In Silico Genome-Wide Analysis of Respiratory Burst Oxidase Homolog (RBOH) Family Genes in Five Fruit-Producing Trees, and Potential Functional Analysis on Lignification of Stone Cells in Chinese White Pear

The accumulation of lignin in fruit has a significant negative impact on the quality of fruit-producing trees, and in particular the lignin formation stimulates the development of stone cells in pear fruit. Reactive oxygen species (ROS) are essential for lignin polymerization. However, knowledge of the RBOH family, a key enzyme in ROS metabolism, remains unknown in most fruit trees. In this study, a total of 40 RBOHs were identified from five fruit-producing trees (Pyrus bretschneideri, Prunus persica, Citrus sinensis, Vitis vinifera, and Prunus mume), and 10 of these sequences came from Pyrus bretschneideri. Multiple sequence alignments revealed that all 10 PbRBOHs contained the NADPH_Ox domain and the six alpha-helical transmembrane domains (TM-I to TM-VI). Chromosome localization and interspecies phylogenetic tree analysis showed that 10 PbRBOHs irregularly distributed on 8 chromosomes and 3 PbRBOHs (PbRBOHA, PbRBOHB, and PbRBOHD) are closely related to known lignification-related RBOHs. Furthermore, hormone response pattern analysis showed that the transcription of PbRBOHs is regulated by SA, ABA and MeJA. Reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptome sequencing analysis showed that PbRBOHA, PbRBOHB, and PbRBOHD accumulated high transcript abundance in pear fruit, and the transcriptional trends of PbRBOHA and PbRBOHD was consistent with the change of stone cell content during fruit development. In addition, subcellular localization revealed that PbRBOHA and PbRBOHD are distributed on the plasma membrane. Combining the changes of apoplastic superoxide (O2.−) content and spatio-temporal expression analysis, these results indicate that PbRBOHA and PbRBOHD, which are candidate genes, may play an important role in ROS metabolism during the lignification of pear stone cells. This study not only provided insight into the molecular characteristics of the RBOH family in fruit-producing trees, but also lays the foundation for studying the role of ROS in plant lignification.


Introduction
Pyrus spp. is one of the world's most important deciduous fruit trees. Asiatic pears or Oriental pears are mainly distributed in East Asia, and the main cultivars include P. bretschneideri, P. sinkiangensis, P. sinkiangensis, P. ussuriensis and P. pyrifolia [1][2][3]. Among them, Pyrus bretschneideri cv. Dangshan Su is the largest diploid (2n = 34) pear species in China. However, 'Dangshan Su' has the defect of a high content of large-diameter stone cell cluster in the fruit, which seriously affects its quality and economic value [4][5][6]. In addition, the large diameter and high content of stone cell clusters are also the "common problems" of some P. ussuriensis and P. pyrifolia varieties [1]. Therefore, reducing the size and content of the stone cell cluster of pear fruit and improving the fruit quality is an urgent problem to be solved in the pear industry.
Stone cells (also known as sclereids) exist in the xylem, phloem, leaves, fruits and seeds of trees, with support and protection functions [7,8]. The stone cells of pears accumulate specifically in the fruit, which is formed by the thickening and lignification of the secondary cell wall of the parenchyma cells [9][10][11]. The multiple aggregated stone cells are composed of stone cell clusters. At present, a large number of studies have shown that the size, content and density of stone cell clusters not only have a significant negative impact on the texture of pear fruit, but also have a close relationship with its sucrose content [6,[12][13][14].
The average content of lignin in pear stone cells is approximately 37%, and the highest is approximately 44% [15]. Pear stone cells can not only undergo lignin-specific color reaction with phloroglucinol-HCl (Wiesner staining), but also have autofluorescence [16,17]. Transmission electron microscopy (TEM) observations revealed a large amount of lignin deposition in the cell corners, compound middle lamellas and secondary cell walls of pear stone cells [18,19]. It can be seen that lignin is one of the key components of pear stone cells, and the biosynthesis of lignin is closely related to stone cell development [20,21]. It is worth noting that lignin synthesis not only has a negative impact on the texture of pear fruit, but also has a significant effect on the intrinsic quality of peach (Prunus persica), loquat (Eriobotrya japonica), pomegranate (Punica granatum), sweet orange (Citrus sinensis), and other fruit trees [22][23][24][25][26][27]. Therefore, studying lignin metabolism is important for fruit-producing trees.
The polymerization mechanism of lignin monomers is still unclear. Previous studies have speculated that lignin monomer-coupled polymeric proteins mainly include dirigent (DIR), peroxidase (POD), laccase (LAC), superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases [8]. Notably, reactive oxygen species (ROS) play an important role in the lignification of plant cells [8,[28][29][30][31]. Plant NADPH oxidase, also known as respiratory burst oxidase homolog (RBOH) [28], is a homolog of the mammalian macrophage NADPH oxidase catalytic subunit gp91 phox [8,32]. It catalyzes the production of O 2 . − , NADP + and H + by O 2 and NADPH. Subsequent O 2 . − can be catalyzed as a substrate for SOD (EC 1.15.1.1) to form hydrogen peroxide (H 2 O 2 ) and molecular oxygen (O 2 ). H 2 O 2 and O 2 participate in the polymerization of lignin monomers as a second substrate for POD and LAC, respectively [8,28,29]. Finally, POD and LAC activate the lignin monomer radicals, resulting in a terminal polymerization of the free radical oligo/monomers with the extension units, and finally the polymerization is coupled to form a long chain of lignin polymer [8,28,33]. Currently, although the functional study of RBOH in biotic and abiotic stress is not uncommon in the field of stress biology, the role of RBOH in plant lignin synthesis and cell wall development has not been well-investigated until now. In addition, the DIR, POD, LAC, and SOD families related to lignin polymerization in pears have been identified [4,[34][35][36], but the study of the RBOH family of pears has not been reported.
To this end, we identified the RBOH family at the genome-wide level of five fruit-producing trees, Pyrus bretschneideri, Prunus persica, Citrus sinensis, Vitis vinifera, and Prunus mume, and systematically analyzed molecular evolution, chromosomal localization, microsynteny, conserved domains, gene structures and other aspects. Secondly, through phylogenetic tree clustering, gene expression profiling and determination of reactive oxygen species, the lignification-related RBOHs in pears were screened and subcellular localization analysis was performed. This study not only clarified the molecular characteristics of the five fruit-producing tree RBOH families, but also laid the foundation for the later study of lignification of pear stone cells and improvement of fruit texture.
The species phylogenetic tree was obtained from Common Taxonomy Tree (https://www.ncbi.nlm. nih.gov/Taxonomy/CommonTree/wwwcmt.cgi). All phylogenetic trees in this study were completed using MEGA v. 5.1 software. The construction parameters are as follows: Statistical Method: Neighbor-joining; Test of phylogeny: Bootstrap method; No. of Bootstrap Replications: 1000; Model: p-distance; Gaps: Pairwise deletion.

RBOH Family Genes' Physical Localization and Gene Duplications
Visualization of the chromosomal location of the RBOHs was done using MapInspect v.1 software [46,47]. The identification of tandem duplication and segmental duplication events is based on the method used as previously described [16].
A sequence of 1500 bp upstream of the start codon of each PbRBOH coding region (CDS) was extracted from the pear genome as a putative promoter. The identification of the type and distribution of cis-acting elements was carried out using the software PLANT CARE v. 1 (http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Plant Materials and Treatments
The annual stem segments, buds, flowers, mature leaves and fruits were sampled from 40-year-old Pyrus bretschneideri cv. Dangshan Su trees were grown in the orchard of Dangshan County, Anhui Province, China. We collected fruits at eight developmental stages: 15 days after flowering (DAF), 39 DAF, 47 DAF, 63 DAF, 79 DAF, 102 DAF and 145 DAF (mature period). For exogenous hormone treatment of pear fruit, pear trees with no pests and diseases, with the same age and plant height, were selected. Hormones were sprayed on the entire surface of the pear fruit using a sprayer at 39 DAF. The concentration of the hormone treatment [0.5 mmol/L abscisic acid (ABA), 0.5 mmol/L methyl jasmonate (MeJA), or 0.2 mmol/L salicylic acid (SA)] was determined according to Cheng et al. (2018 and [4,5]. Samples were collected at 0, 1, 2, and 3 h post-treatment (HPT), and pear fruits of uniform size were harvested at each time point and immediately frozen in liquid nitrogen.

RNA Extraction and Reverse Transcription-Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) Analysis
RNA extraction and reverse transcription were performed according to the instructions in the Plant RNA maxi kit (Biomiga, San Diego, CA, USA) and the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co., Beijing, China), respectively. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis was performed using TransStart Probe qPCR SuperMix (TransGen Biotech Co.) and detected using a CFX96 Touch™ Real-Time PCR Detection System (Bio-rad Laboratories, Inc., Singapore, Singapore). Relative expression levels were calculated using the 2 − Ct method [50]. All RT-qPCR primers are listed in Table S1. In this study, Tubulin (accession No. AB239680.1) was used as an internal reference [4]. The transcriptome sequencing data have been deposited in the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under accession numbers SUB2967341 [6].

Subcellular Localization of PbRBOHs
The specific primers (Table S1) were designed to amplify the PbRBOH (PbRBOHA and PbRBOHD) CDS, and two restriction sites (Xba I, Sma I) were introduced at both ends according to the information provided for the eukaryotic expression vector pCAMBIA1305-GFP. By enzyme digestion and ligation, the plant expression vectors pCAMBIA1304-PbRBOHA/D-GFP were obtained. The vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation, and 4-6-week-old Nicotiana benthamiana were used for the subcellular localization analysis. The method of subcellular localization was based on Cheng et al. (2017) [16].

Identification, Characterization and Genomic Distribution of RBOH Family Genes in Five Fruit-Producing Trees
BLASTp was performed in the genome database of five fruit-producing trees using the amino acid sequence of AtRBOHA~J as the query sequence [51]. After deleting the repeat sequence and the redundant sequence, it was finally determined that there were 10 RBOHs in Chinese white pear (PbRBOH), and 7 RBOHs in peach (PpRBOH), 8 RBOHs in mei (PmRBOH), 7 RBOHs in common grape vine (VvRBOH), and 8 RBOHs in sweet orange (CsRBOH). We named the 40 RBOHs separately and analyzed their amino acid properties ( Table 1). The 40 predicted full-length RBOH proteins varied from 116 (PmRBOHA) to 1067 (PmRBOHE) amino acid residues and the relative molecular mass ranged from 13.74 (PmRBOHA) to 121.09 (PmRBOHE) kDa, with isoelectric points in the range of 4.58-9.37. To expand knowledge of the RBOH family of the five fruit-producing trees, we compared the genome size, chromosome number and RBOH family distribution of the five fruit-producing trees from the common hexaploid ancestor [26]. We constructed a species phylogenetic tree and performed statistics on the number of RBOHs (Figure 1). The results showed that the range of the number (7-10) of RBOH family members among the five species was small. The percentage of putative RBOHs in the total number of predicted genes in five fruit-producing trees was also similar. The number of genes per megabase (Mb) ranged from 0.015 (common grape vine) to 0.034 (mei). This indicates that the number of RBOH family members has not increased due to the size of the species genome. Therefore, the expansion of these five fruit-producing tree RBOH families was not associated with an increase in the genome size and in the total number of predicted genes.

Chromosome Distribution and Duplication Events of RBOHs in Five Fruit-Producing Trees
In order to clarify the distribution of RBOH on the chromosomes of five fruit trees, we used MapInspect v.1 software to map the location of RBOH family members ( Figure S1). The RBOH family members of the five fruit trees showed an irregular distribution on the chromosome and did not form a large number of gene clusters. According to the phylogenetic tree clustering of each RBOH family ( Figure S2) and the rule of determining gene duplication events [16], only two segmental duplication events were identified in the pear RBOH family. The segmental duplicated gene pairs are PbRBOHA/PbRBOHB and PbRBOHH/PbRBOHG, respectively ( Figure S1).

Phylogenetic Relationship, Conserved Motifs and Intron/Exon Structures of RBOHs in Five Fruit-Producing Trees
Through systematic phylogenetic tree analysis, 40 non-repetitive RBOHs derived from five fruit-producing trees can be divided into four subfamilies (I, II, III and IV). In addition, CsRBOF forms an independent clade. Except for subfamily IV, members of subfamily I-III are composed of RBOHs of five fruit-producing trees. Eleven gene pairs were formed among RBOHs with higher bootstrap values (≥99), except for the lower bootstrap values of two pairs: CsRBOHB/VvRBOHD and CsRBOHH/VvRBOHF (Figure 2A).
To investigate the structural diversity of RBOHs, a total of 20 conserved motifs in the RBOHs were captured by MEME v. 5.05 software ( Figure 2B and Table S2). It is worth noting that the type and distribution of the C-terminal domains (motifs 3, 15, 2, 6, 10, 1, and 12) of most RBOHs are similar. However, the type and distribution of the N-terminal domains exhibit a certain subfamily specificity. For example, motif 16 is only present in members of subfamilies I and II. Members of subfamily IV do not contain motif 13. Gene structure analysis showed that the number of introns of RBOHs varied from 3 to 16 ( Figure 2C). The exons of the RBOHs of the five fruit trees are all shorter, which may be characteristic of the RBOH family. Generally, most of RBOH family members in the same subfamilies showed similar motif characteristic and exon-intron structure, which supports their close evolutionary relationship and the classification of subfamilies.

Amino Acid Sequence and Characteristic Domain Analysis of PbRBOHs
We further analyzed the analysis of the characteristic domains of the RBOH family of pear ( Figure 3). The results showed that the characteristic domains of the PbRBOH family members mainly include: NADPH_Ox (PF08414), EFh (IPR002048), Ferric_reduct (PF01794), FAD_binding_8 (PF08022), and NAD_binding_6 (PF08030). As can be seen from Figure 3, NADPH_Ox and EFh are mainly distributed at the N-terminus, while Ferric_reduct, FAD_binding_8, and NAD_binding_6 are distributed at the C-terminus. According to the annotated results of SMART (http://smart.emblheidelberg.de/smart/show_motifs.pl), Ferric_reduct domain is a common region in the transmembrane proteins mammalian cytochrome B-245 heavy chain (gp91-phox). Plant RBOH encode homologs of the mammalian gp91 phox [52], therefore, each PbRBOH contains this domain. NADPH_Ox is a characteristic domain of plant respiratory burst NADPH oxidase proteins, and PbRBOHA-J all have this domain, further demonstrating that the PbRBOH family members we identified are reliable. Interestingly, only PbRBOHJ lacks the NAD_binding_6 domain, which may cause changes in its function. In addition, multiple sequence alignments of 10 PbRBOHs and 10 AtRBOHs were performed according to previously reported methods [32,53] (Figure 4). It can be found that the amino acid sequences of AtRBOH family members and PbRBOH family members are highly conserved, especially the typical domain of the RBOH family, such as two calcium-binding motifs (EF-hands) and six α-helical transmembrane domains (TM-I to TM-VI), and flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate [NAD(P)H]-ribose, and NAD(P)H-adenine conserved binding sites in the C-terminal region. Notably, two amino acid residues (Pro-415 and Asp-500) in gp91 phox are critical for catalytic activity [53]. However, PbRBOHJ lacks these two sites and the tertiary structure changes significantly, suggesting that the catalytic activity of this member will be affected (Figure 4 and Figure S3).

Microsynteny Analysis of PbRBOHs
To explore the evolutionary relationship of RBOHs, we performed microsynteny analysis of PbRBOHs and the coding genes in their surrounding chromosomal regions ( Figure 5). It was found that there are 6 pairs of collinear gene pairs in the pear genome, including Pbr003403.1/Pbr036006.1, Pbr003403.1/Pbr037399.1, Pbr018609.1/Pbr037815.1, Pbr023445.1/Pbr033955.1, Pbr036006.1/ Pbr037399.1, and Pbr038641.1/Pbr038667.1. These 6 pairs of collinear gene pairs did not undergo chromosomal inversion during duplication ( Figure 5). Pear, sweet orange, mei, peach and grape share a common ancestor [4,26]. To further clarify the evolutionary origin and orthologous relationship of the RBOH family among the five fruit-producing trees, we identified the interspecific collinear relationship of these five species (Spreadsheets S1). Our results suggest that 18 collinear gene pairs (7 collinear gene pairs between pear and grape, 1 pair between pear and sweet orange, 3 pairs between sweet orange and grape, 3 pairs between mei and grape, 3 pairs between peach and grape, 1 gene pair is within the peach) were found among pear, sweet orange, mei, peach and grape (Spreadsheets S1). Overall, the degree of interspecies collinearity of CsRBOHs, PpRBOHs, VvRBOHs, and PmRBOHs is lower than intraspecies collinearity. These results indicate that the RBOH family of four species is relatively independent in evolution, and that chromosome duplication, deletion and rearrangement are later than species differentiation.

Comparative Phylogenetic Analysis and Functional Prediction of PbRBOHs
To characterize the evolutionary relationship and possible functions between RBOHs from five fruit trees and other known RBOHs from Arabidopsis, rice and poplar, a neighbor-joining tree was created ( Figure 6).
The results showed that the 70 RBOHs from eight species (pear, peach, grape, mei, sweet orange, Arabidopsis, rice and poplar) could be assigned to 5 phylogenetic groups (I-V). With the exception of Group V, the other four phylogenetic groups are composed of RBOHs from eight species. Ten PbRBOHs were distributed in five phylogenetic groups, suggesting the existence of a diversified RBOH family in pear with diverse functions. Generally, RBOHs from pear have closer relationships with the PpRBOHs or PmRBOHs than that from VvRBOHs and CsRBOHs, which is in accordance with the current understanding of plant evolutionary history. In addition, evolutionary analysis also identified some closely related orthologous RBOHs between peach and mei, indicating that an ancestral set of RBOHs existed prior to the divergence of peach and mei. For members of Group I and III, AtRBOHD and AtRBOHF are considered to be closely related to lignification, and they are also involved in the plant's stress response process, which has a significant impact on the production of ROS in plants [8,28,31,54,55]. Phylogenetic tree clustering results indicated that RBOHA, RBOHB, and RBOHD in pears belonged to the same clades as AtRBOHD and AtRBOHF, suggesting that they may have similar biological functions ( Figure 6).
It has now been demonstrated that AtRBOHC mutations result in inhibition of Arabidopsis root and root hair growth, and the level of ROS in plants is significantly reduced [54]. In addition, AtRBOHA, -B, and -G are all specifically expressed in the root [37]. It is speculated that RBOHs that are closely related to them may play a role mainly in the roots.
The functions of members in Group II and IV are currently poorly understood. AtRBOHH and -J are specifically expressed in pollen, AtRBOHE and OsRbohD are expressed in root and seeds [37]. Therefore, whether PbRBOHs of the same group has similar expression patterns and functions remains to be further verified. Group V contains only 5 RBOHs, while other groups contain at least 11 RBOHs. The five RBOHs in this group are derived from pear, sweet orange, mei and peach, respectively. It is speculated that these five RBOHs (CsRBOHD, CsRBOHF, PbRBOHI, PmRBOHG, and PpRBOHD) are a unique class of these four species, which may have new unknown functions.

Analysis of Cis-Acting Elements in Putative PbRBOH Promoters
Previous studies have shown that RBOH plays an important role in plant stress response, and their expression is regulated by a variety of plant hormones [37,56,57]. To this end, cis-acting elements of 10 PbRBOH promoter regions were identified in this study to explore their possible expression regulation mechanisms (Spreadsheets S2).
Analysis of the results showed that except for PbRBOHG, the other nine members contained G-box elements, which could respond to a variety of hormones, including ABA and MeJA [58]. In addition, W-box elements that can respond to SA, MeJA and ethylene were identified in the promoter regions of PbRBOHG, PbRBOHA, PbRBOHH, and PbRBOHC [5]. We found that 70% of the PbRBOH family members have ABRE (ABA-responsive) in their promoters and 50% of members contain TGACG-motif (MeJA-responsive). In addition to the hormone response elements, we also identified some abiotic stress response elements in the PbRBOH promoter region, such as LTR (low-temperature responsiveness), MBS (drought-inducibility), and ARE (anaerobic induction). Therefore, the transcription of PbRBOH is likely to be multi-regulated by hormones and the environment.

Differentially Expressed PbRBOHs under Hormonal Treatments
Based on the analysis of the cis-acting elements in the promoters of the PbRBOH family members, we found that most of the promoters of PbRBOHs contain a variety of plant hormone response-related elements (Spreadsheets S2). Therefore, we used three exogenous hormone (SA, ABA, and MeJA)-treated pear fruits as materials to analyze the effect of hormone treatment on PbRBOH expression patterns.
For SA-treated pear fruits, it was found that the transcription levels of PbRBOHC and PbRBOHJ were inhibited, and the remaining members peaked at 1 to 3 HPT. Among them, PbRBOHA, PbRBOHE, PbRBOHH and PbRBOHI showed a rise-fall tendency. PbRBOHB, PbRBOHF, PbRBOHG and PbRBOHD peaked at 3 HPT (Figure 7). In the MeJA-treated pear fruits, the transcription levels of PbRBOHE and PbRBOHJ were inhibited, and the expression of other members was induced to varying degrees. Moreover, the transcriptional abundance of most members peaked at 1 HPT and 2 HPT, and decreased at 3 HPT (Figure 7). ABA plays an inducing role in the transcription of 10 PbRBOHs. Among them, the expression levels of PbRBOHA and PbRBOHB reached a peak at 1 HPT, and then basically returned to the pre-treatment level. PbRBOHC, PbRBOHI and PbRBOJ have the highest expression at 2 HPT. The inducing effect of ABA on PbRBOHD lasted for a long time, and the transcript abundance of PbRBOHD from 1 to 3HPT was maintained at a high level. In addition, the transcript levels of PbRBOHG, PbRBOHF, PbRBOHE, and PbRBOHH were gradually increased after ABA treatment, reaching a peak at 3 HPT (Figure 7).

Tissue Specificity and Temporal Expression Patterns in Pear Fruit during Development
Our initial interest in PbRBOHs came from the proposed role of PbRBOHs in cell wall lignification. In order to screen out RBOHs involved in the production of reactive oxygen species during the lignification of stone cells, we analyzed the expression patterns of 10 PbRBOHs in different developmental stages of pear fruit. As can be seen from Figure 9, the transcriptional levels of PbRBOHA, PbRBOHD, and PbRBOHF showed a rise-fall tendency, consistent with changes in the content of stone cells and O 2 . − . This suggests that they may be involved in the production of ROS during the lignification of stone cells.
Subsequently, we used the fruit transcriptome database of two Chinese white pear (Pyrus bretschneideri) cultivars that were previously completed [35] to analyze the transcript abundance of 10 PbRBOHs in different developmental stages of pear fruit ( Figure 10). The fragments per kilobase of exon per million fragments mapped (FPKM) values for each PbRBOH are listed in Table S3. The results showed that although the expression changes of some members in pear fruit were consistent with the content of stone cells and O 2 . − , only PbRBOHA, PbRBOHB, and PbRBOHD had higher transcript abundance in fruits. The remaining members are weakly expressed in the fruit and may not play a major role in the fruit, such as PbRBOHF.  Therefore, combined with the results of phylogenetic tree clustering, trend of expression changes and transcript abundance, this study speculated that PbRBOHA and PbRBOHD are mainly responsible for the supply of ROS during the lignification of pear stone cells.
For buds, stems, leaves and flowers, except for PbRBOHC, the other 9 PbRBOHs expressed the highest in leaves. PbRBOHB and PbRBOHD also have higher levels of transcription in stems ( Figure 11).

Subcellular Localization Analysis of Candidate PbRBOHs for Pear Stone Cell Lignification
Previous studies have shown that the RBOH is mainly localized on the plasma membrane and is responsible for catalyzing the formation of O 2 . − and thus participating in the polymerization of lignin monomers [8,37]. In order to clarify the subcellular distribution of the candidate genes, the eukaryotic expression vectors of PbRBOHA and PbRBOHD were constructed and transiently transformed into tobacco leaves. Green fluorescent protein (GFP) fluorescence was observed by laser scanning confocal microscopy (LSCM) (Olympus, Tokyo, Japan). As can be seen from Figure 12, both PbRBOHA-GFP and PbRBOHD-GFP are localized on the plasma membrane.

Discussion
There are five main types of lignified cells currently known in plants, including tracheary elements (TEs), sclerenchyma cells, endodermal cells, and seed coat cells [8]. Lignified cells are critical to plants, including resistance to external environmental conditions, seed protection, mechanical support, water and nutrient transport [7,8]. However, excessive lignification of the cells can have many negative effects on the fruit quality of trees [18,23,25,59]. Therefore, studying the molecular mechanism of lignification is of great significance for regulating fruit quality.
Numerous studies have shown that ROS play an important role in the lignification process [28,31]. However, the research on the RBOH family of catalytically lignification-related ROS production in fruit trees has rarely been reported. For this reason, 40 RBOHs were screened from the genome database of five fruit trees. Among them, the number of PbRBOH family members was the highest, and the number of PpRBOH family members was the least (Table 1). Phylogenetic tree analysis showed that the RBOH family members of pear, mei and peach were closely related, but they were far from sweet orange and grape, probably because the first three species belonged to Rosaceae (Figure 2). In particular, pear, mei, peach and sweet orange all differentiated into a single group (Group V) RBOH family members. The members of this subfamily are very distantly related to the RBOH members of poplar, rice, grape and Arabidopsis ( Figure 6). It may already have completely different biological functions.
The formation of pear stone cells is a typical lignification process [19]. It provides a good model for the formation of lignification-related ROS. Therefore, this study focused on the content of O 2 . − in pear fruit and the expression pattern of PbRBOHs. The distribution of stone cell clusters in different developmental stages of pear fruit has been revealed in our previous studies [16]. The results show that in the fruit of 39 DAF to 55 DAF, the stone cell cluster accumulates in the pulp between the fruit core and the peels. As expected, NBT staining is the deepest in the pear fruit at this stage of development, suggesting that a large amount of O 2 . − is produced at this time to promote stone cell development and lignification (Figure 8). Peroxidase and laccase catalyzed the formation of monolignol radicals must have H 2 O 2 and O 2 as additional substrates, while O 2 . − is H 2 O 2 and O 2 direct precursor [8,29], so the content of O 2 . − directly affects the lignification polymerization process.
Analysis of cis-acting elements revealed that most of the PbRBOH promoters contained a large number of elements related to hormones and stress response (Spreadsheets S2). This is consistent with previous studies [57]. We selected three hormones (SA, ABA, and MeJA) to treat pear fruit and found that it has a significant effect on the expression of PbRBOHs, and the transcription of most PbRBOHs is induced in a short time (Figure 7). Notably, not all of the 10 PbRBOH promoters have elements that respond to the three hormones. For example, the SA response element has not been found in the PbRBOHI promoter. However, the expression level of PbRBOHI still changed in the fruits treated with SA (Spreadsheets S2 and Figure 7). This may be due to interactions between various plant hormones in plants, which can promote synergy and induce one another [60]. Therefore, spraying a hormone is likely to induce an increase in the content of another hormone, thereby affecting the transcription of the gene. In addition, photoresponsive elements such as Box4, GATA-motif, GT1-motif are also present in the RBOH promoters. This suggests that bagging treatment changes the lignin and stone cell content of pear fruit may be related to the transcription of RBOH [61,62].
Importantly, in order to identify the RBOHs responsible for lignification-related ROS synthesis in pear fruit, we compared the expression patterns of 10 PbRBOHs in pear fruit at 7 developmental stages ( Figure 9). Phylogenetic tree clustering indicated that PbRBOHA, PbRBOHB, and PbRBOHD were most likely related to lignification, and transcriptome sequencing also showed that the transcript abundance of PbRBOHA, PbRBOHB and PbRBOHD was the highest among the 10 members in different pear varieties (Figure 10). It is indicated that these three RBOHs play a role in pear fruit. However, the expression level of PbRBOHB showed a downward trend during the large accumulation period of stone cell (47 DAF and 55 DAF) (Figure 9) [10,16], and the expression level was up-regulated after the stone cell development was basically stopped (63 DAF) [10], which was inconsistent with the process of stone cell development. It is speculated that although this gene has a high expression level in fruits, it is not the major gene involved in the synthesis of ROS during the development of stone cells. In addition, the expression levels of PbRBOHA, PbRBOHD and PbRBOHF in pear fruit showed a rise-fall tendency, which was consistent with the trend of stone cell content ( Figure 9) [10,61,63]. However, transcriptome sequencing showed that PbRBOHF has a weak transcription level in fruits (Figure 10), so it is speculated that this gene has little correlation with stone cell development. Therefore, PbRBOHA and PbRBOHD are considered to be lignification-related RBOH in pears. Notably, the up-regulation of PbRBOHA is most significant at 55 DAF ( Figure 10 and Table S3), suggesting that the gene may be the major gene responsible for lignification-related ROS production.
More strikingly, we summarized the results of this study with the reported pear uridine diphosphate-glycosyltransferases (UGTs), PODs, LACs and DIRs in an ideograph of the polymerization of lignin monomer in pear fruit ( Figure 13) [4,19,35,36]. The monolignols in pear fruit may be catalyzed by PbUGT72AJ1, PbUGT72AJ2 and PbUGT72AK1 to form lignin glycoside, which is then transported across the membrane to cell wall deposition. Subsequently, the lignin oligomers are formed by the catalysis of PbDIR4. Finally, POD and LAC use ROS and O 2 produced by RBOH (PbRBOHA and PbRBOHD) and SOD to catalyze the elongation of lignin polymer chains.

Conclusions
In this study, we identified 10, 7, 8, 8 and 7 members of the RBOH family from the pear, peach, mei, sweet orange and grape genomes, respectively. RBOHs derived from fruit-producing trees can be divided into five subfamilies, and the distribution of conserved motifs and gene structures have a certain subfamily specificity. The 10 RBOH family members of the pear all have six α-helical transmembrane domains, and there are a large number of cis-acting elements related to biotic and abiotic stress in the promoter. Hormone treatment revealed that the transcription of most PbRBOHs was induced by SA, ABA and MeJA. Analysis of spatio-temporal expression patterns showed that PbRBOHA, PbRBOHB, and PbRBOHD had higher transcript abundance in pear fruit, and the expression trends of PbRBOHA and PbRBOHD were consistent with the content of stone cells and O 2 . − . Combined with the results of phylogenetic tree clustering and expression pattern analysis, it is speculated that PbRBOHA and PbRBOHD participate in the production of reactive oxygen species during the formation of stone cells. Subcellular localization analysis indicated that both RBOHs were localized to the plasma membrane.