Genome-Wide Identification and Expression Pattern Analysis of Polyphenol Oxidase Gene Family in Agaricus bisporus

Abstract

Polyphenol oxidase (PPO) is the key enzyme in the melanogenesis pathway of enzymatic browning that converts phenolic substrates to quinones and eventually polymerizes to form melanin. In this study, the genome-wide characterization of the Abppo gene family was performed, and six Abppo genes were identified. These genes were divided into three groups based on sequence alignment and phylogenetic analysis, with members of the same group possessing similar motif structures. Expression analysis showed that the Abppo genes demonstrate diverse expression patterns at different growth stages and postharvest storage. In addition, the expression of Abppo genes could be significantly induced by abscisic acid, salicylic acid, methyl jasmonate, and gibberellic acid 3, indicating their potential roles in response to abiotic stresses. These results provide insights into the potential functions of the Abppo gene family, offering a theoretical reference in the future for mushroom breeders.

1. Introduction

The white button mushroom (Agaricus bisporus) is widely cultivated and consumed in around 90 countries, owing to its high nutritional content, low calories, and therapeutic properties [1,2]. Agaricus bisporus held fourth place in commercial mushroom production worldwide in 2018 [3]. The quality of mushrooms is determined by white color, firm texture, and intense flavor; color is the most influential criterion when consumers purchase mushrooms [4]. Browning is one of the most important causes of quality loss and market value reduction in mushrooms. Mushroom browning is a complex process, which can be affected by internal factors (i.e., water activity, respiration rate, and microbial activity) and external factors (i.e., storage temperature and relative humidity) [5]. Enzymatic browning is the main problem encountered in the further storage of mushrooms due to the high tyrosinase and phenolic content [6].

Mushroom enzymatic browning is usually mediated by different types of copper oxygenases called polyphenol oxidases (PPOs: laccases and tyrosinases) and peroxidases. Laccases play a limited role due to their low activity, and tyrosinases play a major role in mushroom browning [7,8]. PPOs oxidize phenolic substrates [i.e., tyrosine, γ-glutaminyl-4-hydroxybenzene (GHB), γ-glutaminyl-3,4-dihydroxybenzene (GDHB)] into quinones, which are converted into browning polymer pigments [9,10]. PPOs containing two copper atoms are produced in almost all living organisms, including animals, plants, and microorganisms [11]. In mammals, PPOs are involved in the biosynthesis of melanin, which protects their bodies from ultraviolet radiation [12]. In plants, PPOs have been extensively investigated due to their involvement in the enzymatic browning of fruits and vegetables [13,14]. Plant PPOs are usually encoded by a multiple gene family; however, the expression of ppo genes in bread wheat, eggplant, and poplar varies in different vegetative and reproductive tissues [15,16,17,18,19]. In fungi, melanin produced by some pathogenic fungal PPOs is essential for virulence, whereas PPOs produced by edible fungi are considered to be the leading cause of postharvest browning [5,20].

Browning is a major problem in the processing of A. bisporus, because browning not only deteriorates the quality but also seriously affects the shelf life of the mushrooms. The inhibition of PPO activity is critically important for the shelf life (or postharvest quality) of A. bisporus, and more work is needed to identify efficient approaches to retain the storage quality of mushroom. The cross breeding of A. bisporus is difficult, mainly due to the predominantly secondarily homothallic lifecycle of this fungus [21]. Since the genome of A. bisporus has been sequenced, it has become a valuable model for studying the genes and biological processes controlling enzymatic browning during the postharvest of A. bisporus [22]. In A. bisporus, six PPO-encoding genes have been identified [23,24,25,26]; however, no further analysis of Abppo gene family has been conducted. Therefore, in this study, we characterized six Abppo genes in detail, including the phylogenetic relationship, domain conservation, and cis-elements of promoters. Additionally, we studied their expression patterns at different growth stages, during postharvest storage, and in response to abiotic stresses. Finally, the promoter activity of Abppo3 and Abppo4 was measured by fusing with the gene encoding β-glucuronidase (GUS) (Figure 1). Taken together, our results could provide a foundation for further studying the biological functions of the Abppo genes in A. bisporus.

2. Results

2.1. Sequence Analysis of Abppo Genes

The complete sequences of Abppo genes were downloaded from the H97 genome database of JGI (https://mycocosm.jgi.doe.gov/mycocosm/home) (accessed on 17 August 2022) [22,26]. Gene name, gene ID, encoding protein length, molecular weights, isoelectric point, instability index, and grand average of hydropathy are listed in Table S1. The length of the AbPPO proteins ranged from 556 (AbPPO2) to 640 (AbPPO6) amino acids. The molecular weights of AbPPOs were between 63.87 (AbPPO5) and 73.88 kDa (AbPPO6). AbPPO2-6 proteins have instability index values greater than 40, indicating that they are unstable proteins. The total grand average of hydropathicity (GRAVY) of all AbPPO proteins were negative, which implied that these proteins were water-soluble. Sequence alignments revealed that AbPPO3 and AbPPO5 shared 57% similarity for nucleotides and 76.56% for amino acid. AbPPO2 and AbPPO4 share 65.53% nucleotide similarity and 62.95% amino acid similarity (Table S1).

To investigate the molecular evolution and phylogenetic relationships among PPOs in monocotyledonous and eudicotyledonous fungi, 54 PPO protein sequences were aligned by CLUSTALW. A phylogenetic tree was constructed by employing the NJ method using MEGA 7.0 (Figure 2). The analysis divided PPOs into several distinct clades. The nodes at the base of the larger clades were well supported, and nodes at the base of many smaller clades were also robust (bootstrap values > 70%). AbPPO2, 3, 4, and 5 were clustered together. Six PPO protein sequences had several highly conserved regions, such as the copper domains CuA and CuB, which are involved in the copper binding via histidine [7,26]. When analyzed using MEME software version 5.0.5, a total of 10 conserved motifs were found in AbPPO proteins (Figure 3). The members of each subgroup had similar motifs, although the length of the corresponding protein was markedly different. Based on analysis using Pfam and CDD (Conserved Domains Database), we found that Motif 4 and 2 formed the CuA domain and Motif 1 corresponded to CuB.

2.2. Expression Patterns of Abppo Genes

The expression pattern of Abppo genes during fruit body development was analyzed. Abppo1, 3, 4 and 5 exhibited a higher expression level in all stages of the development. In particular, the relative mRNA levels of Abppo1, 3, and 5 were significantly increased from the onset of fruit body development (stage 2 to 3). The expression of Abppo4 was up-regulated gradually during mushroom morphogenesis. The expression level of Abppo2 was generally low and stable during the fruit body development. Abppo6 gene expression was dramatically down-regulated at stage 3, whereas its expression was negligible from stage 4 to 7 (Figure 4). Four genes (Abppo3, 4, 5, and 6) were highly expressed toward the early stage of postharvest storage, suggesting that these genes may be involved in the early browning process. All Abppo genes exhibited a trend of decreased expression during storage (Figure 5).

To explore the role of Abppo genes in response to abiotic stresses, we analyzed the expression patterns of six Abppo genes under different abiotic stressors by using qRT- PCR in A. bisporus mycelia. Under abiotic stresses, all genes were up-regulated to varying degrees except Abppo5 and 6. In particular, SA and MeJA treatment even at the lowest concentration of 50 μM significantly up-regulated the expression of Abppo4. Abppo4 was significantly up-regulated under 100 μM ABA treatment, and its expression level increased approximately two-fold. Abppo6 was up-regulated after ABA treatment, with a maximum expression of approximately 6.36-fold (compared to group CK) at 50 μM. In response to SA treatment, Abppo1 and 2 showed similar expression patterns, of which their expression peaked at 50 μM and decreased at the later treatment. After 50 μM MeJA treatment, the transcript of Abppo5 was decreased, while the other genes were slightly up-regulated. All Abppo genes except for Abppo5 and Abppo6 were slightly up-regulated after GA₃ treatment (Figure 6).

2.3. Activity Analysis of Abppo3 and Abppo4 Promoters

The majority of Abppo genes increased expression levels in response to abiotic stressors, including ABA, MeJA, SA, and GA3. The distribution of different cis-acting elements in the promoter of a gene may indicate the difference in its function and regulation. Therefore, 2-kb genomic sequences upstream of the translation initiation site were analyzed using PlantCARE to investigate the cis-acting elements of the promoter region. Cis-elements related to light responses, abiotic stresses responses, and phytohormone responses were identified. However, no ABA-responsive element was found in Abppo3 promoter region (Figure 7). In order to determine whether there is a potential ABA responsive element, we constructed a reporter system to measure the promoter activity. Since Abppo4 was highly expressed at three concentrations of ABA treatment, the promoters of Abppo3 and Abppo4 were investigated.

GUS activity was detected via measuring blue staining in the mycelium of wild-type As2796 and positive control PCgh-G. The quantitative GUS activity assay showed that GUS staining was not detected in wild-type mycelium while the transformant PCgh-G exhibited a blue staining in the mycelial plug, indicating that PCgh-G not only integrated gus but also expressed this gene (Figure 8A(a,b)). Similar to the wild-type, the GUS staining was not detected in transformants PCgh-ppo3 and PCgh-ppo4 (Figure 8A(c,d)). Moreover, we found that the promoter of Abppo3 and Abppo4 can be induced by the ABA stressor. After ABA treatment, a strong GUS activity was detected in the mycelia of transformants harboring the Abppo3::gus, compared to strain grown under control conditions. GUS activity was elevated with increasing ABA concentration or treatment time in PCgh-ppo3 (Figure 8B). In contrast, GUS activity increased along with the extension of ABA concentration in transformant PCgh-ppo4 treated with ABA for 24 h (Figure 8C(a–c)). However, GUS activity was lowest at a concentration of 50 μM when treated with ABA for 72 h (Figure 8C(d–f)).

3. Materials and Methods

3.1. Strains and Culture Media

The Agaricus bisporus strain As2796 was provided by the Mushroom Genetics & Breeding Laboratory, Zhejiang A&F University. The mycelium was maintained at 25 °C on TYEG medium in a Petri dish to produce fruiting bodies. For the selection and maintenance of transformants, TYEG medium was supplemented with hygromycin (Roche, Mannheim, Germany) at 40 mg/L. Agrobacterium tumefaciens strain AGL1 (self-preservation of laboratory) grown in LB medium (10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) containing 50 mg/mL Rifampicin and 50 mg/mL Kanamycin (Sangon Biotech, Shanghai, China) were used to transform A. bisporus. The expression vector pCAMBIA1301, Escherichia coli glycerol bacteria is a self-preserved strain in our laboratory.

3.2. Plasmids and Vector Construction

A. bisporus genomic DNA was isolated from the tissues of fruiting bodies using a modified CTAB method [28].For cloning the promoters of the Abppo3, Abppo4, gpd-L, gpd-R genes, four pair of primers (Table S2) were designed based on the published genomic library of A. bisporus [22]. The specific primers hpt-F and hpt-R (Table S2) were designed using pCAMBIA1301 plasmid as a template to clone the hpt gene. The PCR product was purified and linked to the pEASY-T1 Cloning Vectors (TransGen Biotech, Beijing, China) named pT-PPO3, pT-PPO4, pT-gpdL, pT-gpdR, and pT-hpt. All of the vectors were transformed to competent cells Trans-T1 (TransGen Biotech, Beijing, China). Positive recombinants were identified via colony PCR, and then the competent cells Trans-T1 were stored in 20% glycerol and sent for sequencing (Sangon Biotech, Shanghai, China). Subsequently, the pT-gpdL and pT-hpt plasmids in Escherichia coli Trans-T1 were extracted using a AxyPrep^TM Plasmid Miniprep Kit (Axygen, Hangzhou City, China), digested by EcoR I and Bgl II restriction enzymes, and then ligated to pMD18-T Vector (TransGen Biotech, Beijing, China) using T4 DNA Ligase (Promega, Madison, WI, USA), named PT-hg. The fusion construct was then inserted into the pCAMBIA1301 Vector backbone after being digested by Xho I and Kpn I, generating the intermediate vector PCgh. The restriction enzymes Sal I and Nco I were then used to digest PCgh, pT-PPO3, pT-PPO4, and pT-gpdR, respectively, the fragments were ligated to form binary expression vectors, PCgh-ppo3::GUS, PCgh-ppo4::GUS, PCgh-G::GUS, with the aid of T4 DNA Ligase (Promega, Madison, WI, USA) (Figure 1). Finally, the ligated products were transformed into Trans-T1 and sequenced (Sangon Biotech, Shanghai, China); all the vectors were introduced into A. tumefaciens strains AGL1 via the freeze–thaw method.

3.3. Agrobacterium-Mediated Transformation of A. bisporus

For transformation experiments, A. tumefaciens was grown in LB solid medium containing rifampicin and Kanamycin at 50 μg/mL for 2 days at 28 °C. Then, plaques were selected and incubated in 5 mL liquid LB with antibiotic for 48 h at 28 °C with the shaking of 150 rpm/min. All of the fresh culture was transferred to 40 mL of liquid LB and grown for 4–6 h at 28 °C to an OD₆₀₀ of 0.5 to 0.8. Bacteria were collected via centrifugation and resuspended in liquid induction medium (IM) containing 200 μM acetosyringone at 28 °C with 120 rpm/min for 4 h to an OD₆₀₀ of 0.5, in order to pre-induce the virulence of A. tumefaciens. Fruiting body tissue pieces were sterilized in Mercuric Chloride for 60 s and rinsed with aseptic water 10 times and then quickly immersed into liquid IM of pre-induced A. tumefaciens. The bacteria–fungus mixture was co-cultivated at 28 °C with shaking at 120 rpm/min for 30 min. Tissue pieces were transferred to selection medium (SM) containing hygromycin at 30 mg/L and cephalosporin at 200 mg/L and maintained at 25 °C for 12 days. Then, colonies growing from the tissue pieces were transferred to SM containing hygromycin at 40 mg/L and cephalosporin at 200 mg/L and maintained at 25 °C for 30 days.

3.4. Gene Bioinformatics Analysis

As the full genome sequence of A. bisporus was completed, sequences of Abppos were downloaded from JGI (https://mycocosm.jgi.doe.gov/Agabi_varbisH97_2/Agabi_varbisH97_2.home.html) (accessed on 5 April 2021). All of the PPO protein sequences were submitted to ExPASy (https://web.expasy.org/protparam/) (accessed on 10 April 2021) for analysis of the physicochemical properties. The conserved motifs were analyzed using MEME 5.0.5 (http://meme-suite.org/tools/meme) (accessed on 10 April 2021) and TBtools [29]. The phylogenetic tree was generated using MEGA 7.0, the neighbor-joining (NJ) method, the Poisson model, the pairwise deletion method, and bootstrap test with 1000 replicates [30]. The PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 10 April 2021) was used to analyze the cis-acting elements [31].

3.5. Cultivation of Mushroom

The mushroom strains were inoculated in wheat grain medium, placed in an incubator at 24 °C, protected from light, and cultured for 20~35 d until the mycelium had grown all over the substrate. The mushroom spawns were planted indoors in trays filled with well-fermented compost using a broadcasting method (the compost was purchased from Zhejiang Longchen Modern Agricultural Science and Technology Co., Hangzhou, China). The mycelium had spread over the compost after three weeks of growth, and then a 2–3 cm layer of casing soil was applied to the compost surface. After casing, the tray should be maintained at a temperature of 14–18 °C and a relative humidity of 80–90% until the first flush of mushrooms appears.

3.6. Sampling and RNA Extraction and Reverse Transcription

To obtain fruiting bodies of different growth stages, mushrooms were grown on a straw-based compost and fruiting bodies were harvested at developmental stages 1 to 7 [27] and flash-frozen in liquid nitrogen and stored at −80 °C. The fruiting bodies of stage 1 were used as control. The effect of low temperature on the Abppos expression of different storage days was assessed. The fruiting bodies were stored at 4 °C and specimens were used at the harvest day (day 0) and on days 1, 2, 3, 4, 5, 6, and 7 postharvest. The fruiting bodies of day 0 were used as control. Under various signaling component treatments, ABA, SA, MeJA, and GA3 were added to liquid TYEG medium to provide final concentrations of 100 μM. A. bisporus mycelia treatment with ethanol was used as control. Total RNA was extracted from these tissue mentioned above using the RNAiso Plus method, and subsequently converted into first-strand cDNA using the PrimeScript^TM RT reagent Kit (TaKaRa, Shiga, Japan). Three biological replicates per sample were used.

3.7. Real-Time RT-PCR Analysis

Real-time RT-PCR with SYBR Green I dye was used for detecting Abppos. The real-time RT-PCR mixture reaction system (20 μL) was composed of 10 μL TB Green Premix Ex Taq II (TaKaRa, Shiga, Japan), 0.4 μL ROX Reference Dye, 2 μL cDNA templates, 0.8 μL 10 μM forward primer, 0.8 μL 10 μM reverse primer, and 6 μL ddH₂O. Each experiment was repeated three times. The real-time RT-PCR was preheated at 95 °C for 30 s. This was followed by 40 cycles with a denaturing temperature of 95 °C for 5 s and an annealing and elongating temperature of 60 °C for 30 s. For each sample, a melting curve was determined to verify a single amplification product. An RNA polymerase II large subunit (Pol II LS) and actin were used as the internal references, and the relative gene expression was calculated as 2^−Δ(ΔCt), where ΔCt = Ct target, i.e., the Ct housekeeping gene, and Δ(ΔCt) = ΔCt-treated control. All statistical analyses were performed with SPSS 19 (IBM Corp., Armonk, NY, USA), and asterisks indicate significant difference, * p < 0.05, ** p < 0.01.

3.8. Phytohormone Induction and Histochemical Staining

All strains were cultured on TYEG medium for 20 d and then transferred to PDA liquid medium with an ABA concentration of 25, 50, and 75 μM for 24 h or 72 h at 25 °C with shaking at 120 rpm/min. For the histochemical staining of specimens, a method based on the protocols described by Jefferson [32] was used. The ABA and treated mycelium were then soaked in dye solution at 37 °C with shaking at 80 rpm/min for 40 h. Then, the mycelium was taken out and immersed in decolorizing solution for 1 h to observe GUS staining. The experiment included both a non-transformed control where the hypha was not induced by ABA and a positive control where the transgenic hypha included a gpd promoter expression vector PCgh g of A. bisporus.

4. Discussion

The PPO gene family, widely distributed in most eukaryotic and prokaryotic organisms, encodes copper-containing metalloproteins that catalyze the oxidation of phenolic compounds to quinones [33]. The brown coloration of fruit, vegetables, and mushrooms during ripening and storage is mainly caused by PPO-mediated melanin formation. With the development of whole genome sequencing, the PPO gene family has been identified and analyzed in many plants, e.g., tomato, potato, eggplant, Fuji apple, and banana, and the distribution and function of PPO proteins are tissue-specific and developmentally controlled [15,16,17,34]. Therefore, the analysis and identification of the PPO gene family at the whole-genome level have practical significance and application value and can provide a theoretical basis for the genetic breeding of further yield traits. In the present study, we conducted a broad study of the PPO genes in A. bisporus, including the investigation of their evolutionary relationships, gene structures, and expression patterns at different growth and storage stages. To our knowledge, this is the first comprehensive analysis of ppo genes in A. bisporus.

This study characterized six ppo genes (Abppo1, Abppo2, Abppo3, Abppo4, Abppo5, and Abppo6) according to the genomic databases and previous study of A. bisporus [26]. Phylogenetic analysis showed that PPO proteins were divided into two major groups, of which fungal PPOs were assigned to group I and plant PPOs were classified as group II. In addition, higher plants have more PPOs than fungi. This might be due to the amount of genomic data as well as the number of repeated sequences [35], suggesting that ppo genes might play a significant role in the species evolution. For example, 19 PPO members in Salvia miltiorrhiza Bunge, a well-known material of traditional Chinese medicine, have the largest SmPPO family in plant species to date [36]. Following sequence alignment and phylogenetic tree construction, the grouping and evolutionary relationships of the A. bisporus PPO gene family were determined. The motif analysis indicated that AbPPO proteins with similar motif compositions are clustered in the same class with AbPPO2, AbPPO4, AbPPO3, and AbPPO5, suggesting that AbPPOs are evolutionarily conserved in A. bisporus.

The potential association of ppo genes with enzymatic browning has been suggested [37]. In the present study, we determined the expression patterns for all putative Abppo genes in different growth stages and during postharvest storage using qRT-PCR. Differences in the expression of the ppo genes in different growth stages are consistent with findings in other species [38,39,40,41]. Abppo3, Abppo4, and Abppo5 exhibited relatively high expression levels during fruit body maturation (from stage 2 to stage 7), indicating that they could play a role in the development of the fruit body. Our results are consistent with previous studies [25,26]. Moreover, Abppo1 was highly expressed at stage 2 to 6, while Abppo2 and Abppo6 were down-regulated at stage 3 to 7. Research elsewhere has indicated that Abppo1 was constitutively expressed from the mycelial aggregate to the sphorophore (stage 3) and Abppo2 showed an up-regulation at fructification and then down-regulated in the expanding sporophore [42]. We conjectured that the transcriptional patterns of the six Abppo genes may be regulated in a complex manner because the temporal transcriptional patterns of the six members differed substantially from each other during growth stages. PPO activity was positively correlated with mushroom browning [43]. During storage, Abppo1 gene expression showed a significant downward trend from the 3rd to 7th days, suggesting that Abppo1 might not be involved in mushroom browning. The results are consistent with previous reports that the transcript level of Abppo1 gene is not correlated with the degree of browning [10]. However, Pbppo1 is involved in the core browning process of ‘Yali’ pears [44]. In strawberry, low temperature storage causes indirect damage to the fruit, induces the accumulation of oxidative substances in the fruit, and activates the expression of ppo genes [45]. In our study, the expression patterns of the Abppo2, Abppo3, Abppo4, and Abppo6 genes showed a rapid increase on the 2nd day and then a slight decrease, suggesting their importance in the initial storage of oxidative substance accumulation.

Previous studies have shown that ppo genes play important roles in plants in response to various biotic and abiotic stresses [34,38,42,45]. In Populus trichocarpa, Ptrppo1, Ptrppo2, and Ptrppo11 are up-regulated in response to methyl jasmonate (MeJA) [46]. In tobacco (Nicotiana tabacum L.), the Ntppo1, Ntppo2, and Ntppo3 genes are differently responsive to MeJA and abscisic acid (ABA) treatments [47]. In Salvia miltiorrhiza, 18 Smppos was significantly up-regulated or down-regulated when exposed to MeJA stress [36]. In the present study, the expression patterns of six Abppos exposed to ABA, MeJA, salicylic acid (SA), and gibberellic acid 3 (GA3) stresses were also investigated using qRT-PCR in mushroom. Consistent with previous results in Populus trichocarpa, the majority of the Abppo genes were responsive to these stressors, exhibiting increased expression profiles, indicating the potential roles of Abppo genes in mushroom adaptation to abiotic stresses. Analyzing the promoters of Abppo genes has revealed that there were multiple hormone-related elements in these promoters: for instance, the MeJA-responsive elements CGTCA and TGACG [48], the ABA-responsive element ABRE [49], and the gibberellin responsive elements P-box [50]. However, no ABRE motif was found in the promoter region of Abppo3, although its expression was significantly up-regulated after ABA treatment. Previous studies have shown that Arabidopsis AtMYC2 and AtMYB2 function as transcriptional activators in the ABA signal transduction pathway under drought stress conditions [51]. Because the binding sites of MYB and MYC were found in the promoter of Abppo3, we hypothesized that the accumulated ABA might stimulate the expression of the MYB/MYC transcription factors to up-regulate the expression of Abppo3. This could explain why Abppo3 could be induced by exogenous ABA without ABA-response elements.

In conclusion, six Abppo genes were identified from the whole A. bisporus genome. Further, the six AbPPO proteins were characterized according to phylogenetic analysis, conserved motifs, and gene expression patterns at different stages and under different stresses (ABA, SA, MeJA, and GA3) via qRT-PCR. Overall, this systematic analysis provides a theoretical foundation and reference for the in-depth exploration of biological function in A. bisporus Abppo gene family and the further genetic breeding analysis of related species.

5. Conclusions

A. bisporus is one of the most widely cultivated edible mushrooms in the world, polyphenol oxidase (PPO) is a key enzyme in the enzymatic browning melanogenesis pathway of A. bisporus. In this study, we characterized six Abppo genes in detail, including the phylogenetic relationship, domain conservation, and cis-elements of promoters. Additionally, we studied their expression patterns at different growth stages, during postharvest storage, and in response to abiotic stresses. Our results could provide a foundation for further studying the biological functions of the Abppo genes in A. bisporus.