Analysis of PRX Gene Family and Its Function on Cell Lignification in Pears (Pyrus bretschneideri)

Class III peroxidases (PRXs) are plant-specific enzymes that play key roles in the responses to biotic and abiotic stress during plant growth and development. In addition, some peroxidases also play roles in plant lignification. In this study, a total of 114 PRX (designated PbPRXs) genes were identified in the pear (Pyrus bretschneideri Rehd) genome based on systematic analysis. These PRX genes were divided into 12 groups based on their phylogenetic relationships. We performed systematic bioinformatics analysis of the PRX genes, including analysis of gene structures, conserved motifs, phylogenetic relationships, and gene expression patterns during pear fruit growth. The PbPRXs are unevenly distributed on the 17 pear chromosomes and some of them on other scaffolds. Gene duplication event analysis indicated that whole-genome duplication (WGD) and segmental duplication play key roles in PRX gene amplification. Ka/Ks analysis suggested that most duplicated PbPRXs experienced purifying selection, with limited functional divergence during the duplication events. Furthermore, the analysis indicated that those highly expressed genes might play significant roles in the lignification of cells to form stone cells in pear fruit. We examined the expression of those highly expressed genes during fruit growth using quantitative real-time PCR (qRT-PCR), verifying differential expression patterns at different stages of fruit. This study provides useful information for further functional analysis of the PRX gene family in pears.


Identification of Class III Peroxidase Genes (PRXs) in Pear
To identify members of the class III peroxidase gene family, multiple database searches were performed. The Arabidopsis class III peroxidase (AtPRXs) gene sequences obtained from the TAIR database were used as queries to perform repetitive blast searches against the Pear Center (http://peargenome.njau.edu.cn/) [19]. Additionally, all protein sequences were then used as queries to perform multiple database searches against proteome and genome files downloaded from these databases. Stand-alone versions of BLASTP and TBLASTN available from the Basic Local Alignment Search Tool were used with the evalue cutoff set to 1 × 10 −3 . All retrieved nonredundant sequences were collected from the phytozome database v9.1, and subjected to domain analysis by using two different domain analysis programs: the Pfam 27.0 and SMART, with the default cut off parameters [20,21]. Genes without PRX-specific peroxidase domains were rejected.

Phylogenetic Analysis, Gene Structure and Conserved Motif Analysis
The PRX family protein sequence alignments and the phylogenetic tree were created by using the Muscle program. The phylogenetic trees for pear class III peroxidase genes were constructed using the maximum likelihood (ML) method in MEGA6.0 and assessed by bootstrap analysis with 1000 resampling replicates.
To determine the exon/intron structures of the PbPRX genes, the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) [22] was used to align their cDNAs with the corresponding genomic DNA sequences. The conserved motifs were detected using the online MEME (Multiple Expectation Maximization for Motif Elicitation) tool (http: //meme.sdsc.edu/meme430/intro.html) [23]. Parameters were set as follows: number of repetitions; optimum motif width set to ≥6 and ≤200; maximum number of motifs set to 20. The conserved motifs were analyzed with the SMART (http://smart.embl-heidelberg.de/) and Pfam (http://pfam.sanger.ac.uk/search) programs.

Chromosomal Location and Synteny Analysis
The chromosomal location information of the PbPRX genes was obtained from genome annotations. The data were then displayed by using Circos. The analysis of synteny in the pear genomes was conducted locally by using a method similar to that developed for the PGDD (http://chibba.agtec.uga.edu/duplication/) [24]. Duplications of PbPRXs were identified using MCScanX software (http://chibba.pgml.uga.edu/mcscan2/) [25]. First, BLASTP was performed to search for potential paralogy gene pairs (E < 1 × 10 −5 , top 5 matches) genomes. Then, these paralogy pairs were used as the input for MCScanX to identify syntenic chains [25]. MCScanX was further used to identify WGD, segmental, tandem duplications in the PbPRX gene family.

Calculating Ka and Ks of the PbPRX Gene Family
The valid gene pairs derived from different gene duplication modes were used to calculate the nonsynonymous (Ka) and synonymous (Ks) substitution rates. KaKs Calculator 2.0 software with default parameters was used to calculate Ka and Ks values, and Ka/Ks ratios based on a model-averaged method [26].
Ka/Ks calculation was applied to estimate the selection pressure of PRX gene pairs. The algorithm was NG.

Gene Ontology Enrichment Analysis
InterPro domains were annotated by InterProScan [27] Release 36.0 and functional assignments were mapped onto Gene Ontology (GO) [28]. Furthermore, the GO classification and draw GO tree using WEGO [29].

Genome-Wide Expression Analysis of PRX Gene Family
To investigate the expression of PRX gene family members, pear fruit samples of the 'Dangshansuli' cultivar on 22 April (15 days after full bloom, DAFB), 13 May (36 DAFB), 27 June (81 DAFB), 28 July (110 DAFB), and 30 August (145 DAFB) were collected in 2011, which included the key stages of pear fruit development from early fruit setting to mature. RNA sequencing libraries of five fruit developmental stages were constructed using an Illumina standard mRNA-Seq Prep Kit (TruSeq RNA and DNA Sample Preparation Kits version 2). The RNA-seq data was downloaded from our center website ( http://peargenome.njau.edu.cn/). Expression values of each gene were logarithm, the cluster analyses were performed using cluster software with the hierarchical cluster method of "complete linkage" and Euclidean distances. Finally, the Treeview program was used to display the results of the cluster. 26 July (112 DAFB) for quantitative real-time PCR (qRT-PCR) analysis. Total genomic RNA was extracted from the pear fruit using the Plant Total RNA Isolation Kit Plus (FOREGENE CO.,LTD, China). A260/A280 ratios of the RNA ranged from 1.9 to 2.1 quantified with a NanoDrop ND1000 spectrophotometer. Finally, about 2 µg of total RNA was used for firststrand cDNA synthesis using REVERTAID 1ST CDNA SYNTH KIT (Fermentas Co.,Ltd, Lithuania) according to the manufacturer's protocol.

Quantitative Real-Time PCR Analysis
The primers used for amplifying PRX genes are listed in Supplementary Table S1. In the present study, the LightCycler 480 SYBR GREEN I Master (Roche, Nutley, NJ, USA) was used according to the manufacturer's protocol. Each reaction mixture contained 10 µL of LightCycler 480 SYBR GREEN I Master, 0.4 µL of each primer, 1 µL of diluted cDNA and 7.4 µL nuclease-free water. The qRT-PCR was performed on the LightCycler 480 (Roche, USA) and all reactions were run as duplicates in 96-well plates. The qRT-PCR reaction conditions were as follows: pre-incubation at 95 • C for 10 min and then 40 cycles of 94 • C for 15 s, 60 • C for 30 s, 72 • C for 30 s, and finally, extension at 72 • C for 3 min, and reading the plate for fluorescence data collection at 60 • C. A melting curve was performed from 60 • C to 95 • C in order to check the specificity of the amplified product. The real-time PCR experiment was carried out three times under identical conditions. Finally, the average threshold cycle (Ct) was calculated per sample, Pyrus Actin was used as the internal control, and the relative expression levels were calculated with the 2 −∆∆Ct method described by Livak et al. [30].

Determination of Stone Cells and Lignin Content
Each fruit was peeled, cored, and diced into cubes. A 100 g sample of pear flesh was stored in the refrigerator at −20 • C for at least 24 h then homogenized with distilled water in a blender for 10 min. The homogenate was then diluted with distilled water. The suspension was incubated at room temperature for 30 min and the supernatant phase was decanted. Finally, the sediment was suspended in 0.5 M HCl for 30 min, decanted, and washed with distilled water. This operation was repeated several times until the stone cells were almost free of extraneous cell debris [31].
The method was carried out as described by Tao et al. [10] with some modifications. Pear flesh was dried in an oven at 65 • C. The dry pear flesh was ground and pestled in 95% ethanol, then the sediment was washed with 95% ethanol and ethanol: hexane (1:2, v/v) three times, respectively, and dried. Dried sediments were digested in 2 mL of 25% (v/v) acetyl bromide in acetic acid and incubated for 30 min at 70 • C. The reaction was terminated by adding 0.9 mL of 2 M NaOH with an extra 5 mL of acetic acid and 0.1 mL of 7.5 M hydroxylamine hydrochloride. The volume was corrected to 10 mL with acetic acid and the absorbance at A280 was measured. The amount of lignin was calculated from a linear calibration curve with commercial alkali lignin (Sigma-Aldrich, St. Louis, MO, USA).

Identification and Construction of Phylogenetic Tree of Class III Peroxidase Gene Family (PRXs) in Pear
In the present study, a total of 114 open reading frames (ORFs) encoding putative PRX proteins were identified in the pear (Pyrus bretschneideri)(cultivar: 'Dangshansuli') genome using the HMMER profile and BLASTp search for further analysis.
Originally, a total of 126 candidate PRX genes were identified in pears. Among these, 12 nontargeted or overlapping protein sequences were manually removed. The results show that all 114 putative pear PRX genes contain a conserved PRX domain; this number is greater than that in Arabidopsis (73) [9]. Finally, on the basis of previous research in Arabidopsis, we assigned names to these PRX genes (PbPRX1-114) according to their chromosomal positions for convenience. The length of the 114 newly identified PRX proteins varies from 84 to 1315 aa, with an average of 336 aa. Other information about the clone number, chromosomal location, molecular weight (Mw), isoelectric point (PI), and exon number of each PbPRX gene/protein is listed in Table 1.
To gain insight into the structure of the PRX genes, the exon and intron boundaries, which are known to play crucial roles in the evolution of multiple gene families, were analyzed. Results showed that exon numbers of 114 PRX genes ranged from one to eighteen ( Figure 1C). Different subfamilies contained different exon numbers, the fact that the PbPRX64 gene has 18 exons, and PbPRX18, PbPRX53, PbPRX83, PbPRX112, and PbPRX113 have only one exon, indicates that both exon gain or loss has occurred during the evolution of the PRX gene family, which might lead to the functional diversity of closely related PRX genes. However, it was found that within each subfamily, genes usually have a similar number of exons.
Phylogenetic analysis of the 114 identified nucleotide sequences of PbPRXs could be classified into 12 subfamilies ( Figure 2). Group I contains 30 members and group I is the biggest subfamily. Group III, IV, VII, and IX are the smallest subfamilies only containing two members. Across the Maximum Likelihood (ML) tree, most bootstrap values were 80 or higher, and 12 nodes of each subfamily clade had a good bootstrap value.

Analysis of Conserved Motifs and Domain
A total of 20 conserved motifs were identified in the pear PRX proteins. Detailed information about the conserved amino acid sequences and lengths of the 20 motifs is shown in Table 2. The conserved motifs obtained from MEME analysis were annotated using the Pfam and SMART programs. Most of the closely related members have the same motif compositions, suggesting that there are functional similarities between PRX proteins within the same subfamily. Figure 2 depicts the structure diagrams of motif 5, which shows that the structure may be the core structure of the function of the PRX gene family in pears. Furthermore, some subfamily-specific motifs with unknown functions were also detected, indicating that these motifs are likely required for subfamily-specific functions. However, some motifs are distributed in nearly every subfamily, although their functions remain unknown (motif 4, 5, 10, 11); these motifs might be important for the functions of PbPRX proteins.
All conserved domains of the PbPRX gene family are shown in Figure 1B. All PRX proteins contain one or more PRX domain, and this is one of the main bases of the gene family screening.

Chromosomal Locations, Gene Duplication, and Collinearity Analyzes
To determine the genome organization and distribution of PbPRXs on different chromosomes in pears, a chromosome map was constructed. The results show that the 99 PbPRX genes are distributed on 17 chromosomes with a nonrandom distribution, and 15 PbPRX genes are mapped onto the other 10 scaffolds, as shown in Table 1. Chromosome 3 contains the most PbPRX genes (13), followed by chromosome 7 (11) and chromosome 8 (11). By contrast, only one PbPRX gene is present on chromosomes 1 and 14. In addition, some chromosomes exhibit a relatively high density of PbPRX genes, such as the bottoms of chromosomes 3 and 7 and the top of chromosome 10. Gene duplication, including segmental or whole-genome duplication (WGD) and tandem duplication, is considered to be one of the primary driving forces in the evolution of genomes [32,33]. During the evolution of a gene family, tandem duplication and WGD/segmental duplication play important roles in generating new members. Therefore, in order to clarify the potential mechanism of evolution of the PRX gene family, both tandem duplication and segmental duplication events were investigated in this study. In this study, 26 related duplicated gene pairs were identified (Table 3), which cover most of the 30 sister pairs. Among the 114 PbPRX genes identified, a total of 26 gene pairs (46 genes) were localized to WGD/segmentally duplicated regions, while there is no gene in tandem repeats. These results indicate that WGD/segmental duplications were the main contributors to the expansion of the pear PRX family. To explore the selection pressures among PbPRX duplicated genes, we calculated the Ka, Ks, and Ka/Ks values for all the PRX gene pairs. The results were not shown except for 26 gene pairs (Table 3). In general, Ka/Ks > 1 indicates positive selection, Ka/Ks = 1 indicates neutral selection, and Ka/Ks < 1 indicates negative or purifying selection. The Ka/Ks ratios of most PbPRX gene pairs were <1, suggesting that these gene pairs evolved under purifying selection in pears. The results of this Ka/Ks analysis suggest that negative or purifying selection was vital to the functional divergence of PbPRX genes.

Chromosomal Locations, Gene Duplication, and Collinearity Analyzes
To determine the genome organization and distribution of PbPRXs on different chromosomes in pears, a chromosome map was constructed. The results show that the 99 PbPRX genes are distributed on 17 chromosomes with a nonrandom distribution, and 15 PbPRX genes are mapped onto the other 10 scaffolds, as shown in Table 1. Chromosome 3 contains the most PbPRX genes (13), followed by chromosome 7 (11) and chromosome 8 (11). By contrast, only one PbPRX gene is present on chromosomes 1 and 14. In addition, some chromosomes exhibit a relatively high density of PbPRX genes, such as the bottoms of chromosomes 3 and 7 and the top of chromosome 10. Gene duplication, including segmental or whole-genome duplication (WGD) and tandem duplication, is considered to be one of the primary driving forces in the evolution of genomes [32,33]. During the evolution of a gene family, tandem duplication and WGD/segmental duplication play important roles in generating new members. Therefore, in order to clarify the potential mechanism of evolution of the PRX gene family, both tandem duplication and segmental duplication events were investigated in this study. In this study, 26 related duplicated gene pairs were identified (Table 3), which cover most of the 30 sister pairs. Among the 114 PbPRX genes identified, a total of 26 gene pairs (46 genes) were localized to WGD/segmentally duplicated regions, while there is no gene in tandem repeats. These results indicate that WGD/segmental duplications were the main contributors to the expansion of the pear PRX family. To explore the selection pressures among PbPRX duplicated genes, we calculated the Ka, Ks, and Ka/Ks values for all the PRX gene pairs. The results were not shown except for 26 gene pairs (Table 3). In general, Ka/Ks > 1 indicates positive selection, Ka/Ks = 1 indicates neutral selection, and Ka/Ks < 1 indicates negative or purifying selection. The Ka/Ks ratios of most PbPRX gene pairs were <1, suggesting that these gene pairs evolved under purifying selection in pears. The results of this Ka/Ks analysis suggest that negative or purifying selection was vital to the functional divergence of PbPRX genes.

Functional Annotation with Gene Ontology
In this study, a total of 109 differentially expressed genes that could be categorized into 25 functional groups were found (Figure 3). The major subcategories were as follows: one for cellular component ('extracellular region'); three for molecular function ('catalytic activity', 'binding', and 'antioxidant activity'); and three for biological process ('metabolic process', 'single-organism process', and 'response to stimulus'). These results indicate that PbPRX genes are mainly functioning in 'catalytic activity', 'binding', 'metabolic process'.

Expression of the PRX Gene Family in Pears
To investigate the transcript pattern of PRX family genes during fruit development, the expression patterns over six developmental stages of the pear fruit, from the early to mature stage, were analyzed using the RNA-seq database available from our previous research [19]. According to publicly available genome-wide transcript profiling data from pear tissues, of the 114 PbPRX genes, only 64 PbPRXs are expressed in fruit. Finally, a hierarchical cluster with the logarithm of average values for the 64 PRX family members was generated. As shown in Figure 4, PRX family genes can be divided into four major groups based on their expression profiles. Group A contained seven PRX genes, all of them exhibited preferential expression in the first two stages, indicating that those genes may play important roles in lignin formation during early fruit development. In addition, 23 PRX genes belong to group B, which showed high expression in the first three stages, among them, PbPRX55 and PbPRX111 had the highest expression levels in the first stage of fruit development. Group C included 13 PRX genes, which showed high expression in the S3 stages, but were almost all lower than the expression of group A and B. Group D consisted of 21 PRX genes that displayed higher expression in the last three stages, and with low expression during the foregoing stage of fruit development.

Functional Annotation with Gene Ontology
In this study, a total of 109 differentially expressed genes that could be categorized into 25 functional groups were found (Figure 3). The major subcategories were as follows: one for cellular component ('extracellular region'); three for molecular function ('catalytic activity', 'binding', and 'antioxidant activity'); and three for biological process ('metabolic process', 'single-organism process', and 'response to stimulus'). These results indicate that PbPRX genes are mainly functioning in 'catalytic activity', 'binding', 'metabolic process'.

Stone Cells and Lignin Content of Pear Fruit during Pear Fruit Development
To investigate the formation of stone cells, we determined the content of stone cells from early fruit set to maturity. We found that the content of stone cells reached a maximum at 49 DAFB, and after that, the stone cells number was reduced. The content of stone cells was at a minimum when the fruit was mature ( Figure 5). Plants 2021, 10, x FOR PEER REVIEW 13 of 18  To investigate the formation of stone cells, we determined the content of stone cells from early fruit set to maturity. We found that the content of stone cells reached a maximum at 49 DAFB, and after that, the stone cells number was reduced. The content of stone cells was at a minimum when the fruit was mature ( Figure 5).
To investigate the formation of lignin during pear fruit development, we determined the content of lignin in the pulp powder of pear. We found that the content of lignin reached its maximum at 35 DAFB ( Figure 6).

Verification of Gene Expression by qRT-PCR
On the basis of the RNA-seq database which combines the content of stone cells and lignin, we found that the expression levels of nine PRX (PbPRX2, PbPRX3, PbPRX6, PbPRX17, PbPRX25, PbPRX27, PbPRX53, PbPRX55, and PbPRX110) genes were closely related to lignin formation during pear fruit development, and may play a more important role than other genes. In order to verify whether these genes were associated with lignin content during pear fruit development, the expression levels of these eleven genes were analyzed by qRT-PCR. We chose the first four stages of pear fruit development which lignin is mostly formed in. Finally, the results of the qRT-PCR analysis indicated that the expression levels of all these nine PRX genes are closely tied with the content change of  To investigate the formation of lignin during pear fruit development, we determined the content of lignin in the pulp powder of pear. We found that the content of lignin reached its maximum at 35 DAFB ( Figure 6).

Stone Cells and Lignin Content of Pear Fruit during Pear Fruit Development
To investigate the formation of stone cells, we determined the content of stone cells from early fruit set to maturity. We found that the content of stone cells reached a maximum at 49 DAFB, and after that, the stone cells number was reduced. The content of stone cells was at a minimum when the fruit was mature ( Figure 5).
To investigate the formation of lignin during pear fruit development, we determined the content of lignin in the pulp powder of pear. We found that the content of lignin reached its maximum at 35 DAFB ( Figure 6).

Verification of Gene Expression by qRT-PCR
On the basis of the RNA-seq database which combines the content of stone cells and lignin, we found that the expression levels of nine PRX (PbPRX2, PbPRX3, PbPRX6, PbPRX17, PbPRX25, PbPRX27, PbPRX53, PbPRX55, and PbPRX110) genes were closely related to lignin formation during pear fruit development, and may play a more important role than other genes. In order to verify whether these genes were associated with lignin content during pear fruit development, the expression levels of these eleven genes were analyzed by qRT-PCR. We chose the first four stages of pear fruit development which lignin is mostly formed in. Finally, the results of the qRT-PCR analysis indicated that the expression levels of all these nine PRX genes are closely tied with the content change of

Verification of Gene Expression by qRT-PCR
On the basis of the RNA-seq database which combines the content of stone cells and lignin, we found that the expression levels of nine PRX (PbPRX2, PbPRX3, PbPRX6, PbPRX17, PbPRX25, PbPRX27, PbPRX53, PbPRX55, and PbPRX110) genes were closely related to lignin formation during pear fruit development, and may play a more important role than other genes. In order to verify whether these genes were associated with lignin content during pear fruit development, the expression levels of these eleven genes were analyzed by qRT-PCR. We chose the first four stages of pear fruit development which lignin is mostly formed in. Finally, the results of the qRT-PCR analysis indicated that the expression levels of all these nine PRX genes are closely tied with the content change of lignin during pear fruit development (Figure 7), none of them were different from RNA-seq data, supporting the reliability of our RNA-seq data. lignin during pear fruit development (Figure 7), none of them were different from RNAseq data, supporting the reliability of our RNA-seq data.

Discussion
Class III peroxidases are plant-specific enzymes that play key roles in the responses to biotic and abiotic stress during plant growth and development, as well as being involved in plant lignification. While systematic and comprehensive whole-genome analyses of PRX gene families in Arabidopsis thaliana, Oryza sativa, and Populus trichocarpa have been reported [7][8][9], a systematic whole-genome study of this family has not previously been reported in pears. The full pear genome sequence serves as a useful tool for analyzing the pear PRX gene family to predict its evolutionary history and function [19]. In this study, we performed a comprehensive analysis of the PRX family genes in pears, including analysis of their phylogeny, gene structures, conserved motifs, chromosomal locations, gene duplication, and expression profiles. The number of PRX genes in pear (114) is higher than that in Arabidopsis (73) and Poplar (93) but slightly lower than that in rice (138), similar to that in maize(119), which indicates that the PRX genes in pear have expanded compared to those in Arabidopsis and poplar. Gene duplications are one of the primary driving forces in the evolution of genomes and genetic systems [33]. An increasing number of studies have shown that segmental duplication was largely responsible for the expansion of pear gene families. In this study, the number of PbPRX genes involved in segmental duplication is much more than that involved in tandem duplication, suggesting that segmental duplications are the main contributors to the expansion of the pear PRX family. By contrast, tandem duplication has contributed significantly to the expansion of

Discussion
Class III peroxidases are plant-specific enzymes that play key roles in the responses to biotic and abiotic stress during plant growth and development, as well as being involved in plant lignification. While systematic and comprehensive whole-genome analyses of PRX gene families in Arabidopsis thaliana, Oryza sativa, and Populus trichocarpa have been reported [7][8][9], a systematic whole-genome study of this family has not previously been reported in pears. The full pear genome sequence serves as a useful tool for analyzing the pear PRX gene family to predict its evolutionary history and function [19]. In this study, we performed a comprehensive analysis of the PRX family genes in pears, including analysis of their phylogeny, gene structures, conserved motifs, chromosomal locations, gene duplication, and expression profiles. The number of PRX genes in pear (114) is higher than that in Arabidopsis (73) and Poplar (93) but slightly lower than that in rice (138), similar to that in maize(119), which indicates that the PRX genes in pear have expanded compared to those in Arabidopsis and poplar. Gene duplications are one of the primary driving forces in the evolution of genomes and genetic systems [33]. An increasing number of studies have shown that segmental duplication was largely responsible for the expansion of pear gene families. In this study, the number of PbPRX genes involved in segmental duplication is much more than that involved in tandem duplication, suggesting that segmental duplications are the main contributors to the expansion of the pear PRX family. By contrast, tandem duplication has contributed significantly to the expansion of this gene family in poplar [8]. According to the above analysis, we speculate that the expansion of the PRX gene family differed between monocotyledons and eudicotyledons.
The Ka/Ks ratios of the 26 duplicated pairs show that purifying selection may be largely responsible for maintaining the functions of pear PRX proteins.
Phylogenetic analysis of the PbPRX gene family revealed that the exon/intron structures and motif compositions of these genes are relatively conserved. The 114 PbPRX genes contain different numbers of introns/exons, with most PRX genes containing more than two introns, indicating that there is some diversity in the pear PRX gene family. It is well known that the structural diversity of genes drives the evolution of multigene families. Furthermore, the differences in these characteristics detected between different subfamilies suggest that pear PRX members are functionally diversified.
Many studies have shown that introns were specifically inserted into plants and were retained in the genome during the course of evolution [34]. Therefore, we speculate that introns were gained or lost from the PRX coding region in a subfamily-specific manner.
Furthermore, MEME analysis revealed that different conserved motifs are present in each of the pear PRX proteins. However, some motifs with unknown functions are present in nearly every subgroup, these motifs might play important roles in the PbPRX family.
PRXs can catalyze the reduction of H 2 O 2 by moving electrons to it and receiving electrons from various donor molecules. and are involved in several important physiological and developmental processes, including lignin and suberin formation, the cross-linking of cell wall components, wound healing, the removal of H 2 O 2 , the oxidation of toxic reductants, and defense against pathogen or insect attack. The result of Gene Ontology (GO) analysis indicates that 'catalytic activity', 'antioxidant activity', and 'response to stimulus' are the main functions of the PbPRX gene family, which corresponds to the acknowledged function of the peroxidase enzyme.
Gene expression patterns can provide important clues about gene function. We used publicly available genome-wide transcript profiling data from pear tissues as a resource to investigate the expression patterns of PbPRXs [19]. Of the 114 PbPRX genes, only 64 PbPRXs are expressed in fruit. According to the hierarchical cluster, 64 PbPRXs can be divided into ABCD four groups. We speculate that there are functional divergences in the four groups. PRX genes in Group A and B are smainly expressed at the first three stages of fruit development, which are major stages of lignification. So we speculate these genes are more important in lignification.
The content of stone cells in our study reached a peak at 49 DAFB, which agrees with the study carried out by Tao etc. [10]. The content of lignin increased at the earlier stage, the peak value was 35 DAFB, which is ten days earlier than that of stone cells. As is known to all, Stone cells mainly consist of lignin and cellulose. Lignin was accumulating in the cell wall, the secondary cell wall was then thickened constantly and eventually turned into stone cells, so that the content of stone cells reached a maximum after ten days. According to the RNA-seq database and qPCR, we found that PbPRX2, PbPRX3, PbPRX6, PbPRX17, PbPRX25, PbPRX27, and PbPRX55 are likely to be involved in the lignification of pear stone cells.

Conclusions
In summary, the PRX family contains a large group of genes with essential functions in various developmental processes in plants. This study provides a foundation for further studying the functions of PbPRX genes, particularly for members with potentially important functions in lignification. However, further experiments should be conducted to directly examine the functions of PbPRX genes and their potential regulatory factors, including external cultivation and internal genetic factors.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10091874/s1, Table S1 List of PbPRX and internal control genes primers used for qPCR gene expression.
Author Contributions: S.T. contributed to the experimental design and management, data analysis, and paper review. Z.X. and W.R. contributed to the performance of laboratory work and manuscript preparation. Y.Y., X.S. prepared the plant materials and performed tissue collection. X.L., X.G. and J.B. contributed to the data analysis and laboratory materials preparation during the experiments. S.Z. and K.S. provided suggestions and reviewed and modified this paper. All authors read and approved the published version of the manuscript.