Nitric oxide is involved in regulating cytochrome P450 gene family and mitochondrial related genes of Pleurotus eryngii in response to cadmium stress: preliminary expression patterns analysis

ABSTRACT   Nitric oxide (NO) plays an important role in fungal response to abiotic stress, but little is known about whether or not NO is involved in regulating cytochrome P450 (CYP) gene family and mitochondrial related genes (MRG) of Pleurotus eryngii in response to cadmium (Cd) stress. In this study, 125 CYP members and 127 differentially expressed MRG were identified. The analysis of gene length and exon number revealed the complexity of CYP gene family. After adding exogenous NO, the expression patterns showed that PleCYP2 and 4-hydroxybenzoate polyprenyltransferase were significantly up-regulated, the functions of membrane and membrane components were enhanced, and the mitochondrial splicing apparatus component gene was activated, which might be the potential mechanism of NO regulating P. eryngii resistance to Cd stress. This study lays a foundation for further revealing the mechanism of NO in regulating P. eryngii response to abiotic stress and promotes the potential application in fungal cultivation.


Introduction
Toxic contaminants such as heavy metals are accumulating in the environment, as a result of various natural and anthropogenic activities like industrial activities [1][2][3][4][5].As a common heavy metal, cadmium (Cd) is highly toxic even at a very low level due to its carcinogenic and neurotoxic effects on animals and humans, which can easily enter vegetables, edible fungi, etc. [6][7][8][9].Excessive Cd can directly or indirectly cause damage to edible fungi [10].Pleurotus eryngii (P.eryngii), an edible fungus and belonging to basidiomycotina, has the highest production in China and around the world [11].It is popular edible fungus among consumers because it has excellent taste and rich in nutrients [12,13].Bag cultivation is the main planting mode for P. eryngii, which usually uses a variety of raw materials, including agricultural and sideline products or industrial waste [14].Once the raw materials in the growth substrates for cultivating P. eryngii contain excessive Cd, it will cause stress damage to P. eryngii, leading to poor growth and decline in yield [10].Due to the sessile nature of P. eryngii, it cannot escape the stress of Cd in its growth substrates [15].Therefore, it is necessary to enhance the stress response of P. eryngii to Cd. Nitric oxide (NO) is an important signaling molecule with various signaling roles including the regulation of fungal response to stress, which can alleviate the toxicity of heavy metals to organisms and play an important regulatory role in the responses of animals, plants and fungi to stress [16][17][18][19].In recent years, the role of NO has been gradually studied, and it was found that exogenous NO contributes to the resistance of fungi to abiotic stress through various physiological and genetic level changes [20][21][22].For instance, Kong et al. demonstrated that NO can regulate trehalose accumulation in P. eryngii when subjected to abiotic stresses [23].Nitrate reductase-dependent NO might be related to the hydrogen gas-alleviated Cd toxicity in Ganoderma lucidum [19].Our previous work revealed that exogenous NO can enhance the responses of P. eryngii to Cd stress by upregulating the expression of dehydrogenase, transcription factors, etc [15].However, the mechanism of NO in regulating CYP and MRG as response of P. eryngii to Cd stress at the gene level needs further investigation.
Cytochrome P450 (CYP) gene family is widely distributed in fungi and plays an important role in regulating fungal responses to abiotic stress [24,25].CYP gene family can regulate the response of fungi to abiotic stresses mainly by participating in various physiological reactions, for example, the biosynthetic pathways of some fungal compounds involve the modification of CYP [25][26][27].In addition, mitochondria are exquisitely sensitive to environmental stress, and the mitochondrial related genes (MRG) can encode a series of respiratory chain complexes to maintain the energy supply of cells in response to external stress [28,29].It can be seen that fungi can cope with external stresses by maintaining cell homeostasis through the regulation of CYP gene family and MRG, thus it is necessary to simultaneously analyze the regulatory role of CYP gene family and MRG [28,30].A previous study demonstrated that NO can induce alternative oxidase gene expression to participate in regulating the production of reactive oxygen species, enhancing the resistance of Pleurotus ostreatus to heat stress [22].However, the knowledge about the participation of NO in regulating CYP gene family and MRG of P. eryngii in response to Cd stress is limited.
In this study, sodium nitroprusside (SNP) could produce NO after decomposition and is used as exogenous NO donor [10,15].In order to verify whether NO was involved in regulating the CYP gene family and MRG of P. eryngii to cope with Cd stress, its expression patterns were analyzed.Our results will help to better understand the role and mechanisms of NO in regulating the response of P. eryngii to abiotic stress and lay a foundation for the potential application of NO in fungal cultivation.

P. eryngii cultivation and sample collection
P. eryngii mycelia were isolated from a cultivated strain, which were identified by molecular and morphological analysis and stored in the Sichuan Academy of Agricultural Sciences in Sichuan, China [15].The mycelium of P. eryngii was initially inoculated into potato dextrose agar (PDA) medium at 28°C for 5 days, and then five pieces of mycelium (punch diameter: 5 mm) were transferred into 150 mL of liquid PDA and incubated in shaker at 28°C and 110 rpm for cultivation.In the preliminary experiments, the mycelia of P. eryngii were subjected to Cd stress using 50 µM CdCl 2 (Cd), and the control group (0 µM CdCl 2 ).In the second part of the experiment, 150 µM SNP were added into 50 µM Cd-stressed mycelia (Cd_SNP).The potassium ferrocyanide (K 4 [Fe(CN) 6 ]), an analog of SNP that does not release NO, was used as negative control of SNP [15].All cultures were placed in shaking incubator at 28°C and 110 rpm for 5 days, and then the mycelia were harvested and used for subsequent analysis.Each treatment contains three biological replicates.

Identification and phylogenetic analysis of CYP gene family in P. eryngii
The cytochrome P450 (CYP, pf00067) seed sequence was downloaded from Pfam (http://pfam.xfam.org/)database and was used as probe.BLASTp was used to search the genomic data of P. eryngii ATCC 90,797 v1.0 (https://genome.jgi.doe.gov/pages/search-for-genes.jsf?organism=Pleery1) to obtain homologous protein sequence.Then SMART and Pfam were used to identify the domain of protein sequence of P. eryngii gene family, and all members of CYP gene family of P. eryngii were obtained [31].The amino acid sequences of CYP gene family proteins of P. eryngii were aligned by ClustalX1.8software, and the phylogenetic tree was constructed by MEGA 6.0 software with neighbor joining algorithm.The phylogenetic tree of CYP gene family in P. eryngii was classified with 1000 times bootstrap repeat value.

Analysis of gene structure and protein domain of CYP gene family in P. eryngii
The map of gene structure map and protein functional domain of CYP gene family in P. eryngii was darwn by TBtools (https://github.com/CJ-Chen/TBtools),which included the location information of exon and intron, and the motif information of gene sequence [32].

Analysis of CYP gene family expression based on RNA-seq data
The total RNA of P. eryngii mycelium was extracted according to the methods of previous research [15].After high throughput sequencing, the quality of sequencing data was controlled, then the gene expression was analyzed by FPKM software, and the differential expression was analyzed by DESeq [33,34].The screening threshold was Padj < 0.05, and the p-value obtained from the original hypothesis test was corrected.Finally, the heat map of CYP gene family expression pattern comparison in CK (0 µM CdCl 2 ) and Cd (50 mM CdCl 2 ) was drawn, and CK1, 2, 3 and Cd1, 2, 3 represent triplicate experiment.

Differential expression analysis of mitochondrial related genes in P. eryngii
The mycelia of P. eryngii treated with 0 µM (CK), 50 µM CdCl 2 (Cd) and 50 µM CdCl 2 +150 µM SNP (Cd_SNP) were harvested after 5 days and sequenced by RNAseq.Briefly, RNA-seq includes the following sequential steps: library preparation, clustering, sequencing, quality control, transcriptome assembly, quantification of gene expression levels and differential expression analysis, and the specific information and operational procedure can be obtained from our previous work [153535, Gene expression levels were estimated by RSEM, and differential expression analysis was performed using the DESeq R package (1.10.1).DESeq provides statistical routines for determining differential expression in digital gene expression data using a model, which is based on the negative binomial distribution [15,33].The p-value of the original hypothesis test was corrected, and the heat map of mitochondrial-related genes expression pattern comparison in CK, Cd and Cd_SNP group was drawn.
Gene ontology (GO) term annotation (biological process, cellular component and molecular function) and enrichment analysis of mitochondrial related genes (CK and Cd samples; Cd and Cd_SNP samples) were carried out by the GOseq R packages that were based on a Wallenius non-central hyper-geometric distribution, which can adjust for gene length bias in DEGs [15,36].

Phylogenetic tree, gene structure and protein functional domain analysis of CYP gene family in P. eryngii
The phylogenetic tree of CYP gene family in P. eryngii is shown in Figure 1.A total of 125 CYP gene family members were identified from the genomic data of P. eryngii, and these members had a certain degree of differentiation, which could be divided into subgroups (5 different colors).Subgroup I has the least total number of members, only one CYP protein that has a long genetic distance from other subgroups.Subgroup II includes 63 CYP proteins of P. eryngii, and the proportion is the highest (50.4%).Subgroup III includes 51 CYP gene family proteins of P. eryngii, which reaches 40.8% of the total.Subgroup IV includes seven CYP proteins of P. eryngii, accounting for 5.6%.Subgroup V includes three CYP protein of P. eryngii, and the total proportion is only 2.4%.The gene structure and protein functional domains of CYP gene family members in P. eryngii are different to some extent.As shown Figure 2, the lengths of CYP genes of P. eryngii ranged from 479 bp to 4852 bp, the number of exons was 2-24.Among which, the length of most CYP genes ranged from 1.1 kb to 2.7 kb.It can be seen that the gene length among CYP gene family members varies greatly, and the number of exons also shows significant differences.CYP Gene family members with relatively close genetic distance in phylogenetic tree have similar gene structure and exon number (Figure 2a), and their protein functional domains are relatively conservative (Figure 2b).The length of CYP gene family members was relatively large, and the number of exons also exhibited great differences, suggesting that these CYP gene family members of P. eryngii were associated with the process of adapting to Cd stress [27,37].In addition, the results of conserved domain analysis showed that 15 conserved domains were detected in 125 proteins of P. eryngii CYP gene family (Figure 2b), and the number of domains for each gene was 1-15.The genes with close phylogenetic relationship have similar functional domains, while some gene family members with close phylogenetic status lack regularity in gene length and exon number variation, which reveals that the gene family of P. eryngii has a certain degree of complexity [38,39].

NO regulates the expression of CYP gene family in response to Cd stress
NO can regulate the expression of some genes in plants and fungi to cope with various abiotic stresses [22,40].Li et al also revealed that exogenous NO contributed to the enhancement of P. eryngii response to extremely high levels of heavy metals, which by upregulation of dehydrogenase, transcription factors gene expression induced by NO [15].CYP gene family in fungi is closely associated with the adaptation to abiotic stress [41,42].However, our understanding of whether or not NO regulates CYP gene family in P. eryngii as defense mechanism against Cd stress is limited.In this work, in order to study the response of CYP gene family to Cd stress in P. eryngii, the expression patterns of CYP gene family in P. eryngii under Cd stress were analyzed.As shown in Figure 3, the heatmap of clustering analysis of CYP gene family in P. eryngii exhibited diverse expression patterns, which suggested that CYP gene might play a regulatory role for P. eryngii response to Cd stress, and some gene family members were responsive to Cd stress [42,43].One of the Cd treatment replicates (Cd2) showed abnormal expression, which may be due to the impact of sampling time and other factors, thus Cd2 group with abnormal expression did not exhibit comparative analysis of gene expression patterns.The diverse expression patterns may reflect the complex responses of CPY gene family under Cd stress in P. eryngii [44].As shown in Figure 3, the expression heatmap of P. eryngii CYP gene family in CK and Cd was divided into four groups (Group I-Group IV).Compared with the control sample, Group I genes were up-regulated after 50 mM CdCl 2 treatment, mainly including PleCYP2, PleCYP101, PleCYP102, etc. Group II genes, mainly including PleCYP21, PleCYP26, PleCYP116, etc., showed no significant difference in the expression level when P. eryngii was exposed to Cd stress, which indicated that this group of genes had no significant effect on the resistance of P. eryngii to Cd stress.Compared with the control sample, the expression of group III genes in P. eryngii decreased significantly under Cd stress, such as PleCYP87, PleCYP50, PleCYP14, etc.The group IV genes exhibited no expression in P. eryngii, such as PleCYP110, PleCYP13, PleCYP96, etc.The lack of expression of group IV genes under Cd stress indicated no participation in the resistance to Cd stress or the possibility of being silent genes in P. eryngii.After adding exogenous NO, the expression of PleCYP2 gene was significantly up-regulated, indicating that NO could enhance the tolerance of P. eryngii to Cd stress by regulating the expression of PleCYP2 gene [45].Further investigation on PleCYP2 gene from CYP gene family can help in the directional cultivation of highly stress resistant P. eryngii.The specific physiological functions of PleCYP2 gene need to be further analyzed, moreover, the detailed expression patterns and characteristics of these up-regulated genes in P. eryngii in response to abiotic stress of P. eryngii also require further research.

NO is involved in regulating MRG of P. eryngii in response to Cd stress
The expression of MRG plays an important role in coping with abiotic stress [46,47].The expression patterns of MRG in P. eryngii are shown in Figure 4, a total of 127 MRG were differentially expressed in mycelium samples of P. eryngii (CK, Cd and Cd_SNP), of which eight genes were encoded by mitochondrial genome and 119 genes by nuclear genome.Of the 127 differentially expressed genes, 126 were differentially expressed in Cd and CK, suggesting that Cd treatment could significantly affect the expression of MRG in P. eryngii.Eighty-eight genes, including mitochondrial endomembrane peptidase complex, mitochondrial tricarboxylic acid, mitochondrial GTPase, mitochondrial respiratory chain complex IV, mitochondrial respiratory chain complex III, ATP synthase subunit 9, were significantly up-regulated in P. eryngii under Cd stress, which indicated that mitochondria enhanced energy supply to cope with heavy metal stress [48].Compared with the Cd treatment alone, six MRG were differentially expressed in Cd and Cd_SNP, and five genes were differentially expressed in Cd and CK, while the mitochondrial splicing apparatus component gene was activated only in the presence of NO, which enhanced the tolerance of P. eryngii to Cd stress.In addition, the expression levels of tRNA (guanine26-N 2 )-dimethyltransferase, Cytochrome c peroxidase, mitochondrial chaperone BCS1-B OS and hydroxymethylglutaryl coenzyme A synthetase were significantly decreased after adding NO, indicating that NO could affect the expression of these genes [49].
Most notably, the expression of 4-hydroxybenzoate polyprenyltransferase gene was significantly up regulated by NO, indicating it played an important role in NO-mediated tolerance enhancement of P. eryngii to Cd stress.Subsequent research will focus on the specific regulatory mechanism of the mitochondrial splicing apparatus component gene and 4-hydroxybenzoate polyprenyltransferase gene.GO enrichment analysis showed that the differentially expressed MRG in Cd and CK were mainly enriched in cellular process, metabolic process and single-organism process (Figure 5A).In the category of cellular component, the functions of enrichment are mainly concentrated in cell, cell part, organelle, etc.In the category of molecular function, the enriched functions mainly focus on catalytic activity, binding, transport activity, etc.These enriched functions were mostly enhanced when the P. eryngii was subjected to heavy metal stress, and the most significant enhancement was in the functions of cellular processes, metabolic processes, and catalytic activity [30].But the functions of multi-organism process and extracellular region were reduced when P. eryngii was subjected to Cd stress.After adding exogenous NO, the function of enrichment was mainly concentrated in cell process and metabolic process (Figure 5B).In the category of cellular component, the function of enrichment was mainly concentrated in membrane and membrane components.In the category of molecular function, the function of enrichment was concentrated in binding and catalytic activity.In conclusion, the function of cellular process, metabolic process, binding and catalytic activity was decreased, while the functions of membrane and membrane part were enhanced after the addition of exogenous NO, which might be the mechanism of NO enhancing the response of P. eryngii to Cd stress [23,24,50].These results laid a foundation for further revealing the mechanism of NO in enhancing the response of fungi to abiotic stress.

Conclusions
In summary, Cd stress can significantly affect the expression of CYP gene family and MRG in P. eryngii.Preliminary expression patterns analysis reveals that NO is involved in strengthening the response of P. eryngii to Cd stress by enhancing the functions of membrane and membrane components, as well as regulating the upregulation of PleCYP2 gene, mitochondrial splicing apparatus component gene and 4-hydroxybenzoate polyprenyltransferase gene, which might be the potential mechanism of NO regulating the resistance of P. eryngii to Cd stress.This study will help to better reveal the mechanism of NO in regulating the response of fungi to abiotic stress and promote the potential application of NO in fungal cultivation.

Figure 2 .
Figure 2. Gene structure (a) and Protein functional domain (b) of Cytochrome P450 family in P. eryngii.

Figure 3 .
Figure 3. Heatmap of clustering analysis of gene expression pattern comparison of Cytochrome P450 gene family in P. eryngii.CK, control sample; Cd, P. eryngii mycelium treated with 50 mM CdCl 2 .Each treatment contains three biological replicates, CK1, 2, 3 and Cd1, 2, 3 represent triplicate experiment, respectively.The gene expression level increased with color from green to red.

Figure 4 .
Figure 4. Heatmap of clustering analysis on gene expression pattern comparison of mitochondrial related genes in P. eryngii.CK, control sample; Cd, P. eryngii mycelium treated with 50 mM CdCl 2 ; Cd_SNP, samples treated with 50 mM CdCl 2 and 150 mM NO donor.Each treatment contains three biological replicates.The gene expression level increased with color from green to red.

Figure 5 .
Figure 5. GO enrichment analysis of mitochondrial related genes in (A) CK and Cd samples (B) Cd and Cd_SNP samples of P. eryngii.CK, control sample; Cd, P. eryngii mycelium treated with 50 mM CdCl 2 ; Cd_SNP, samples treated with 50 mM CdCl 2 and 150 mM NO donor.Each treatment contains three biological replicates.