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Chemical and Biological Technologies in Agriculture
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Genome-wide identification and expression analysis of watermelon (Citrullus lanatus) SSR2 gene during fruit development

Chemical and Biological Technologies in Agriculture volume 11, Article number: 100 (2024) Cite this article

Abstract

Background

Sterol side-chain reductase 2 (SSR2) is a key enzyme in the synthesis of plant cholesterol pathway. Despite the importance of watermelon as a horticultural cash crop, the SSR2 gene in watermelon has not been previously studied or reported.

Results

In this study, 28 SSR2 genes were identified in the watermelon genome. The physicochemical properties of 28 ClaSSR proteins were predicted by bioinformatics methods, and the gene structure, conserved motif, chromosome localization, phylogenetic analysis, cis-acting elements, expression patterns, promoter activity analysis and subcellular localization of ClaSSRs were studied. The 28 ClaSSRs were unevenly distributed on 11 chromosomes, and phylogenetic analysis showed that they could be grouped into 4 groups with other related Cucurbitaceae homologous genes. Analysis of gene structure and motifs revealed similarities in exons/introns and motifs between members of the same group, further supporting phylogenetic results. The RT–qPCR results showed variations in ClaSSRs expression during watermelon fruit development. The analysis of promoter activity for ClaSSR25 showed strong activity. Subcellular localization studies confirmed that ClaSSR25 is mainly located in the cytoplasm, which aligns with the predicted outcomes. We additionally estimated the network of protein–protein interactions for ClaSSR25 and analyzed proteins that could potentially interact with ClaSSR25 in melon and Arabidopsis thaliana.

Conclusions

We conducted bioinformatics analysis and expression analysis of members of the watermelon SSR2 gene family in this work, and the outcomes set the stage for further investigations into the watermelon SSR2 gene.

Graphical Abstract

Introduction

All eukaryotes need sterols, and while plant sterols play a significant role in the structure of plant cell membranes, certain sterols have also been linked to the growth and development of plants as well as their ability to respond to biotic and abiotic stresses [1,2,3,4,5,6]. Phytosterols are also involved in the formation of lipid rafts, a special structure that helps establish cell polarity, signaling, and plant–pathogen interactions [6,7,8]. Cholesterol, which is an important unsaturated sterol, can be present as a component of lipids on the leaf surface or in a free form in plant cell membranes [6, 9, 10]. In Solanaceae plants, such as tomatoes and potatoes, cholesterol functions as a precursor molecule for steroidal glycoalkaloids (SGAs) [11,12,13,14]. In addition, its derivative, 7-dehydrocholesterol (7-DHC), is an essential precursor for the synthesis of vitamin D3. When exposed to UVB radiation at 290–315 nm, 7-DHC is transformed into vitamin D3 [15, 16].

7-DHC holds great potential for creating vitamin D3 biofortified crops, and scholars have used CRISPR/Cas9 to silence 7-dehydrocholesterol reductase 2 (7-DR2), leading to the overaccumulation of 7-DHC, which resulted in successful vitamin D3 biofortified tomatoes [17]. Whereas cholesterol is not only a component of plant cell membranes but also of interest as a precursor for the synthesis of SGAs, scientists used RNA interference (RNAi) to silence sterol side-chain reductase 2 (SSR2), which inhibits the synthesis of cycloartanol and significantly reduces cholesterol levels, lowering the accumulation of SGAs that would be detrimental to the plant if accumulated in excessive amounts, which effectively reduces the amount of SGAs in potatoes [18]. The cholesterol derivative 7-DHC is both a precursor for vitamin D3 synthesis and an important intermediate product in the plant cholesterol synthesis pathway, so we think it is valuable to study the plant cholesterol synthesis pathway even though SGAs are rarely studied in species other than Solanaceae.

Phytosterol metabolism and plant cholesterol production pathways overlap. As demonstrated in Fig. 1, depending on the enzyme that serves as a catalyst, cycloartenol may be involved in the phytosterol metabolic pathway or the pathway that produces cholesterol [11]. Sterol C-24 methyltransferase (SMT1) metabolizes cycloartenol, causing it to enter the phytosterol metabolic pathway. A cascade of enzymes then catalyzes the production of plant sterols, including stigmasterol, campesterol, and sitosterol. Alternatively, cycloartenol enters the cholesterol production pathway under the catalytic action of SSR2 and progressively synthesizes cholesterol with the help of several additional enzymes [11, 18]. In 2021, Zheng used CRISPR–Cas9 to modify StSSR2, and potato SGA levels were correspondingly drastically decreased [19]. The lowering of SGAs by RNA interference (RNAi) study and this one both demonstrate that SSR2 is an essential enzyme in the plant cholesterol synthesis pathway [18, 19].

Fig. 1
figure 1

Schematic diagram of plant cholesterol synthesis and phytosterol metabolism pathways. The red color block represents the cholesterol synthesis pathway, while the blue color block represents the phytosterol metabolism pathway. The single arrow represents one reaction step, and the multiple arrows represent multiple reaction steps. CAS cycloartenol synthase, SSR2 sterol side chain reductase 2, 7-DHC 7-dehydrocholesterol, 7-DR2 7-dehydrocholesterol reductase 2, SMT sterol C-24 methyltransferase

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Ranked among the top ten fruits in the world, watermelon (Citrullus lanatus) is known for its sweet, and delicious flavor. Nearly 100 million tons of watermelons were produced worldwide in 2022, according to the Food and Agriculture Organization of the United Nations (https://www.fao.org/statistics/zh/) [20]. Studies have revealed that free vitamin D3 is present in zucchini, but watermelon, which is a member of the same Cucurbitaceae family, has not been the subject of any pertinent research [16]. In the process leading to the synthesis of cholesterol in plants, SSR2 is an essential enzyme [18, 19]. Knowing how it works is crucial for the synthesis of critical derivatives, such as vitamin D3, as well as for the generation of cholesterol. However, current research on SSR2 in watermelon is limited, and information about its gene family has not yet been discovered and elucidated. To close this gap, the goal of this work was to find watermelon members of the SSR2 gene family using bioinformatics techniques. Gene covariance, expression patterns, promoter activity, subcellular localization, conserved motifs, cis-acting elements, gene structure, chromosome distribution, and evolutionary relationships are all included in the research. The results of this study lay the foundation for future research on the watermelon SSR2 gene.

Materials and methods

Plant materials

The watermelon materials used are all Huajing No. 18, taken from Henan Luoyang Agricultural Development Agricultural Biotechnology Co., Ltd. The variety is early maturing, with small fruit, red flesh, green skin, and soluble solids of about 12.5%. All pulp samples were frozen with liquid nitrogen immediately after harvest and refrigerated at – 80 ℃.

Identification of ClaSSRs in watermelon

Watermelon (97103_pep_v2.5), cucumber (Cucumis sativus L., Chinese long v3), melon [Cucumis melo L., DHL92 v4 (http://cucurbitgenomics.org/v2/ftp/genome/melon/DHL92/v4.0)] and pumpkin (Cucurbita moschata, Cpepo v4.1) protein sequence files and genomic files were obtained from the Cucurbita Genomics Database (CuGenDB, http://cucurbitgenomics.org/v2/). The conserved domain FAD_binding_4 of the ClaSSR proteins and its corresponding Pfam were identified from Uniprot (https://www.uniprot.org/). The Hidden Markov Model (HMM) profiles of the FAD_binding_4 domain (Pfam:PF01565) were downloaded from the PFAM database (http://pfam.xfam.org/). Candidate genes of the ClaSSR family were obtained using HMM profiles with an e-value < 10–3. In addition, the SSR2 protein sequences of potato was downloaded from NCBI (https://www.ncbi.nlm.nih.gov) as the query sequence and BLASTp aligned with the protein sequence of watermelon [19]. The SSR2 genes identified from the two ways were taken together. All candidate gene sequences obtained are submitted to the CDD tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and PFAM (https://www.ebi.ac.uk/interpro/), and genes containing FAD_binding_4 domains are retained.

Analysis of the basic characteristics and phylogenetic analysis of ClaSSRs

Number of amino acid, molecular weight, pI, and grand average of hydropathicity of the ClaSSRs were analyzed using the Protein Paramter Calc program in TBtools (v2.075) [21]. The subcellular localization of 28 ClaSSRs was predicted with Cell-Ploc (http://www.csbio.sjtu.edu.cn/bioinf/plant/). The SSR2 genes of melon, cucumber and pumpkin were identified by the same method as watermelon, and a phylogenetic tree was constructed by combining the above SSR2 genes with the 28 identified ClaSSRs. The phylogenetic tree of these SSR2 genes was constructed with MEGA X and the JTT model in the maximum likelihood method employed, all other parameters are the default values. Furthermore, the tree was modified on the chiplot website (https://www.chiplot.online/).

Chromosomal distribution analysis, structure, motif analysis and protein three-dimensional structure prediction of ClaSSRs

Information of the chromosome length and the position of genes on the chromosomes was obtained from the watermelon gene structure annotation file (GFF3), and the ClaSSRs were mapped to the chromosome through the Gene Location Visualize from GTF/GFF in TBtools (v2.075). The conserved motifs of ClaSSRs were analyzed using the MEME website (https://meme-suite.org/meme/tools/meme), and the parameters were set to the optimal motif width of 6–12; maximum motif set to 20. The structures of ClaSSRs were analyzed by Gene structure view (advanced) in TBtools (v2.075) [21]. The protein sequences of Class were input and the tertiary structures of ClaSSRs were predicted by homology modeling in SWISS–MODEL (https://swissmodel.expasy.org/).

Prediction of cis-acting elements, and collinearity analysis of ClaSSRs family

The obtained ClaSSRs were analyzed using the online prediction tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict all obtained upstream 2000 bp sequences of ClaSSRs and the major cis-acting elements. The collinearity relationships between ClaSSRs were analyzed and visualized using Advanced Circos in TBtools (v2.075) [21].

Expression analysis of ClaSSRs at different developmental stages of watermelon fruit and its correlation with other genes

Watermelon samples were collected at 7 days, 14 days, 21 days, 28 days and 35 days after pollination, and were frozen and ground in liquid nitrogen for watermelon RNA extraction. The total RNA of Huajing XVIII watermelon fruits was extracted with the Tiangen Polysaccharide Polyphenol Plant Total RNA Extraction Kit. FPKM values for Huajing were derived from transcriptome sequencing data [18], and the complete data are shown in Supplementary Table S1. Heatmaps were generated using HeatMap in TBtools (v2.075) [21]. According to the KEGG module database (https://www.kegg.jp/kegg/module.html), the genes related to plant steroid biosynthesis were screened and calculated by combining their expression with ClaSSRs [22].

Quantitative real-time PCR

Vazyme HiScript II First Strand cDNA Synthesis Kit was used to reverse transcribe the above RNA into cDNA, which was used as a template for RT–qPCR. DNA was cloned from the leaves using the FastPure Plant DNA Isolation Mini Kit to clone the ClaSSR promoters. The primers for the ClaSSRs were designed using Premier 5, and actin was used as the housekeeping gene. The relative expression of each gene was calculated using 2−∆∆CT algorithm, and each experiment was repeated three times. All primers for RT–qPCR are shown in Supplementary Table S2.

Promoter activity analysis

The promoter expression vector (0390-35S-GUS) was constructed using a homologous recombination method. Primer sequences are shown in Supplementary Table S3. When tobacco (Nicotiana benthamiana L.) grew for 4–6 weeks, transient transformation of tobacco leaves was performed by vacuum expansion. The GUS vector without promoter was used as a negative control and the GUS vector under the control of CaMV35S promoter was used as a positive control. GUS staining was performed after one days of incubation under normal light, and then decolorization with 70% ethanol was performed to observe the staining status.

Subcellular localization

The open reading frame (ORF) of ClaSSR25 without stop codon was cloned into the EcoR-I and BamH-I restriction sites of vector 101LYFP to form a fusion structure co-expressing ClaSSR25 and YFP (yellow fluorescent protein) protein (35S: ClaSSR25-YFP). After successful sequencing, the recombinant plasmid was transferred to GV3101 and transfected into Nicotiana benthamiana leaves [23, 24]. After normal culture for 2–3 days, the fluorescence signal was observed using a laser scanning confocal microscope [25].

Prediction of protein–protein interaction network for ClaSSR25

The protein interaction network was predicted using the STRING online server (https://cn.string-db.org/). Interaction protein prediction was carried out in two species, Arabidopsis thaliana and Cucumis melo, respectively. The minimum protein–protein interaction score threshold is 0.9, and all other values are the default.

Results

Identification of ClaSSRs in watermelon

Using Uniprot (https://www.uniprot.org/), we first determined the conserved domain of the watermelon SSR2 gene to be FAD_binding_4 (Pfam: PF01565) to discover members of the watermelon SSR2 gene family. After screening the SSR2 gene family members from the watermelon genome using HMM search and BLASTp, we gave them the names ClaSSR1 ~ ClaSSR28 based on where on the chromosomes they were found. We also conducted a physicochemical analysis, the findings of which are shown in Table 1. The majority of the amino acid sequence lengths fell between 500 and 800, and the isoelectric points ranged from 5.45 (ClaSSR5) to 9.56 (ClaSSR20). Approximately 64% of the ClaSSR proteins have an isoelectric point larger than 7, indicating a potential basic state. The isoelectric point of the remaining proteins is less than 7, suggesting that they are acidic. The ClaSSR proteins ranged in molecular weight from 239,811.64 (ClaSSR1) to 57,353.16 (ClaSSR14). Moreover, all 28 ClaSSR proteins had negative grand averages for hydropathicity, indicating that they were all hydrophilic proteins. Of the 28 ClaSSRs, about 68% are predicted to be localized in the cytoplasm, 25% in the cell membrane, and some genes in the nucleus, chloroplasts, etc.

Table 1 Chromosomal location and amino acid numbers, putative molecular weight, pI, grand average of hydropathicity, and subcellular localization predictions for ClaSSRs
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Chromosomal distribution analysis and phylogenetic analysis of ClaSSRs in watermelon

Watermelon has eleven chromosomes in total. Since chromosome 6 lacked a ClaSSR, the analysis only showed the remaining 10 chromosomes. It was found that 28 ClaSSRs were randomly distributed on these chromosomes. Remarkably, chromosome 3 included 14 ClaSSRs, or more than half of the total, while the other chromosomes only had one or two ClaSSRs. While chromosomes 2, 8, 9, and 11 only had one ClaSSR, which made up less than 4% of the total, chromosomes 1, 4, 5, 7, and 10 each had two ClaSSRs, making up roughly 7% of the total (Fig. 2A).

Fig. 2
figure 2

Chromosome distribution of ClaSSRs and phylogenetic tree of melon, cucumber, pumpkin and watermelon. A Scale on the left represents the length of the chromosome in Mb. The thin vertical bars on the right represent the chromosomes of watermelon, the chromosome number is shown on the left side of each chromosome, and the internal color represents the gene density on the chromosome, the bluer the color, the lower the gene density at that position, and the redder the color, the higher the gene density at that position. B Tree contains a total of 122 protein sequences containing FAD_binding_4 domains, of which the yellow square symbol indicates the melon sequence, the light blue diamond represents the pumpkin sequence, the dark blue triangle represents the cucumber sequence, and the red circle indicates the watermelon sequence

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To investigate the evolutionary relationship of SSR2, we used the maximum likelihood method to create a phylogenetic tree with ClaSSRs and the SSR2 genes from cucumber, melon, and pumpkin (Fig. 2B). The 122 SSR2 genes were split up into four subgroups. The SSR2 genes of the four species mentioned above were spread rather evenly throughout the remaining two groupings, with the exception of group III and group IV, which were devoid of pumpkin SSR2 genes. Group I comprised 26 sequences: 6 for melon and watermelon, and 7 for cucumber and pumpkin. Group II included of 39 sequences, with 15 pumpkins and 8 of each type of melon, cucumber, and watermelon. Group III comprised six watermelon sequences and nine melon and cucumber sequences total. There were 33 sequences in Group IV, including 8 for watermelon, 12 for cucumber, and 13 for melon. A close association was shown by the placement of very comparable sequences in the same group with relatively high bootstrap values.

Conserved motifs, gene structure analysis and protein three-dimensional structure prediction of ClaSSRs

To identify the conserved and diverse ClaSSR proteins, the conserved motifs of ClaSSRs were predicted utilizing the online website MEME (https://meme-suite.org/meme/tools/meme). The 28 ClaSSRs can be categorized into 3 groups based on the phylogenetic tree, composition, and distribution of motifs (Fig. 3A). Each subgroup exhibits genes with similar motif composition and distribution. For instance, ClaSSR7, ClaSSR9, ClaSSR10, ClaSSR12, ClaSSR13, and ClaSSR15 in group I share 13 motifs, all with identical types and distribution positions. However, there are variations in motif composition within the same group of genes. For instance, ClaSSR28, of the group III, lacks motif 4 and motif 15, while ClaSSR17 is the sole gene in the group III that includes motif 16. The resemblance in motif composition suggests gene similarity, whereas differences in motif composition may contribute to functional diversity among genes [26] (Fig. 3B).

Fig. 3
figure 3

Phylogenetic relationships, motifs, gene structures and three-dimensional structure of ClaSSRs. A Rectangular phylogenetic tree constructed for ClaSSR protein sequences; B Colored rectangular boxes represent the conserved motifs of ClaSSRs; C Green rectangles and black lines indicate CDS (coding sequence) and introns, respectively, yellow rectangles indicate UTR (untranslated region), and black lines in horizontal coordinates indicate the relative length of genes. D Three-dimensional structure of some proteins of the ClaSSR family

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Analyzing the genetic structural distribution of exons and introns is essential to understanding phylogenetic processes and identifying phylogenetic relationships more accurately [27]. This study examined the DNA sequences of ClaSSRs to ascertain the amount of UTRs, CDSs, and introns in each. It was found that 28 ClaSSRs had different numbers of CDSs, spanning from 1 to 19, by comparing the numbers and locations of these elements (Fig. 3C). ClaSSRs contain several single CDS genes, such as ClaSSR2, ClaSSR6, and ClaSSR9. The number of introns in the remaining ClaSSRs ranged from 1 to 18, and ClaSSR1 had multiple larger intron insertions, resulting in significantly longer gene lengths than other genes in the family (Fig. 3C). Variations in the quantity of CDSs and intron insertions could account for the variations in the composition and capabilities of various ClaSSR members. The findings of the phylogenetic study were supported by the minor differences within the same group, which suggests functional similarity between genes within the same group.

The tasks that proteins carry out are intimately associated with their three-dimensional structure [28]. Eight ClaSSR proteins' three-dimensional structures were predicted using the homology modeling approach and SWISS–MODEL. The model with the best degree of confidence was chosen, and the resulting predictions are displayed in Fig. 3D. These included the structurally similar ClaSSR1 and ClaSSR5, ClaSSR12 and ClaSSR14, and ClaSSR16 and ClaSSR18. The phylogenetic alignment results were in line with the aforementioned findings. The evolutionary tree showed that ClaSSR21 and ClaSSR25 belonged to separate families, and their three-dimensional protein architectures differed as well, indicating potential differences in the roles they play in watermelon.

Covariance analysis of the ClaSSRs

Plants are more able to adapt to their surroundings as a result of massive segment duplication and tandem repeats amplification of gene families [27]. We used intra-species collinear analysis of ClaSSRs to investigate the evolutionary connection of the SSR2 gene in watermelon. The analysis revealed no tandem duplication events, only one segmental duplication pair involving ClaSSR18 and ClaSSR26 (Fig. 4A), suggesting that fragment repeat events are a key driver of ClaSSRs gene diversity [29].

Fig. 4
figure 4

Covariance analysis of ClaSSRs. A Colinearity in the watermelon genome. The gray line in the background represents all co-linear blocks of the watermelon genome, the red line represents ClaSSRs gene pairs of duplicated fragments, the GC ratio of chromosomes is represented by the heat map, and N ratio is represented by the point graph. B Colinearity between Arabidopsis, Citrullus lanatus and Cucumis melo genomes. The gray line in the background represents all collinear blocks of the genome between the two species, and the red line represents the gene pairs that are colinear with the ClaSSRs

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As shown in Fig. 4B, we examined the gene collinearity of Arabidopsis thaliana, melon, and watermelon to further ascertain the homologous link of SSR2 genes in watermelon with other plants. The findings demonstrated that 26 melon genes and 15 Arabidopsis thaliana genes were collinear with 20 ClaSSRs and 10 ClaSSRs, respectively (Fig. 4B). Among these, ten genes were discovered to be identical in melon and Arabidopsis thaliana genes (ClaSSR2, ClaSSR6, ClaSSR16, ClaSSR18, ClaSSR21, ClaSSR22, ClaSSR24, ClaSSR26, ClaSSR27, and ClaSSR28), indicating their important involvement in the evolution of ClaSSRs [30].

Analysis of cis-acting elements in ClaSSR promoters

For cells to accomplish effective and accurate transcriptional level regulation of gene expression, the promoter is a crucial component [31]. To clarify the types and distribution of cis-acting elements contained in the promoter of ClaSSRs, the upstream 2000 bp sequence of the gene was extracted and prediction analysis was performed using Plant Care. The two primary kinds of cis-acting elements found in the ClaSSR family are abiotic stress and hormonal response. Hormone response elements include the following: methyl jasmonate (TGACG-motif and CGTCA-motif), auxin (TGA-element and AuxRR-core), gibberellin (TATC-box, P-box, and GARE-motif), abscisic acid (ABRE), salicylic acid (TCA-element), and zeatin (O2-site) (Fig. 5A). Light response elements (GT1-motif, G-Box, AE-box, MRE, ACE, and Box 4), low temperature response element (LTR), and anaerobic induction-related (ARE) are among the abiotic stress response elements. Furthermore, components implicated in wound-responsiveness (WUN-motif), defense and stress response (TC-rich repeats), and endosperm expression (GCN4_motif) were identified. Light-responsive element Box 4 was found to be abundant in the promoters of ClaSSR8 and ClaSSR25, whereas the ClaSSR24 gene had 11 light-responsive elements G-box and 14 abscisic acid response elements ABRE (Fig. 5B). This implies that hormones including auxin, gibberellin, MeJA, abscisic acid, and others, as well as light, are probably responsible for controlling ClaSSRs.

Fig. 5
figure 5

Prediction of regulatory cis-acting elements in ClaSSRs promoters. A Different colors inside the box represent different cis-acting elements, and the promoter length is 2000 bp. B Statistics on the number of different regulatory cis-acting elements contained in the ClaSSRs promoters

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Expression pattern, RT–qPCR and analysis of ClaSSRs during watermelon fruit development

To gain a preliminary understanding of the function of ClaSSRs, we analyzed RNA-Seq data and identified 13 ClaSSRs with relatively high expression out of a total of 28 ClaSSRs. The expression of these 13 ClaSSRs is presented using a heatmap (Fig. 6A). 7 days, 14 days, 21 days, 28 days and 35 days denote 7, 14, 21, 28 and 35 days after pollination of watermelon, respectively. These genes were clustered according to their expression patterns, and genes within the same cluster showed similar expression patterns. For example, ClaSSR1, ClaSSR12, ClaSSR21, and ClaSSR23 showed similar expression patterns over time after pollination, and they were all roughly down-regulated. In contrast, the expression of ClaSSR4, ClaSSR16, and ClaSSR19 was progressively up-regulated. The expression patterns of ClaSSR17, ClaSSR20, ClaSSR22, and ClaSSR24 were similar, showing an early up-regulation of expression, a peak at 21 days, and a subsequent down-regulation. Furthermore, ClaSSR27 exhibited stable expression throughout all timepoints, peaking at 21 days and showing minimal variation in expression during other times. When compared to the other 12 genes, ClaSSR25 was significantly more expressed practically all the time, peaking at 7 days. It was subsequently down-regulated at first, up-regulated at 28 days, then down-regulated again, but at 35 days, its expression level was still higher than the other ClaSSRs.

Fig. 6
figure 6

Expression patterns and real-time quantitative PCR of ClaSSRs. A Thirteen genes with relatively high expression were selected from all ClaSSRs for expression pattern analysis; B Expression analysis of 8 ClaSSRs at diferent periods. Transcript levels were measured by RT–qPCR. Values are represented as mean ± standard deviation from three biological replicates. The asterisks indicate the significant level based on a Bartlett’s test (*0.05 ≤ P < 0.01, **0.01 ≤ P < 0.001, ***P ≤ 0.001)

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Real-time quantitative PCR was conducted on eight ClaSSRs from Huajing XVIII fruits at different developmental stages, and the results are presented in Fig. 6B. To determine whether there were any appreciable differences in the expression of these eight genes at other times, the relative expression at 7 days was utilized as a reference. Comparing 35–7 days, six ClaSSRs (ClaSSR1, ClaSSR4, ClaSSR17, ClaSSR19, ClaSSR20, and ClaSSR22) exhibited noticeably greater expression. On the other hand, ClaSSR17 displays a distinct pattern of expression, up-regulating gradually during predevelopment, down-regulating abruptly around 28 days, and then up-regulating again. The pattern was marginally different from the other ClaSSRs. After 7 days the relative expression of ClaSSR25 peaked, was markedly down-regulated at 14 days, and then steadily increased after 21 days. The expression level was still noticeably lower at 35 days than it was at 7 days, though. Comparably, after 21 days, the relative expression of ClaSSR27 dropped sharply and subsequently climbed gradually; at 35 days, the relative expression level was noticeably lower than at 7 days. While there were some discrepancies, overall there were parallels between the expression trajectories of these 8 ClaSSRs and the matching RNA-seq data.

Correlation of ClaSSRs with cholesterol biosynthesis

We examined the co-expression of pertinent genes involved in the plant steroid biosynthesis with ClaSSRs to explore the connection between ClaSSRs and plant cholesterol synthesis. The expression values of ClaSSRs and identified genes relevant to plant steroid production were used to compute Pearson’s coefficients. The ClaSSRs were divided into two groups, labeled a and β (Fig. 7). There were nine ClaSSRs (e.g., ClaSSR16, ClaSSR19, and ClaSSR20) in group a, which were strongly positively correlated with genes encoding enzymes, including NSDHL-1 (Reticulon-like protein), SMO1 (C-4 sterol methyl oxidase), CPI1 (cycloeucalenol cycloisomerase), CYP51 (sterol C-14 demethylase), and CAS [32,33,34]. It is noteworthy that ClaSSR25 in group a has positive correlations with genes, such as NSDHL-2 and 7-DR2, as well as positive correlations with CYP51, SMO1, CAS, and NSDHL-1, indicating that it might play a role in the steroid biosynthesis pathway.

Fig. 7
figure 7

Correlation of ClaSSRs with plant steroid biosynthesis. The degree of correlation is indicated by the size of the circle; a larger circle denotes a stronger connection, a smaller one a weaker correlation, a blue circle denotes a negative correlation, and a red circle denotes a positive correlation

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Promoter activity analysis and subcellular localization of ClaSSR25 in tobacco leaves

We assessed the expression of the ClaSSRs gene at each stage, its co-expression with other enzymes in the pathway, and its fundamental physicochemical features based on the aforementioned data, and ultimately we decided to concentrate on ClaSSR25. We cloned an area upstream of ClaSSR25, about 1200 bp, to examine the promoter activity of ClaSSR25. After applying the promoter to tobacco leaves under vacuum utilizing an Agrobacterium infection, the staining state was noted. We employed a GUS empty vector devoid of a promoter as a negative control and a GUS vector containing 35S as a positive control. According to the staining results, leaves with the ClaSSR25 promoter showed a normal blue stain that was lighter than that of the 35S-positive control (Fig. 8A). This may indicate that the promoter of ClaSSR25 is quite active.

Fig. 8
figure 8

Analysis of ClaSSR25 promoter activity and subcellular localization. A Illustration of the promoter activities driven by promoter of ClaSSR25 (Cla97C09G166970.1) in tobacco leaves. GUS as negative control on the left, CaMV35S::GUS as positive control on the right. B (a–d) and (e–h) represent the fluorescence of the constructed vector (35S-ClaSSR25-YFP) and the control (YFP) respectively. Bars = 20 μm

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We created a fusion expression vector of 35S-ClaSSR25-YFP to gain a deeper understanding of the function of ClaSSR25. The empty vector of 35S-YFP was used as a negative control. After transferring these vectors to tobacco leaves, a × 20 laser confocal microscope was used to detect the fluorescence. While the fluorescent signal of 35S-ClaSSR25-YFP is expressed in the cytoplasm and substantially fused in the cytoplasm marker, the negative signal of YFP is expressed in both the cytoplasm and nucleus (Fig. 8B). This aligns with the anticipated outcomes.

Protein–protein interaction network analysis of ClaSSR25

To understand the interactions of ClaSSR25 with other proteins and to better understand its potential functions, we selected the model plant Arabidopsis thaliana and the cucumber as reference species, and constructed gene networks using STRING database, respectively (Fig. 9). The results showed that DIM [delta (24)-sterol reductase] was the protein with the highest match to ClaSSR25 in Arabidopsis, while DWF5 (7-dehydrocholesterol reductase), the CPI1 (cycloeucalenol cycloisomerase), and SMT1 (cycloartenol-C-24-methyltransferase), and other proteins in the reciprocal network are the proteins that may interact with ClaSSR25. The prediction results in melon showed that A0A5D3CFT7 [delta (24)-sterol reductase] was the most suitable protein for ClaSSR25, while A0A5A7TLX5 (sterol 14-demethylase, CYP51), A0A5A7UXH5 (7-DR2), A0A5A7VAE6 [delta(7)-sterol-C5(6)-desaturase-like] and other exhibited proteins may interact with ClaSSR25.

Fig. 9
figure 9

Protein–protein interaction analysis of ClaSSR25 protein. Each node represents a protein, and each edge represents an interaction, colored by evidence type

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Discussion

The pathway of cholesterol production in plants has attracted significant attention, leading to extensive studies in the Solanaceae family [17, 19, 35,36,37]. By silencing StSSR2, Sawai et al. showed the significance of SSR2 for plant cholesterol production, particularly in potatoes [18]. Zheng et al. employed CRISPR technology in 2021 to silence StSSR2 once more. Their findings were in line with previous research, so establishing SSR2's function as a crucial enzyme in the pathway leading to the creation of cholesterol in plants [19]. 28 ClaSSRs were found in this investigation using the most recent watermelon genome. We carried out a number of bioinformatics analyses, such as physicochemical property characterization, phylogenetic relationship exploration, conserved motif analysis, gene structure investigation, chromosomal localization, covariate prediction, cis-acting element analysis, and co-expression analysis. The majority of the 28 identified ClaSSR proteins were found to have isoelectric points within the basic range, indicating a high concentration of basic amino acids throughout the watermelon ClaSSR family. The majority of the ClaSSR proteins were found to be localized in the cytoplasm, according to the results of the subcellular localization prediction (Table 1). 28 ClaSSRs were analyzed for their chromosomal location, and it was found that 14 of the ClaSSRs were found on chromosome 3, with the remaining ClaSSRs being spread out throughout other chromosomes (Fig. 2A). Building phylogenetic evolutionary trees is a popular technique for examining the gene-to-gene affinity. Based on their affinity, the watermelon, pumpkin, melon, and cucumber SSR2 proteins were divided into four subfamilies according to the results of the phylogenetic tree analysis (Fig. 2B). ClaSSRs were discovered to be most frequently distributed in Group II and Group IV within these subfamilies. Each subfamily has a different number of members, which is thought to be related to the processes of evolution, loss of function, and replication. The emergence of polygenic families has fueled the diversification of gene structures, including promoters, exons, introns, and protein sequences. Genes belonging to the same subclade share conserved motifs, indicating that their activities are likely comparable. ClaSSRs were divided into three groups according to the anticipated conserved motif type and distribution (Fig. 3). When examining the evolutionary history of gene families, the arrangement of exons and introns as well as their quantity can yield important insights [42, 44]. A study of the gene structures of ClaSSRs showed that the coding sequences (CDSs) of 28 genes ranged from 1 to 19. Genes in the same subgroup exhibited similar CDS numbers and locations, pointing to a possible commonality in function. The findings demonstrate the evolutionary conservation of ClaSSRs.

The interspecies collinearity analysis of ClaSSRs revealed that ten genes, including ClaSSR2, ClaSSR6, ClaSSR16, ClaSSR18, ClaSSR21, ClaSSR22, ClaSSR24, ClaSSR26, ClaSSR27, and ClaSSR28, played a significant role in the evolution of ClaSSRs. The collinearity analysis of ClaSSRs revealed that there were two collinearity genes in watermelon and no tandem repeat events (Fig. 4). Cis-acting elements, which are essential for gene transcription and important in controlling gene expression, are found in the promoter region of genes [45, 46]. This work revealed the presence of many cis-acting elements, such as GT1-motif, G-Box, AE-box, MRE, ACE and Box 4 that are linked to light-responsive regulation. This implies that light signaling controls ClaSSRs with these components. In addition, ClaSSRs also possess elements, such as TGA-element, AuxRR-core, P-box, and TGACG-motif, indicating that they may be regulated by phytohormones, such as auxin, gibberellin, abscisic acid, MeJA, etc. (Fig. 5). We investigated the expression of ClaSSRs in various developmental stages of watermelon. The result revealed that most ClaSSRs were expressed during all developmental stages (Fig. 6A). To further explore this, we utilized RNA-Seq data to identify 13 genes with high expression. Heat maps were generated to visualize the expression patterns of these genes at different timepoints. In addition, we selected 8 genes for quantitative analysis (Fig. 6B). Our findings demonstrated that the expression of these 8 ClaSSRs varied across different developmental stages, and the results obtained from RNA-Seq were broadly consistent with the results of fluorescence quantification. Co-expression analysis showed correlation of ClaSSRs with several annotated steroid synthesis-related genes. Among them, CYP51, SMO1, CAS, NSDHL-1, NSDHL-2, and 7-DR2 were all positively correlated with ClaSSR25 (Fig. 7). We ultimately decided to focus on ClaSSR25 as the problematic gene after considering the expression of ClaSSRs and the aforementioned analytical results. After analysis, it was discovered that ClaSSR25's promoter activity was very active (Fig. 8A). Tobacco leaves were used for the subcellular localization of ClaSSR25, which was discovered in the cytoplasm (Fig. 8B). According to the prediction of the protein–protein interaction network, DIM, CPI1, SMT1, CYP51, 7-DR2, and other enzymes may interact with ClaSSR25. These findings could be paired with the findings of the correlation analysis, which further demonstrated the close relationship between ClaSSR25 and the aforementioned enzymes (Fig. 9).

Conclusion

In watermelon, a total of 28 ClaSSRs were found in this study, distributing irregularly across 10 chromosomes. It was discovered by subcellular localization prediction that roughly 68% of ClaSSRs were localised in the cytoplasm. Phylogenetic analysis of SSR2 genes from four species, watermelon, pumpkin, melon, and cucumber, were carried out and divided into four subfamilies. The structures, conserved motif sequences and cis-acting regions of the ClaSSRs were investigated and their expression patterns were characterized. The distinct expression patterns of eight ClaSSRs may suggest that these genes play distinct roles in the growth and development of watermelon. Based on their conserved motifs and gene structures, the 28 ClaSSRs can be generally classified into three subfamilies. The expression of ClaSSRs is regulated by light signals and phytohormones, including as gibberellins, growth factors, abscisic acid, and MeJA. Expression pattern analysis, quantitative fluorescence analysis, promoter activity analysis, subcellular localisation and protein interaction network prediction provided insights into SSR2 genes in watermelon. This work lays the foundation for more detailed studies of the watermelon SSR2 genes in the future.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

7-DR2:

7-Dehydrocholesterol reductase 2

CAS:

Cycloartenol synthase

cDNA:

Complementary DNA

CDS:

Coding sequence

CPI1:

Cycloeucalenol cycloisomerase

CYP51:

Sterol C-14 demethylase

DIM:

Delta (24)-sterol reductase

DWF5:

7-Dehydrocholesterol reductase

FPKM:

Fragments per Kilobase Million

KEGG:

Kyoto Encyclopedia of Genes and Genomes

MeJA:

Methyl jasmonate

NCBI:

National Center for Biotechnology Information

NSDHL:

Reticulon-like protein

ORF:

Open reading frame

pI:

Isoelectric point

SMO1:

C-4 sterol methyl oxidase

SSR2:

Sterol side-chain reductase 2

UTR:

Untranslated regions

UVB:

Ultraviolet radiation B

RT-qPCR:

Quantitative real-time PCR

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Acknowledgements

The authors acknowledge the financial support of the Funding of Joint Research on Agricultural Variety Improvement of Henan Province [2022010503], Key Scientific and Technological Projects of Luoyang City (Top of the list) [2301024A].

Funding

Funding of Joint Research on Agricultural Variety Improvement of Henan Province [2022010503], Key Scientific and Technological Projects of Luoyang City (Top of the list) [2301024A].

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Authors and Affiliations

  1. College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang, 471023, China

    Jing Zhang, Hao-Ting Sun, Ding-Ding Zuo, Yang Qiao, Rui Sun, Jia-Lin Xing & Da-Long Guo

  2. Henan Engineering Technology Research Center of Quality Regulation of Horticultural Plants, Luoyang, 471023, China

    Jing Zhang, Hao-Ting Sun, Ding-Ding Zuo, Yang Qiao, Rui Sun, Jia-Lin Xing & Da-Long Guo

  3. Luoyang Nongfa Agricultural Biotechnology Co. Ltd, Luoyang, 471100, Henan Province, China

    Yan-Ge Li, Zhong-Hou Zhu & Xue-Jie Zhu

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Da-Long Guo designed the study, supervised the bioinformatics analysis and revised the manuscript; Jing Zhang performed the experiments, analyzed the data, bioinformatics analysis and wrote the paper; Yan-Ge Li, Hao-Ting Sun, Ding-Ding Zuo, Jia-Lin Xing, Zhong-Hou Zhu and Xue-Jie Zhu participated in the experiments participated in the experiment; Yang Qiao and Rui Sun participated in bioinformatics analysis.

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Zhang, J., Li, YG., Sun, HT. et al. Genome-wide identification and expression analysis of watermelon (Citrullus lanatus) SSR2 gene during fruit development. Chem. Biol. Technol. Agric. 11, 100 (2024). https://doi.org/10.1186/s40538-024-00624-7

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