Genome-Wide Identification of PEBP Gene Family in Solanum lycopersicum

The PEBP gene family is crucial for the growth and development of plants, the transition between vegetative and reproductive growth, the response to light, the production of florigen, and the reaction to several abiotic stressors. The PEBP gene family has been found in numerous species, but the SLPEBP gene family has not yet received a thorough bioinformatics investigation, and the members of this gene family are currently unknown. In this study, bioinformatics was used to identify 12 members of the SLPEBP gene family in tomato and localize them on the chromosomes. The physicochemical characteristics of the proteins encoded by members of the SLPEBP gene family were also examined, along with their intraspecific collinearity, gene structure, conserved motifs, and cis-acting elements. In parallel, a phylogenetic tree was built and the collinear relationships of the PEBP gene family among tomato, potato, pepper, and Arabidopsis were examined. The expression of 12 genes in different tissues and organs of tomato was analyzed using transcriptomic data. It was also hypothesized that SLPEBP3, SLPEBP5, SLPEBP6, SLPEBP8, SLPEBP9, and SLPEBP10 might be related to tomato flowering and that SLPEBP2, SLPEBP3, SLPEBP7, and SLPEBP11 might be related to ovary development based on the tissue-specific expression analysis of SLPEBP gene family members at five different stages during flower bud formation to fruit set. This article’s goal is to offer suggestions and research directions for further study of tomato PEBP gene family members.


Introduction
A highly conserved protein known as the phosphatidylethanolamine-binding protein (PEBP) gene family has been found in a variety of biological tissues. These proteins were initially isolated from the brains of cows and were given the name phosphatidylethanolaminebinding protein due to their strong affinity for phospholipids [1,2]. The structure of a number of discovered PEBP-like family members reveals that PEBP has a conserved domain with a sizable central β-sheet. A small β-sheet and an α-helix make up this large β-sheet [3,4].
The PEBP gene family controls a wide range of biological processes. Some studies have found that PEBP genes can be involved in regulating flowering and growth-related signaling pathways [5,6], and play an important role in plant type differentiation [7]. The PEBP gene family in plants can be divided into three distinct subgroups: the FLOWERING LO-CUS T-like gene, which is thought to be involved in encoding florigen [8,9]; the TERMINAL FLOWER1 (TFL1)-like gene related to inhibiting flowering [7,10]; and the MOTHER OF FT AND TFL1 (MFT)-like gene related to regulating seed germination [11,12]. The Arabidopsis FT, TSF, and tomato SFT genes are FL-like genes. SFT has been demonstrated to produce a signal to guide flowering and play a regulatory role in plant meristem growth [13,14]. TFL1, ATC, and BFT in Arabidopsis and the SP gene of tomato are TFL1-like genes, and SP

Identification of PEBP Gene Family Members in Tomato and Analysis of the Physicochemical Properties of Proteins
Through HMMER 3.0 software analysis and screening with HMMER and SMART online software, 12 PEBP family members were analyzed and named SLPEBP1-12. Basic information about the SLPEBP gene family and the physicochemical properties of its encoded proteins is shown in Table 1. In addition, the named genes are also shown in the table. The shortest SLPEBP gene family coding sequence length is SLPEBP8 at 423 bp and the longest is SLPEBP2 at 699 bp; the average length is 527 bp. SLPEBP family genes are distributed across seven tomato chromosomes. The physicochemical properties of the proteins were predicted by ExPASy online software. The smallest molecular weight was 16.06 for SLPEBP8, and the largest was 24.80 for SLPEBP2. The theoretical isoelectric point ranged from 5.26 (SLPEBP7) to 9.63 (SLPEBP2), and the instability index ranged from 28.46 (SLPEBP10) to 50.26 (SLPEBP9). The maximum aliphatic index value was 92.36 (SLPEBP8), and the minimum value was 72.71 (SLPEBP3). The results of hydrophilicity and hydrophobicity analysis indicated that all SLPEBP family proteins are hydrophilic proteins. The results of subcellular localization of the proteins analyzed using online software showed that all SLPEBP family proteins are cytoplasmic proteins.

Chromosome Localization and Collinearity Analysis of PEBP Family Members in Tomato
The relationship between the location of the SLPEBP gene family on the chromosomes and the collinearity within the species is shown in Figure 1. Members of the SLPEBP family are only partially distributed on the chromosomes of tomato and are scattered, with 12 genes distributed across seven tomato chromosomes, mostly relatively close to the telomeric region of the chromosome. Only SLPEBP5 among the 12 SLPEBP family members is located away from the telomeric region. Among the 12 SLPEBP family members, only SLPEBP5 was in a region with low gene density. The analysis of collinearity between genes demonstrated the replication relationships between genes, and these relationships within species are shown in Figure 1. SLPEBP gene family members have only one collinear relationship within species, i.e., SLPEBP1 with SLPEBP9.
To determine the collinear relationships of the 12 SLPEBP family genes among other species, collinearity analysis was performed using Arabidopsis, potato, and pepper with PEBP family members from tomato. The results of the analysis are displayed in Figure 2. Tomato had 11 collinearities with potato, 10 collinearities with pepper, and 4 collinearities with Arabidopsis. SLPEBP2 and SLPEBP5 had no collinearity with genes from other species. Among the three species, SLPEBP is most closely related to STPEBP. Comparative analysis of the SLPEBP family with other species can provide a research reference for analyzing the genetic relationships and gene functions of species. Collinearity relationship of tomato SLPEBP gene with potato, pepper and Arabidopsis thaliana can be found in Table S1. 12 genes distributed across seven tomato chromosomes, mostly relatively close to the telomeric region of the chromosome. Only SLPEBP5 among the 12 SLPEBP family members is located away from the telomeric region. Among the 12 SLPEBP family members, only SLPEBP5 was in a region with low gene density. The analysis of collinearity between genes demonstrated the replication relationships between genes, and these relationships within species are shown in Figure 1. SLPEBP gene family members have only one collinear relationship within species, i.e., SLPEBP1 with SLPEBP9. To determine the collinear relationships of the 12 SLPEBP family genes among other species, collinearity analysis was performed using Arabidopsis, potato, and pepper with PEBP family members from tomato. The results of the analysis are displayed in Figure 2. Tomato had 11 collinearities with potato, 10 collinearities with pepper, and 4 collinearities with Arabidopsis. SLPEBP2 and SLPEBP5 had no collinearity with genes from other species. Among the three species, SLPEBP is most closely related to STPEBP. Comparative analysis of the SLPEBP family with other species can provide a research reference for analyzing the genetic relationships and gene functions of species. Collinearity relationship of tomato SLPEBP gene with potato, pepper and Arabidopsis thaliana can be found in Table S1

Conserved Motif and Gene Structure Analysis of PEBP Family Members in Tomato
A phylogenetic tree containing only SLPEBP family genes with subfamilies divided into three groups was constructed by MEGA-X, consistent with the previous evolutionary tree results, and based on this tree, we performed conserved motif and gene structure analysis of SLPEBP gene family members ( Figure 4).

Conserved Motif and Gene Structure Analysis of PEBP Family Members in Tomato
A phylogenetic tree containing only SLPEBP family genes with subfamilies divided into three groups was constructed by MEGA-X, consistent with the previous evolutionary tree results, and based on this tree, we performed conserved motif and gene structure analysis of SLPEBP gene family members ( Figure 4). In the gene structure analysis, six SLPEBP family genes contained four exons and two introns, accounting for 50% of the overall SLPEBP gene family members. Nine genes contained four exons, accounting for 75% of the family members. Seven genes contained two In the gene structure analysis, six SLPEBP family genes contained four exons and two introns, accounting for 50% of the overall SLPEBP gene family members. Nine genes contained four exons, accounting for 75% of the family members. Seven genes contained two introns, accounting for 58.3% of the family members. Nine genes contained introns, accounting for 75% of the family members. The majority of SLPEBP gene family members contained four exons, and most of its members contained one or two introns. In the analysis of conserved motifs, 10 conserved motifs were found and named motifs 1-10. All genes except SLPEBP3, SLPEBP6, SLPEBP8, SLPEBP10, and SLPEBP12 contained all 10 conserved motifs, and the 10 motifs were arranged in the same order. Only motifs 3 and 4 are shared by all 12 SLPEBP gene family members. The most complete conserved motif is found in the TFL1 subfamily, followed by the FT subfamily.

Prediction of the Three-Dimensional Structures of SLPEBP Proteins
The structure of the protein encoded by a gene is related to the function of the gene. Homology modeling is a method to construct the three-dimensional structure of a target protein by using the amino acid sequence of the protein as a template for the three-dimensional structure of a protein that has been experimentally validated. Using the homology modeling method, we predicted the 3D structures of 12 SLPEBP proteins. The 3D structures obtained by the homology modeling method for all 11 genes except SLPEBP10 were evaluated as accurate by online software detection and evaluation. As shown in Figure 5, all 11 of these proteins contain a large β-fold, and 10 of them (the exception is SLPEBP8) contain two α-helices. protein by using the amino acid sequence of the protein as a template for the three-dimensional structure of a protein that has been experimentally validated. Using the homology modeling method, we predicted the 3D structures of 12 SLPEBP proteins. The 3D structures obtained by the homology modeling method for all 11 genes except SLPEBP10 were evaluated as accurate by online software detection and evaluation. As shown in Figure 5, all 11 of these proteins contain a large β-fold, and 10 of them (the exception is SLPEBP8) contain two α-helices.

Figure 5.
Three-dimensional structure model of SLPEBP family member proteins. Yellow represents β-fold, pink represents α-helix, cyan represents β-corner, and blue represents irregular curl. The translucent part shows the outer surface of the protein.

Prediction of Cis-Acting Elements of SLPEBP Family Members
Cis-acting elements are important for gene transcription, and in this study, a 2000 bp sequence upstream of each gene was used to predict cis-acting elements in the 12 genes of the SLPEBP family ( Figure 6). A total of 36 cis-acting elements were detected and classified Three-dimensional structure model of SLPEBP family member proteins. Yellow represents β-fold, pink represents α-helix, cyan represents β-corner, and blue represents irregular curl. The translucent part shows the outer surface of the protein.

Prediction of Cis-Acting Elements of SLPEBP Family Members
Cis-acting elements are important for gene transcription, and in this study, a 2000 bp sequence upstream of each gene was used to predict cis-acting elements in the 12 genes of the SLPEBP family ( Figure 6). A total of 36 cis-acting elements were detected and classified into six categories, developmental-associated elements (18), environmental stressrelated elements (27), hormone-responsive elements (84), light-responsive elements (141), promoter-associated elements (1166), and site-binding-associated elements (13), for a total of 1448 predicted cis-acting elements. All SLPEBP genes except the SLPEBP2 gene contained G-box. All family members except SLPEBP4 contained Box 4. Among the cis-acting elements in the promoter region of the SLPEBP family members, the number of lightresponsive elements (141) was the highest. The number of development-related elements was the lowest (18); SLPEBP7 did not contain any development-related elements. Details of cis-acting elements in the promoter region of SLPEBP genes can be found in Table S2.  Thirty-eight kinds of cis-acting elements were divided into six categories. The number in the grid represents the number of elements, and the color from blue to red represents the number of elements from fewer to more.

Expression of SLPEBP Genes in Different Tissues and Organs of Tomato
The results of the analysis are shown in Figure 7. Four genes were significantly expressed in the buds, flowers, and roots, and two genes were significantly expressed in the leaves. Some genes (SLPEBP3, SLPEBP12) were expressed in several tissues, while some genes (SLPEBP8, SLPEBP10) were not found to be expressed in any of the tissues examined. Average FPKM value of SLPEBP genes transcriptome in different tissues of tomato can be found in Table S3.

Expression of SLPEBP Genes in Different Tissues and Organs of Tomato
The results of the analysis are shown in Figure 7. Four genes were significantly expressed in the buds, flowers, and roots, and two genes were significantly expressed in the leaves. Some genes (SLPEBP3, SLPEBP12) were expressed in several tissues, while some genes (SLPEBP8, SLPEBP10) were not found to be expressed in any of the tissues examined. Average FPKM value of SLPEBP genes transcriptome in different tissues of tomato can be found in Table S3.

Analysis of qRT-PCR Gene Expression in the SLPEBP Gene Family
We determined the gene expression of all 12 genes, SLPEBP1 to SLPEBP12, at five different periods of flowering, and the results are shown in Figure 8. Among them, the expression of SLPEBP1 and SLPEBP12 was extremely low and is therefore not shown in the figure. Among the 12 genes, SLPEBP3, SLPEBP5, SLPEBP6, SLPEBP8, SLPEBP9, and SLPEBP10 were expressed at high levels during the flowering period and might be involved in flower opening. The other genes may be related to ovary formation and may play a regulatory role in tomato yield.

Analysis of qRT-PCR Gene Expression in the SLPEBP Gene Family
We determined the gene expression of all 12 genes, SLPEBP1 to SLPEBP12, at five different periods of flowering, and the results are shown in Figure 8. Among them, the expression of SLPEBP1 and SLPEBP12 was extremely low and is therefore not shown in the figure. Among the 12 genes, SLPEBP3, SLPEBP5, SLPEBP6, SLPEBP8, SLPEBP9, and SLPEBP10 were expressed at high levels during the flowering period and might be involved in flower opening. The other genes may be related to ovary formation and may play a regulatory role in tomato yield.

Discussion
The type of plant growth (determinate, indeterminate, and semi-determinate), the time of flowering, the number of flowers, and the development of flower organs are closely related to the yield of tomato. Currently, mechanized and automatic harvesting and processing are gradually replacing traditional manual work, further improving production efficiency and saving time and cost. To adapt to mechanized harvesting, it is necessary to study tomato plant types. Members of the PEBP gene family have obvious functions in plant growth type, flowering period, flower organ development, seed germination, root morphogenesis, and even abiotic stress resistance. Mutants of its genes in this family have been reported to cause changes in tomato flowering, flower morphology, plant growth type, root morphology, etc. However, no article has provided a comprehensive study and summary of the PEBP gene family in tomato to examine the common characteristics of its family members. We predicted 12 genes containing PEBP conserved domains through the tomato genome database, which is consistent with the results of Cao et al. [35] and Moreira et al. [30]. Through data analysis, studies found that the genes in this family are involved in transcriptional regulation; moreover, stress and signal transduction have strong homology and a large gene family [36][37][38], while the genes involved in basic functions have weak homology and a small gene family [39]. The SLPEBP gene family has only 12 members. It can be inferred that the gene function of the SLPEBP gene family is relatively basic. Using the same method, we predicted the PEBP gene family members of

Discussion
The type of plant growth (determinate, indeterminate, and semi-determinate), the time of flowering, the number of flowers, and the development of flower organs are closely related to the yield of tomato. Currently, mechanized and automatic harvesting and processing are gradually replacing traditional manual work, further improving production efficiency and saving time and cost. To adapt to mechanized harvesting, it is necessary to study tomato plant types. Members of the PEBP gene family have obvious functions in plant growth type, flowering period, flower organ development, seed germination, root morphogenesis, and even abiotic stress resistance. Mutants of its genes in this family have been reported to cause changes in tomato flowering, flower morphology, plant growth type, root morphology, etc. However, no article has provided a comprehensive study and summary of the PEBP gene family in tomato to examine the common characteristics of its family members. We predicted 12 genes containing PEBP conserved domains through the tomato genome database, which is consistent with the results of Cao et al. [35] and Moreira et al. [30]. Through data analysis, studies found that the genes in this family are involved in transcriptional regulation; moreover, stress and signal transduction have strong homology and a large gene family [36][37][38], while the genes involved in basic functions have weak homology and a small gene family [39]. The SLPEBP gene family has only 12 members. It can be inferred that the gene function of the SLPEBP gene family is relatively basic. Using the same method, we predicted the PEBP gene family members of Arabidopsis, potato, and pepper, and carried out the same analysis. Seven members of the Arabidopsis PEBP gene family were found. The results of 10 potato PEBP gene family members are different from those of the 15 PEBP gene family members detected by Zhang et al., due to the different potato reference genomes [40]. The results of 11 pepper PEBP gene family members are slightly different from those of the 9 PEBP gene family members detected by Niu et al. due to different reference genomes and methods [41]. We found that the 12 genes of the SLPEBP gene family were unevenly distributed across seven tomato chromosomes (Figure 1), and all of them except SLPEBP5 were distributed at the far end of the chromosome.
Gene replication events often occur in plants. In this way, plants can evolve with higher efficiency [42]. The members of the gene family are all from the same ancestor, and a group of genes that encode similar protein products with similar sequence and structure are formed by mutation, domestication, and selection through gene replication events or copies produced by whole-genome duplication (WGD) [43]. Gene duplication events can be divided into whole-genome duplications and single-gene duplications [44][45][46]. Repeated genes produced by whole-genome duplication are often lost or silenced after a period of time, and plants return to the diploid state quickly [47]. Single-gene duplication can occur in many ways, such as tandem duplication (TD), proximate duplication (PD), diffuse duplication (TRD), and separated duplication (DSD) [48]. More than 60% of the genes in the tomato genome are derived from gene replication events [45]. Intraspecific collinearity analysis showed that only the SLPEBP1 and SLPEBP9 genes had collinearity, indicating that SLPEBP gene family members had fewer copies, or that their copies were discarded more during evolution. At the same time, to further analyze the homologous relationships of SLPEBP gene family members in plants, we carried out interspecific collinearity analysis on tomato, potato, pepper, and Arabidopsis. The highest to lowest collinearity observed is potato (11), pepper (10), and Arabidopsis (4). This result is consistent with genetic distance [48]. SLPEBP2 and SLPEBP5 have no collinear relationship with the other three species and may be unique to tomato. SLPEBP4, SLPEBP6, and SLPEBP9 and potato, pepper, and Arabidopsis all have a collinear relationship. These three genes may play an important role in the evolution of the PEBP gene family, or have irreplaceable functions. The phylogenetic tree of PEBP gene family members of tomato, Arabidopsis, pepper, and potato was constructed using MEGA-X. The evolutionary tree divides these members into three subgroups, namely TFL1, FT, and MFT ( Figure 3). In the TFL1 branch, Arabidopsis TFL1 (AT5G03840) and FT are antagonistic. TFL1 has been shown to move in the apical meristem (SAM), regulate the floral transition, and regulate the expression of hundreds of genes [49]. Arabidopsis ATC (AT2G27550) can inhibit floral transition, and its expression is induced under short-day conditions [50]. Tomato SP3C (SLPEBP4) was found to be able to promote seed germination after being knocked out, and its overexpression can improve the tolerance of seedlings to water stress, and is related to root morphogenesis [30]. Puneli et al. found that SP (SLPEBP9) is homologous to CEN and TFL1 [15]. The mutation of a single amino acid in the SP gene changed tomato from indeterminate to determinate growth. The Solanum pennellii SP9D (SLPEBP11) infiltration system may be associated with semi-determinate growth [29]. In the FT branch, Arabidopsis FT (AT1G65480) is a kind of floral integratorgene that plays a role in the floral transition process [51]. Arabidopsis BFT (AT5G62040) was found to play a role in the development of axillary inflorescence and regulate floral transition under high salt and drought conditions [52,53]. Arabidopsis TSF (AT4G20370) interacts with FT, but the direction of action is unknown at present [54]. Tomato SP5G (SLPEBP7) was found to be related to the photoperiod sensitivity of tomato. It is a kind of flowering replicator, and its variation enables the cultivated tomato to be planted in a wider area without being affected by the photoperiod [55]. Zhang et al. found that the photosensitive change in wild tomato from long-day plant to cultivated tomato was caused by the loss of 52 bp in the enhancer of the SP5G 3 untranslated region [34]. Tomato SP3D/SFT (SLPEBP5) is an important gene controlling flower formation, responsible for encoding florigen and regulating the transformation of tomato from vegetative growth to reproductive growth [56]. When the SFT function loss allele is heterozygous, it can increase the yield by more than 60% and has strong heterosis [33]. By adjusting the balance between florigen and anti-florigen, the yield of tomato can be adjusted and optimized slightly to maximize the yield [57]. Potato SP6A (PGSC0003DMT400060057) controls the tuber vascular bundle transport signals and forms a complex with IT1 to regulate the formation of potato tubers. The absence of its PEBP domain leads to the failure of Solanum etuberosum to produce potatoes [58]. In the MFT branch, the MFT (AT1G18100) gene of Arabidopsis was found to promote flowering and regulate the germination of Arabidopsis seeds through the ABA and GA signaling pathways [11,59]. Through collinearity and phylogenetic tree analysis, it was found that the function of the PEBP gene family is related to the synthesis of florigen, the regulation of floral transition, light sensitivity, plant growth and development, and some abiotic stress responses. In addition, the function of the PEBP gene family in different species varies greatly. It is obvious that Arabidopsis is a short-day plant, while tomato is a long-day plant. The response results of the PEBP gene family in these two plants are similar, but the regulatory modes are opposite.
In the analysis of the conserved motifs of the SLPEBP genes, only motifs 6 and 8 of the 10 conserved motifs were present in all 12 SLPEBP genes. All genes except for SLPEBP3, SLPEBP6, SLPEBP8, SLPEBP10, and SLPEBP12 contain all 10 conserved motifs, and the 10 motifs are arranged in the same order. At present, there are no detailed reports on the functions of these five genes. Among the genes containing all 10 conserved motifs, SLPEBP1 and SLPEP2 have not been found to have specific functions. SLPEBP3, SLPEBP6, and SLPEBP10 belong to the MFT subfamily. The FT and TFL1 subfamilies are believed to have evolved from the genes of this subfamily, which may be the reason why the conserved motifs of these three genes are incomplete. The prediction of the conserved motifs of the SLPEBP gene family provides a theoretical basis for gene classification and gene function prediction. The gene structure of the SLPEBP gene family is basically composed of four exons, with 75% of family members containing four exons, while there is loss of introns in the gene family, with the family members containing at most two introns. SLPEBP8 has five exons, and SLPEBP10 contains only two exons. SLPEBP12 contains eight exons, and the gene structure is in good agreement with the conserved motif analysis, so it is likely that two genes were spliced together. The three-dimensional structure of proteins determines how they interact with other molecules, and determines their role in cells. The abundant three-dimensional structure of proteins allows them to perform a considerable number of functions in cells, and the three-dimensional structure of proteins can be inferred from their amino acid sequences [60]. From the three-dimensional structure prediction of proteins, we can see that the SLPEBP gene family is composed of a similar domain, among which SLPEBP1, SLPEBP2, and SLPEBP4 have very similar three-dimensional structure prediction results, and they belong to the same subfamily. The predicted results are in line with the characteristics of a gene family protein, and all have similar conserved domains. In the process of protein transcription, the gene promoter and its cis-acting element play an important regulatory role. The core promoter of the gene encoding the protein usually contains a "TATA-box". The cis-acting element and core promoter can become the binding sites of transcription and regulate the specific binding sites of the protein [61][62][63]. Through the analysis of cis-acting elements, it was found that all 12 members of the SLPEBP gene family contain the "TATA-box". The analysis results of cis-acting elements can be divided into six categories: 18 development-related elements (8 kinds), 27 environmental stressrelated elements (5 kinds), 84 hormone-response elements (9 kinds), 141 photo-response elements (20 kinds), 1166 promoter-related elements (4 kinds), and 13 site-binding related elements (4 kinds). Light-responsive elements were the most abundant in the SLPEBP gene family. In addition to cis-acting elements related to basic functions (promoter-related elements and site-binding related elements), the number of light-responsive elements is also the greatest, which proves that the functions of SLPEBP gene family members are related to light response, which is consistent with the results discussed earlier. In addition, SLPEBP gene family members are also involved in plant growth and development, abiotic stress response, hormone regulation, and other functions. This analysis result can also be verified in the previous discussion. After identifying the cis-acting elements in the promoter region, we found that the cis-acting elements were irregularly scattered in the promoter region ( Figure 6B). The cis-acting elements distributed in different positions are conducive to regulating the functions of proteins under different environmental stimuli. Some members of the SLPEBP gene family, such as SP9D, SP, SP5G, and SP3D, have been shown to be expressed in many tissues and organs of tomato [29]. However, no study has been conducted to analyze the expression of all 12 SLPEBP genes in different tissues and organs of tomato. The expression and regulatory mechanisms of these 12 SLPEBP gene family members have been studied more clearly only for SP, SFT, and SP5G. By analysis of RNA-seq data, we found that some members of the SLPEBP gene family have tissuespecific expression (Figure 7). Among them, SLPEBP1, SLPEBP2, SLPEBP9, and SLPEBP11 were specifically expressed in tomato roots, which may be related to root development or growth regulation, and interestingly, they all belong to the TFL1 subfamily. SLPEBP4, SLPEBP6, and SLPEBP12 were specifically expressed in buds and flowers, which may be related to flower differentiation and development. SLPEBP5 was specifically expressed in flowers, and SLPEBP3 was specifically expressed in buds. SLPEBP8 and SLPEBP10 were not found to be expressed in any of the tissues and organs tested, and their expression may not be involved in the tested organs of tomato or they are not expressed in Heinz 1706. We selected five different stages of tomato from flowering to fruit setting for tissue-specific expression analysis, hoping to identify at which stage the SLPEBP gene family plays a role in flowering. SLPEBP3, SLPEBP5, SLPEBP6, SLPEBP8, SLPEBP9, and SLPEBP10 have high expression in buds or incompletely opened flowers, and we speculate that they may be involved in the regulation of flowering in tomato. The other genes had higher variation in expression during flowering or fruit formation, so it is speculated that they may be involved in the regulation of ovary development.

Tomato Growth and Treatment
The cultivated tomato Ailsa Craig served as the experimental material. The plant material was planted in April 2022 in a solarium at Northeastern Agricultural University and sampled in October (126.916 E, 45.773 N). Tomato flower buds, incompletely opened flowers, completely opened flowers, and organs with 3 and 5 days of fruit set were collected to examine gene expression.

Total RNA Extraction and cDNA Synthesis
Total RNA was extracted from tomato materials using TaKaRa RNA extraction kit (Takara Bio, Beijing, China). cDNA synthesis and gDNA removal were performed using reagents produced by Vazyme (Vazyme, Nanjing, China). The obtained cDNA was stored at −80 • C.

Relationship between Chromosome Position and Replication of PEBP Family Genes
The chromosome position data, including chromosome length and gene starting and ending positions, were obtained from Solanaceae Genomics Network (SGN) (https: //solgenomics.net/, accessed on 8 September 2021). A chromosome distribution map of the SLPEBP genes was drawn using TBtools [70]. Multiple collinear scanning toolkits (MCScanX) were used to analyze gene replication events, and the default values were adopted [71]. The collinear relationships within the SLPEBP gene family and the collinear relationships with genes in other species homologous to the SLPEBP gene family were plotted by Dual Systeny Plotter software [70].

Multiple Sequence Alignment and Phylogenetic Analysis
The PEBP family members in Arabidopsis, potato, and pepper were identified by the same method as that used to identify the PEBP family members in tomato, and seven PEBP family members in Arabidopsis, ten PEBP family members in potato, and eleven PEBP family members in pepper were identified by screening conserved structural domains using online software. A total of 7 PEBP family members from Arabidopsis, 10 PEBP family members from potato, 11 PEBP family members from pepper, and 12 PEBP family members from tomato were sequenced using MEGA-X, and the results were analyzed by phylogenetic analysis. The analysis was performed using neighbor-joining (NJ) and the protein evolution model JTT (Jones-Taylor-Thornton), as well as 1000 bootstrap replicates, and other default values.

Gene Structure and Conserved Motif Analysis
Gene Structure Display Server 2.0 (GSDS) (http://gsds.gao-lab.org/, accessed on 15 September 2021) was used to construct the gene structure map [72]. The conserved motifs in the SLPEBP family genes were predicted by MeMe (https://meme-suite.org/meme/ tools/meme, accessed on 15 September 2021). The predicted number of motifs was 10, and the other was the default value.

Prediction of Three-Dimensional Protein Structure of Tomato PEBP
SWISS-MODEL (https://swissmodel.expasy.org/, accessed on 7 October 2021) was used to predict the three-dimensional structure of the protein by homology modeling [73][74][75][76][77]. A structure with sequence consistency greater than 30% was selected as the template, and the protein model was evaluated by SAVES v6.0 (https://saves.mbi.ucla.edu/, accessed on 7 October 2021) [78]. The template that passes three or more assessments was selected as the final template. Structural visualization was carried out by the 3D protein structure visualization software VMD (http://www.ks.uiuc.edu/Research/vmd, accessed on 7 October 2021) [79].

Analysis of Cis-Acting Elements of the SLPEBP Gene Family Members
The sequence of 2000 bp upstream of the transcription initiation site was extracted as the promoter sequence of the SLPEBP gene, and PlantCARE (http://bioinformatics.psb. ugent.be/webtools/plantcare/html/, accessed on 6 October 2021) was used to predict the potential cis-acting elements on the promoter sequence [80]. The prediction results were visualized, classified, and analyzed.

Analysis of SLPEBP Gene Expression in Different Tissues of Tomato
To investigate the differences in expression of the SLPEBP gene in different tissues of tomato, FPKM (fragment per million exons mapping) data for tomato variety Heinz 1706 with TFGD (http://ted.bti.cornell.edu/, accessed on 2 April 2023) accession number D004 were used [81]. Twelve SLPEBP genes were screened using these data and analyzed expression in buds; fully open flowers; leaves; roots; 1 cm, 2 cm, and 3 cm fruits; and mature green, breaker, and breaker +10 fruits of tomato cultivar Heinz 1706 [81]. To measure gene expression levels, the total number of FPKM values for each gene was calculated based on the length of the gene and the count of reads mapping to that gene. The data for each row were normalized and plotted according to the average FPKM value for each gene. The Euclidean distance between the genes was also analyzed and they were clustered. The R package pheatmap was used to generate the heatmap.

Analysis of SLPEBP Family Member Gene Expression
Specific primers were designed using NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 4 September 2022) and synthesized by BGI Genomics Co., Ltd. Detailed primer sequence information can be found in Table S4. AceQ qPCR SYBR Green Master Mix (Without ROX) reagent from Nanjing Vazyme Company was used, configured as a 20 µL system according to the instructions, and qRT-PCR analysis was performed using the German Analytik Jena quantitative PCR instrument qTOWER3G. Detailed procedures: Stage 1, 94 • C 2 min for 1 cycle; Stage 2, 94 • C 15 s, 57 • C 15 s, 72 • C 30 s for 40 cycles (scan); melting curve 60 to 90 • C, 10 s with ∆T 0.5 • C (scan). The Actin gene was used as the internal reference gene as the control for data standardization. The final calculation 2 −∆∆ Ct was used to analyze the relative expression of genes. GraphPad Prism 9 was used to analyze the variance, and one-way ANOVA (and nonparametric or mixed) was used to analyze the significance.

Conclusions
In this study, 12 SLPEBP gene family genes were identified and analyzed. There is a collinear relationship between SLPEBP1 and SLPEBP9. Eleven collinear relationships of tomato with potato, ten collinear relationships with pepper, and four collinear relationships with Arabidopsis were found. Through the analysis and construction of the evolutionary tree, the PEBP gene family was divided into three subfamilies, TFL1, FT, and MFT, and the characteristics of the subgroups were summarized. The gene structure and conserved motifs of the 12 genes were analyzed, and their general characteristics were summarized. The protein models of 11 members of the SLPEBP gene family were predicted. They all contain a similar conserved domain. Through the prediction of cis-acting elements, the function of SLPEBP gene family members was further analyzed. We speculate that SLPEBP gene family members have functions in plant growth and development, light response, hormone regulation, and some abiotic stress responses. Tissue-specific expression analysis showed changes in the expression of 10 members of the SLPEBP gene family in five different stages, and verified their role in plant growth and development. Six genes (SLPEBP3, SLPEBP5, SLPEBP6, SLPEBP8, SLPEBP9, and SLPEBP10) are hypothesized to be associated with tomato flowering and four (SLPEBP2, SLPEBP3, SLPEBP7, and SLPEBP11) may be associated with ovary development. The above analysis can provide research directions and ideas for future research on SLPEBP gene family members.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are openly available in the Tomato Functional Genomics Database (TFGD) (cornell.edu), reference number D004.