Comparative Genomic Analysis of PEBP Genes in Cucurbits Explores the Interactors of Cucumber CsPEBPs Related to Flowering Time

The family of phosphatidylethanolamine-binding proteins (PEBPs) participates in various plant biological processes, mainly flowering regulation and seed germination. In cucurbit crops, several PEBP genes have been recognized to be responsible for flowering time. However, the investigation of PEBP family members across the genomes of cucurbit species has not been reported, and their conservation and divergence in structure and function remain largely unclear. Herein, PEBP genes were identified from seven cucurbit crops and were used to perform a comparative genomics analysis. The cucurbit PEBP proteins could be classified into MFT, FT, TFL, and PEBP clades, and further, the TFL clade was divided into BFT-like, CEN-like, and TFL1-like subclades. The MFT-like, FT-like, and TFL-like proteins were clearly distinguished by a critical amino acid residue at the 85th position of the Arabidopsis FT protein. In gene expression analysis, CsaPEBP1 was highly expressed in flowers, and its expression levels in females and males were 70.5 and 89.2 times higher, respectively, than those in leaves. CsaPEBP5, CsaPEBP6, and CsaPEBP7 were specifically expressed in male flowers, with expression levels 58.1, 17.3, and 15.7 times higher, respectively, than those of leaves. At least five CsaPEBP genes exhibited the highest expression during the later stages of corolla opening. Through clustering of time-series-based RNA-seq data, several potential transcription factors (TFs) interacting with four CsaPEBPs were identified during cucumber corolla opening. Because of the tandem repeats of binding sites in promoters, NF-YB (Csa4G037610) and GATA (Csa7G64580) TFs appeared to be better able to regulate the CsaPEBP2 and CsaPEBP5 genes, respectively. This study would provide helpful information for further investigating the roles of PEBP genes and their interacting TFs in growth and development processes, such as flowering time regulation in cucurbit crops.


Introduction
Phosphatidylethanolamine-binding proteins (PEBPs) are widespread in plants and contain a conserved domain peculiar to eukaryotes, despite sharing an ancient functional unit with PEBPs in bacteria, archaea, and animals [1,2].In the model plant Arabidopsis, AtPEBP proteins are mainly classified into three evolutionary branches, including FLOW-ERING LOCUS T (FT)-like, TERMINAL FLOWER1 (TFL1)-like, and MOTHER OF FT AND TFL1 (MFT)-like clades.In the AtFT-like clade, overexpression of both FT and TWIN SISTER OF FT (TSF) promotes earlier flowering [3].Among the three members of the TFL1like clade, TFL1 is a major negative regulator of inflorescence meristem development that considerably delays flowering time, while BROTHER OF FT AND TFL1 (BFT) play a role by acting redundantly with it [4].Also, ARABIDOPSIS THALIANA CENTRORADIALIS (ATC), an additional paralogous of TFL1, exhibits a weak ability to complement the early flowering and terminal flower formation of tfl1-1 mutant phenotypes, implying that ATC has a comparable function to TFL1 and BFT.MFT-like genes, which are considered to be the evolutionary origin of FT-like and TFL-like clade genes, regulate the transcripts of several key genes during seed germination but also have a mild FT-like activity to encourage flowering in Arabidopsis [5,6].In any case, PEBP family proteins are pivotal participants in controlling the plant flowering time.
FT and TFL1 proteins, with the opposite functions on flowering, have two highly conserved short motifs, DPDxP and GxHR, which presumably contribute to the conformation of the ligand binding pocket [1,7].The amino acid residues encoded by the fourth exons of FT and TFL1 that are essential for conferring biological specificity on the two proteins can be divided into four segments (Segment A-D).Segment B is especially important for the determination of functional specificity between FT and TFL1, as is the adjacent segment C that contains the LYN/IYN triplet motif [8].Additionally, the distal CCAAT box (TGTG(N2-3)ATG) and proximal CORE1/2 domains in the FT promoter region are found to be required cis-elements for binding by CONSTANS (CO) involved in photoperiodic pathways [9,10].These findings offer valuable information for investigating FT-and TFL1-like specific interactors in other crops.
The Cucurbitaceae members (cucurbits) contain many important vegetable and fruit crops, such as cucumber (Cucumis sativus L.), melon (Cucumis melo L.), watermelon (Citrullus lanatus L.), pumpkin (Cucurbita moschata L.), cushaw (Cucurbita argyrosperma L.), zucchini (Cucurbita pepo L.), and winter squash (Cucurbita maxima Duch.), etc. [11].The flowering time of the cucurbit crops is strongly correlated with early maturity and production, which is of great significance to the global or local economy [12].To date, only a few floweringrelated genes have been characterized in several cucurbit crops, including CsFT and CsTFL1 in cucumber, CmoFTL1 and CmoFTL2 in Chinese pumpkin, and FT in watermelon, all of which belong to the PEBP family [13][14][15][16].However, the regulatory mechanisms of the FT and TFL-like genes have not been well studied, and the roles of other PEBP members in regulating flowering time remain unclear.
The aim of this study was to identify the PEBP family genes in the seven cucurbit crops: cucumber, melon, watermelon, pumpkin, cushaw, zucchini, and winter squash.This investigation intends to comparatively analyze their chromosomal locations, phylogenetic relationships, and protein-conserved domains.Clustering of time-series-based RNA-seq data and promoter cis-element analyses were performed to explore the functions of the cucumber PEBP genes and their potential interacting transcription factors (TFs).These results could enhance our understanding of the cucurbit PEBP family members and provide available clues for delving deeper into their roles, especially in flowering time regulation.

Evolution of PEBP Genes in Arabidopsis and Seven Cucurbit Crops
To investigate the evolutionary relationships of the PEBP proteins, a comparative Neighbor-Joining (NJ) tree was conducted using the amino acid sequences encoded by the 72 PEBP genes from Arabidopsis and seven cucurbit crops (Figure 2, Table S1).According to the NJ tree, a total of 72 PEBPs fall into four major branches, which were designated as MFT clade, FT clade, TFL clade, and PEBP clade.Further, the TFL clade could be divided into BFT-like, CEN-like, and TFL1-like subfamilies (Figure 2).Both MFT-like and TFL1-like subfamilies consisted of 17 cucurbit PEBP members, including at least two PEBPs from each cucurbit crop.The FT/TSF-like subfamily was comprised of one ClaPEBP, one CmePEBP, and one CsaPEBP, as well as two CarPEBPs, two CmaPEBPs, and two CmoPEBPs.The member number of the FT/TSF-like subfamily seemed to be consistent with the ploidy of different crops.The remaining BFT-like, CEN-like, and PEBP_bact-like subfamilies only had one PEBP protein from each of the seven cucurbits, except for the absence of any CpePEBP in the BFT-like subgroup (Figure 2).
A syntenic map illustrated the conserved syntenic blocks across the seven cucurbit crops, and 62 of the 65 PEBP genes were mapped to these orthologous blocks, indicating a high degree of macrosynteny (Figure 3, Table S3).In total, 289 orthologous gene pairs were identified, with the number of genes matched varying from one to four.Among them, most one-to-one orthologous genes belonged to the PEBP genes from MFTlike, BFT-like, or PEBP-like subfamilies, e.g., CmaPEBP5/6/8/9, CmoPEBP4/5/7/8, and CpePEBP2/4/6/10.The one-to-four orthologous genes mainly come from the TFL1-like subfamily.The FT/TSF-like members matched one or two PEBPs as orthologous genes.Interestingly, CmePEBP3-CsaPEBP6 seemed to be a unique orthologous gene pair (Figure 3, Table S3), which likely resulted from an independent whole-genome duplication event between melon and cucumber.
Additionally, 28 orthologous gene pairs were identified between Arabidopsis and seven cucurbit crops, including five in cucumber, three in melon, five in watermelon, three in pumpkin, four in cushaw, five in zucchini, and three in winter squash (Figure S1, Table S4).Except for ClaPEBP1-AtATC/AtTFL and CsaPEBPE8-AtATC/AtTFL, the rest were oneto-one orthologous gene pairs.All of these gene pairs fall into the MFT, TFL (BFT-like, CEN-like, and TFL-like), and PEBP clades, indicating that they might be derived from a common ancestor of Arabidopsis and seven cucurbit crops.A syntenic map illustrated the conserved syntenic blocks across the seven cucurbit crops, and 62 of the 65 PEBP genes were mapped to these orthologous blocks, indicating a high degree of macrosynteny (Figure 3, Table S3).In total, 289 orthologous gene pairs were identified, with the number of genes matched varying from one to four.Among them, most one-to-one orthologous genes belonged to the PEBP genes from MFT-like, BFT-like, or PEBP-like subfamilies, e.g., CmaPEBP5/6/8/9, CmoPEBP4/5/7/8, and CpePEBP2/4/6/10.The one-to-four orthologous genes mainly come from the TFL1-like subfamily.The FT/TSF-like members matched one or two PEBPs as orthologous genes.Interestingly, CmePEBP3-CsaPEBP6 seemed to be a unique orthologous gene pair (Figure 3, Table S3), which likely resulted from an independent whole-genome duplication event between melon and cucumber.Additionally, 28 orthologous gene pairs were identified between Arabidopsis and seven cucurbit crops, including five in cucumber, three in melon, five in watermelon, three in pumpkin, four in cushaw, five in zucchini, and three in winter squash (Figure S1, Table S4).Except for ClaPEBP1-AtATC/AtTFL and CsaPEBPE8-AtATC/AtTFL, the rest were oneto-one orthologous gene pairs.All of these gene pairs fall into the MFT, TFL (BFT-like,

Comparative Analysis of the PEBP Family Gene Structure
Multiple sequence alignments showed that all the cucurbit PEBP proteins from the MFT clade, FT clade, and TFL clade had conserved PEBP domains and DPDxP motifs (Figures 4 and S2).Also, the 85th amino acid 'Y' of AtFT, marked with a green rectangle, could clearly distinguish the MFT-like (W), FT-like (Y), and TFL-like (H) proteins, which was a key amino acid residue that determined FT-like and TFL-like functions.Most cucurbit FT-like and TFL-like proteins had amino acid residues marked with a red rectangle, indicating they might interact with the 14-3-3 protein.The other two amino acid sequences, 'LGRQTVYAPGWRQN' and 'L/IYN' were found to be highly conserved in the segments B and C of each cucurbit FT-like protein (Figures 4 and S2), which were the critical motifs for determining the FT activity and the FT/TFL1 function.These results suggested that the FT-like clade from seven cucurbits was the most conserved, followed by the TFL-like clade, while the MFT-like clade was more variable.In gene structure analysis, the 58 cucurbit PEBP genes from the MFT, FT, and TFL clades were identified to possess a similar number of exons, varying between three and six.Among them, approximately 75% (44) of PEBP genes had four exons (Table S1).The PEBPs ranged from 233 bp to 6958 bp in gene length.From these, it could be inferred that the variations in the length and number of exons and introns may lead to the functional diversity of PEBP family members.

Cis-Acting Elements in the PEBP Promoter Regions
Within the putative promoter regions (2000 bp upstream from the transcription start site) of PEBP genes, there were five categories of cis-acting elements predicted, including the defensive or stress-related element, the developmental-related element, the hormoneresponsive element, the light-responsive element, and the TF binding element (Figure 5, Table S5).Among them, 40 of the 65 cucurbit PEBP promoters had no developmentalrelated element, indicating that they might be specific PEBP members involved in growth and development regulation.Exactly like the CsaPEBP1 promoter, it was the only one of eight cucumber PEBP members with this element (CAT-box), which functioned in meristem expression (Table S5).Moreover, the TF binding elements (e.g., AP-1, MYB, MYC, and HD-Zip) were identified in each PEBP promoter, accounting for almost 50% of the total (Figure 5, Table S5), indicating that they might have the potential to regulate PEBP genes.
FT is a critical integration factor in the pathway of flowering initiation signal (florigen) in Arabidopsis, and the distal CCAAT box and proximal CORE domains in the promoter region are required for its proper expression [10].Herein, we extracted the 7000-bp region of 11 FT/TSF-like genes from seven cucurbits and searched for the CCAAT box and CORE1/2 domain.Results showed that seven to sixteen CCACA boxes were present in all FT/TSF-like promoters, and the CORE1/2 domain was detected except for CmePBEP1, CmoPEBP2, and CpePEBP8 (Figure S3).The above cis-acting regulatory elements would provide some information for the tissue-specific expression and interaction models of the In gene structure analysis, the 58 cucurbit PEBP genes from the MFT, FT, and TFL clades were identified to possess a similar number of exons, varying between three and six.Among them, approximately 75% (44) of PEBP genes had four exons (Table S1).The PEBPs ranged from 233 bp to 6958 bp in gene length.From these, it could be inferred that the variations in the length and number of exons and introns may lead to the functional diversity of PEBP family members.

Cis-Acting Elements in the PEBP Promoter Regions
Within the putative promoter regions (2000 bp upstream from the transcription start site) of PEBP genes, there were five categories of cis-acting elements predicted, including the defensive or stress-related element, the developmental-related element, the hormoneresponsive element, the light-responsive element, and the TF binding element (Figure 5, Table S5).Among them, 40 of the 65 cucurbit PEBP promoters had no developmentalrelated element, indicating that they might be specific PEBP members involved in growth and development regulation.Exactly like the CsaPEBP1 promoter, it was the only one of eight cucumber PEBP members with this element (CAT-box), which functioned in meristem expression (Table S5).Moreover, the TF binding elements (e.g., AP-1, MYB, MYC, and HD-Zip) were identified in each PEBP promoter, accounting for almost 50% of the total (Figure 5, Table S5), indicating that they might have the potential to regulate PEBP genes.

Spatio-Temporal Expression Patterns of CsaPEBP Genes
Cucumber is a typical model for unisexual flower development, and two PEBP family genes, CsaPEBP2 (CsFT) and CsaPEBP5 (CsTFL1), have been reported to influence flowering time [14,15].To further investigate the roles of cucumber PEBP members, the expression patterns of eight CsaPEBPs were analyzed in various tissues (leaf, stem, tendril, male, female, and fruit).Reverse transcription-polymerase chain reaction (RT-qPCR) analysis indicated that each CsaPEBP gene displayed distinct expression patterns.In detail, Csa-PEBP2 and CsaPEBP8 were expressed in all tissues (Figure 6b,h).CsaPEBP1 was highly expressed in flowers, and its expression levels in females and males were 70.5 and 89.2 times higher, respectively, than those in leaves (Figure 6a).CsaPEBP5, CsaPEBP6, and Csa-PEBP7 were specifically expressed in male flowers, with expression levels 58.1, 17.3, and 15.7 times higher, respectively, than those of leaves (Figure 6e-g).CsaPEBP3 and Csa-PEBP4 exhibited higher expression levels in the stem (Figure 6c,d).Furthermore, the FT is a critical integration factor in the pathway of flowering initiation signal (florigen) in Arabidopsis, and the distal CCAAT box and proximal CORE domains in the promoter region are required for its proper expression [10].Herein, we extracted the 7000-bp region of 11 FT/TSF-like genes from seven cucurbits and searched for the CCAAT box and CORE1/2 domain.Results showed that seven to sixteen CCACA boxes were present in all FT/TSF-like promoters, and the CORE1/2 domain was detected except for CmePBEP1, CmoPEBP2, and CpePEBP8 (Figure S3).The above cis-acting regulatory elements would provide some information for the tissue-specific expression and interaction models of the cucurbit PEBP genes.

Spatio-Temporal Expression Patterns of CsaPEBP Genes
Cucumber is a typical model for unisexual flower development, and two PEBP family genes, CsaPEBP2 (CsFT) and CsaPEBP5 (CsTFL1), have been reported to influence flowering time [14,15].To further investigate the roles of cucumber PEBP members, the expression patterns of eight CsaPEBPs were analyzed in various tissues (leaf, stem, tendril, male, female, and fruit).Reverse transcription-polymerase chain reaction (RT-qPCR) analysis indicated that each CsaPEBP gene displayed distinct expression patterns.In detail, CsaPEBP2 and CsaPEBP8 were expressed in all tissues (Figure 6b,h).CsaPEBP1 was highly expressed in flowers, and its expression levels in females and males were 70.5 and 89.2 times higher, respectively, than those in leaves (Figure 6a).CsaPEBP5, CsaPEBP6, and CsaPEBP7 were specifically expressed in male flowers, with expression levels 58.1, 17.3, and 15.7 times higher, respectively, than those of leaves (Figure 6e-g).CsaPEBP3 and CsaPEBP4 exhibited higher expression levels in the stem (Figure 6c,d).Furthermore, the expression levels of CsaPEBP1, CsaPEBP2, CsaPEBP5, CsaPEBP6, and CsaPEBP7 were found to match the RNAseq data reported by Li et al. [17], indicating that these genes could play stable roles during the growth and development of cucumber, especially in the development of floral organs.In cucumber, the male or female corolla development could be divided into fo stages, including green bud, green-yellow bud, yellow bud, and flowering based on t color and shape (Figure 7a).To explore the roles of several CsaPEBP genes in the proce of male and female flower opening, RT-qPCR analysis was performed to examine th expression levels at each stage.As shown in Figure 7b, during the opening of the m flower, the expression levels of CsaPEBP1 and CsaPEBP7 had obviously increased at flo ering (M4), CsaPEBP5 and CsaPEBP6 showed higher expression at the yellow bud (M while CsaPEBP2 maintained a high expression level at both M3 and M4.In the progressi of female corolla opening, the expression of CsaPEBP1 and CsaPEBP5 exhibited a simil gradually upregulated trend, with a peak at flowering (C4), the peak expression of C PEBP2 occurring at the yellow bud (C3), while CsaPEBP6 and CsaPEBP7 were express at a higher level at C3 and C4.Unfortunately, the expression patterns of the five CsaPE Figure 6.Expression profiles of CsaPEBP1-CsaPEBP8 genes (a-h) in cucumber leaf, stem, tendril, male, female and ovary.Grey columns indicate the relative gene expression levels determined using RT-qPCR, and red lines represent the transcript abundance changes calculated using the Reads Per Kilobase per Million mapped reads (RPKM) method.Lowercase letters (for example, a, b, c, etc.) above the columns represent significant differences at the 0.05 level based on Tukey's test.
In cucumber, the male or female corolla development could be divided into four stages, including green bud, green-yellow bud, yellow bud, and flowering based on the color and shape (Figure 7a).To explore the roles of several CsaPEBP genes in the process of male and female flower opening, RT-qPCR analysis was performed to examine their expression levels at each stage.As shown in Figure 7b, during the opening of the male flower, the expression levels of CsaPEBP1 and CsaPEBP7 had obviously increased at flowering (M4), CsaPEBP5 and CsaPEBP6 showed higher expression at the yellow bud (M3), while CsaPEBP2 maintained a high expression level at both M3 and M4.In the progression of female corolla opening, the expression of CsaPEBP1 and CsaPEBP5 exhibited a similar, gradually upregulated trend, with a peak at flowering (C4), the peak expression of CsaPEBP2 occurring at the yellow bud (C3), while CsaPEBP6 and CsaPEBP7 were expressed at a higher level at C3 and C4.Unfortunately, the expression patterns of the five CsaPEBP genes in the ovary did not exhibit sufficient regularity.These results suggested that CsaPEBP1, CsaPEBP2, CsaPEBP5, CsaPEBP6, and CsaPEBP7 might be potential factors involved in regulating cucumber flowering, mainly functioning at the later stages: the yellow bud and flowering.

Screening for TFs That Interact with CsaPEBPs
FT or FT-like proteins, belonging to the PEBP family are well-known integrators in the flowering networks in multiple plants.Also, they can be directly activated by some TFs, or form complexes with TFs to trigger the onset of flowering [18].In the current work, the clustering of time-series analysis between 1251 differentially expressed TFs and 4 CsaPEBP genes (CsaPEBP1/2/5/7) was performed from the published RNA-seq data of the corolla opening.Results showed that the expression patterns of CsaPEBP1, CsaPEBP2, CsaPEBP5, and CsaPEBP7 were consistent with those of 66, 20, 50, and 23 TFs, respectively (Figure 8a, Table S6), with correlation coefficients varying from 0.8 to 1.0 (Figure 8b).Further, the TFs with a strong positive correlation (correlation index > 0.99) were selected for screening interactors of CsaPEBPs (Table S6).

Screening for TFs That Interact with CsaPEBPs
FT or FT-like proteins, belonging to the PEBP family are well-known integrat the flowering networks in multiple plants.Also, they can be directly activated by TFs, or form complexes with TFs to trigger the onset of flowering [18].In the current the clustering of time-series analysis between 1251 differentially expressed TFs and PEBP genes (CsaPEBP1/2/5/7) was performed from the published RNA-seq data corolla opening.Results showed that the expression patterns of CsaPEBP1, CsaPEBP2 PEBP5, and CsaPEBP7 were consistent with those of 66, 20, 50, and 23 TFs, respec (Figure 8a, Table S6), with correlation coefficients varying from 0.8 to 1.0 (Figure 8b ther, the TFs with a strong positive correlation (correlation index > 0.99) were select screening interactors of CsaPEBPs (Table S6).Multiple TF binding sites (TFBSs) were identified in the promoter regions of th PEBP1, CsaPEBP2, CsaPEBP5, and CsaPEBP7 genes, which could correspond to the TFs mentioned above (Figure 8c, Table S7).Based on the sequence similarity, the Multiple TF binding sites (TFBSs) were identified in the promoter regions of the CsaPEBP1, CsaPEBP2, CsaPEBP5, and CsaPEBP7 genes, which could correspond to the most TFs mentioned above (Figure 8c, Table S7).Based on the sequence similarity, the TFBSs with 100% similarity to Arabidopsis ones were retained, as follows: one ERF, three NAC, and one HSF in the CsaPEBP1 promoter; eight TALE in the CsaPEBP2 promoter; two Dof, and five NF-YB TALE in the CsaPEBP5 promoter; eight WRKY and 79 GATA in the CsaPEBP7 promoter (Figure 8d).These elements provided critical evidence for whether TFs directly regulate CsaPEBP genes.Especially, the tandem repeats of NF-YB and GATA might enhance the binding abilities of Csa4G037610 and Csa7G064580 to the promoters of CsaPEBP2 and CsaPEBP5, respectively, thereby regulating their expression.

Discussion
Proper flowering time for crops such as cucurbits means a greater yield and more robust adaptation [19].Mining the vital genes related to flowering time would be beneficial to gain a better understanding of their genetic regulatory mechanisms, facilitating the cultivation of cucurbit varieties with superior growth periods and adaptations.
Comparative genomics analysis is acknowledged as a powerful approach to exploring the evolution and function of genes.In the current work, we examined the inter-and intra-specific differences between seven cucurbit species using a comparative genomics analysis of their PEBP genes.By integrating transcript expression profiles with promoter element prediction, these findings would empower us to discover the novel genes and their potential interactors involved in cucumber flowering.

Conservation of the PEBP Family in Seven Cucurbits
At the whole-genome level, eight CsaPEBPs, nine CmePEBPs, seven ClaPEBPs, 10 CmoPEBPs, 11 CarPEBPs, 10 CpePEBPs, and 10 CmaPEBPs were identified from cucumber, melon, watermelon, pumpkin, cushaw, zucchini, and winter squash, respectively.The number of PEBP family members in seven cucurbits was similar to Arabidopsis (7) but fewer than rice (19) and maize (25) [20,21].This validated the theory that monocots had three to four times more PEBP family members than dicots did [22].In actuality, cucurbits have a slightly higher number of PEBP genes than Arabidopsis, perhaps as a result of having a larger genome.Interestingly, out of the seven cucurbits, the tetraploid ones (pumpkin, cushaw, zucchini, and winter squash) have a larger number of PEBP genes than the diploid cucurbit ones (cucumber, melon, and watermelon), which is similar to the findings from Yi et al. [19].
According to Arabidopsis, a total of 65 PEBP proteins from seven cucurbits could be grouped into four main clades as follows: MFT, FT, TFL (including BFT-like, CEN-like, and TFL1-like subclades), and PEBP clades.Among these, the PEBPs from the MFT, FT, and TFL clades exhibited high sequence homology with Arabidopsis PEBPs, sharing the conserved PEBP domains and DPDxP motifs.Significantly, the MFT-like, FT-like, and TFL-like proteins may be distinguished by the 85th amino acid of AtFT, which explains why FT and TFL1 homologs functioned differently in several plants [22].Consistently, the cucumber FT gene (CsaPEBP2) has been reported to be a key gene controlling early flowering, but CsTFL1 (CsaPEBP5) inhibited terminal flower formation [14,15,23].For the gene structure, most cucurbit PEBP genes contained four exons and had large variations in gene length, which might lead to the functional diversity of PEBP family members.
Gene duplication events are an important way for plant evolution to have high efficiency [12].For the PEBP family in the seven cucurbits, whole-genome duplication might contribute more to gene diversity.Together with interspecies collinearity analysis and evolutionary relationships, the orthologous gene pairs with Arabidopsis were conducive to investigating the biological functions of PEBP genes in cucurbits.Notably, all of these gene pairs belonged to the MFT, TFL, and PEBP clades, indicating that they may have shared an ancestor with Arabidopsis and seven cucurbit crops.Moreover, the majority of collinear gene pairs belonged to one-to-one relationships, which was comparable to other crops, such as tomato [24].Additionally, it was also noticed that the members from the PEBP clade, which encoded the PEBP domain present in bacteria and archaea, were distinct in both the conserved domain and gene structure, unlike PEBP members from other clades (Figure S4, Table S1).Nevertheless, their phylogenetic relationships and conserved sequence regions provided evidence for the ancient common origin of a basic protein functional unit.

Functional Diversification of Cucumber PEBP Genes
Despite extensive sequence conservation, PEBP genes are well-known to be the key regulators of various biological processes, especially FT/TFL-like genes in the regulation of floral transition [1,25].Considering that the exact molecular function of the cucurbit PEBP genes was, in most cases, still unclear, CsaPEBP genes from cucumber, a well-established model system for unisexual flower development, were chosen for expression pattern profiles.Both in RNA-seq and RT-qPCR analyses, eight CsaPEBP genes exhibited divergent expression levels in six tissues of plants.This resembled the reports on tomato, bamboo, and Rosaceae tree species [22,24,26].More particularly, CsaPEBP5, CsaPEBP6, and CsaPEBP7 in male flowers, as well as CsaPEBP3 and CsaPEBP4 in the stem, appeared to exhibit strong tissue-specific high expression.Even if the genes belonging to the same clade might function differently, such as CsaPEBP1 and CsaPEBP6 from the MFT-like subclade and CsaPEBP5 and CsaPEBP8 from the TFL-like subclade.For the genes expressed in either male or female flowers, CsaPEBP1, CsaPEBP2, CsaPEBP5, CsaPEBP6, and CsaPEBP7 might act at the later stages of corolla opening.CsaPEBP2 and CsaPEBP5 have been reported to influence flowering in cucumber, namely CsFT and CsTFL1 [14,15], and correspondingly, their expression trends were reversed during the female corolla opening process.Summarily, these variable expression patterns suggested functional diversification of CsaPEBP genes, which was supported by the variations in their gene structure.

Novel TFs That Potentially Interact with CsaPEBPs
In the plant flowering regulatory network, TFs generally function in conjunction with flowering-related genes to regulate flowering time.CO (Zinc finger), CIB1 (bHLH), and FLC (MADS-box) have been recognized as the classical TFs that directly bind to the FT and its homolog promoter and induce their expression [27], and recently several novel TFs have also been confirmed to do so.For example, Arabidopsis B3-Domain TF VAL1 regulates the floral transition by repressing FT [28].StABL1, a TF central to abscisic acid signaling, binds to FT homologs to promote flowering and, consequently, a short life cycle in potatoes [29].This demonstrated that other TFs also have the potential to directly interact with FT or its homologs.Considering this, a strategy combining clustering of time-series and promoter cis-element analysis was carried out.After screening, several TFs were obtained as possible roles for directly binding FT or other PEBPs during flowering response.Except for the ERF TF reported in Arabidopsis, there is still little information on the direct interaction between FT and the other TFs [30].
In addition, the tandem repeats of NF-YB and GATA binding sites occurred in the promoter regions of CsaPEBP2 and CsaPEBP5, which may mean a stronger binding ability between TFs and CsaPEBPs.Although these findings need to be further verified, they can still provide novel evidence for understanding the flowering regulatory mechanism in cucurbit crops.

Multiple Alignments, Phylogenetic and Synteny Relationships Analysis
Using the MUSCLE method with default settings, the PEBP family members from all seven cucurbit crops, as well as Arabidopsis, were sequenced by parameters.The outcome file was imported into the GeneDoc (version 2.7.0) software for visualization [33].Based on the multiple sequence alignment, an NJ phylogenetic tree was conducted using MEGA 11 (version 11.0.13)software in accordance with the p-distance model, as well as 1000 bootstrapping replicates and other default options [34].The NJ tree was displayed and optimized using an online tool iTOL [35].
Conserved syntenic blocks within and between the genomes of seven cucurbit species and Arabidopsis were identified and visualized using MCScanX (version 1.1) and TBtools-II (version 2.0) software [36,37].

Gene Localization and Structure Analysis
The chromosomal localization and exon/intron organization of the PEBP genes were investigated based on the GFF/GTF annotation files of seven cucurbit crop genomes from CuGenDB.The gene localization information was visualized using an advanced Circos plot in TBtools-II [37].Then the exon number and length of each PEBP gene were counted.

Investigation of Cis-Elements in the Promoter Region
The 2000 bp DNA sequences upstream at the transcription start site (ATG) were extracted from the CuGenDB database as the promoter regions of PEBP genes.These sequences were uploaded to the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 7 November 2023) to identify the putative cisacting elements.To predict TFBSs, the FASTA file of the promoter sequences was submitted to the PlantPAN4.0website (http://plantpan.itps.ncku.edu.tw/plantpan4/index.html, accessed on 19 February 2023) [38].

Plant Sampling, RNA Preparation and RT-qPCR Analysis
The inbred cucumber line 'D1008' was used as the experimental material and was planted at a greenhouse in the Horticulture Experiment Station, Northeast Agricultural University (Harbin, China).The cultivation conditions were maintained at 28 • C/18 • C (day/night) and a 16 h day/8 h night cycle.At the fruiting stage, a total of five tissues (leaf, stem, tendril, female flower, and male flower) were separately collected.Otherwise, female and male flowers were sampled from 1, 3, 4, and 5 days after labeling (DAL; labeling was made when an ovary became visible), which represented the four stages of cucumber flowering: green bud, green-yellow bud, yellow bud, and flowering.Five individual plants were pooled to form one biological replicate, and three biological replicates were prepared for each tissue.All samples were stored at −80 • C for gene expression detection.
Extraction of total RNA was performed using the RNAprep Pure Plant Kit with DNase I (Tiangen, Beijing, China), and then the qualified RNA was used for the synthesis of first-strand cDNA using the HiScript 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).For each gene, the RT-qPCR protocol with cross-intron primers was executed at a qTOWER3 system (Analytik Jena, Jena, Germany) in triplicate (Table S8).The cucumber elongation factor 1 alpha (CsEF1α) gene, as an internal control, was used to calculate the relative transcription levels of target genes using the 2 −∆∆Ct method.The statistical analysis of gene expression was performed by using SPSS software (Version 18.0).

Clustering of Time-Series and Correlation Analysis Based on RNA-Seq Data
The RNA-seq data published by Sun et al. [40] were acquired from the NCBI Gene Expression Omnibus database (GEO accession: GSE76358), which referred to the corollas at 1, 3, 4, and 5 DAL from the normal ovary.All protein sequences of the differentially expressed genes were submitted as queries to the PlantTFDB database [41] to screen the TFs.Also, the CsPEBP genes in this study were matched in the RNA-seq data using a BLASTp search.Finally, a total of 8 cucumber PEBP genes and 1251 TF genes were identified and shown in Table S9.Gene expression levels were measured using transcripts per million (TPM) values.
Clustering of time-series analysis was performed using the Short Time-series Expression Miner (STEM) program [42], and the maximum number of model profiles was set to fifty.
Pearson correlation coefficients of expression levels between CsPEBPs and TFs were estimated using GraphPad Prism 9 and visualized as a radar chart.

Conclusions
In short, 65 PEBP genes were comparatively identified and characterized in seven cucurbit crops, including their evolutionary, structural conservation, and functional diversification.A distinct PEBP clade was noticed, encoding the conserved domain in bacteria and archaea.CsaPEBP1 was highly expressed in male and female flowers, while CsaPEBP5, CsaPEBP6, and CsaPEBP7 were active in cucumber males.All four genes might function at the later stages of corolla opening.Furthermore, several cucumber TFs were inferred to be partners of FT or other PEBPs; of these, Csa4G037610 and Csa7G64580 might have more vital binding ability due to the tandem repeats of NF-YB and GATA in CsaPEBP2 and CsaPEBP5 promoters.Together, these outcomes enrich our understanding of the primary characteristics of the PEBP family in cucurbit crops and provide novel thoughts for flowering regulation through TFs.

Figure 1 .
Figure 1.Localization and synteny of the PEBP genes in seven cucurbit crops.(a) ClaPEBPs in the watermelon genome.(b) CmePEBPs in the melon genome.(c) CsaPEBPs in the cucumber genome.(d) CarPEBPs in the cushaw genome.(e) CmaPEBPs in the winter squash genome.(f) CmoPEBPs in the pumpkin genome.(g) CpePEBPs in the zucchini genome.Gene pairs with a syntenic relationship were joined by the red lines.Tandem duplicated genes were laid in a red box.

Figure 1 .
Figure 1.Localization and synteny of the PEBP genes in seven cucurbit crops.(a) ClaPEBPs in the watermelon genome.(b) CmePEBPs in the melon genome.(c) CsaPEBPs in the cucumber genome.(d) CarPEBPs in the cushaw genome.(e) CmaPEBPs in the winter squash genome.(f) CmoPEBPs in the pumpkin genome.(g) CpePEBPs in the zucchini genome.Gene pairs with a syntenic relationship were joined by the red lines.Tandem duplicated genes were laid in a red box.

Figure 2 .
Figure 2. Evolutionary tree of FT/PEPB-related proteins in Arabidopsis and seven cucurbit crops.

Figure 3 .
Figure 3.A syntenic map of PEBP genes among seven cucurbit crops.

Figure 4 .
Figure 4. Pattern diagram of PEBP family proteins from seven cucurbit crops.Red rectangle indicates amino acid residues that interact with 14-3-3 protein.The green rectangle indicates a key amino acid residue that determines MFT-like, FT-like and TFL-like proteins.Black boxes represent the conserved DPDxP, GxHR motif and L/IYN.Underlines represent segments A, B, C and D, respectively.

Figure 4 .
Figure 4. Pattern diagram of PEBP family proteins from seven cucurbit crops.Red rectangle indicates amino acid residues that interact with 14-3-3 protein.The green rectangle indicates a key amino acid residue that determines MFT-like, FT-like and TFL-like proteins.Black boxes represent the conserved DPDxP, GxHR motif and L/IYN.Underlines represent segments A, B, C and D, respectively.

Figure 5 .
Figure 5. Prediction of cis-acting elements in the cucurbit PEBP gene promoters.

Figure 5 .
Figure 5. Prediction of cis-acting elements in the cucurbit PEBP gene promoters.
Int. J. Mol.Sci.2024,25,  x FOR PEER REVIEW 9 of expression levels of CsaPEBP1, CsaPEBP2, CsaPEBP5, CsaPEBP6, and CsaPEBP7 we found to match the RNA-seq data reported by Li et al.[17], indicating that these gen could play stable roles during the growth and development of cucumber, especially in t development of floral organs.

Figure 6 .
Figure 6.Expression profiles of CsaPEBP1-CsaPEBP8 genes (a-h) in cucumber leaf, stem, tend male, female and ovary.Grey columns indicate the relative gene expression levels determined us RT-qPCR, and red lines represent the transcript abundance changes calculated using the Reads P Kilobase per Million mapped reads (RPKM) method.Lowercase letters (for example, a, b, c, e above the columns represent significant differences at the 0.05 level based on Tukey's test.
Int. J. Mol.Sci.2024,25,  x FOR PEER REVIEW 10 of 17 genes in the ovary did not exhibit sufficient regularity.These results suggested that Csa-PEBP1, CsaPEBP2, CsaPEBP5, CsaPEBP6, and CsaPEBP7 might be potential factors involved in regulating cucumber flowering, mainly functioning at the later stages: the yellow bud and flowering.

Figure 7 .
Figure 7. Expression patterns of CsaPEBP genes in the process of cucumber flowering.(a) Progression of corolla opening in the normal male (M; top) and female (C, corolla and O, ovary; bottom), including four stages: green bud (1), green-yellow bud (2), yellow bud (3), and flowering (4).Bar = 1 cm.(b) Heatmap (left) showing different expression levels of five CsaPEBP genes in male and female flowers at four developmental stages.Changes in gene expression levels are shown in color as the scale.Mini Line Chart (right) shows the mean expression trend of each gene.

Figure 7 .
Figure 7. Expression patterns of CsaPEBP genes in the process of cucumber flowering.(a) Progression of corolla opening in the normal male (M; top) and female (C, corolla and O, ovary; bottom), including four stages: green bud (1), green-yellow bud (2), yellow bud (3), and flowering (4).Bar = 1 cm.(b) Heatmap (left) showing different expression levels of five CsaPEBP genes in male and female flowers at four developmental stages.Changes in gene expression levels are shown in color as the scale.Mini Line Chart (right) shows the mean expression trend of each gene.

Figure 8 .
Figure 8. Screening for TFs that interact with CsaPEBPs.(a) Time-series clusters of CsaPEB TFs based on RNA-seq data.The horizontal axis represents four developmental stages in the las of a female flower: green bud (C1), green-yellow bud (C2), yellow bud (C3), and flowerin Trend lines are color-encoded with gray shades denoting high membership values of genes b ing to the time-series cluster, and the red lines indicate the expected trend.(b) Radar chart Pearson correlation coefficient between expression levels of TFs and CsaPEBPs.(c) Number o scription factor binding sites (TFBSs) in CsaPEBP gene promoters.(d) Putative TFs interactin CsaPEBPs.

Figure 8 .
Figure 8. Screening for TFs that interact with CsaPEBPs.(a) Time-series clusters of CsaPEBPs and TFs based on RNA-seq data.The horizontal axis represents four developmental stages in the corollas of a female flower: green bud (C1), green-yellow bud (C2), yellow bud (C3), and flowering (C4).Trend lines are color-encoded with gray shades denoting high membership values of genes belonging to the time-series cluster, and the red lines indicate the expected trend.(b) Radar chart of the Pearson correlation coefficient between expression levels of TFs and CsaPEBPs.(c) Number of transcription factor binding sites (TFBSs) in CsaPEBP gene promoters.(d) Putative TFs interacting with CsaPEBPs.