Genome-Wide Identiﬁcation and Characterization of the SBP Gene Family in Passion Fruit ( Passiﬂora edulis Sims)

: The SQUAMOSA promoter binding proteins (SBPs) gene family plays important roles in plant growth and development. The SBP gene family has been identiﬁed and reported in many species, but it has not been well studied in passion fruit. In this study, a total of 14 SBP genes were identiﬁed in passion fruit and named from PeSBP1 to PeSBP14 based on their chromosomal distribution. The phylogenetic tree, gene structure, conserved motifs, collinearity analysis, and expression patterns of the identiﬁed SBP members were analyzed. We classiﬁed the PeSBP genes into eight groups (I to VIII) according to the phylogenetic tree, gene structure, and conserved motifs. Synteny analysis found that 5 homologous gene pairs existed in PeSBP genes and 11 orthologous gene pairs existed between passion fruit and Arabidopsis. Synonymous nucleotide substitution analysis showed that the PeSBP genes were under strong negative selection. The expression pattern of PeSBP genes in seed, root, leaf, and ﬂower showed that nine of the PeSBP genes displayed high expression in the leaf and the ﬂower. The expression patterns of PeSBP3 / 6 / 8 / 9 / 10 were further detected by qRT-PCR. In addition, differences in the expression levels occurred for each gene in the different ﬂower organs and at the different developmental stages. There were large differences among SBPs based on transcriptional levels under cold, heat, salt, and osmotic stress conditions. Altogether, this study provides an overview of SBP genes in passion fruit and lays the foundation for further functional analysis.


Introduction
Transcriptional regulation is an important part of eukaryotic gene expression regulation, and transcription factors (TFs) play pivotal roles in plant growth and development processes [1]. TFs, including DNA binding domains, transcriptional activation domains, nuclear localization signals, and oligomerization sites [2], function by binding to corresponding gene promoters, thus activating or inhibiting the transcription of target genes [3]. TFs can be divided into different families based on their DNA binding domains [4]. In plants, 64 TF families have been identified [5].
The SQUAMOSA promoter binding (like) proteins (SBPs/SPLs) gene family is part of a class of important transcription factor families that regulates the growth and the development of green plants under stress. The SBP TFs have a conserved SBP domain containing about 79 amino acid residues that are highly conserved [6]. The SBP domain includes two typical zinc-binding sites (Cys-Cys-Cys-His, Zn1 and Cys-Cys-His-Cys, Zn2) and a highly conserved nuclear localization signal (NLS) at the C-terminal partially overlapping with the Zn2 zinc finger structural sequence [7]. The SBP protein localizes in the nucleus with the nuclear localization signal serving as a guide to regulate the transcriptional expression of

Identification and Characterization of SBP Genes in Passion Fruit
To identify PeSBP genes in passion fruit, HMMER software and CDD online tools were used. A total of 14 SBP gene sequences were identified and named from PeSBP1 to PeSBP14 according to the location of the genes on the chromosomes (Supplementary Materials Table  S1). Sequence alignment was performed with 14 SBP proteins in passion fruit to predict the SBP domain. The results showed that all PeSBP proteins had typical characteristics of the SBP domain, including two zinc domains (Zn1 and Zn2) and a nuclear localization signal (NLS) (Figure 1). With CDS ranging from 552 to 2943 bp, the full length of the 14 PeSBP proteins varied from 183 (PeSBP2) to 981 (PeSBP7) amino acid residues, with the molecular mass ranging from 20.57 (PeSBP4) to 108.28 (PeSBP5) kDa and isoelectric points ranging from 5.93 (PeSBP5) to 9.64 (PeSBP4). The total average hydrophilicity (GRAVY) values were all negative, indicating that all PeSBPs are hydrophilic. The subcellular localization results showed that thirteen of the fourteen PeSBP proteins were located in the nucleus (Supplementary Materials Table S1), while the PeSBP12 was located in the chloroplast. That may be because the nuclear localization sequence of chloroplast localization proteins will be removed after the protein enters the chloroplast. So, the protein will not move to the nucleus from the chloroplast. Furthermore, we identified the orthologous gene of PeSBPs in Arabidopsis. The orthologous gene and the function in Arabidopsis are shown in Supplementary Materials Table S1.
with the molecular mass ranging from 20.57 (PeSBP4) to 108.28 (PeSBP5) kDa and isoelectric points ranging from 5.93 (PeSBP5) to 9.64 (PeSBP4). The total average hydrophilicity (GRAVY) values were all negative, indicating that all PeSBPs are hydrophilic. The subcellular localization results showed that thirteen of the fourteen PeSBP proteins were located in the nucleus (Supplementary Materials Table S1), while the PeSBP12 was located in the chloroplast. That may be because the nuclear localization sequence of chloroplast localization proteins will be removed after the protein enters the chloroplast. So, the protein will not move to the nucleus from the chloroplast. Furthermore, we identified the orthologous gene of PeSBPs in Arabidopsis. The orthologous gene and the function in Arabidopsis are shown in Supplementary Materials Table S1. and lightblue backgrounds indicate entire conservative residues, 75% conservative residues, and 50% conservative residues respectively.

Phylogenetic Analysis of Passion Fruit SBP Gene Family
To analyze the phylogeny of the SBP gene family, a total of 301 SBP homologs were selected from 18 representative plants from seven green plant families. Among these plants, the soybean had the maximum number of SBP genes, with 46 members. However, only one SBP gene in group I was found in Ostrecocus lucimarinus and Ostrecocus sp. rcc809 ( Figure 2). To further understand the phylogenetic relationship of the PeSBP gene family, a phylogenetic tree was constructed using SBP proteins of passion fruit, Arabidopsis, grape, sorghum, and rice. Among them, passion fruit, Arabidopsis, and grape belong to eudicots, while sorghum and rice belong to monocots. According to the unrooted phylogenetic tree, 14 PeSBPs could be divided into 8 groups (I to VIII). Four PeSBPs belonged to group II, while none belonged to group III. There was only one member of PeSBP in groups I, V, and VIII. The homology between the PeSBPs gene and the VvSBPs was relatively high, and the similarity between their protein sequences indicates that the SBP gene of these two species may have similar biological functions ( Figure 3).

Figure 1.
Multiple sequence alignment of SBP domains of PeSBP proteins. The darkblue, purple, and lightblue backgrounds indicate entire conservative residues, 75% conservative residues, and 50% conservative residues respectively.

Phylogenetic Analysis of Passion Fruit SBP Gene Family
To analyze the phylogeny of the SBP gene family, a total of 301 SBP homologs were selected from 18 representative plants from seven green plant families. Among these plants, the soybean had the maximum number of SBP genes, with 46 members. However, only one SBP gene in group I was found in Ostrecocus lucimarinus and Ostrecocus sp. rcc809 ( Figure 2). To further understand the phylogenetic relationship of the PeSBP gene family, a phylogenetic tree was constructed using SBP proteins of passion fruit, Arabidopsis, grape, sorghum, and rice. Among them, passion fruit, Arabidopsis, and grape belong to eudicots, while sorghum and rice belong to monocots. According to the unrooted phylogenetic tree, 14 PeSBPs could be divided into 8 groups (I to VIII). Four PeSBPs belonged to group II, while none belonged to group III. There was only one member of PeSBP in groups I, V, and VIII. The homology between the PeSBPs gene and the VvSBPs was relatively high, and the similarity between their protein sequences indicates that the SBP gene of these two species may have similar biological functions ( Figure 3).

Gene Structure and Conserved Motif Analysis of SBP Genes in Passion Fruit
The structure of each gene, especially the number and distribution of exon and intron, may be closely related to evolution. To better understand the diversity of PeSBP gene structure, the coding sequence of each gene was compared with its corresponding genomic sequence. The number of exons varied from 2 to 12. The number of introns in group II was the maximum, while all PeSBP genes in group VI only contained 1 intron, which was the lowest number. Group VII contained two to five introns, and group V contained 4 introns ( Figure 4). In group II, PeSBP5, PeSBP7, PeSBP 11, and PeSBP14 in the evolutionary tree may have been cleaved and polymerized during the evolutionary process. The results showed that the number of exons and introns was highly variable in different groups, while the number of exons was nearly consistent in the same group.
The number and types of conserved motifs contained in each protein sequence are different, which may reveal the different functions of each gene. A total of 10 motifs were identified in the PeSBP proteins and named motifs 1 to 10 (Supplementary Materials Figure S1). The results showed that all SBP members contained Motif 1 and Motif 2, suggesting that Motif 1 and Motif 2 constituted the conserved SBP domain (Supplementary  Materials Table S2). PeSBP members in group II contained the maximum number of conservative motifs. PeSBP proteins clustered in the same group tended to have similar motif numbers. In contrast, some motifs were specific in some groups of PeSBP proteins-Motif 8, Motif 10, Motif 6, Motif 5, Motif 9, and Motif 7 were only found in group II, and Motif 4 was only found in PeSBP10, suggesting that some proteins may have specific biological functions under specific conditions ( Figure 5). The majority within the same group exhibited similarity in motif compositions. Besides, the exon-intron structure of SBP proteins supported the phylogenetic analysis of SBP family genes, and the differences among the different groups indicate their diverse functions. tron, may be closely related to evolution. To better understand the diversity of PeSBP gene structure, the coding sequence of each gene was compared with its corresponding genomic sequence. The number of exons varied from 2 to 12. The number of introns in group II was the maximum, while all PeSBP genes in group VI only contained 1 intron, which was the lowest number. Group VII contained two to five introns, and group V contained 4 introns (Figure 4). In group II, PeSBP5, PeSBP7, PeSBP 11, and PeSBP14 in the evolutionary tree may have been cleaved and polymerized during the evolutionary process. The results showed that the number of exons and introns was highly variable in different groups, while the number of exons was nearly consistent in the same group.  Table S2). PeSBP members in group II contained the maximum number of conservative motifs. PeSBP proteins clustered in the same group tended to have similar motif numbers. In contrast, some motifs were specific in some groups of PeSBP proteins-Motif 8, Motif 10, Motif 6, Motif 5, Motif 9, and Motif 7 were only found in group II, and Motif 4 was only found in PeSBP10, suggesting that some proteins may have specific biological functions under specific conditions ( Figure 5). The majority within the same group exhibited similarity in motif compositions. Besides, the exon-intron structure of SBP proteins supported the phylogenetic analysis of SBP family genes, and the differences among the different groups indicate their diverse functions.

Synteny Analysis of PeSBP Genes
The distribution analysis of PeSBP genes on the chromosomes indicated that 13 PeSBP genes map to seven chromosomes, except LG2 and LG8, while PeSBP14 was located on P_eduliaContig 140022932.g. Since the passion fruit chromosome assembly and anchoring rate was only 96.07%, the PeSBP14 (P_ eduliaContig140022932.g) was not anchored to any chromosome but was assembled to Contig14. Among them, LG1 had the maximum number of PeSBP genes, including 5 PeSBP members.
LG4 and LG5 chromosomes contain 2 PeSBP genes. The remaining four chromosomes had only 1 PeSBP gene ( Figure 6). MCScanX software was used to analyze the collinearity of SBP genes between passion fruit and Arabidopsis. The blue line represents the collinearity of SBP genes between passion fruit and Arabidopsis, and the red line represents the synteny gene pairs of SBP genes between different chromosomes of passion fruit. A total of 16 pairs of collinear genes were identified, including 5 pairs of collinear genes within passion fruit and 11 pairs of synteny gene pairs between passion fruit and Arabidopsis. It can also be seen from the chromosome distribution diagram that there is still a collinearity relationship between PeSBP14 and other genes on chromosomes. However, PeSBP14 was located on the contig

Synteny Analysis of PeSBP Genes
The distribution analysis of PeSBP genes on the chromosomes indicated that 13 PeSBP genes map to seven chromosomes, except LG2 and LG8, while PeSBP14 was located on P_eduliaContig 140022932.g. Since the passion fruit chromosome assembly and anchoring rate was only 96.07%, the PeSBP14 (P_ eduliaContig140022932.g) was not anchored to any chromosome but was assembled to Contig14. Among them, LG1 had the maximum number of PeSBP genes, including 5 PeSBP members. LG4 and LG5 chromosomes contain 2 PeSBP genes. The remaining four chromosomes had only 1 PeSBP gene ( Figure 6). MCScanX software was used to analyze the collinearity of SBP genes between passion fruit and Arabidopsis. The blue line represents the collinearity of SBP genes between passion fruit and Arabidopsis, and the red line represents the synteny gene pairs of SBP genes between different chromosomes of passion fruit. A total of 16 pairs of collinear genes were identified, including 5 pairs of collinear genes within passion fruit and 11 pairs of synteny gene pairs between passion fruit and Arabidopsis. It can also be seen from the chromosome distribution diagram that there is still a collinearity relationship between PeSBP14 and other genes on chromosomes. However, PeSBP14 was located on the contig rather than the chromosome ( Figure 6). It indicated that segmental duplication events might play a more important role in the evolution of the passion fruit SBP gene family. To further understand the evolutionary relationship of the SBP genes, we calculated Ka and Ks values to predict selection pressure for synteny gene pairs. Ka/Ks = 1 indicates neutral selection for homologous genes, Ka/Ks > 1 indicates that these genes evolved under positive selection, and Ka/Ks < 1 indicates that these genes had purification selection [30]. The results showed that the Ka/Ks values of all the SBP homogenous gene pairs within passion fruit and between passion fruit and Arabidopsis were less than 1, indicating that they may play a role in purification selection in the evolutionary process (Supplementary Materials Table S3).

Expression Profiles of PeSBP Genes in Different Tissues
Recent studies in various species have indicated that the SBP gene plays an important regulatory role in processes associated with plant flowering and inflorescence development [31]. To investigate the tissue-specific expression pattern of PeSBP genes in passion fruit flower development, we analyzed RNA-sequencing data from NGCB to determine the tissue-specific expression patterns of different passion fruit flower tissues (including the mixed flower, bract, sepal, petal, corona filament, stamen, stigma, and ovule at different development processes) and vegetative tissues (including the seed, root, and leaf).
According to their expression patterns, we found that the PeSBP genes showed a tissue-specific expression pattern. For instance, PeSBP13 had high expression during pollen development, while PeSBP9 had specifically high expression in the stigma, and PeSBP4 had increased expression in the sepal and early stigma stages ( Figure 7A, Supplementary Materials Table S4). The expression level of PeSBP6, PeSBP8, and PeSBP13 genes were

Expression Profiles of PeSBP Genes in Different Tissues
Recent studies in various species have indicated that the SBP gene plays an important regulatory role in processes associated with plant flowering and inflorescence development [31]. To investigate the tissue-specific expression pattern of PeSBP genes in passion fruit flower development, we analyzed RNA-sequencing data from NGCB to determine the tissue-specific expression patterns of different passion fruit flower tissues (including the mixed flower, bract, sepal, petal, corona filament, stamen, stigma, and ovule at different development processes) and vegetative tissues (including the seed, root, and leaf).
According to their expression patterns, we found that the PeSBP genes showed a tissue-specific expression pattern. For instance, PeSBP13 had high expression during pollen development, while PeSBP9 had specifically high expression in the stigma, and PeSBP4 had increased expression in the sepal and early stigma stages ( Figure 7A, Supplementary Materials Table S4). The expression level of PeSBP6, PeSBP8, and PeSBP13 genes were higher in flowers and leaves but lower in seeds and roots ( Figure 7B, Supplementary Materials Table S4), suggesting that these three genes may play an important role in passion fruit flower and leaf development. The expression levels of PeSBP7, PeSBP10, PeSBP11, and PeSBP12 were high in all tissues, indicating that they might be involved in various physiological processes of passion fruit flower development. Strangely, the expression levels of PeSBP3 in all tissues were significantly lower than in other genes. These results suggest that diverse PeSBPs might be involved in the different floral tissues of passion fruit.

In Silico Promoter Analysis and Expression Profiles of PeSBP Genes in Response to Abiotic Stresses
To explore the mechanisms of PeSBPs in response to various stresses, we analyzed cis-elements of PeSBPs promoters. Two kb upstream genomic DNA sequences with transcriptional start site (ATG) from 14 PeSBP genes were analyzed. Four different classes of cis-elements were found in PeSBPs promoter regions, including stress responsiveness, light responsiveness, growth and development, and hormone responsiveness (Figure 8). The distribution of cis-elements in PeSBP genes is different, indicating that the expression of PeSBPs may vary in stressful, developmental, and hormonal contexts. Most PeSBP Furthermore, to validate the expression patterns of the PeSBP genes in the root, leaf, seed, and flower tissues, five PeSBP genes (PeSBP3, PeSBP6, PeSBP8, PeSBP9, and PeSBP10) were randomly selected to test their expression level in these four tissues by qRT-PCR. The qRT-PCR results were consistent with our RNA-sequencing results ( Figure 7C), suggesting that all the PeSBP gene expression patterns in different tissues are reliable.

In Silico Promoter Analysis and Expression Profiles of PeSBP Genes in Response to Abiotic Stresses
To explore the mechanisms of PeSBPs in response to various stresses, we analyzed cis-elements of PeSBPs promoters. Two kb upstream genomic DNA sequences with transcriptional start site (ATG) from 14 PeSBP genes were analyzed. Four different classes of cis-elements were found in PeSBPs promoter regions, including stress responsiveness, light responsiveness, growth and development, and hormone responsiveness (Figure 8). The distribution of cis-elements in PeSBP genes is different, indicating that the expression of PeSBPs may vary in stressful, developmental, and hormonal contexts. Most PeSBP genes contained regulatory elements ABRE, ERE, CGTCA-motif, MBS, and W-box (Supplementary Materials Table S5). These results indicated that various types of cis-element in the same type of PeSBP genes might have different functions. ABRE is involved in abscisic acid responsiveness [32], while CGTCA-motif is involved in MeJA-responsiveness, and MBS (MYB binding site) is involved in drought inducibility. W-box is an important class of cis-element found in the promoter of plant defence response-related genes [33]. Gene ontology (GO) enrichment analysis of the PeSBPs was performed to understand their possible functions at the molecular level, i.e., molecular function (MF), biological process (BP), cellular component (CC). In MF class, PeSBPs enrichment in DNA binding, transcription regulator activity, nucleic acid binding, and so on (Supplementary Materials Figure S2). Five terms were identified as belonging to the CC class (Supplementary Materials Figure S2). Most promoters of PeSBPs genes were significantly enriched in the binding sites of transcription factors involved in the BP class in terms of regulation of nucleic acid-templated transcription, regulation of DNA-templated transcription, and regulation of gene expression (Supplementary Materials Figure S2). The information regarding the enrichment annotation results of GO terms for MF, CC, and BP is provided in Supplementary Materials Table S6. These results show that SBP gene family members may play an important role in different regulations.  To further confirm whether different abiotic stresses influence the expression of PeSBP genes, qRT-PCR experiments were performed to analyze the SBP gene expression patterns in response to different treatments, including cold, heat, and osmotic stress. As shown in Figure 9, seven SBP genes were selected for transcription-level detection. Under cold treatment, five genes (PeSBP6/7/8/9/10) had similar expression patterns that initially decreased and subsequently increased during the course of the treatment, while the relative expression level PeSBP3/12 first increased and then decreased with the treatment time. During the heat treatment, three genes (PeSBP3/6/8) showed a continuous decrease with the treatment time, and the other four genes (PeSBP7/9/10/12) first decreased and then increased their expression with the treatment time. To further confirm whether different abiotic stresses influence the expression of PeSBP genes, qRT-PCR experiments were performed to analyze the SBP gene expression patterns in response to different treatments, including cold, heat, and osmotic stress. As shown in Figure 9, seven SBP genes were selected for transcription-level detection. Under cold treatment, five genes (PeSBP6/7/8/9/10) had similar expression patterns that initially decreased and subsequently increased during the course of the treatment, while the relative expression level PeSBP3/12 first increased and then decreased with the treatment time.
During the heat treatment, three genes (PeSBP3/6/8) showed a continuous decrease with the treatment time, and the other four genes (PeSBP7/9/10/12) first decreased and then increased their expression with the treatment time. The cis-elements in promoter regions are essential for regulating the gene expression of different stress response pathways. As shown above, we found some low-temperature responsiveness elements (LTR) in the promoter of PeSBP6, PeSBP7, and PeSBP8 ( Figure  8). Additionally, the qRT-PCR results showed that the LTR regulates the expression of PeSBP6, PeSBP7, and PeSBP8 in response to temperature stress. Similarly, the MYB binding site involved in drought-inducibility was found in the PeSBP3/8/10/12 promoter region. After osmotic stress, the transcription of PeSBP8/12 continuously decreased with the treatment time, while the expression of PeSBP3/10 first showed an increase and then decreased with the treatment time. It indicates that the MBS is important as it participates in regulating the expression of PeSBP3/8/10/12 response to osmotic stress. After salt treatment, the expression level of PeSBP8/9/10 increased gradually, while the expression of PeSBP6/7/12 was repressed, and the expression of PeSBP3 increased within 24 h and then decreased 48 h after treatment. Altogether, different SBP genes display differential expression patterns after stress treatment, indicating that these genes might function in response to abiotic stresses.

Discussion
The SBP gene family is a plant-specific transcription factor family widely found in green plants, and it plays a crucial role in plant growth and development, physiological and biochemical activities, and environmental response [34]. Passion fruit is a famous tropical and subtropical crop which has extensive economic value [35]. However, only a few studies on the SBP gene family in passion fruit exist. In this study, a total of 14 SBP genes were identified from the passion fruit genome via the bioinformatics method, which was similar in number to other eudicot plants, like Arabidopsis thaliana (16), Arabidopsis lyrata (15), and Vitis vinifera (17), but fewer in number than Glycine max (46) and Populus The cis-elements in promoter regions are essential for regulating the gene expression of different stress response pathways. As shown above, we found some low-temperature responsiveness elements (LTR) in the promoter of PeSBP6, PeSBP7, and PeSBP8 ( Figure 8). Additionally, the qRT-PCR results showed that the LTR regulates the expression of PeSBP6, PeSBP7, and PeSBP8 in response to temperature stress. Similarly, the MYB binding site involved in drought-inducibility was found in the PeSBP3/8/10/12 promoter region. After osmotic stress, the transcription of PeSBP8/12 continuously decreased with the treatment time, while the expression of PeSBP3/10 first showed an increase and then decreased with the treatment time. It indicates that the MBS is important as it participates in regulating the expression of PeSBP3/8/10/12 response to osmotic stress. After salt treatment, the expression level of PeSBP8/9/10 increased gradually, while the expression of PeSBP6/7/12 was repressed, and the expression of PeSBP3 increased within 24 h and then decreased 48 h after treatment. Altogether, different SBP genes display differential expression patterns after stress treatment, indicating that these genes might function in response to abiotic stresses.

Discussion
The SBP gene family is a plant-specific transcription factor family widely found in green plants, and it plays a crucial role in plant growth and development, physiological and biochemical activities, and environmental response [34]. Passion fruit is a famous tropical and subtropical crop which has extensive economic value [35]. However, only a few studies on the SBP gene family in passion fruit exist. In this study, a total of 14 SBP genes were identified from the passion fruit genome via the bioinformatics method, which was similar in number to other eudicot plants, like Arabidopsis thaliana (16), Arabidopsis lyrata (15), and Vitis vinifera (17), but fewer in number than Glycine max (46) and Populus trichocarpa (28), suggesting that SBP genes in different eudicot species underwent different gene duplication events [36]. There was only one SBP gene in Ostrecocus lucimarinus and Ostrecocus sp. rcc809 in group I, leading us to speculate that the SBP gene of group I might be their ancestor. We found that the number of SBP genes increased in terrestrial plants, and gene expansion was intensified strongly in angiosperms, which may reflect the flowering plants' effectiveness in adapting to the different unstable environmental conditions [37]. According to phylogenetic tree analysis, the SBP gene family of passion fruit could be divided into eight groups. This result is consistent with previous SBP gene studies in other plants [30]. No PeSBP gene belonging to group III was identified. Whether these genes were lost in the evolution of passion fruit remains to be explored. Most PeSBP genes were clustered closely with the SBP family genes of grape and Arabidopsis but were far away from the SBP genes of rice. This finding is consistent with the fact that passion fruit, grape, and Arabidopsis are eudicot plants. Compared with the lineage that led to the formation of monocots, it is closer to the divergence in evolution from a common ancestor.
The isoelectric points of PeSBPs were between 5.93-9.64, indicating that most PeSBP proteins are rich in basic amino acids, and they may play a role in an acidic subcellular environment. Genes clustered in an evolutionary clade may have similar genetic structures and functions and often have closer relationships [38]. The gene structure of PeSBPs was relatively simple, and the number of exons ranged from 2 to 12. Most PeSBP genes in the same group shared a similar exon-intron structure. The exon -intron structure varied significantly among different groups, suggesting that the gene structure is closely related to the function. Motif identification is particularly important to explore new members of the gene family. Conserved motif analysis results showed that all PeSBP proteins contained motif 1 and motif 2, suggesting that motif 1 and motif 2 constitute the SBP domain. There were some differences in the number of motifs among different groups, with group II possessing ten motifs and groups V, VI, IV, and VII only having two motifs (motif 1 and motif 2). In general, gene structure and conserved motif analysis can support the grouping of evolutionary trees and are associated with specific biological functions [39].
Gene duplication events (segmental and tandem) are the major driving forces for finding novel genes and gene family expansion which can support the adaptation of organisms to different complex environments [40]. Collinearity analysis mainly evaluates the remaining and lost duplication genes through homology comparison. It can provide an important basis for studying the evolutionary history of genes by comparing the homologous gene sequences of different plant genomes [41]. Colinear analysis revealed the evolutionary relationship between homologous gene pairs within passion fruit and between passion fruit and Arabidopsis. A total of 5 segmental duplication events of collinear genes were found within passion fruit, and 11 duplication synteny gene pairs were found between passion fruit and Arabidopsis, indicating segmental duplication events produced the diversity of PeSBPs. The Ka/Ks values of SBP collinearity gene pairs in all identified homologous gene pairs were less than 1, suggesting that SBP collinearity gene pairs underwent purification selection in the evolution process with no functional change.
Plant SBP transcription factors play an important role in plant development, including flower development [42], fruit development [36], plant hormone signal transduction [43], and vegetative to reproductive phase transition [44,45]. An analysis of cis-acting elements of the PeSBP gene promoters showed that the promoters of SBP genes contained multiple cis-acting regulatory elements, which regulate multiple different growth and development and stress tolerance processes. The regulatory elements of different SBP genes in passion fruit were different, which might be closely related to the functional diversity of SBP genes. In Arabidopsis, AtSPL6 is a positive regulator of defence gene expression [46]. The phylogenetic tree results showed that AtSPL6 are orthologous to PeSBP1 and PeSBP10 in group IV. Interestingly, the cis-element analysis showed that PeSBP1 and PeSBP10 contain multiple defence and stress-responsive elements, such as plant defence response, defence and stress responsiveness, and the MYB binding site involved in drought-inducibility. We speculate that PeSBP1 and PeSBP10 might also be positive regulators of defence gene expression in passion fruit. However, the role of cis-elements in the PeSBP gene promoter regions in stress response requires further investigation. To further understand the possible role of PeSBP genes, we examined the expression levels of 14 SBP genes in different tissues of passion fruit. The results showed tissue-specific SBP gene expression during the growth and development of passion fruit, and the expression levels of the same gene were different in different tissues. In addition, most PeSBP genes were highly expressed in leaf and flower tissues. These results were similar to SBP genes of other species and were highly expressed in the shoot development, apical buds inflorescences, and flower buds [47]. For instance, PeSBP8 in group V was orthologous to AtSPL2, AtSPL10, and AtSPL11 [48] and expressed highly in the passion fruit leaves, suggesting that PeSBP8 plays an important role in the regulation of leaf development. The mutant of AtSPL1 and AtSPL12 inflorescences displayed hypersensitivity to heat stress, whereas the overexpression of AtSPL1 or AtSPL12 enhanced the thermotolerance in Arabidopsis [49]. PeSBP5, PeSBP7, and PeSBP14 were orthologous to AtSPL1 and AtSPL12, expressed highly in the passion fruit ovule and flower stages. The cis-element analysis also showed that PeSBP5, PeSBP7, and PeSBP14 promoters contained many growth, development, and stress response related elements, such as binding sites for transcription factors involved in the regulation of endosperm expression, meristem expression, LTR, and defence and stress responsiveness (Figure 8, Supplementary Materials Table S5). These results may improve the cognition of PeSBPs and furnish potential clues for further studies of SBP family genes in passion fruit.

Plant Materials
Two-month-old plants were used for different treatments. Passion fruit cultivar Tainong No. 1 seeds were germinated in the greenhouse at 30 ± 1 • C, with a relative humidity of 70%, and a 16-h light/8-h dark photoperiod. At two true leaves stages, the seedlings were transferred to the growth chamber and grown for an additional two months. The materials were provided by the passion fruit breeding group at Fujian Agriculture and Forestry University.

Sequence Alignments and Phylogenetic Analysis
MUSCLE software was used to compare the amino acid sequences of the SBP gene family. To study the evolutionary relationship of SBP genes, a phylogenetic tree was constructed among passion fruit, Arabidopsis, grape, sorghum, and rice using MEGA (v6.0, Tokyo Metropolitan University, Hachioji, Tokyo, Japan) with the maximum likelihood method. The following parameters were used: JTT model, pairwise deletion, and the bootstrap test, which were replicated 1000 times. The phylogenetic tree was imported into the Interactive tree of life (iTOL, https://itol.embl.de/ (accessed on 12 December 2021)) [53].

Gene Structure and Conserved Motif Analysis
The Gene Structure Display Server (GSDS, http://gsds.gao-lab.org/https://itol.embl. de/ (accessed on 15 December 2021)) was used to determine the exon-intron structure of passion fruit SBP genes based on their genome DNA sequence and CDS sequence [54]. Then, the protein sequence of the passion fruit SBP gene family was submitted to the MEME online tool (https://meme-suite.org/meme https://itol.embl.de/ (accessed on 15 December 2021)) [55] for conserved motif prediction, and the number of motif discovery was set to 10 with default parameters. TBtools was used to visualize the prediction motif results of the SBP gene family in passion fruit [56].

Chromosomal Locations and Synteny Analysis
The 14 passion fruit SBP family genes were located on the chromosomes using the information annotated in the passion fruit genome. The physical location of PeSBPs on the chromosomes was visualized using Circos software (v0.69, http://www.circos. ca/ (accessed on 19 December 2021)). A synteny analysis of the SBP gene family in the passion fruit genome was conducted using MCScanX software (http://chibba.pgml. uga.edu/mcscan2 (accessed on 19 December 2021)). Circos software also was used to visually map synteny genes. The easy_Kaks calculation program (https://github.com/ tangerzhang/FAFU-cgb/tree/master/easy_KaKs (accessed on 19 December 2021)) was used to calculate the non-synonymous (Ka), synonymous (Ks), and Ka/Ks substitution ratios of the homologous gene pairs of passion fruit and Arabidopsis to estimate the selection and substitution rates.

Cis-Elements Analysis of PeSBPs Promoters
The promoter sequence of PeSBP genes was 2 kb upstream of the transcriptional start site (ATG). The cis-elements of the promoter of the PeSBP genes were predicted using the Plant Cis-Acting Regulatory Element (PlantCARE, http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/ (accessed on 19 December 2021)) [57].

Expression Patterns Analysis
The RNA-sequencing data of the different floral tissues, including bract, sepal, corona filament, petal, stamen, stigma, and ovule, were obtained from China National Gen-Bank (CNGB) (accession number CNP0002747), and the RNA-sequencing results of the root, flower, leaf, and seed tissues were downloaded from (https://ngdc.cncb.ac.cn/gsa/ browse/CRA003773 (accessed on 19 December 2021)). The heatmap was then constructed using the pheatmap package of R software.

RNA Extraction and Quantitative Real-Time PCR
High-quality RNA was extracted using plant total RNA extraction kits (Omega Bio-Tek, Shanghai, China) and then reverse-transcribed into cDNA using a reverse transcription kit (TaKaRa, Beijing, China). A SYBR Premix Ex Taq kit (TaKaRa, Beijing, China) was used for qRT-PCR to verify the expression levels of each sample. Three replicates were designed for each sample, and the EF1a gene was used as a reference to calculate the relative expression levels of SBP genes in various tissues. Primers were designed to amplify sequences in PrimerQuest tools (Supplementary Materials Table S7).

PeSBPs Response to the Abiotic Stresses
Two-month-old passion fruit plants (variety in Tainong No. 1) in soil were analyzed in response to the abiotic stress treatment. For cold (4 • C) and heat (45 • C) stress, seedlings in soil were transferred to the incubator at 16/8 h, day/night. For salt and osmotic stress treatment, the passion fruit plants were first grown in the liquid 1 2 MS medium for 7 days and then transferred to fresh liquid 1 2 MS medium with 200 mM NaCl or 200 mM mannitol for stress treatment. Leaves were collected from at least three independent plants at 12 h, 24 h, and 48 h after treatment, and the controls were not subjected to any stress treatments. Three replicates were performed for each treatment. The collected samples were immediately stored in liquid nitrogen before total RNA extraction. The expression profiles of PeSBPs were detected using qRT-PCR.

Conclusions
In the present study, a total of 14 SBP genes were identified in passion fruit. Based on the phylogenetic, gene structure, and conserved motifs analysis, all PeSBPs were classified into eight groups. The major gene expansion of the PeSBP gene family was segmental duplication and under negative selection. Cis-element analyses showed that PeSBPs responded to different stresses and plant hormones. In addition, the study of expression patterns under various stress types suggested that PeSBPs function across multiple stress responses. Taken together, our comprehensive analyses are helpful in selecting candidate SBP genes to further understand the classification and function of the SBP gene family in passion fruit.