Nitric Oxide Negatively Regulates the Rapid Formation of Pleurotus ostreatus Primordia by Inhibiting the Mitochondrial aco Gene

Nitric oxide (NO) is as a signaling molecule that participates in the regulation of plant development and in a number of physiological processes. However, the function and regulatory pathway of NO in the growth and development of edible mushrooms are still unknown. This study found that NO played a negative role in the transformation of Pleurotus ostreatus from vegetative growth to reproductive growth by the exogenous addition of NO donors and scavengers. Further studies showed that NO can inhibit the gene expression and enzyme activity of aconitase (ACO). Moreover, the overexpression (OE) of mitochondrial aco and RNA interference (RNAi) confirmed that ACO participates in the regulation of the primordia formation rate. The effects of aco OE and RNAi on the tricarboxylic acid (TCA) cycle and energy metabolism were further measured. The results showed that RNAi-aco mutant strains can affect the enzyme activities of isocitrate dehydrogenase of mitochondria (ICDHm) and α-ketoglutarate dehydrogenase (α-KGDH) in the TCA cycle, thereby reducing the production of nicotinamide adenine dinucleotide (NADH) in the TCA cycle, decreasing the contents of adenosine triphosphate (ATP) and hydrogen peroxide (H2O2), and negatively regulating the rapid formation of primordia. In addition, H2O2 was significantly increased during the transformation from vegetative growth to reproductive growth of P. ostreatus. Additionally, the exogenous addition of H2O2 and its scavengers further confirmed the positive regulation by H2O2 in primordia formation. This study shows that during the growth and development of P. ostreatus, NO can inhibit the expression of the mitochondrial aco gene and ACO protein in the TCA cycle, reduce the production of ATP and H2O2 in the respiratory chain, and negatively regulate the rate of primordia formation.


Introduction
Pleurotus ostreatus is a typical heterothallic edible fungus that is widely cultivated all over the world [1,2]. It is rich in nutrients and has economic and ecological value and medicinal properties [3]. Recently, the growth and development of mushrooms has become a trending topic in mycological research [4]. Many functional genes, transcription factors, and signal transduction pathways related to mushroom development have been found and studied in edible fungi. In Ganoderma lucidum, nicotinamide adenine dinucleotide phosphate oxidase genes (NoxA and NoxB) can not only regulate mycelial branching, fruiting development, and the production of reactive oxygen species (ROS), but also participate in the regulation of ganoderic acid biosynthesis [5]. In Coprinus cinereus, the dark stipe1 (dst1) gene encodes a putative photoreceptor for blue light, which is involved

Determination of NO Content
Intracellular NO content was measured using the NO assay kit (Beyotime, Shanghai, China). First, the mycelium was frozen in liquid nitrogen and ground. Then, 20 mg was placed into a centrifuge tube, and 200 µL of lysate was added. After incubation in an ice bath for 10 min and centrifugation at 14,000× g for 10 min, the supernatant was collected. The NO concentration was detected according to the instructions of the kit. In this experiment, the culture bottles were divided into 4 groups, with 10 bottles per group. Fruiting body was induced in the end of spawn running. One group was used as the control, and 1 mL of SNP (100 µM) or cPTIO (250 µM) was added to the two other groups. The same amount of SNP + cPTIO was added to the last group. After treatment for 24 h, the exogenous additive was removed. The formation rate of primordia in different treatment groups was observed and photographed.

Determination of ACO Activity
The protein concentration in different samples was determined by a Bradford protein quantification kit (Vazyme, Nanjing, China). The ACO activity in the cytoplasm and mitochondria in different samples was determined using an ACO activity detection kit (Solarbio, Beijing, China).

H 2 O 2 Content Determination
The protein concentration and H 2 O 2 content in different samples were determined by a Bradford protein quantification kit (Vazyme, Nanjing, China) and H 2 O 2 quantitative assay kit (Sangon Biotech, Shanghai, China).

Measurement of the Activities of Isocitrate Dehydrogenase of Mitochondria (ICDHm) and α-Ketoglutarate Dehydrogenase (α-KGDH)
ICDHm catalyzes the formation of α-ketoglutarate from isocitric acidin in the TCA cycle, and this process simultaneously reduces NAD + to NADH. ICDHm is one of the rate-limiting enzymes of the TCA cycle, and its catalytic reaction is one of the main sources of cellular NADH. The α-KGDH is widely distributed in the mitochondria of animals, plant microorganisms, and cultured cells. It is one of the key enzymes regulating the α-oxidative decarboxylation of ketoglutarate to succinyl coenzyme in the TCA cycle [30,31]. The activity of ICDHm and α-KGDH in each sample was detected according to the instructions of the kit (Solarbio, Beijing, China).

NADH and NAD + Content Determination
The NADH content and NAD + /NADH ratio in different samples were determined with an NAD + /NADH assay kit (Beyotime, Shanghai, China).

Adenosine Triphosphate (ATP) Content Determination
The enhanced ATP assay kit (Beyotime, Shanghai, China) was used to measure the changes in ATP content in differently treated samples.

Western Blot Analysis
According to a previous study [32], the expression of mitochondrial ACO protein in P. ostreatus was detected by Western blot. ACO and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were synthesized by a company (GenScript, Nanjing, China). Briefly, total protein was extracted from different samples, and the protein concentration was determined by a detergent-compatible Bradford protein quantification kit (Vazyme, Nanjing, China). Then, 20 µg of total protein from different samples was separated on a 12% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes. Finally, ACO antibodies were used for Western blot analysis, with GAPDH antibodies as a reference. The chemiluminescent signal was revealed using ECL substrate for detection [33].

Quantitative Real-Time PCR (qPCR)
RNA was extracted as previously described [34]. The expression of aco in differently treated samples was detected according to Wang et al. [29]. In brief, total RNA was first extracted from different samples, and cDNA was synthesized for qPCR analysis. In this study, the β-actin gene was used as a reference. The qPCR amplification procedure was as follows: 95 • C for 3 min, 40 cycles of 95 • C for 3 s, and 60 • C for 32 s, and a final extension at 72 • C for 30 s. The relative expression level of genes was calculated according to the 2 −∆∆CT method. The primers are shown in Table 1. The culture bottles were divided into 5 groups, with 10 bottles per group. Fruiting body was induced when the spawn running completed. One group served as the control (adding 1 mL of H 2 O), and 1 mL of 25 mM or 50 mM H 2 O 2 was added to the two other groups. Then, 1 mL of 25 mM or 50 mM DMTU was added to the final two groups. After treatment for 24 h, the exogenous additive was removed. The formation rate of primordia in different treatment groups was observed and photographed.

Data Analysis
GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS statistics 17.0 (SPSS Inc., Chicago, IL, USA) were used for statistical analysis. The values were reported as the means ± SEs, and were analyzed by one-way ANOVA according to Duncan's test. A p value < 0.05 was considered significant.

NO Content in P. ostreatus Varies in Different Developmental Stages
NO, as a signaling molecule, plays an important role in the growth and development of many organisms. In this study, the changes in NO content in different developmental stages were explored. Figure 1A shows the different developmental stages (mycelia, primordia, young fruiting body, fruiting body, and spores) of P. ostreatus. Figure 1B shows the changes in the NO content in different developmental stages of P. ostreatus. The results showed that the NO content in the mycelia of the culture bottles was significantly higher than that in the plates; this content was increased by 16.8-fold. After the formation of primordia, the NO content in the primordia decreased by 56.4% compared to that in the culture bottles. It is suggested that NO may play a negative role in primordia formation. After that, the content of NO in the young fruiting body and the fruiting body was 0.107 and 0.021 µmol/µg protein, respectively. The content of NO in spores increased slightly to 0.237 µmol/µg protein.

Data Analysis
GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA) and SPSS statistics 17.0 (SPSS Inc., Chicago, IL, USA) were used for statistical analysis. The values were reported as the means ± SEs, and were analyzed by one-way ANOVA according to Duncan's test. A p value < 0.05 was considered significant.

NO Content in P. ostreatus Varies in Different Developmental Stages
NO, as a signaling molecule, plays an important role in the growth and development of many organisms. In this study, the changes in NO content in different developmental stages were explored. Figure 1A shows the different developmental stages (mycelia, primordia, young fruiting body, fruiting body, and spores) of P. ostreatus. Figure 1B shows the changes in the NO content in different developmental stages of P. ostreatus. The results showed that the NO content in the mycelia of the culture bottles was significantly higher than that in the plates; this content was increased by 16.8-fold. After the formation of primordia, the NO content in the primordia decreased by 56.4% compared to that in the culture bottles. It is suggested that NO may play a negative role in primordia formation. After that, the content of NO in the young fruiting body and the fruiting body was 0.107 and 0.021 µmol/µg protein, respectively. The content of NO in spores increased slightly to 0.237 µmol/µg protein.

NO Plays a Negative Role in Primordia Formation
In this study, an NO donor (SNP) and the NO scavenger, cPTIO, were added to verify the function of NO. Figure 2 shows that SNP affects the rapid formation of primordia, prolongs the time of primordia formation, and then affects the development cycle. However, it does not affect the formation of the fruiting body. There was no significant effect on the development of the fruiting body when cPTIO was added.

NO Plays a Negative Role in Primordia Formation
In this study, an NO donor (SNP) and the NO scavenger, cPTIO, were added to verify the function of NO. Figure 2 shows that SNP affects the rapid formation of primordia, prolongs the time of primordia formation, and then affects the development cycle. However, it does not affect the formation of the fruiting body. There was no significant effect on the development of the fruiting body when cPTIO was added.

NO Inhibits ACO Enzyme Activity and Mitochondrial aco Gene Expression
There are two subtypes of ACO in organisms. The mitochondrial ACO (mACO) subtype is a component of the TCA cycle, and the cytoplasmic ACO (cACO) subtype participates in the glyoxylic acid cycle. Figure 3A,B show that the activity of cytoplasmic ACO and mitochondrial ACO increased significantly when the mushroom bottles were transferred to the mushroom production room for 48 h. This result suggests that the TCA cycle is hastened during primordia formation in P. ostreatus. Interestingly, the addition of an exogenous NO donor (SNP) can significantly inhibit the activity of the ACO enzyme in the mitochondria and cytoplasm of P. ostreatus. However, cPTIO completely blocked the effect of the NO donor on the activity of ACO. As shown in Figure 3C, mitochondrial ACO enzyme activity was 5.7-fold higher than the cytoplasmic ACO enzyme activity. Therefore, mitochondrial ACO was selected for further study. Figure 3D shows that compared to that of the full-bottle mycelia (CK), the expression level of the mitochondrial aco gene in the mycelia of CK (48 h) was significantly increased by 2.9-fold. The addition of exogenous SNP inhibited the expression of the aco gene, as it was decreased by 25.3%. In contrast, the addition of the NO scavenger cPTIO promoted the expression of the aco gene, as it was increased by 13.3%.

NO Inhibits ACO Enzyme Activity and Mitochondrial aco Gene Expression
There are two subtypes of ACO in organisms. The mitochondrial ACO (mACO) subtype is a component of the TCA cycle, and the cytoplasmic ACO (cACO) subtype participates in the glyoxylic acid cycle. Figure 3A,B show that the activity of cytoplasmic ACO and mitochondrial ACO increased significantly when the mushroom bottles were transferred to the mushroom production room for 48 h. This result suggests that the TCA cycle is hastened during primordia formation in P. ostreatus. Interestingly, the addition of an exogenous NO donor (SNP) can significantly inhibit the activity of the ACO enzyme in the mitochondria and cytoplasm of P. ostreatus. However, cPTIO completely blocked the effect of the NO donor on the activity of ACO. As shown in Figure 3C, mitochondrial ACO enzyme activity was 5.7-fold higher than the cytoplasmic ACO enzyme activity. Therefore, mitochondrial ACO was selected for further study. Figure 3D shows that compared to that of the full-bottle mycelia (CK), the expression level of the mitochondrial aco gene in the mycelia of CK (48 h) was significantly increased by 2.9-fold. The addition of exogenous SNP inhibited the expression of the aco gene, as it was decreased by 25.3%. In contrast, the addition of the NO scavenger cPTIO promoted the expression of the aco gene, as it was increased by 13.3%.  Figure 4A,B show the expression levels of aco genes and ACO proteins at different developmental stages. Figure 4A shows that the aco gene in P. ostreatus was significantly upregulated during its growth and development. Compared to the aco gene expression level in the mycelia stage, those in the stages of primordia, fruiting bodies, and spores were increased by 5.0-fold, 16.0-fold, and 33.0-fold, respectively. Since the assembly from gene expression to mature protein is a very complex process, the formation of the ACO protein in different developmental stages was detected by Western blot. The results are shown in Figure 4B. The results showed that the ACO protein was detected in primordia, young fruiting bodies, and fruiting bodies, but not in other developmental stages. It is speculated that ACO protein was not detected because of its low expression levels in mycelia. In spores, a high expression of the aco gene was detected at the mRNA level, but no visible amount of ACO protein was detected.   Figure 4A,B show the expression levels of aco genes and ACO proteins at different developmental stages. Figure 4A shows that the aco gene in P. ostreatus was significantly upregulated during its growth and development. Compared to the aco gene expression level in the mycelia stage, those in the stages of primordia, fruiting bodies, and spores were increased by 5.0-fold, 16.0-fold, and 33.0-fold, respectively. Since the assembly from gene expression to mature protein is a very complex process, the formation of the ACO protein in different developmental stages was detected by Western blot. The results are shown in Figure 4B. The results showed that the ACO protein was detected in primordia, young fruiting bodies, and fruiting bodies, but not in other developmental stages. It is speculated that ACO protein was not detected because of its low expression levels in mycelia. In spores, a high expression of the aco gene was detected at the mRNA level, but no visible amount of ACO protein was detected.

The aco Gene Is Involved in Primordia Formation
To further explore the function of the aco gene in the development of P.ostreatus, the WT, OE-aco, and RNAi-aco strains were used for cultivation experiments. As shown in Figure 4, there was no significant difference in the growth rate of the different strains on PDA plates. However, the results of the cultivation experiment showed that the different To further explore the function of the aco gene in the development of P.ostreatus, the WT, OE-aco, and RNAi-aco strains were used for cultivation experiments. As shown in Figure 4, there was no significant difference in the growth rate of the different strains on PDA plates. However, the results of the cultivation experiment showed that the different strains showed differences in the formation stages of mycelia and primordia. As shown in Figure 4C, after 26 days of inoculation, the primordium was formed in both the OE-aco and WT strains. However, in the RNAi-aco strains, no primordia formation was observed. When the cultivation experiment was carried out for 29 days, the RNAi-aco strains also formed the primordium. This finding shows that aco gene interference prolonged the time of primordium formation. Then, when the cultivation experiment lasted for 32 days, the WT strain and OE-aco strains formed mature fruiting bodies, and the RNAi-aco strains formed young mushrooms. It can be concluded that RNAi of the aco gene can prolong the developmental cycle of the fruiting body.
strains also formed the primordium. This finding shows that aco gene interference prolonged the time of primordium formation. Then, when the cultivation experiment lasted for 32 days, the WT strain and OE-aco strains formed mature fruiting bodies, and the RNAi-aco strains formed young mushrooms. It can be concluded that RNAi of the aco gene can prolong the developmental cycle of the fruiting body.

aco Gene Interference Affects Energy Metabolism and Regulates H2O2 Production and Accumulation
Mitochondrial ACO is an important TCA cycle enzyme. In this study, mitochondrial aco-transformed strains were used to further explore the function of this gene in P. ostreatus. The culture bottles were transferred to the mushroom production room for 48 h, and then, mycelial samples of different strains were collected to detect the effects of the aco gene on the TCA cycle and energy metabolism ( Figure 5). ICDHm and α-KGDH are

aco Gene Interference Affects Energy Metabolism and Regulates H 2 O 2 Production and Accumulation
Mitochondrial ACO is an important TCA cycle enzyme. In this study, mitochondrial aco-transformed strains were used to further explore the function of this gene in P. ostreatus. The culture bottles were transferred to the mushroom production room for 48 h, and then, mycelial samples of different strains were collected to detect the effects of the aco gene on the TCA cycle and energy metabolism ( Figure 5). ICDHm and α-KGDH are key enzymes in the TCA cycle. Figure 5A,B show that aco OE can significantly increase the activities of ICDHm and α-KGDH, whereas RNAi-aco strains showed the opposite effect. This suggests that OE of aco can accelerate the TCA cycle.
an average increase of 8.4%. The ATP content in aco-interfering strains decreased by 30.5%. Interestingly, the significant upregulation of NADH content in OE-aco strains did not cause significant changes in ATP content. Figure 5F shows that the H2O2 content in OE-aco strains was significantly upregulated, and the H2O2 content in the OE-aco 18 and OE-aco 2 strains was upregulated by 36.2% and 59.4%, respectively. These findings are consistent with the change trend of the NADH content in OE-aco strains. The H2O2 content in RNAi-aco 76 and RNAi-aco 15 decreased by 24.5% on average. Therefore, OE of aco can further regulate the TCA cycle, affect energy metabolism, and promote H2O2 accumulation.

H2O2 Plays an Important Role in P. ostreatus Development
H2O2 has many biological functions as a signaling molecule [37]. In this study, H2O2 was detected in different developmental stages of P. ostreatus, and the content of H2O2 showed a specific change trend ( Figure 6A,B). Previous studies have shown that H2O2 is abundant at the edge of colonies on PDA plates. It is speculated that the presence of H2O2 may regulate the elongation of apical mycelia [38]. The results of this study showed that H2O2 accumulated gradually during the transformation from mycelia to primordia in P. ostreatus ( Figure 6A,B). Figure 6B shows that in the mycelial, primordial, and young Nicotinamide adenine dinucleotide exists in oxidized (NAD + ) and reduced (NADH) forms in cells, and can participate in cell energy metabolism [35]. As shown in Figure 5C,D, the average NADH content in the OE-aco strains was 2.6-fold higher than that of the WT strain, and the average NAD + /NADH ratio in these strains decreased by 67.8%. In contrast, the average NADH content of the RNAi-aco strains decreased by 44.5% compared to that in the WT strain, and the NAD + /NADH ratio increased by 47.0%. Therefore, it is further speculated that OE of the aco gene can increase NADH content in the TCA cycle.
These small molecules of NAD participate in various biological processes, including energy metabolism, redox metabolism, and biosynthesis [36]. Figure 5E shows that during the formation of primordia, the ATP content in OE-aco strains increased slightly, with an average increase of 8.4%. The ATP content in aco-interfering strains decreased by 30.5%. Interestingly, the significant upregulation of NADH content in OE-aco strains did not cause significant changes in ATP content. Figure 5F shows that the H 2 O 2 content in OE-aco strains was significantly upregulated, and the H 2 O 2 content in the OE-aco 18 and OE-aco 2 strains was upregulated by 36.2% and 59.4%, respectively. These findings are consistent with the change trend of the NADH content in OE-aco strains. The H 2 O 2 content in RNAi-aco 76 and RNAi-aco 15 decreased by 24.5% on average. Therefore, OE of aco can further regulate the TCA cycle, affect energy metabolism, and promote H 2 O 2 accumulation.

H 2 O 2 Plays an Important Role in P. ostreatus Development
H 2 O 2 has many biological functions as a signaling molecule [37]. In this study, H 2 O 2 was detected in different developmental stages of P. ostreatus, and the content of H 2 O 2 showed a specific change trend ( Figure 6A,B). Previous studies have shown that H 2 O 2 is abundant at the edge of colonies on PDA plates. It is speculated that the presence of H 2 O 2 may regulate the elongation of apical mycelia [38]. The results of this study showed that H 2 O 2 accumulated gradually during the transformation from mycelia to primordia in P. ostreatus ( Figure 6A,B). Figure 6B shows that in the mycelial, primordial, and young fruiting body stages, the content of H 2 O 2 increased significantly. It started to decrease in the fruiting body stage, and the content was the lowest in the spores. fruiting body stages, the content of H2O2 increased significantly. It started to decrease in the fruiting body stage, and the content was the lowest in the spores.

H2O2 Promotes the Rapid Formation of Primordia
In this study, different concentrations of H2O2 and H2O2 scavenger (DMTU) were added to explore the function of H2O2 in P. ostreatus. Figure 7 shows that 26 days after inoculation, the experimental group with exogenous addition of 25 mM and 50 mM H2O2 formed primordia faster than the control group. Therefore, it is speculated that the addition of exogenous H2O2 promoted the transformation from vegetative growth to reproductive growth in P. ostreatus. In contrast, the rate of primordia formation was slowed down after the exogenous addition of DMTU. After 32 days of inoculation, mature fruiting bodies formed in the control group and the exogenous H2O2 addition experimental group. Unexpectedly, some P. ostreatus in the DMTU experimental group formed young fruiting bodies, and some did not form primordia. It is speculated that the addition of the H2O2 scavenger seriously affected the formation of primordia.

H 2 O 2 Promotes the Rapid Formation of Primordia
In this study, different concentrations of H 2 O 2 and H 2 O 2 scavenger (DMTU) were added to explore the function of H 2 O 2 in P. ostreatus. Figure 7 shows that 26 days after inoculation, the experimental group with exogenous addition of 25 mM and 50 mM H 2 O 2 formed primordia faster than the control group. Therefore, it is speculated that the addition of exogenous H 2 O 2 promoted the transformation from vegetative growth to reproductive growth in P. ostreatus. In contrast, the rate of primordia formation was slowed down after the exogenous addition of DMTU. After 32 days of inoculation, mature fruiting bodies formed in the control group and the exogenous H 2 O 2 addition experimental group. Unexpectedly, some P. ostreatus in the DMTU experimental group formed young fruiting bodies, and some did not form primordia. It is speculated that the addition of the H 2 O 2 scavenger seriously affected the formation of primordia.
J . Fungi 2022, 8, 1055 10 of 16 fruiting body stages, the content of H2O2 increased significantly. It started to decrease in the fruiting body stage, and the content was the lowest in the spores.

H2O2 Promotes the Rapid Formation of Primordia
In this study, different concentrations of H2O2 and H2O2 scavenger (DMTU) were added to explore the function of H2O2 in P. ostreatus. Figure 7 shows that 26 days after inoculation, the experimental group with exogenous addition of 25 mM and 50 mM H2O2 formed primordia faster than the control group. Therefore, it is speculated that the addition of exogenous H2O2 promoted the transformation from vegetative growth to reproductive growth in P. ostreatus. In contrast, the rate of primordia formation was slowed down after the exogenous addition of DMTU. After 32 days of inoculation, mature fruiting bodies formed in the control group and the exogenous H2O2 addition experimental group. Unexpectedly, some P. ostreatus in the DMTU experimental group formed young fruiting bodies, and some did not form primordia. It is speculated that the addition of the H2O2 scavenger seriously affected the formation of primordia.

Discussion
As an important and universal signaling molecule, NO can participate in a variety of developmental processes [39,40]. In plants, NO causes a delay in floral transition, which indicates that NO plays a negative role in the transformation from vegetative growth to reproductive growth [41]. Compared to plants, the function of NO in edible mushrooms has not been fully studied. In this study, the results showed that the content of NO decreased gradually during the transformation from vegetative growth to reproductive growth. It is speculated that NO may play a negative role in this process. Subsequently, experiments with exogenous NO donors and scavengers further confirmed that NO played a negative role in the primordium formation stage. The effects of NO found in this study are similar to those found in plants. In addition, studies in plants showed that NO regulates flowering through different pathways. Previous studies have shown that NO in plants regulates the activities of sugar metabolism enzymes through S-nitrosation, affects the synthesis of reducing polysaccharides, and reduces ATP levels [42,43]. It is speculated that NO is closely related to the regulation of energy metabolism. Additionally, ACO can participate in the regulation of cellular metabolism [44]. Previous studies have shown that mitochondrial ACO is one of the main targets of NO [45,46]. In this study, the addition of an exogenous NO donor (SNP) significantly inhibited the activities of cytoplasmic ACO and mitochondrial ACO enzymes in P. ostreatus during the transformation from vegetative growth to reproductive growth. Additionally, the expression of the mitochondrial ACO coding gene was also significantly inhibited (Figure 3), which shows that NO inhibits the gene expression and enzyme activity of mitochondrial ACO during the formation of the P. ostreatus primordium. This result is similar to previous research results, which further indicated that ACO is one of the targets of NO in P. ostreatus [19]. It is interesting that NO has a much greater effect on ACO activity than on the level of aco gene expression. Previous studies have shown that in Ganoderma lucidum, NO can directly act on ACO through S-nitrosation [46]. It is speculated that in P. ostreatus, NO can directly affect ACO enzyme activity. However, as a signaling molecule, NO regulates the expression of nuclear genes, which is a complex regulatory network.
The expression patterns of ACO were different in different developmental stages. The results showed that in Arabidopsis thaliana, the expression of the aco gene was low in most developmental stages, but significantly increased in seeds and pollen [47]. This study showed that the relative expression of ACO in P. ostreatus at different developmental stages was also different. Interestingly, although the mitochondrial aco gene was abundantly expressed in spores, a large number of proteins were not detected at this stage. According to the literature, mitochondrial ACO has two major functions in eukaryotes: as an enzyme in the TCA cycle, and as a biosensor for ROS and iron [48,49]. Therefore, it is speculated that the mRNA of aco transcription may not be translated into mature ACO proteins in spores, and its specific biological function needs further study.
Recently, genetic transformation technology has been used in edible fungi [5,50]. To explore the regulatory pathway by which NO inhibits ACO to slow down the rate of primordium formation, mushroom experiments were carried out with OE-aco and RNAi-aco strains. At present, research on aco function in microorganisms is also gradually increasing. For example, in Staphylococcus aureus, the inactivation of ACO inhibited growth after the exponential period, and increased the survival rate during the stable period [51]. In Streptomyces coelicolor, the aco disruption mutant did not grow on minimal glucose media in the absence of glutamate [52]. In Xanthomonas campestris, the loss of aco affects the transcription and activity of some extracellular enzymes, and reduces the amount of extracellular polysaccharide produced by the organism [53]. A Streptomyces viridochromogenes Tü494 strain expressing a mutant aco gene cannot form any aerial mycelia, spores, or phosphinothricin tripeptide [27]. However, in P. ostreatus, RNAi of the mitochondrial aco gene did not affect the formation of aerial mycelia. However, it did affect the time of primordia formation and prolong the developmental cycle of fruiting bodies. The reason for this phenomenon might be that RNAi only reduce gene expression level, rather than disrupt the specific genes. In addition, the results of this study showed that the aco gene and ACO protein expression levels were significantly increased in the primordia compared with the mycelia stage ( Figure 4A,B). It is speculated that ACO plays an important role in primordia formation. Therefore, the OE-aco and RNAi-aco strains showed differences in the time of primordia formation. It can be inferred that there are differences in the function of mitochondrial aco in different organisms. Furthermore, ACO is involved in a variety of abiotic stress response pathways in plants, such as heat stress [54] and oxidative stress [55]. In the process of P. ostreatus cultivation, the mycelium needs to be stimulated by low temperatures to form the primordium [56]. In other words, low temperature stimulation is equivalent to cold stress for the mycelium. Moreover, an increase in aco gene expression in the primordia was detected. It is suggested that low temperature stimulation may affect the upregulation of aco gene expression in the primordium, and then promote the differentiation of the primordium. Moreover, the aco gene extended the time of primordium formation after interference, which further suggested that the aco gene participated in the development of P. ostreatus.
The TCA cycle is distributed throughout mitochondria, and is the final metabolic pathway and connection hub of sugars, lipids, and amino acids [57]. It is closely related to energy metabolism [58]. Considering that ACO is the key enzyme in the TCA cycle, it is speculated that ACO gene interference may reduce the TCA cycle rate and affect the metabolism of carbohydrates, lipids, and amino acids. Thus, it can reduce energy metabolism and affect the development of the fruiting body. ICDHm and α-KGDH are the rate-limiting enzymes in the TCA cycle [59]. In this study, the activity of ICDHm and α-KGDH was significantly increased in the OE-aco strains, and the opposite results were observed in the RNAi-aco strains. Additionally, the interference of aco also significantly reduced the content of NADH and ATP. In plants, studies have shown that NO can inhibit ATP synthase activity and reduce ATP synthesis through S-nitrosation [42]. The current results were similar to those in plants. In a word, NO can regulate ATP production in plants and edible fungi, but its regulatory pathway is different. H 2 O 2 has a variety of signaling functions, which are very important for the growth and development of plant systems [60,61]. In this study, H 2 O 2 accumulated gradually during the transformation from mycelia to primordia in P. ostreatus, which may be due to exposure to cold stress and light stress in the mushroom room. It is suggested that H 2 O 2 plays an important role in the transformation from vegetative growth to reproductive growth in P. ostreatus. Further results showed that aco OE or RNAi could also regulate the production and content of H 2 O 2 . In plants, H 2 O 2 can not only inhibit the development of leaves, but can also enhance the expression of flower-related genes. Thus, it can promote plant reproductive development [62]. The current results were similar to those in plants, indicating that H 2 O 2 can play an active regulatory role in reproductive growth as a signaling molecule.

Conclusions
The current data suggested that during the growth and development of P. ostreatus, NO signaling molecules can negatively regulate the rapid formation of primordia by inhibiting ACO, affecting the TCA cycle rate, reducing the production of NADH, and affecting the contents of ATP and H 2 O 2 ( Figure 8). This study preliminarily analyzed the function and regulatory pathway of NO in the growth and development of P. ostreatus. In the future, the mechanism by which H 2 O 2 regulates the formation of the P. ostreatus primordium will be further studied.    Data Availability Statement: All data generated or analyzed during this study are included in this published article.