significantly enriched in glycosphingolipid biosynthesis-globo series, galactose metabolism, sphingolipid metabolism, glycerolipid metabolism, pentose and glucuronate interconversion, and starch and sucrose metabolism (Figure 2a), whereas the downregulated DEGs were significantly enriched in protein processing in the endoplasmic reticulum, fatty acid biosynthesis, and fatty acid metabolism (Figure 2b). These results suggest that the expression patterns of genes involved in lipid metabolism significantly changed during the incubation period when the mycelia grew at 25 °C in the dark.

GO analyses showed that the upregulated DEGs were significantly enriched in the biological processes of carbohydrate metabolism, phospholipid biosynthesis, proteolysis, and thiazole biosynthesis and the molecular functions of raffinose alpha-galactosidase activity and hydrolase activity (Figure 3a), whereas downregulated DEGs were related mainly to the biological processes of oxidation-reduction, response to stress, oxalate metabolism, cellular metabolism, organic substance metabolism, and primary metabolism; the molecular functions of flavin adenine dinucleotide binding, oxidoreductase, D-arabinono-1,4-lactone oxidase, enoyl-[acyl-carrier-protein] reductase, L-gulonolactone oxidase, aspartic-type endopeptidase, prenyltransferase, nutrient reservoir, holo-[acyl-carrier-protein] synthase, oxidoreductase, and protein domain-specific binding; and the cellular components of the fatty acid synthase complex and membrane (Figure 3b). The results were similar to those from the KEGG analyses.

Heatmap analyses demonstrated that among these 13 DEGs enriched significantly in KEGG terms, only one gene coding for fatty acid synthase was significantly downregulated, whereas the other 12 DEGs were significantly upregulated (Figure 4a). A total of 58 DEGs were enriched significantly in GO terms, of which the significantly downregulated genes encoded fatty acid synthase, NADP-binding protein, and NADPH2 dehydrogenase, whereas 20 significantly upregulated genes encoded galactan endo-β-1,3-galactanase, mannan endo-1,4-β-mannosidase, UPF0271 protein, alcohol dehydrogenase, propanol-preferring, thiamine thiazole synthase, D-xylose reductase, L-glyceraldehyde reductase, bacterial leucyl aminopeptidase, carboxypeptidase D, 3-oxoacyl-[acyl-carrier protein] reductase, β-mannosidase, β-D-xylosidase 4, D-xylulose reductase, Gly-Xaa carboxypeptidase, phosphatidylserine decarboxylase, and α-galactosidase (Figure 4b). Some of the key DEGs selected via KEGG analysis were the same as those selected via GO analysis.

3.3. The Lipid Components Changed Significantly During the Incubation Period

Because transcription analysis revealed that lipid metabolism was most active during the mycelial culture period, the changes in the relevant lipid components during the incubation period were further analyzed. The results showed that the triglycerides (Figure 5a), unsaturated free fatty acids (Figure 5b), sphingosine (Figure 5i), and some types of phospholipids, including phosphatidic acid (Figure 5d), phosphatidylglycerol (Figure 5e), phosphatidylinositol (Figure 5f), and phosphatidylethanolamine (Figure 5h), significantly decreased in T3, as compared to T1. However, the saturated fatty acids and phosphatidylcholine (Figure 5g) significantly increased during the incubation period. The proportion of unsaturated free fatty acids was much greater than that of saturated fatty acids (Figure 5c). Phosphatidylglycerol and phosphatidylcholine were the main components of the phospholipids involved in mycelial growth in the dark.

3.4. The Effect of Environmental Stimulation on Metabolic Pathways in Mycelia of Various Incubation Days

Free fatty acids and sphingosine are precursors to the synthesis of sphingolipids, which are essential structural substances of cell membranes and signaling molecules for cell fate determination. Transcription data showed that the process of the response to stress is significantly downregulated during mycelial incubation in the dark. Therefore, we compared the metabolic pathway differences after mycelia cultured for various incubation days were stimulated by light and low temperature. A total of 2660 DEGs changed in the mycelia cultured for 4 days in the dark followed by stimulation (T1 vs. T2); however, in mycelia cultured for 6 days followed by environmental stimulation, the transcriptions of 2143 genes were changed (T3 vs. T4) (Figure S2). GO classification showed that the mycelia cultured for different incubation periods presented marked differences in the transcript levels of genes involved in location, cellular component organization, membranes, transporter activity, and nucleic acid binding transcription factor activity (Figure 6a,d). KEGG enrichment analyses indicated that mycelial culture for 4 d followed by environmental stimulation induced the upregulation of 1264 DEGs that were significantly enriched in the nitrogen, starch, sucrose, and glycerolipid metabolic pathways and the monobactam-, steroid-, sesquiterpenoid-, triterpenoid-, and longevity-regulating pathways (Figure 6b), whereas the 1396 DEGs with decreased expressions were related mainly to ribosomes; aminoacyl-tRNA biosynthesis; the degradation of valine, leucine, and isoleucine; tryptophan metabolism; etc. (Figure 6c). A total of 870 DEGs, whose expressions were increased by environmental stimulation in mycelia cultured for 6 days in the dark, were significantly enriched in tyrosine metabolism, amino sugar and nucleotide sugar metabolism, glycerophospholipid metabolism, and phenylalanine and tryptophan metabolism (Figure 6e), whereas a total of 1273 DEGs whose expressions were downregulated were involved mainly in the metabolism of glycine, serine, threonine, cysteine, methionine, and histidine; the biosynthesis of valine, leucine, and isoleucine; and the metabolism of fructose and mannose (Figure 6f). These results demonstrated that changes in the number and transcriptional level of DEGs during incubation affected the mycelial response to environmental stimuli.

Heatmap analyses revealed that the DEGs in the longevity-regulating pathway and MAPK signaling pathway were significantly upregulated in the mycelia that were cultured for 4 days and then stimulated (T1 vs. T2). An opposite change in gene transcription of the transcriptional enhancer factor Tec1 was observed in mycelia cultured for 6 days (T3 vs. T4); however, other genes encoding the transcription factor RLM1, Sho1 osmosensor, protein kinase A, and another transcriptional enhancer factor Tec1 presented no obvious transcriptional changes (Figure 7a). The DEGs enriched in lipid metabolism according to the KEGG database were selected for analysis. The genes encoding delta-9 desaturase, delta-12 desaturase, and 3-oxoacyl-[acyl-carrier protein] reductase, which are related to the biosynthesis of unsaturated fatty acids in mycelia cultured for 6 days and then stimulated (T3 vs. T4), were significantly downregulated; however, the genes encoding phosphatidylserine decarboxylase, phosphatidylserine synthase 2, phospholipase D, and lysophospholipase I, which are enriched in glycerophospholipid metabolism, were significantly upregulated. Most genes encoding alcohol dehydrogenase, aldehyde dehydrogenase (NAD⁺), glutaryl-CoA dehydrogenase, acetyl-CoA acetyltransferase, and enoyl-CoA hydratase, which are involved in fatty acid degradation, were significantly downregulated in the mycelia cultured for 4 days and then stimulated (T1 vs. T2), whereas the genes involved in steroid biosynthesis, including C-4 methyl sterol oxidase and squalene monooxygenase, were significantly upregulated (Figure 7b).

3.5. Validation of Key DEGs

To confirm the quality of RNA-seq data and the expression patterns of DEGs in mycelia that were cultured for different incubation periods followed by light and low-temperature stimulation, six DEGs involved in signaling pathways and 14 DEGs involved in lipid metabolism were selected to validate their expression trends via RT-qPCR (Figure 8). The results from RT-qPCR validation showed that the expression trends of most selected genes were consistent with those detected by transcriptome sequencing, except for four genes encoding squalene monooxygenase (newGene 1706), transcriptional enhancer factor Tec1 (newGene 4823), SHO1 osmosensor (P1A1038), and protein kinase A (newGene 3847), whose expression trends in the mycelia cultured for 6 days and then stimulated were opposite to those demonstrated by transcriptome analyses. These results suggest that our RNA-seq data are reliable.

4. Discussion

Pleurotus tuoliensis is a species of low-temperature fructifying edible mushroom. Mycelia can only form primordia after accumulating sufficient nutrients during the growth phase and then being subjected to low temperature and light stimulation [14]. In this study, we investigated the correlation between the duration of mycelial incubation at 25 °C in the dark and the primordium formation of P. tuoliensis on the basis of analyses of the transcriptome and targeted metabolome.

4.1. Lipid Metabolism Was Most Active During the Incubation Period

Our studies suggested that lipid metabolism, including glycosphingolipid biosynthesis-globo series, glycerolipid, and sphingolipid metabolism, were active during the incubation period. Studies on Hypsizygus marmoreus revealed that the glycerolipid metabolic pathway was upregulated during the hyphal knot phase [28], which is consistent with the results of the present study. The decreased expression of fatty acid synthase during mycelial incubation in the dark from 4 days to 6 days and decreased levels of triacylglycerol and free fatty acid demonstrated the slowdown of fatty acid and triacylglycerol biosynthesis. Previous studies have suggested that triglyceride and phospholipid levels are significantly reduced after the silencing of fas1, which can affect growth and development [29]. Phosphatidylserine decarboxylase plays important roles in phospholipid homeostasis, filamentous fungal growth, and morphogenesis [30]. Our study revealed significantly increased transcriptional levels of phosphatidylserine decarboxylase genes during mycelial growth in the dark and in mycelial response to environmental stimulation. The deletion of FgPsd2 resulted in significant reductions in the mycelial growth rate and conidial number of Fusarium graminearum. FgPsd2 mutants grew better when they were supplied with exogenous ethanolamine, which demonstrated that the PE content produced by FgPsd2 was essential for mycelial growth [31].

Targeted lipidomic analysis revealed that the lipid composition, which included triglycerides, free fatty acids, phosphatidylcholine, phosphatidylglycerol, and sphingosine, significantly changed during the incubation period. Recent studies suggest that the TAG stores in lipid droplets release FAs that act as signals to influence the transcriptional control of gene expression [32]. The process of synthesizing or degrading TAG produces lipid intermediates, such as phosphatidic acid and diacylglycerol, which may serve as activators or inhibitors of signaling pathways [33]. The present study revealed that phosphatidylglycerol and phosphatidylcholine were the main phospholipids involved in mycelial growth in the dark. The level of phosphatidylcholine gradually increased during the stage of mycelial growth and was the most abundant phospholipid. Previous studies have shown that phosphatidylcholine is usually the most abundant phospholipid in eukaryotic cells, comprising about 40–50% of the total cellular phospholipids [34]. Phosphatidylcholine is predicted to participate in organellar and cellular biogenesis, as well as the formation of vesicles for the transport of proteins and lipids within cells [35]. GO enrichment analyses also revealed that DEGs whose expressions changed during incubation were enriched mainly in integral components of the membrane, extracellular regions, and membrane parts in the cellular component category. Sphingosines belong to a structurally diverse group of lipids, which are abundant in membranes. In our study, a significantly greater level of sphingosine was detected in mycelia cultured for 4 days than in that cultured for 6 days. Numerous studies have shown that sphingosine can regulate the phosphorylation of many proteins in the MAPK pathway, in addition to its inhibitory effects on protein kinase C [36].

4.2. Lipids Are Components of the Eukaryotic Cellular Membrane and Influence the Mycelial Response to Environmental Stimuli

Lipids not only maintain the homeostasis of the intracellular environment and participate in the formation of cell membranes but also act as signaling molecules in the eukaryotic response to physiological signals and multiple types of stress [37,38]; thus, we hypothesized that the change in lipid metabolism during the incubation period would cause differences in the mycelial response to environmental stimulation, thus affecting primordium formation. The accumulation of TAG can activate the stress pathways of the transcription factors Msn2/4 and superoxide dismutase and significantly prolong lifespan [39]. In the present research, the expression of protein kinase A, which is involved in the longevity-regulating pathway, was significantly increased after the mycelia were cultured for 4 days and then stimulated with light and low temperature. RNA-seq data showed that the transcription levels of three kinds of transcription factors and the osmosensor SHO1, which is involved in the MAPK signaling pathway, significantly changed during the response to stimulation of mycelia of various incubation periods. MAPK activation results in the modification of transcription factors, allowing cellular responses to adapt to external stimuli. Sho1 is an osmosensor in the HKR1 subbranch of the HOG pathway. High external osmolarity induces structural changes in Sho1, which lead to Hog1 activation. Numerous non-osmotic stressors are also known to activate the HOG pathway, including cold stress, the inhibition of glycosylphosphatidylinositol anchor synthesis, and the inhibition of sphingolipid synthesis [40]. Sho1 mutants are defective in the filamentous growth Kss1 MAPK signaling pathway, which might indicate that Sho1 serves a scaffold function in the filamentous growth pathway [41]. Sko1, a transcriptional repressor of the ATF/CREB family, is doubly regulated by Hog1 and PKA to regulate the transcription of a subset of stress-responsive genes upon osmotic stress. Compared with wild-type Candida albicans, the sko1 mutant of C. albicans has an increased ability to filament [42]. The transcription factor Tec1 is an activator of G1 cyclin and vegetative adhesin genes. In Saccharomyces cerevisiae, Tec1, which is involved in the Kss1 MAPK signaling pathway, is necessary for morphological changes leading to pseudohyphal growth [43]. In Ustilago maydis, Tec1 was found to be a crucial factor for normal mating and basidiocarp development [44]. In our study, Tec1, Sho1, and PKA presented opposite transcriptional patterns in mycelia incubated for different periods followed by environmental stimulation, suggesting that they have different influences on osmolyte synthesis, antioxidant synthesis, and filamentation. Rlm1 is a downstream target of the MAPK signaling pathway. The absence of RLM1 alters the cell wall content, specifically the chitin and mannan layers [45]. The present study demonstrated that the upregulated DEGs in mycelia that were cultured for 6 days in the dark and then stimulated were significantly enriched in amino sugar and nucleotide sugar metabolism, and the downregulated DEGs were significantly enriched in fructose and mannose metabolism, which were different from the responses of the mycelia cultured for 4 days to environmental stimulation.

Our results showed that if mycelial incubation time in the dark is insufficient, glycerolipid metabolism and steroid biosynthesis would be strengthened after the mycelia are subjected to environmental stimulation, which would have a passive influence on primordium formation. Glycerolipid metabolism is critical for the homeostasis of cellular lipid stores and membranes. Glycerol kinase is a key enzyme in the glycerol metabolic pathway. Mogly1 is associated with carbon source utilization and glycerol catabolism in Magnaporthe oryzae, and the single knockout mutant Mogly1 reduces aerial mycelium [46]. Steroids play indispensable roles in the process of eukaryotic endocytosis by regulating membrane fluidity and permeability. Deletion of the gene encoding squalene monooxygenase results in an ergosterol decrease and inhibits growth in Sporisorium scitamineum [47]. The ectopic overexpression of AaSMO1 causes an increase in total sterol content and improves germination and growth in subsequent generations [48]. Steroid biosynthesis is an oxygen-dependent process, and half of the enzymatic reactions require molecular oxygen [49]. Increased steroid biosynthesis increases oxygen consumption.

5. Conclusions

Our research indicated that various incubation times resulted in changes in the levels of lipid components and the expression of genes involved in lipid metabolism, which have an influence on the primordium formation of P. tuoliensis (Figure 9). The present study contributes to a better understanding of the molecular mechanisms underlying the primordium formation of P. tuoliensis. The functional validation of key genes was further performed to elucidate the regulatory pathways, and low-temperature treatment increased the sphingolipid content involved in the growth and development of P. tuoliensis.