Two pal genes of Pleurotus ostreatus participate in primordium formation and heat stress response

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is the first key enzyme in the phenylpropanoid pathway. The pal gene is widely studied in plants and participates in plant growth, development and defense systems. However, the biological function of pal in P. ostreatus development and abiotic stress has not been reported. In this study, we cloned and characterized the pal1 (2232 bp) and pal2 (2244 bp) from basidiomycete Pleurotus ostreatus CCMSSC 00389. The pal1 and pal2 genes are interrupted by 6 and 10 introns and encode proteins of 743 and 747 amino acids, respectively. Furthermore, prokaryotic expression experiments showed that PAL enzymes catalyzed the conversion of L-phenylalanine to trans-cinnamic acid. The function of pal1 and pal2 was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the two pal genes had similar expression patterns during different developmental stages. The expression of pal genes was higher in the reproductive growth stage than in the vegetative growth stage. And the overexpression of pal1 and pal2 resulted in the formation of primordia earlier. The results of heat stress assays showed that the RNAi-pal1 strains had enhanced mycelial tolerance to high temperature, while the RNAi-pal2 strains had enhanced mycelial resistance to H2O2. These results indicate that two pal genes may play a similar role in the development of P. ostreatus fruiting bodies, but may alleviate stress through different regulatory pathways under heat stress.


Abstract Background
Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is the first key enzyme in the phenylpropanoid pathway. The pal gene is widely studied in plants and participates in plant growth, development and defense systems. However, the biological function of pal in P. ostreatus development and abiotic stress has not been reported.

Results
In this study, we cloned and characterized the pal1 (2232 bp) and pal2 (2244 bp) from basidiomycete Pleurotus ostreatus CCMSSC 00389. The pal1 and pal2 genes are interrupted by 6 and 10 introns and encode proteins of 743 and 747 amino acids, respectively. Furthermore, prokaryotic expression experiments showed that PAL enzymes catalyzed the conversion of L-phenylalanine to trans-cinnamic acid. The function of pal1 and pal2 was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the two pal genes had similar expression patterns during different developmental stages. The expression of pal genes was higher in the reproductive growth stage than in the vegetative growth stage. And the overexpression of pal1 and pal2 resulted in the formation of primordia earlier. The results of heat stress assays showed that the RNAi-pal1 strains had enhanced mycelial tolerance to high temperature, while the RNAi-pal2 strains had enhanced mycelial resistance to H2O2.

Conclusions
These results indicate that two pal genes may play a similar role in the development of P.
ostreatus fruiting bodies, but may alleviate stress through different regulatory pathways under heat stress. Background series of structural and defensive phenolic compounds, such as lignin, phenolic acid and hydroxybenzoic acid, flavonoids and stilbene [17]. The pal gene is widely studied in plants and participates in plant growth, development and defense systems [18,19], including lignin synthesis in cell walls, nutrient transport, and the regulation of seed color [20]. In addition, plants can induce PAL enzymes under abiotic stresses (ultraviolet-B (UV-B) light, high and low temperature, injury, salt, etc.), leading to the accumulation of phenolic compounds such as flavonoids and phenolic acids [21]. Under salt stress, the antioxidant capacity of plants has been shown to be enhanced by increasing PAL activity [22]. Under UV-B stress, the roots and leaves of soybeans increased their salicylic acid (SA) content by increasing PAL activity and subsequently became stress resistant [23]. The pal gene has also been studied in mushrooms in recent years, such as in Flammulina velutipes, where the pal gene was cloned and characterized. The different expression patterns of the F. velutipes pal gene and its activity in different organs of the mushroom indicated that pal is associated with mushroom growth [24]. In Tricholoma matsutake, transcriptome analysis revealed a pattern of pal gene expression that was dependent on the developmental stage, suggesting that pal has many physiological functions in this mushroom [25]. In several basidiomycete fungi, a metabolic pathway for the metabolism of phenylalanine via cinnamic, benzoic, p-hydroxybenzoic, and protocatechuic acids has been reported that is similar to that observed in plants [26]. However, the biological function of pal in P. ostreatus development and abiotic stress has not been reported.
At present, many studies have used molecular and genetic methods to silence the pal gene and study its biological function in plant growth, development and environmental stress [20,27]. In recent years, RNAi and OE technologies have been widely used to study of gene function in P. ostreatus . For example, the overexpression of a methionine sulfoxide reductase A gene enhances stress tolerance in P. ostreatus [28], which provides a more effective method for studying the function of genes in P. ostreatus. In this study, we searched and cloned the pal genes from the P. ostreatus genome. On the basis of describing their characteristics, we studied the role of pal genes in fruiting body development and heat stress using RNAi and OE technologies.

Results
Cloning and bioinformatics analysis of pal Two pal genes were identified in the P. ostreatus genome and were named pal1 and pal2, the full-length cDNA sequences which are 2232 and 2244 bp, respectively. DNA sequence analysis showed that 7 exons are interrupted by 6 introns in pal1, while 11 exons are interrupted by 10 introns in pal2 (Fig. 2B). The two sequences were deposited in GenBank with the accession numbers MK207023 and MK207024, respectively.
To understand the phylogenetic relationship between the PAL proteins and other fungal PALs, phylogenetic analysis was performed. Phylogenetic analysis of 19 PAL sequences showed that PAL can be divided into two distinct branches ( Fig. 2A). The phylogenetic tree showed that PAL1 and PAL2 have higher similarities to the protein sequences of other mushrooms or fungi than to each other. The cladogram revealed the variation in the PAL protein sequence among fungi.
The pal1 and pal2 sequenceswere bioinformatically analyzed to determine their physicochemical properties and possible structure. The pal1 gene encodes a putative 743amino-acid polypeptide with an approximate molecular weight and calculated pI of 79.845 kDa and 5.28, respectively. The pal2 gene encodes a putative 747-amino-acid polypeptide of 79.946 kDa with a predicted isoelectric point of 6.11 [29]. An online analysis revealed a Pfam lyase aromatic domain in both pal1 and pal2,whereas only pal2 was observed to have a SCOP d1qj5a_domain. The gene models for pal1 and pal2 from different organisms are shown in Fig. 3A, which primarily describes the amino acid identities and similarities among pal genes in different organisms. The pal motif is labeled with a red box, and the conserved active-site motif (Ala-Ser-Gly) and specific amino acids are also shown in Fig.   3A. The conserved active-site motif is labeled with circles under the specific amino acids, which can be converted into an MIO (4-methylidene-imidazole-5-one) prosthetic group (Fig. 2C). The other active-site residues are labeled with red circles in Fig. 3A. The 3-D structure of PAL (Fig. 2C) showed that it is composed of an MIO domain, a core domain and an inserted shielding domain [30]. Thus, the PAL amino sequences are highly conserved with other characterized PAL proteins in fungi.
To investigate the activity of the PAL proteins in vitro, the transformant strains (E. coli) were induced to express the PAL protein by the addition of IPTG. Subsequently, the proteins were purified by nickel column affinity chromatography, and analyzed by SDS-PAGE. The results showed that the purified proteins (PAL1 and PAL2) had molecular weights of approximately 75 kDa (Fig. 3B), which is consistent with predictions. The activity of the purified enzymes was determined by spectrophotometry (Fig. 3C). The results showed that the activity of PAL1 (24.469 ± 2.296 u/mgprotein) was significantly lower than that of PAL2 (43.387 ± 2.551 u/mgprotein).
Expression of pal1 and pal 2 during different P. ostreatus developmental stages To investigate the expression patterns of pal1 and pal2 during P. ostreatus development, the expression of these genes during different developmental stages and different parts of the fruiting bodies of the WT strain were assessed (Fig. 4). The results showed that compared with that in mycelia, the expression of pal1 was significantly upregulated in primordia (3.5-fold), fruiting bodies (19.3-fold) and spores (11.8-fold) (Fig. 4A). In addition, the expression of pal2 was upregulated significantly and continuously during P. ostreatus development stages and was higher than that of mycelia in primordia (7-fold), fruiting bodies (15.2-fold), and spores (68-fold) (Fig. 4B).  Fig. 5F and G show the changes in the mycelial total respiration rate and relative ion leakage under different temperature stresses. The results showed that with increasing heat stress time, the total respiration rate of mycelia increased temporarily and then decreased rapidly. At the same time, the relative ion leakage increased significantly with increasing stress time, indicating that the degree of mycelial damage increased.

Generation of pal OE and RNAi strains
Gene transformation with a gene knockout vector is a useful approach to explore the function of genes in fungi [24]. To study the roles of pal1 and pal2 in P. ostreatus, two RNAi-pal silencing vectors and two OE-pal OE vectors were constructed containing the hyg gene as a selectable marker (Fig. 6). The efficiency of RNAi and OE of the transformants was further confirmed by qPCR analysis. The transcription of pal1 in theOE strains (OE-pal1 7.11-11 and OE-pal1 7.11-9) was approximately 4-fold higher than that of the WT strain, whereas pal1 transcription in the RNAi strains (RNAi-pal1 8.1-26 and RNAi-pal1 8.1-38) decreased by more than 50 %. Therefore, these strains were selected for further study ( Fig. 6 A). The transcription of pal2 in the OE strains (OE-pal2 7.11-7 and OE-pal2 7.  and RNAi strains (RNAi-pal2 7.18-1 and RNAi-pal2 7. [18][19] were significantly different that observed of the WT strain. The pal2 gene expression of the overexpression strains was approximately 3-fold higher than that in the WT strain, while the expression in the RNAi strains decreased to 20 % (Fig. 6 B). The PAL enzyme activity in the tested strains was also assessed. The results showed that the PAL activity in the OE-pal1 7.11-11 strain was 1.7-fold greater than that of the WT strain, and the PAL activity in the OE-pal2 7.11-7 was 1.8-fold greater than that of the WT strain ( showed that OE and RNAi of pal1 had no visible phenotypic effects. However, compared with the colony diameter of the WT strain, that of the OE-pal2 strains was slightly larger, while the growth rate of the RNAi strains was significantly lower (Fig. B and C). In mushroom production experiments, we observed that OE-pal strains formed primordia earlier than WT strain, while the RNAi strains exhibited the opposite phenotype (Fig. 7A).
Correspondingly, the period of mushroom cultivation was shortened by pal overexpression and prolonged by RNAi. To further explore the biological role of pal in the development of fruiting bodies, the expression of pal1 and pal2 was assessed in the WT, OE and RNAi strains at different developmental stages by qPCR. Fig. 7 D and E shows that the pal gene expression patterns in the OE-pal and RNAi-pal strains at different developmental stages were similar to those of WT strain. Except for the spores of the RNAi-pal1 strains, the pal gene expression in the other strains during the reproductive growth stage was higher than that observed during the vegetative reproductive stage. In summary, pal1 and pal2 are involved in the formation of P. ostreatus primordia, and the overexpression and interference of the two pal genes have no significant effect on their gene expression patterns.
Pal1 and pal2 participate in the regulation of the mycelial response to heat stress strains to H 2 O 2 was higher than that of the WT strain, especially when at an H 2 O 2 concentration of 10 mM (Fig. 8B). In addition, the resistance of other strains to H 2 O 2 did not change significantly under the tested conditions ( Fig. 8B and D). In summary, the interference of the pal2 gene reduced the sensitivity of mycelia to H 2 O 2 .

Discussion
PAL plays an important role in the acquisition of secondary metabolites. The role of PAL in plants has been extensively studied. However, the biological function of PAL in fungi, which has important research significancehas, yet to be fully elucidated. The number and structure of pal genes varies greatly in different organisms, and there are several pal genes in fungi. In Rhodosporidium toruloides, pal is encoded by a single gene. In the genomes of Aspergillus oryzae RIB 40 and Aspergillus nidulanFGSC A4, four and two pal genes are encoded, respectively. In this study, two pal genes were identified within the P.
ostreatus genome, which is generally consistent with that observed in other basidiomycetes. For example, two pal genes were identified in Coprinopsis cinerea and Schizophyllum commune. Our phylogenetic tree also supports this result. Po-PAL1 and Po-PAL2 clustered together with PALs from Coprinopsis cinerea and Schizophyllum commune, respectively. In addition, the P. ostreatus pal1 and pal2 genescontained 6 and 10 introns, respectively, differing greatly in genetic structure. Previous studies have shown that the number of introns in Basidiomycota pal genes ranges from 0-13 introns, and our results are consistent with these observations [31]. In phylogenetic trees, the PAL1 and PAL2 sequences were not phylogenetically closely related to each other, suggesting that PAL1and PAL2 did not undergo a simple gene duplication.
PAL proteins participates in the growth and development of plants, playing different roles in different species. In Arabidopsis, Antje et al. reported that pal1 and pal2 mutants had no obvious morphological phenotype, but rather became sterile [20]. Junli et al. showed that three independent pal1 and pal2 double mutants generated yellow seeds due to the lack of condensed tannin pigments in the seed coat [32]. In fungi, many pal genes have been cloned, but little research has been performed to elucidate their biological function.
In this study, the results showed that the gene expression patterns of pal1 and pal2 during different developmental stages were essentially the same, with pal1 and pal2 expression increasing during the transformation from the vegetative to the reproductive growth stages. The gene expression of pal1 and pal2 was also consistent in different parts of the fruiting body. At the same time, the overexpression of pal1 and pal2 resulted in the formation of primordia earlier, suggesting that these genes may play a similar role in the development of fruiting bodies. In Flammulina velutipes, the expression of pal in the stipe increased significantly, suggesting that pal may be involved in stipe elongation [33]. We observed that pal gene expression in stipe was significantly lower than that observed in fruiting the body, possibly because the long stipe is not a beneficial trait during P. ostreatus development. The transcription pal in the cap was significantly higher than that in the stipe. Considering that phenolic compounds in plants are synthesized through activity of pal in the phenylpropanoid pathway, pal expression in the P. ostreatus cap may also be involved in the production of phenolic compounds, which may improve the antioxidant capacity of mushrooms. In this study, different levels of pal1 and pal2 transcription were observed in spores. Previous studies have shown that the transcription of tmpal2 is the highest in the gill in Tricholoma matsutake [34]. Our results also indicate that pal2 transcription in spores is significantly higher than that of pal1, which may indicate that pal2 may play a major role in spore-related progress.
In this study, pal1 gene expression increased significantly after heat stress, but the RNAi-pal1 strains showed a significant growth advantage over the WT strain at 32 °C. The OE-pal2 and RNAi-pal2 strains showed no significant difference at 32 °C, but showed obvious resistance to exogenous H 2 O 2 . Previous biochemical studies using isotope feeding demonstrated that a number of plants can synthesize SA from cinnamate, synthesized by PAL from phenylalanine [35,36]. Furthermore, some studies have shown that the accumulation of SA can promote the production of H 2 O 2 , which leads to the production of ROS and cell death [37,38]. Thus, we speculated that the increase in pal1 gene expression may lead to ROS generation by regulating SA production. In plants, oxidative stress is produced as a secondary stress during the heat stress response, which results in the abundant production of ROS [39]. ROS poses a serious threat to cell function by damaging lipids and proteins [40]. ROS  more sensitive to UV-B radiation but were more resistant to drought stress [42]. In Brachypodium, no significant difference in UV-B radiation and drought resistance was observed between RNAi-pal and WT plants [43].

Conclusions
In summary, in this study, two pal genes were cloned and the structural characteristics of the encoded proteins was studied. Through a qPCR analysis, we observed that the gene expression patterns of pal1 and pal2 were essentially the same during P. ostreatus different developmental stages. In addition, this study confirmed that pal overexpression could promote the formation of primordia. These results indicate that pal genesare involved in the development of P. ostreatus fruiting bodies. In addition, this study assessed the role of pal in heat stress, providing a basis for exploring the role of the phenylpropanoid pathway in the development and stress response of P. ostreatus.

Methods
Strains, plasmids and media cetyltrimethylammonium bromide (CTAB), respectively. The first-strand cDNA was synthesized using a PrimeScript™ RT-PCR kit (Vazyme). The amplified products were purified and cloned into the vector pGEM-T (Promega, Madison, WI, USA) for sequencing.
All primers used in the experiment are shown in Table 1 The cultured cells were centrifuged at 4 °C and 5000 rpm for 5 min, washed with PBS buffer, and then suspended in the lysis buffer. After the cells were lysed by ultrasonication, the enzymes were retained in the supernatant after centrifugation. The supernatant was loaded onto an Ni-NTA column (Qiagen, Duesseldorf, Germany) that was preequilibrated with binding buffer. Subsequently, the column was eluted with binding buffer, washing I buffer, washing II buffer, elution I buffer and elution II buffer. Finally, the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [45].

OE and RNAi vector construction
The original pCAMBIA1300 vector was modified to harbor the hyg phosphotransferase gene (hyp), which was expressed under the control of the upstream lac promoter [28,46]. The pal gene OE cassettes were constructed as follows. The P. ostreatus gpd promoter was PCR amplified, after which the pal1 and pal2 cDNA was obtained. The two cassettes were individually cloned into vector to generate the pal gene expression cassette driven by the P. ostreatus gpd promoter (Fig. 1A, B, C). Finally, the vector was introduced into A.
tumefaciens GV3101. RNAi-F and a RNAi-R fragments were obtained by PCR, after which the two amplicons were invidiously inserted into the vector to construct the interference vectors ( Fig. 1D, E, F). Finally, the interference vectors were transferred into P. ostreatus by A. tumefaciens GV3101. The primers used to construct the vectors are shown in Table   1.

Quantitative real-time PCR (qPCR)
To analyze the expression of pal at different developmental stages, samples were collected from the mycelia, primordia, fruiting body and spore stages. The levels of genespecific mRNA expressed by the WT, OE-pal and RNAi-pal strains were analyzed using qPCR according to ourprevious study [6], with the β-actin gene used as a reference. Determination of relative ion leakage and total respiratory rate Ten pellet pieces (5 mm) were inoculated into 100 mL of potato dextrose broth medium for 5 days at 28 °C with shaking at 180 rpm. Heat stress was then applied for different times at 40 °C (0, 3, 6, 12, 24, and 48 h). The conductivity of mycelial pellets (C1) was measured by washing electrolytes attached to the surface with deionized water and then putting them into 20 mL of deionized water at 28 °C for 2 h. Then, the sample was autoclaved for 30 min to determine the total conductivity (C2). The relative ion leakage rate (%) = C1/C2 100 [49]. The respiration rate was determined by measuring the production of carbon dioxide with a carbon dioxide meter (MultiRAE IR PGM-54) in sealed containers. The total respiratory rate was measured according to previous studies [9].