MAC Family Transcription Factors Enhance the Tolerance of Mycelia to Heat Stress and Promote the Primordial Formation Rate of Pleurotus ostreatus

Abstract

Pleurotus ostreatus is a typical tetrapolar heterologous edible mushroom, and its growth and development regulatory mechanism has become a research hotspot in recent years. The MAC1 protein is a transcription factor that perceives copper and can regulate the expression of multiple genes, thereby affecting the growth and development of organisms. However, its function in edible mushrooms is still unknown. In this study, two transcription factor genes, PoMCA1a and PoMAC1b, were identified. Afterwards, PoMAC1 overexpression (OE) and RNA interference (RNAi) strains were constructed to further explore gene function. The results showed that the PoMAC1 mutation had no significant effect on the growth rate of mycelia. Further research has shown that OE-PoMAC1a strains and RNAi-PoMAC1b strains exhibit strong tolerance under 32 °C heat stress. However, under 40 °C heat stress, the OE of PoMAC1a and PoMAC1b promoted the recovery of mycelial growth after heat stress. Second, the OE of PoMAC1a can promote the rapid formation of primordia and shorten the cultivation cycle. In summary, this study indicated that there are functional differences between PoMAC1a and PoMAC1b under different heat stresses during the vegetative growth stage, and PoMAC1a has a positive regulatory effect on the formation of primordia during the reproductive growth stage.

1. Introduction

Pleurotus ostreatus is rich in various active ingredients and has multiple functions, such as immune regulation [1], hypoglycemia, hypolipidemia [2], antitumor [3], antioxidant [4], antibacterial [5], and anti-inflammatory [6]. It is one of the most widely cultivated edible mushrooms in the world [7]. The growth and development of edible mushrooms can be divided into the vegetative growth stage (mycelial growth stage) and reproductive growth stage (mushroom emergence stage) [8]. During the vegetative growth stage of mushrooms, they are most likely to be affected by high-temperature environments [9,10]. Previous studies have shown that high-temperature-treated P. ostreatus can induce cell apoptosis, and cell death can be inhibited by adding chemicals or ROS scavengers that can block the mitochondria-induced apoptosis process [11]. Subsequently, many studies have explored the mechanism of the heat stress response in P. ostreatus through proteomics and transcriptomics. For example, heat stress promotes the degradation of unsaturated fatty acids and nucleotides in P. ostreatus, increases the content of amino acids and vitamins, and accelerates glycolysis and the tricarboxylic acid cycle [12]. Furthermore, with the mature application of genetic transformation technology in edible mushrooms, the functions of many genes and transcription factors have been reported. For example, interference with the phenylalanine ammonia lyase (PAL) gene enhances the tolerance of mycelia to high temperature in P. ostreatus [13]. Overexpression of the methionine sulfoxide reductase gene enhances the stress tolerance of P. ostreatus [14]. Many genes and transcription factors have also been proven to play an important role in the emergence stage of mushrooms [15,16]. For example, the PDD1 transcription factor positively regulates the emergence of Flammulina filiformis and can increase their yield [17]. The transcription factor LFC1 negatively regulates the growth, development, and yield of F. filiformis [18]. Interfering with the PAL gene can delay the formation of mushroom primordia [13]. However, compared with animals and plants, there is currently a lack of basic research on edible mushrooms, and the response mechanism to heat stress in P. ostreatus is still not perfect.

Many enzymes require metal ions as cofactors to function, and common metal enzyme cofactors include iron, zinc, and copper [19]. The copper sensing transcription factor MAC1 is mainly involved in regulating the absorption of Cu ions in yeast cells. The MAC1 protein contains DNA-binding domains and activation domains [20]. There is a highly conserved copper fist structure in its DNA-binding domain, which plays a crucial role in binding to the copper ion-responsive elements of downstream gene promoters [21]. There are one or two cysteine-rich motifs (REPs) in the activation domain of MAC1, which are involved in the binding of copper ions. In Saccharomyces cerevisiae, there is also a copper-sensing transcription factor, ACE1, which has the same copper fist structure and REP motif as MAC1 [22]. In S. cerevisiae, MAC1 and ACE1 are two opposite transcription regulatory factors that strictly control the level of free Cu⁺. When copper is excessive, ACE1 activates the detoxification and storage genes of copper. Therefore, the Cu⁺ levels detected by ACE1 set an upper limit for free copper in yeast cells. In contrast, when copper levels are low, MAC1 activates the copper uptake gene, so the Cu⁺ level perceived by MAC1 represents the lower limit of copper ions [23]. The first reported copper-sensitive transcription factor in basidiomycetes is the activating protein ACE1, expressed by Ruben et al. in Pseudomonas aeruginosa, which activates CUP1 [24] expression [25]. In the filamentous fungus Aspergillus fumigatus, the AfMAC1 deletion mutation resulted in slow growth and incomplete conidia, including short-chain conidia and defective melanin [26]. AnMAC1 and its regulated Cu transporters have been shown to be necessary for the growth and conidial development of neutral Aspergillus nidulans during copper starvation [27]. Studies have also shown that under high-temperature stress conditions, NO activates MAC1, making cells resistant to stress, and increases SOD1 activity through CTR1-bound Cu⁺ ions [28,29]. However, little is currently known regarding the roles of MAC1 in modulating the growth, development and thermotolerance of edible mushrooms.

In this study, two MAC1 coding genes of P. ostreatus were cloned. Subsequently, the functions of the two MAC1 coding genes in response to heat stress and the growth and development of P. ostreatus were further explored by constructing RNA interference (RNAi) and overexpression (OE) mutants, laying the foundation for further exploration of the regulatory mechanism of MAC1.

2. Materials and Methods

2.1. Fungal Strains and Culture Conditions

The P. ostreatus strain CCMSSC 00389 was obtained from the China Center for Mushroom Spawn Standards and Control. The WT and mutant strains were cultured on potato dextrose agar (PDA) plates, Escherichia coli (DH5α) was cultured in Luria–Bertani (LB) medium containing 50 ng/mL kanamycin, and Agrobacterium tumefaciens (GV3101) was cultured in LB containing 50 ng/mL kanamycin and 25 ng/mL rifampicin. The PDA culture medium was purchased from Beijing Boidee Biotechnology Co., Ltd. (Beijing, China), Sensory cells purchased from TransGen Biotech (Beijing, China). Restrictive endonuclease was purchased from New England Biolabs (NEB) (Beverly, MA, USA), and DNA polymerase, reverse transcription kits, and DNA gel extraction kits were purchased from Vazyme (Nanjing, China). Primer synthesis and DNA sequencing were completed by Tsingke Biotech (Xian, China). The plasmid pBI121-EGFP was purchased from Miaoling Biology (Wuhan, China).

2.2. Identification, Cloning, and Sequence Analysis of PoMAC1 Genes

The MAC1 gene sequence was obtained from the website of the National Biotechnology Information Center (https://www.ncbi.nlm.nih.gov/gene/855035) (accessed on 15 August 2014) of S. cerevisiae [29], and the gene ID was 855035. Then, the sequence of this gene was used to BLAST against the CCMSSC 00389 genome database [30] to identify two PoMAC1 genes. Two PoMAC1 protein sequences are available through online websites (http://www.bioinformatics.org/sms/index.html) (accessed on 4 August 2023). A phylogenetic tree was constructed with the MEGA11 program using the neighbor joining method with a 1000 bootstrap value [31]. The MEME website (https://memesuite.org/meme/tools/meme) (accessed on 4 August 2023) was used to predict the motif of the PoMAC1 proteins. DNAMAN 6.0 software was used for multiple sequence alignment. The molecular weight, distribution of amino acids, isoelectric point (pI), and signal peptide of PoMAC1 were predicted using the online ProtParam (http://web.ExPASy.org/protparam/) (accessed on 4 August 2023). The structural domains of the PoMAC1 proteins were analyzed online (http://smart.embl-heidel-berg.de/) (accessed on 4 August 2023). The online URLs (https://swissmodel.ExPASy.org/interactive) (accessed on 4 August 2023) were used to predict the tertiary structure of the PoMAC1 proteins. The nuclear localization signals (NLS) of the PoMAC1 proteins were analyzed online (https://www.novopro.cn/tools/nls-signal-prediction.html) (accessed on 25 November 2023). All primers used in this study are shown in Table S1.

2.3. Subcellular Localization of PoMAC1a and PoMAC1b

The PoMAC1a and PoMAC1b genes were cloned and inserted into the vector pBI121-EGFP through homologous recombination and then introduced into A. tumefaciens EHA105, with empty space as the control. Afterwards, A. tumefaciens EHA105 containing the target plasmid was cultured, collected and suspended in a concentrated solution (150 mM acetylsyringone, 10 mM MES monohydrate, 10 mM magnesium chloride, and pH 5.6) to achieve a final OD value of 1.0. Finally, A. tumefaciens was injected into tobacco leaves with good growth and cultured for 3 days. The results were observed and recorded using a confocal microscope.

2.4. Construction of OE and RNAi Plasmids

The PoMAC1a and PoMAC1b gene OE cassettes were constructed as follows. The original OE plasmids stored in the laboratory were digested with SpeI and PspOMI [13]. Then, the cDNA of PoMAC1a and PoMAC1b was obtained through PCR and cloned and inserted into the vector to generate OE plasmids containing the PoMAC1a and PoMAC1b genes. Acquisition of RNAi plasmids: The original RNAi plasmids stored in the laboratory were first digested with SpeI and BglII, and the cloned PoMAC1a-sence and PoMAC1b-sence fragments were homologously recombined onto the plasmids. Subsequently, the obtained plasmids were digested using SpeI and PspOMI, and the cloned PoMAC1a-anti and PoMAC1b-anti fragments were inserted to construct the interference vectors. Finally, the vectors were introduced into A. tumefaciens GV3101. The primers used to construct the vectors are shown in Table S1.

2.5. Acquisition of Mutant Strains

First, P. ostreatus mycelia were inoculated onto PDA plates and cultured at 28 °C for 5 days. Then, the mycelial pellets were cut to a 5 mm diameter at the edge of the colony. Then, the mycelial pellets were placed into CYM medium and cultivated at 28 °C for 2 days. A. tumefaciens containing OE and RNAi plasmids were expanded and cultured, and the A. tumefaciens were collected in sterile tubes (50 mL capacity) by centrifugation at 4500 rpm and 4 °C for 10 min. Then, the A. tumefaciens cells were suspended in induction medium (IM) and incubated for 5 h. After that, the mycelial pellets were placed onto IM medium (containing A. tumefaciens) and cocultured at 28 °C for 5 h without shaking. Afterwards, the mycelial pellets were transferred to IM solid medium at 28 °C for 3 days and then transplanted onto CYM medium with 90 μg/mL hyg and 50 μg/mL cef. Transformants were obtained after 20–30 days [13].

2.6. Mushroom Production

The WT and mutant strains were cultured on PDA medium at 28 °C for 7 days. Then, the mycelia were inoculated into cottonseed shell culture medium and incubated in darkness at 25 °C for 25 days. When the mycelia were full, the culture bottles were transferred to the mushroom production box to control the production conditions. For fruiting body production, the temperature was set at 18 °C (day, 12 h at 500 lux) and at 10 °C (night, 12 h dark), and the ‘s humidity was maintained at 95% [32]. Primordia, fruiting bodies, spores, and liquid nitrogen were collected for rapid freezing, and then stored at −80 °C.

2.7. Heat Stress Treatment

To assess the function of PoMAC1 in the mycelial response to heat stress, the WT, OE and RNAi strains were cultured on PDA plates at different temperatures 32 for 7 days. Afterwards, the function of PoMAC1 was tested under 40 °C heat stress. First, P. ostreatus mycelia were cultured at 28 °C for 4 days, transferred to 40 °C for 24 h, and then returned to 28 °C to recovery growth for 3 days. Afterwards, the recovery growth rate of different strains of mycelia was recorded, and the growth inhibition rates of different strains were calculated [33].

2.8. Growth Susceptibility Assay

To assess the susceptibility of the test strains to oxidative stress, mycelial tip pellets with a 5 mm diameter were inoculated onto PDA plates supplemented with 5 mM H₂O₂. The diameters of the strains were measured after incubation at 28 °C for 7 days.

2.9. Quantitative PCR (qPCR)

To analyze the expression pattern of PoMAC1 under different treatments, samples from different growth stages (mycelia, primordia, fruiting bodies, spores) and different periods of heat stress were collected for gene expression detection. Using the β-tubulin gene as the internal reference, qPCR was used to analyze the specific mRNA expression level of the MAC1 gene. The qPCR amplification program was as follows: amplification at 95 °C for 3 min, amplification at 95 °C for 3 s, amplification at 60 °C for 32 s, 40 cycles, and amplification at 72 °C for 3 s. The relative gene expression was analyzed according to the 2^−ΔΔCT method.

2.10. Data Analysis

SPSS 26 software was used for statistical analysis. The values are reported as the means ± SEs and were analyzed by one-way ANOVA, with a p value of <0.05 considered significant. GraphPad Prism 11 and Photoshop 2023 software were used for figure analysis.

3. Results

3.1. Cloning and Bioinformatics Analysis of PoMAC1a and PoMAC1b

Two MAC1 genes were obtained and identified in the genome of P. ostreatus, named PoMAC1a and PoMAC1b, with total cDNA lengths of 1776 bp and 1275 bp, respectively (Figure 1A). DNA sequence analysis showed that five exons of PoMAC1a were interrupted by four introns, and four exons of PoMAC1b were interrupted by three introns (Figure 1A). Bioinformatics analysis was performed on PoAMC1a and PoMAC1b sequences to determine their physicochemical properties and possible structures. The PoMAC1a gene encodes a peptide of 591 amino acids, with a molecular weight and pI of 63.03 kDa and 7.55, respectively. The PoMAC1b gene encodes a polypeptide of 424 amino acids, with a molecular weight and pI of 44.50 kDa and 9.03, respectively. To determine the evolutionary relationship between PoMAC1 identified in P. ostreatus and MAC1 homologous sequences reported in other fungal species, a detailed phylogenetic analysis and conservative motif comparison were conducted. The results showed that the two PoMAC1 proteins were distributed on different branches, suggesting that there might be differences in function between PoMAC1a and PoMAC1b (Figure 1B). Conservative motif analysis showed that motifs 1 and 3 existed in all MAC1 amino acid sequences. It is speculated that these two motifs may be related to the MAC1-binding site. Figure 1B shows that both PoMAC1a and PoMAC1b contain conserved motifs 1, 2, 3 and 9. However, PoMAC1a alone exhibits motifs 4, 5, 6, 8, 10, 11, and 12, and PoMAC1b alone exhibits motif 7. The amino acid comparison results showed that PoMAC1a and PoMAC1b exhibit typical Cu-sensing transcription factor characteristics, with a conserved “Cu-fist” structure at the N-terminus, which plays a crucial role in MAC1 binding to DNA. In addition, cysteine-rich motifs are widely present at the C-terminus and participate in sensing copper and stabilizing DNA binding. In Figure 2A, the Cu-fist structure is marked with a red box, while the Cys-rich motif is marked with blue. However, there is only one cysteine-rich motif, REP-Ⅰ, in the PoMAC1a, while there are two cysteine-rich motifs, REP-Ⅰ and REP-Ⅱ, in the PoMAC1b (Figure 2B). This means that different Cys-rich motifs may lead to differences in the functions of the PoMAC1a and PoMAC1b genes.

3.2. Subcellular Localization of PoMAC1

Transcription factors synthesized in the cytoplasm can only function by transferring to the nucleus [34]. We can verify whether PoMAC1 has nuclear localization signals through tobacco transient expression experiments. The results showed that GFP signals were present in the nucleus and cytoplasm of the positive control group, while the PoMAC1a-GFP and PoMAC1b-GFP fusion proteins were located in the nucleus (Figure 3). The research results indicated that PoMAC1 has a nuclear localization signal.

3.3. Acquisition of PoMAC1a and PoMAC1b Mutant Strains

To investigate the functions of PoMAC1a and PoMAC1b, OE and RNAi transformants were constructed. Figure 4A,B show the OE and RNAi plasmid maps. In the maps, the hyg gene was used as a selection marker to further confirm the RNAi and OE efficiency of the transformants. Then, the OE and RNAi plasmids were transferred to the mycelia of P. ostreatus through Agrobacterium-mediated genetic transformation. Figure 4C shows that the hyg fragment can be amplified in the mutant strains, with a size of approximately 1000 bp. Furthermore, the mutant strains were further identified by detecting gene expression levels. The results showed that compared to WT, the relative expression levels of PoMAC1a in OE-PoMAC1a-1 and OE-PoMAC1a-2 were approximately 2.9-fold and 2.7-fold higher, respectively. The PoMAC1a expression levels in RNAi-PoMAC1a-1 and RNAi-PoMAC1a-2 were reduced by 53% and 41%, respectively. The relative expression levels of PoMAC1b in OE-PoMAC1b-1 and OE-PoMAC1b-2 were approximately 3.2-fold and 3.6-fold higher, respectively, while the PoMAC1b expression levels in the RNAi strains (RNAi-PoMAC1b-1 and RNAi-PoMA1b-2) were reduced by 67% and 62%, respectively, compared to that in the WT strain (Figure 4D). Therefore, these mutant strains were selected for further research.

3.4. PoMAC1 Participates in the Regulation of the Mycelial Response to Different Heat Stresses

MAC1 plays an important role in various stresses. Previous studies have shown that Saccharomyces cerevisiae MAC1 can confer stress resistance to yeast cells under high-temperature stress [20]. In this study, we tested the function of PoMAC1a and PoMAC1b under 32 °C heat stress. The results showed that compared with the WT strain, there was no significant difference in the colony size of the RNAi-PoMAC1a strains grown at 32 °C for 7 days, while the colony diameter of the OE-PoMAC1a mutant strain significantly increased. The growth rates of the OE-PoMAC1a-1 and OE-PoMAC1a-2 strains increased by 35.83% and 23.27%, respectively, compared with the WT strain (Figure 5A,B). It is speculated that PoMAC1a may play a positive regulatory role under 32 °C heat stress. In the PoMAC1b mutant strains, OE-PoMAC1b-1 and OE-PoMAC1b-2 showed stronger heat resistance, and compared with the WT strain, their mycelial growth rates increased by 35.83% and 23.27%, respectively (Figure 5A,B). It is speculated that PoMAC1b may play a negative regulatory role under 32 °C heat stress. Further research has shown that PoMAC1a is a factor in H₂O₂ tolerance, as its silencing reduces growth; PoMAC1b is a factor for sensitivity to this oxidizing agent, as its silencing results in increased growth in the mutant. (Figure S2). In summary, it can be concluded that there are differences in the functions of PoMAC1a and PoMAC1b under heat stress at 32 °C.

To further investigate the functions of PoMAC1a and PoMAC1b in P. ostreatus, the expression patterns of PoMAC1 under heat stress were detected. The results showed that there were significant differences in the expression patterns of PoMAC1a and PoMAC1b under 40 °C heat stress at different times. It can be inferred that there are also differences in functionality between PoMAC1a and PoMAC1b. In the early stage of 40 °C stress (1 h), the expression level of the PoMAC1a gene significantly increased and reached its peak. In contrast, with the prolongation of heat stress time, the expression level of PoMAC1b gradually increased and reached its peak in the later stage of stress (48 h) (Figure 6A). The growth zones of the OE-mutants of PoMAC1a and PoMAC1b were visibly more prominent than those of the control (WT) and the silenced mutants. This is evident in the peripheral zone of the colony (approximately 0.5 cm), with a finer and more exploratory mycelium showing recovery from heat stress. This exploratory growth edge is not as clearly visible in the WT or the silenced mutants, which exhibit colonies with compact edges (Figure 6B). Subsequently, the recovery growth rates of the PoMAC1a and PoMAC1b mutants were tested after heat stress at 40 °C for 24 h. The results showed that compared to the WT strain, the mycelial recovery rates of the OE-PoMAC1a and OE-PoMAC1b strains were significantly accelerated. Compared to the WT strain (80.59%), the growth inhibition rates of the OE-PoMAC1a and OE-PoMAC1b strains were reduced to 51.17% and 64.26%, respectively. In contrast, the recovery growth rates of the RNAi-PoMAC1a and RNAi-PoMAC1b strains were not significantly slower than that of the WT strains. However, the growth inhibition rates of the RNAi-PoMAC1a and RNAi-PoMAC1b strains were increased to 87.16% and 88.65%, respectively (Figure 6C). This indicates that PoMAC1a and PoMAC1b play a positive regulatory role in the response of mycelia to 40 °C heat stress.

3.5. PoMAC1a Positively Regulates the Formation of Primordia

To further investigate the function of the PoMAC1a and PoMAC1b genes in the growth and development of P. ostreatus, the expression levels of PoMAC1a and PoMAC1b in different developmental stages (mycelia, primordia, fruiting body, spores) were detected. The results showed that compared with the mycelia stage, the expression patterns of PoMAC1a and PoMAC1b were similar in both the fruiting body and spore stages, with significant downregulation of expression levels during the fruiting body stage and a significant increase in expression levels in spores. Interestingly, compared to the mycelial stage, the expression level of PoMAC1a in the primordia was significantly upregulated, while the expression level of PoMAC1b was not significantly changed (Figure 7A,B). It is speculated that PoMAC1a plays a key role in primordial formation. To further investigate whether PoMAC1a plays an important role in primordial formation, a mushroom production experiment was conducted. The results showed that compared with the WT strain, the OE-PoMAC1a strains promoted the primordia formation rate and shortened the growth and development cycle of fruiting bodies. In contrast, PoMAC1a interference prolonged the time needed for primordial formation and extended the developmental cycle of fruiting bodies. However, compared to the WT strain, the PoMAC1b mutant strains did not show any differences compared with the WT strain (Figure 7C,D). These results indicated that PoMAC1a can positively regulate the formation of primordia.

4. Discussion

MAC1 plays an important role in maintaining the intracellular copper ion concentration, and its function and mechanism of action in fungal yeasts have been widely studied. However, there are few reports on basidiomycetes, which have important research significance. In this study, based on a whole-genome search of P. ostreatus, two MAC1 genes were identified, with PoMAC1a and PoMAC1b having 4 and 3 introns, respectively. The amino acid sequence consistency between PoMAC1a and PoMAC1b was only 22.84% (Figure S1), and in the phylogenetic tree, the genetic relationship between the two MAC1 genes was not close, indicating that PoMAC1a and PoMAC1b are not simple gene duplications. The MAC1 protein is a copper-sensing transcription factor that participates in regulating genes essential for copper ion transport, thereby maintaining intracellular Cu concentrations [20,35]. The PoMAC1 protein has a highly conserved Cu-fist structure located in the N-terminal DNA-binding domain, and there is a cysteine-rich REP motif in its C-terminal activated domain, which is consistent with other species of MAC1 proteins. In the amino acid sequence of PoMAC1a, there is only one cysteine-rich motif, REP-I, while PoMAC1b has two different cysteine-rich motifs, REP-I and REP-II. This is similar to the study by Zhengdong Cai et al. [36]. The A. fumigatus copper-sensing transcription factor MAC1 homolog contains two identical Cys-rich motifs REP-Ⅰ (CXCX₃CXCX₂CX₂H) at the C-terminus, while ScMAC1 in S. cerevisiae contains two identical Cys-rich motifs, REP-II (CXCX₄CXCX₂CX₂H). In Schizosaccharomyces pombe, there is only one Cys-rich motif, REP-Ⅰ [36,37]. CaMAC1 in Candida. albicans contains two different Cys-rich motifs, REP-Ⅰ and REP-Ⅱ, at the C-terminus [38]. The REP-I of PoMAC1a and PoMAC1b in P. ostreatus is composed of five cysteines and one histidine, which is similar to previous research. However, the REP-II motif in PoMAC1b is composed of six cysteines and one histidine (CXCX₄CX₁CX₃CX₃CX₂H), which is the difference between the REP-II of the MAC1 in P. ostreatus and the REP-II of MAC1 in other fungi [39,40]. The above results indicate that the MAC1 transcription factor is highly conserved at the N-terminus, while different changes may occur at the C-terminus, which may lead to its genes performing different functions.

MAC1 protein is involved in the growth and development of fungi and various stress responses. However, the biological function of MAC1 in edible mushrooms has not been reported. This study found that during the vegetative growth stage, there are differences in the functions of PoMAC1a and PoMAC1b in P. ostreatus under different heat stresses. Under mild heat stress at 32 °C, PoMAC1a and PoMAC1b exhibit opposite functions. The OE-PoMAC1a and RNAi-PoMAC1b strains promoted the growth rate of mycelia and exhibited strong heat resistance. Under extreme heat stress (40 °C), the expression patterns of PoMAC1a and PoMAC1b were similar, showing a trend of first increasing, then decreasing, and then increasing. However, PoMAC1a showed the highest expression in the early stage of stress (40 °C stress for 1 h), while PoMAC1b reached its peak in the later stage of stress (40 °C stress for 12 h). In addition, the OE of PoMAC1a and PoMAC1b can promote the recovery growth rate of mycelia after heat stress. Therefore, it is speculated that PoMAC1a and PoMAC1b may participate in regulating the response of mycelium to 40 °C heat stress at different times. Previous studies have shown that the NO produced by yeast cells under high-temperature stress activates the MAC1-induced CTR1 gene, leading to an increase in intracellular copper levels. Then, Cu⁺ activates SOD1 to resist high-temperature stress [29]. Therefore, it is speculated that under 40 °C heat stress, the high expression of two PoMAC1 genes in the mycelia of P. ostreatus may improve the activity of the SOD enzyme, enhance the tolerance of mycelia to reactive oxygen species, and thus promote the recovery and growth of mycelia after heat stress. In addition, the function of MAC1 has been extensively studied in other species; for example, in yeast, the functional defect mutation of MAC1 can lead to an increase in yeast cell thermal sensitivity [20] In Arabidopsis, there is a copper sensing transcription factor ACE1, which has a highly similar structure to MAC1. Moreover, OE-ACE1 transgenic plants have a higher survival rate under copper stress than WT plants, while increasing the activity of SOD and POD [16]. The results of this study are similar to those of previous studies, indicating that MAC1 can participate in various abiotic stress response pathways. However, it is interesting that there are significant differences in the functions of PoMAC1a and PoMAC1b in P. ostreatus under different stresses.

During the reproductive growth stage, PoMAC1a positively regulates primordial formation and shortens the developmental cycle of fruiting bodies. However, there was no significant difference between the PoMAC1b mutant strains and the WT strain during the reproductive stage. Previous studies have found that in A. fumigatus, the AfMAC1-deficient mutant not only significantly slows its growth rate, but also has incomplete conidia, including short chains and melanin deficiency [26]. In S. cerevisiae, the loss-of-function mutation of MAC1 may manifest defects in the activity of plasma membrane Cu²⁺ and Fe³⁺ reductases, slow growth, and respiratory defects [20]. Our research results are similar to those of our predecessors. These results all indicate that MAC1 plays an important role in growth and development processes. At present, the regulatory mechanism of PoMAC1 in the growth and development of P. ostreatus is still unclear. In future work, further exploration of its possible regulatory mechanism will be conducted through omics techniques.

5. Conclusions

In summary, this study cloned two PoMAC1 genes from P. ostreatus and analyzed their protein structures. The function of PoMAC1a and PoMAC1b in the growth and development of P. ostreatus was explored by constructing OE and RNAi strains. During the vegetative growth stage, both PoMAC1a and PoMAC1b play a positive regulatory role under extreme heat stress (40 °C); under mild heat stress (32 °C), PoMAC1a plays a positive regulatory role, while PoMAC1b plays a negative regulatory role. During the reproductive growth stage, only PoMAC1a plays a positive role in the primordial formation process. This study provides a basis for exploring the role of the copper-sensitive transcription factor MAC1 in edible mushrooms.