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Article

Genome-Wide Characterization of Shi-Related Sequence Gene Family and Its Roles in Response to Zn2+ Stress in Cucumber

by
Xinhui Zhang
1,
Bilal Ahmad
2,†,
Shuang Zeng
1,†,
Yuhan Lan
1,
Xin Hu
1,
Lingling Fu
1,
Tian Hu
1,
Jinhua Li
1,
Xingguo Zhang
1,
Yu Pan
1,* and
Dan Du
1,*
1
Key Laboratory of Agricultural Biosecurity and Green Production in the Upper Reaches of the Yangtze River, Ministry of Education/College of Horticulture and Landscape Architecture, Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China
2
National Key Laboratory of Tropical Crop Breeding, Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(11), 1154; https://doi.org/10.3390/horticulturae10111154
Submission received: 30 August 2024 / Revised: 21 October 2024 / Accepted: 28 October 2024 / Published: 31 October 2024
(This article belongs to the Special Issue Advancements in Genetic Improvement of Stress Tolerance in Vegetable Crops)
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Abstract

:
Shi-related sequence (SRS) proteins, which consist of the RING-like zinc finger domain and IGGH domain, are plant-specific transcription factors that have been well-studied in several plant species. However, information about SRS genes and their roles in cucumber (Cucumis sativus L.) is limited. Therefore, we performed detailed bioinformatic analysis of the SRS gene family, including gene numbers and positions, genes structures, conserved motif distribution patterns, phylogenetic analysis, and promoter cis-element analysis. Eight SRS genes were identified in cucumber and distributed on all seven cucumber chromosomes. SRS genes are conserved in plants and divided into two groups in cucumber based on their protein sequence. In silico analysis predicted that most genes may function in response to abiotic stresses and phytohormones. Gene ontology analysis predicted the possible involvement of genes in development and reproduction, and DNA and protein binding on a molecular level. Furthermore, the differential expression pattern of SRS genes in leaf, stem and root under the condition of Zn2+ stress suggested their roles in response to Zn2+ stress. Furthermore, our metal tolerance assay suggested that CsSRS2 and CsSRS5 mediated enhanced tolerance to Zn2+ stress in Escherichia coli cells. Our study provides a foundation for the functional study of SRS genes in cucumber.

1. Introduction

The Shi-related sequence (SRS) gene family members belong to the plant-specific C3HC4 (RING-HC) zinc finger protein family. SRS family proteins are specific for two conserved domains, RING-like zinc finger domain and IGGH domain [1], including LATERAL ROOT PRIMORDIA 1 (LPR1), STYLISH1 (STY1), STYLISH2 (STY2), and SRS7 four members [1,2,3]. The conserved region of the RING-like zinc finger motif, also called the RING domain (CX2CX7CX4CX2C2X6C) [1,3], is one type of typical C3H2C3 or C3HC4 motif and was first identified to function as a DNA binding protein in Xenopus laevis [4]. Except for this, the RING-like domain also includes modified domains such as RING-V, RING-D, RING-S/T, RING-G, and RING-C2 [5,6]. Zinc finger domains, which are involved in DNA/RNA binding and protein–protein recognition, are widely distributed in eukaryotic genomes [7]. Zinc finger proteins have been well studied in different plants for their important functions in photoinduction, peroxidase formation, seed, and root development. In addition, some zinc finger proteins can respond to environmental stresses, such as salt, heat, drought and low-temperature stress as well [8]. The RING domain can bind to different substrates (RNA, proteins, and lipids), which have been implicated in transcriptional regulation and the ubiquitination-mediated degradation of targeted protein [1]. However, the IGGH domain is required for homodimerization [9] and its specific function is unknown yet [10].
The SRS proteins are important developmental regulators and have been reported to function in various developmental processes. In Arabidopsis, the reported members of the SRS gene family include SHI, STY1, STY2, LRP1, SRS3 to SRS8, most of which have been reported to function in plant development. STY1 upregulates auxin biosynthesis-related genes expression to increase auxin concentrations [11], and STY1 and other SHI/STY members function in the architectural development of the shoot apical meristem though auxin levels [9]. Furthermore, LRP1 forms a protein complex with SHI, STY1, SRS3, SRS6, and SRS7 to regulate lateral root development by regulating auxin signal transduction and chromatin modification [12,13,14]. On the contrary, SHI acts as a repressor of the GA response to downregulate the expression of auxin biosynthesis-related genes [1,15]. However, CAMTA3/SR1 has been reported in response to drought stress tolerance and ABA signaling [16]. In soybean, GmSRS18 has been involved in drought and salt stresses [17]. In cotton, salt-inducible gene GhSRS21 is a negative regulator of salt stress response [18]. In common bean, Pvul-SRS-1, Pvul-SRS-4, and Pvul-SRS-10 genes have been identified to respond to salt stress as well [19]. GRMZM2G077752 might participate in ABA signaling and activate carbohydrate remobilization during leaf aging in maize [20]. In rice, OsSHI1 functions pleiotropically in plant architecture establishment [21]. In barley, Vrs2 has a role in spike development and lks2 influences the spike morphology by regulating awn elongation and pistil development [22]. However, SRS genes in cucumber (Cucumis sativus L.) have not been characterized.
Cucumber is an important economic vegetable crop that is susceptible to various abiotic and biotic stresses [23]. Abiotic stresses, like heat, drought, and high salinity, cause great issues for crop growth and yields. For example, under the salt stress condition, the content of K+ and chlorophylls in cells, membrane stability index and fruit yield of all genotypes significantly decreased [24]. In the drought stress condition, the content of chlorophylls in leaves was reduced significantly [25]. Although SRS transcription factors have been reported to function in many biological and stress tolerance processes in other plant species, research about the SRS gene family in stress response is highly limited in cucumber. Furthermore, stress-resistant gene characterization is one of the effective methods to breed the robust cucumber cultivar. This study has identified eight SRS genes in cucumber and detailed bioinformatic and phylogenetic analyses of SRS genes have been characterized. In addition, the expression levels of SRS genes in leaf, stem and root have been analyzed under the Zn2+ stress condition. The putative functions of SRSs in Zn2+ stress response in cucumber have been analyzed and two genes have shown enhanced resistance to a high concentration of Zn2+ stress by metal tolerance assay in E. coli cells. The results provided a general understanding of SRS genes in cucumber, providing new candidate genes and an established foundation for further functional analyses of SRS genes.

2. Materials and Methods

2.1. Isolation of SRS Family Genes in Cucumber

Ten SRS proteins of Arabidopsis from the TAIR database (http://www.arabidopsis.org (Accessd on 29 August 2022)) were obtained. To acquire all cucumber SRS genes, the HMM search program (HMMER v3.0, http://hmmer.org/ (Accessd on 10 September 2022)) and BLASTP alignment were carried out in CuGenDB (http://www.icugi.org/ (Accessd on 15 September 2022)) using Geneious 4.8.5 software (http://www.geneious.com/ (Accessd on 17 September 2022)) with the SRS protein sequences from Arabidopsis as queries. The protein sequences of the SRS family with the e-value < 1 × 10−10 were downloaded from the CuGenDB (recourse or version). Subsequently, all candidate SRS genes were confirmed using SMART (http://smart.embl-heidelberg.de/ (Accessd on 27 September 2022)) and Pfam (http://pfam.xfam.org (Accessd on 27 September 2022)). The coding sequences (CDSs) were determined by performing BLASTn in CuGenDB. The physicochemical properties of the proteins including the molecular weight (MW), isoelectric point (pI), and grand average of hydropathy (GRAVY) values were deduced using the ExPASy-ProtParam tool (http://web.expasy.org/protparam/ (Accessd on 28 September 2022)). The SRS sequences of tomato, rice, and maize were collected from the Phytozome database (https://phytozome.jgi.doe.gov (Accessd on 29 September 2022)). Furthermore, all the sequences were evaluated according to the same criteria as used for cucumber.

2.2. Protein Sequence and Phylogenetic Analysis of the SRS Family Genes in Cucumber

Phylogenetic trees were constructed to evaluate the phylogenetic relationships of SRS proteins in plants. Two phylogenetic trees were constructed. One contains SRS proteins from 5 species including Arabidopsis, rice, maize, tomato, and cucumber, and the other contains eight SRS proteins from cucumber. All SRS protein sequences were aligned by ClustalW2.1 software with the default settings [26]. Then, the aligned sequences were used for the construction of phylogenetic tree. The two phylogenetic trees of SRS proteins were constructed by MEGA7 using the neighbor-joining (NJ) method with 1000-bootstrap replicates.

2.3. Gene Duplication, Structure, and Conservation Analysis

To find out the distribution of the SRS genes in the genome, the chromosomal positions of eight SRS genes in cucumber were identified following the annotation of the CuGenDB, then the physical chromosome maps of the SRS gene family were drafted with MapChart v2.0. Subsequently, the exon/intron structures of cucumber SRS genes were identified by GSDS v2.0 (https://gsds.gao-lab.org/Gsds_help.php (Accessd on 27 September 2022)). The conserved motifs of SRS genes were predicted using MEME v5.0.3 (http://meme-suite.org/tools/meme (Accessd on 6 October 2024) [27] with the setting parameters (optimum motif widths of 6–300 residues and a maximum of 10 motifs with an e-value < 1 × 10−10) [28].

2.4. Subcellular Location Prediction, Cis-Element and GO Analysis

Subcellular locations of proteins and GO analysis were performed using the online server (http://cello.life.nctu.edu.tw/cello2go/ (Accessd on 19 September 2022)) [29]. For cis-element analysis, 2 kb upstream sequence of each gene was downloaded from Phytozome 12 (https://phytozome.jgi.doe.gov/pz/portal.html# (Accessd on 20 September 2022)) and was uploaded in Plant CARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html (Accessd on 20 September 2022)).

2.5. Plant Materials and Zn2+ Treatment

The 6-week-old cucumber plants were cultured in a nutrient medium containing a mixture of nutrient soil, vermiculite, and perlite (3:1:1) with different concentrations of zinc sulfate (0.05, 0.1, 0.5, 1.0, and 2.0 mmol/L). The leaf, stem, and root samples of cucumber were collected at different time points (0 h, 6 h, 12 h, 24 h, 48 h, and 96 h). All the tissues were cut immediately to put in the liquid nitrogen, and stored at −80 °C in a refrigerator for further use.

2.6. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA was extracted from samples using RNA preparation pure plant kit (TIANGEN, Beijing, China), following the manufacturer’s instructions. The integrity of RNA was detected by 1.0% agarose gel in a 1/2 TAE electrophoresis solution. The concentration and quality of RNA were detected by nucleic acid concentration analyzer. The cDNA was synthesized using 1 μg of total RNA by RNA PCR Kit v3.0 (Takara Bio, Dalian, China) according to the manufacturer’s guidelines. Gene-specific primers were designed using the Primer Premier 5.0. software. qRT-PCR was performed using the CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA) and SYB qPCR master (Vazyme, Nanjing, China). The PCR conditions were 94 °C for 15 s, followed by 42 cycles of 94 °C for 5 s and 59–63 °C (Depending on the specific primer) for 30 s. Actin gene was used as an internal reference and each experiment was repeated three times. The 2−ΔΔCT method was used to examine relative expression levels [30]. The t-test was performed to evaluate the overall significance (* p < 0.05, ** p < 0.01) of the results. As CsSRS1 failed to be cloned in our lab, the expression of CsSRS1 was not carried out by qRT-PCR. Specific primers of SRS genes for qRT-PCR analysis are listed in Table S1.

2.7. Zn2+ Tolerance Assay

CsSRS2 and CsSRS5 were cloned to pET32a plasmid and transformed into E. coli strain BL21, respectively, for the expression of the heterologous recombinant proteins with Trx-tagged, and the empty pET32a vector was used as control. The cells with recombinant plasmid were grown at 37 °C, 200 rpm to an OD600 of 0.6, and protein expression was induced with 1 mM isopropyl β-D-thiogalactoside (IPTG). After growing for an additional 3.5 h at 37 °C, cultures were adjusted to OD600 to about 0.8. The cells went through serial dilutions (1:1; 1:50 and 1:500) and were spotted onto LB plates with 1 mM ZnSO4. The results were photographed after being incubated for 16 h at 37 °C.

3. Results

3.1. Eight Genes from SRS Family Were Identified in Cucumber

A total of eight SRS genes were identified in cucumber genomes that were named CsSRS1-CsSRS8 based on their locations on the chromosomes. Eight SRS genes are unevenly distributed on seven pairs of chromosomes (Figure 1). Except for two genes (CsSRS6 and CsSRS7) on chromosome 6, there is only one gene on each chromosome. All eight genes were located on the telomeric ends of the chromosomes. The upstream and downstream region of the CDS and the exon–intron distribution patterns are shown in Figure S1a. The number of exons and introns in eight SRS genes was the same (Figure S1a).
The gene locus ID, physical location on chromosomes, and gene length of eight SRS genes are listed in Table 1. Protein secondary structure analysis of eight SRS genes identified the complete information about their molecular properties, including the isoelectric point (pI), molecular weight (MW), and grand average of hydropathy (GRAVY) values (Table 1). The length of SRS protein members varied from 263 aa (CsSRS4) to 365 aa (CsSRS6). The molecular weight alters from 28.68 KDa (CsSRS4) to 37.66 KDa (CsSRS6). Furthermore, the pI values of eight SRS proteins were predicted to range from 6.24 (CsSRS4) to 8.90 (CsSRS1). Furthermore, all members of the cucumber SRS family are predicted to be unstable hydrophobic proteins. Thus, the results above suggested that SRS genes are relatively conserved in cucumber during the evolutionary process.

3.2. The SRS Proteins Are Relatively Conserved in Plants

The type and pattern of domain distribution can provide some clues about gene functions. The RING-like zinc finger and IGGH domain have been identified as being conserved in all members, except for one amino acid change in CsSRS8 and CsSRS4 (Figure S1b). Furthermore, according to protein motif analysis, CsSRS1, CsSRS5 and CsSRS6 just have the three main conserved motifs that are available in all SRS genes in cucumber (Figure 2). Among them, Motif 1 (red box) corresponds to the zinc finger domain (CX2CX7CX4CX2C2X6C), the green box indicates the second conserved motif, while the blue boxes represent the IGGH domain, of which differences in motif size (27 to 50 aa) and distribution patterns are shown (Figure 2). Meanwhile, except for CsSRS1, CsSRS5 and CsSRS6, all others have the motif (yellow box) on their C-terminus, and CsSRS2, CsSRS3 and CsSRS8 have the extra conserved motifs on their N-terminus (Figure 2). At the same time, the phylogenetic tree of eight SRS proteins from cucumber showed that CsSRS1, CsSRS5 and CsSRS6 were clustered in the same subgroup while the other five were clustered together (Figure 3a), which is consistent with the protein motif analysis in Figure 2.
Phylogenetic tree was constructed with 38 SRSs from rice, maize, Arabidopsis, tomato, and cucumber to better understand their phylogenetic relationships (Figure 3b). According to the phylogenetic tree, 38 SRSs were clustered into three main branches; CsSRS1, CsSRS5 and CsSRS6 were clustered together with 10 SRS proteins from four species in Group 1, while the other five were clustered with 19 SRS proteins from Arabidopsis and tomato in Group 2 (Figure 3b). No SRSs from cucumber were found in Group 3 containing SRSs from monocot only.
Cis-elements can provide inklings about gene expressions and functions. In this study, light-related cis-elements (BOX4, G-Box and GATA motif) and anaerobic respiration (ARE) were distributed in the promoters of all genes (Figure 4). Hormone response-related elements, including ERE (ethylene response), TCA element (salicylic acid response), and ABRE (abscisic acid response) were found in most of the genes. In addition, environmental stress-related elements (LTR and ERE) were found in all promoters of SRS genes except for CsSRS5. Development-related elements (ABRE, LTR, TCA element, CGTCA motif, TGACG motif and GATA motif) were also present in the promoter regions of almost all genes (Figure 4). All SRS proteins were predicted to be located in the nucleus except for three (CsSRS1, CsSRS5 and CsSRS6), which were predicted to be located in both the nucleus and extracellular organelles (Table 2). The possible mediated biological process prediction suggested that they might function in anatomical structure development and reproduction. Moreover, SRSs were predicted to be involved in DNA and protein binding at the molecular level. Thus, it is reasonable to predict that SRS proteins may function as TFs in cucumber.

3.3. Expression Pattern of SRS Genes in Cucumber Under Zn2+ Stress

Our lab has long focused on research on heavy metal stress response. Thus, we consider whether SRS genes are involved in heavy metal stress response. Heavy metals pose a threat to environmental sanitation and food security. To explore the involvement of SRS genes in Zn2+ stress tolerance in cucumber, the expression profile of SRS genes in tissues (roots, stems, and leaves) under different concentrations of zinc solution was carried out by qRT-PCR analysis.
The results showed that SRS genes demonstrated differential expression levels in leaves, stems and roots under Zn2+ treatment. CsSRS2 showed a higher expression in all tissues at the beginning of Zn2+ treatment and was downregulated at 24 h, 48 h and 96 h after Zn2+ treatment under almost all concentrations of ZnSO4 (Figure 5). Except for stem, the expressions of CsSRS2 in root and leaf were higher compared with those without Zn2+ treatment at all different time points. However, the expression of CsSRS3 cannot be induced in all tissues at all time points under Zn2+ treatment (Figure 5). The expression of CsSRS4 increased significantly in roots at the beginning of treatment but decreased rapidly afterwards. However, the expression of CsSRS4 varied greatly at different concentrations of Zn2+ treatment, indicating that CsSRS4 may not function in metal stress response (Figure 5). CsSRS5 was not activated at the beginning of Zn2+ treatment, but it was significantly downregulated after 12 h of treatment at almost all concentration levels of Zn2+ (Figure 5). As for CsSRS6, it showed an elevated expression in root but was inhibited in stems, and no significant expression change was noticed in leaves (Figure 5). CsSRS7 showed a significantly increased expression at the beginning of treatments in roots and stems, but was not significantly expressed in leaves (Figure 5). Lastly, CsSRS8 showed a tissue-specific expression pattern and was expressed highly only in roots (Figure 5). Thus, taking together, it is reasonable to speculate that CsSRS2 and CsSRS5 may function in Zn2+ stress response.

3.4. Overexpressing CsSRS Genes Enhanced Zn2+ Tolerance in E. coli

To verify whether CsSRS2 and CsSRS5 were involved in Zn2+ response, CsSRS2 and CsSRS5 were overexpressed in BL21 E. coli cells, respectively. The CsSRS2- and CsSRS5-containing cells were induced when expressing in LB medium containing 1 mM IPTG. The cells were subjected to a series of multiple dilutions (1:1, 1:50, and 1:500) on Zn2+ containing LB solid medium to observe its performance (Figure 6). There were no obvious differences in growth between the control (containing empty vector) and the cells containing CsSRS2 and CsSRS5 genes, respectively, and genes without a dilution of E. coli cells. However, E. coli cells overexpressing CsSRS2 showed slightly better growth compared with those containing empty vectors, while E. coli cells overexpressing CsSRS5 grew vividly even after diluting 500 times. Thus, the expressions of CsSRS2 and CsSRS5 in E. coli cells enhanced their tolerance to Zn2+ stress.

4. Discussion

Transcription factors (TFs) are not only important for plant growth and development, but are also involved in the regulation of biotic and abiotic stress response, such as drought, cold, salt, and heat [31]. In cucumber, many TFs have been confirmed to play important roles in development and stress response. The CBF genes are involved in the regulation of ABA pathways in their response to low and high temperatures [32]. Heat shock transcription factor (HSF) functions in the regulation of thermal response genes in cucumber [33]. NAC family TFs play essential roles in fruit trichome development and heat defense response [34]. The GRF family may take part in plant organ development and stress response [35]. Although some SRS genes have been identified and characterized in Arabidopsis [1,3,15,36,37,38], soybean [17], maize [20], rice [21], barley [39], and lotus (japonicas) [20], no SRS genes have been identified or characterized in cucumber. Critical roles of SRS genes in other species rationalize a comprehensive bioinformatic and expression patten analysis of the gene family in cucumber.
In this study, a total of eight SRS genes were identified in cucumber that are disputed on all seven chromosomes. The number of SRS genes identified in cucumber is more than that identified in rice (n = 6), but less than that identified in Arabidopsis (n = 11) and maize (n = 11) [3,20,40]. All eight SRS genes have two exons and one intron, and all have upstream and downstream regions except for only downstream regions, which were found in CsSRS4 and CsSRS8. Interestingly, substitutions were found in IGGH domains in CsSRS4 and CsSRS8. Generally, SRS proteins contained two distinct conserved domains, the RING-like domain (CX2CX7CX4CX2C2X6C) and IGGH domain [1]. Although the IGGH domain is present in all members of SRS genes, the RING-like domain may or may not be present in the monocot plant maize [20]. For example, the RING-like zinc finger domain was lost in GRMZM2G179021, AC195955.2-FG006 and AC206191.3 [20]. However, all SRS proteins in the cucumber contained RING-like zinc finger domain and IGGH domain, which indicate that SRS proteins are more conserved in cucumber. Our results are consistent with the previous studies of SRS genes in rice [40] and maize [20]. The diversity in conserved domains leads to the functional differentiation of proteins. A relatively conserved motif was found in CsSRS2, CsSRS3 and CsSRS8 on their N-terminal side, while another one was found on the C-terminus of all SRSs except for CsSRS1, CsSRS5 and CsSRS6. The protein motif analysis is consistent with the phylogenetic studies. CsSRS1, CsSRS5 and CsSRS6 were clustered together in Group 1, while others were in Group 2 with SRSs from dicot plants only. Our results are consistent with the previous study [41]. Furthermore, according to the phylogenetic tree, Group I contained SRS proteins from all species, while Group II and Group III consisted of SRS proteins all from dicots (Arabidopsis, cucumber and tomato) and monocots (maize and rice), respectively. These results suggest that SRS genes are generally conserved and the evolutionary process of the SRS family in some species may be faster than the differentiation of dicot and monocot in plants by gaining or losing some domains to cope with the changing environment.
It has been proposed that Zn2+ can directly bind to SRS proteins due to the presence of the RING-like zinc finger domain. The reports about functions of SRS proteins in zinc stress alleviation have been studied in maize and soybean [17,20], but no related research has been reported in cucumber. Our results on the metal tolerance assay in cucumber suggest that the induced overexpression of CsSRS2 or CsSRS5 enhanced the tolerance of E. coli cells to Zn2+ stress (Figure 5). The presence of environmental stress-related elements in cucumber indicated their involvement in stress tolerance, especially LTR and ERE motifs. The availability of ABRE cis-elements in all promoters of SRS genes in cucumber suggest their involvement in abscisic acid signaling pathways. The critical role of ABA in the regulation of SRS genes in stress responses has been well studied in many plant species. For example, GRMZM2G07752 responded to the ABA signal by activating the reactivation of carbohydrates in senescence leaves in maize [20]. GmSRS18 participated in both drought and salt responses either through ABA-dependent or -independent pathways in soybeans [17]. Research concerning zinc stress and SRS genes has not been reported yet. The differential expression of CsSRS2 and CsSRS5 in response to different concentrations of ZnSO4 treatments suggests their roles in zinc stress tolerance. In addition, Zn2+ toxicity experiments in E. coli indicated their roles in Zn2+ stress response in E. coli. Heavy metals can cause damage to enzyme activity, cell membranes and other physiological processes in both microorganisms and plants. Thus, it is speculated that toxicity effects may be similar in both microorganisms and plants, and CsSRS2 and CsSRS5 may function in a similar way in response to Zn2+ stress [42,43,44]. Previously, the ectopic expression of GmSRS18 in Arabidopsis increased the sensitivity of plants to drought and salt stresses [17]. Our study provides a detailed bioinformatic analysis of all eight SRS genes in cucumber and inklings about their functions in Zn2+ stress tolerance. This study characterized the SRS gene family and the expression patterns in resistance to metal stress, providing a basis for the functional study of SRS genes in cucumber.

5. Conclusions

There are eight putative SRS genes in cucumber which are conserved in the RING-like zinc finger domain except for substitutions, which were found in IGGH domains in CsSRS4 and CsSRS8. In addition, they were divided into two groups by phylogenetic study, consistent with protein motif analysis. Promoter cis-element analysis showed that SRS genes may function in both stress and development processes. The results of qRT-PCR and the zinc stress assay in E. coli indicated that CsSRS2 and CsSRS5 may function in metal stress tolerance in cucumber.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae10111154/s1. Figure S1: Gene structure and protein sequence alignment. (a) Exon–intron structures. Yellow boxes referred to as exons and introns are marked as black lines. (b) The protein sequence alignment of eight SRS proteins in cucumber. Table S1: Quantitative primers and annealing temperature.

Author Contributions

Y.P., X.Z. (Xinhui Zhang) and D.D. designed the structure and concept of the article; S.Z. and B.A. provided the methods and bioinformatic analysis; S.Z., L.F. and Y.L. constructed the phylogenetic trees; T.H. and X.H. carried out the qRT-PCR; X.Z. (Xinhui Zhang), Y.P. and D.D. wrote the article; B.A., J.L. and X.Z. (Xingguo Zhang) revised the article; Y.P., D.D. and X.H. provided the funding support. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the China Postdoctoral Science Foundation (2022M712627), the Chongqing Postdoctoral Science Foundation (CSTB2022NSCQ-BHX0037), the National Natural Science Foundation of China (No. 31772320), and the Natural Science Foundation of Chongqing (2024NSCQ-MSX3674).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Distribution of SRS gene family members on cucumber chromosomes.
Figure 1. Distribution of SRS gene family members on cucumber chromosomes.
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Figure 2. Protein motif analysis of 8 SRS genes in cucumber. The distributions of conserved motifs in the SRS proteins are identified by MEME v5.0.3 and shown in different colored boxes.
Figure 2. Protein motif analysis of 8 SRS genes in cucumber. The distributions of conserved motifs in the SRS proteins are identified by MEME v5.0.3 and shown in different colored boxes.
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Figure 3. Unrooted phylogenetic trees. (a) Phylogenetic analysis of eight SRS proteins in cucumber. Different colored boxes indicate different subfamilies. Numbers next to the branches represent bootstrap values. (b) Phylogenetic analysis of 38 SRS proteins. Different proteins are denoted with different colored symbols; green stars denote cucumber proteins; yellow stars denote maize proteins; red squares denote tomato; blue dots denote Arabidopsis proteins, and purple dots denote rice proteins. Numbers near the branches denote bootstrap values.
Figure 3. Unrooted phylogenetic trees. (a) Phylogenetic analysis of eight SRS proteins in cucumber. Different colored boxes indicate different subfamilies. Numbers next to the branches represent bootstrap values. (b) Phylogenetic analysis of 38 SRS proteins. Different proteins are denoted with different colored symbols; green stars denote cucumber proteins; yellow stars denote maize proteins; red squares denote tomato; blue dots denote Arabidopsis proteins, and purple dots denote rice proteins. Numbers near the branches denote bootstrap values.
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Figure 4. Predicted cis-elements in cucumber SRS gene promoters. The 2000 bp sequences of 8 cucumber SRS genes were analyzed with Plant CARE.
Figure 4. Predicted cis-elements in cucumber SRS gene promoters. The 2000 bp sequences of 8 cucumber SRS genes were analyzed with Plant CARE.
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Figure 5. Relative expression levels of SRS family genes of cucumber in leaves, roots, and stems under Zn2+ stress; values represent means ± SD (n = 3). The value of “0 mmol treatment” at each time point is used as control for p-value (for asterisks) calculation. Asterisk (*) indicates p < 0.05; asterisk (**) indicates p < 0.01; asterisk (***) indicates p < 0.001; asterisk (****) indicates p < 0.00001 (2-way ANOVA method and Dunnett’s multiple comparisons test were used for statistical significance analysis).
Figure 5. Relative expression levels of SRS family genes of cucumber in leaves, roots, and stems under Zn2+ stress; values represent means ± SD (n = 3). The value of “0 mmol treatment” at each time point is used as control for p-value (for asterisks) calculation. Asterisk (*) indicates p < 0.05; asterisk (**) indicates p < 0.01; asterisk (***) indicates p < 0.001; asterisk (****) indicates p < 0.00001 (2-way ANOVA method and Dunnett’s multiple comparisons test were used for statistical significance analysis).
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Figure 6. Growth of E. coli cells containing CsSRS2, CsSRS5 and empty vector, respectively, on LB medium (left) and LB medium containing 1 mM ZnSO4 (right) in a series of multiple dilutions (1:1, 1:50, and 1:500).
Figure 6. Growth of E. coli cells containing CsSRS2, CsSRS5 and empty vector, respectively, on LB medium (left) and LB medium containing 1 mM ZnSO4 (right) in a series of multiple dilutions (1:1, 1:50, and 1:500).
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Table 1. Physical and molecular properties of eight SRS proteins in cucumber.
Table 1. Physical and molecular properties of eight SRS proteins in cucumber.
Gene Locus IDGene NumberStartStopGene Bases
(bp)		CDS
(bp)		Amino Acids
(aa)		MW (Da)pI
Csa1G628000.1CsSRS124,761,04724,763,733268792430731,146.628.90
Csa2G364580.1CsSRS217,580,60417,582,0961493104434737,386.306.45
Csa3G865400.1CsSRS336,176,11036,177,603149490930233,877.297.65
Csa4G043960.1CsSRS43,461,1113,462,133102379226328,682.786.24
Csa5G623840.1CsSRS525,062,87825,064,891201491230332,073.748.89
Csa6G432270.1CsSRS620,428,91320,430,8001888109836537,662.427.15
Csa6G504650.2CsSRS725,599,24025,601,517156099633135,897.297.00
Csa7G051450.1CsSRS83,302,3273,303,550113495431735,221.947.13
Table 2. Subcellular localization and GO analysis in biological process and molecular function.
Table 2. Subcellular localization and GO analysis in biological process and molecular function.
Protein NameLocalization PredictionMolecular FunctionBiological Process
CsSRS1Extracellular and nuclearDNA and protein bindingAnatomical structure development
Reproduction
CsSRS2NuclearDNA and protein bindingAnatomical structure development
CsSRS3NuclearDNA and protein bindingAnatomical structure development
CsSRS4NuclearDNA and protein bindingAnatomical structure development
CsSRS5Extracellular and nuclearDNA and protein bindingAnatomical structure development
CsSRS6Extracellular and nuclearDNA and protein bindingAnatomical structure development
Reproduction
CsSRS7NuclearDNA and protein bindingAnatomical structure development
CsSRS8NuclearDNA and protein bindingAnatomical structure development
Note: The methods used are available in “Section 2”.
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Zhang, X.; Ahmad, B.; Zeng, S.; Lan, Y.; Hu, X.; Fu, L.; Hu, T.; Li, J.; Zhang, X.; Pan, Y.; et al. Genome-Wide Characterization of Shi-Related Sequence Gene Family and Its Roles in Response to Zn2+ Stress in Cucumber. Horticulturae 2024, 10, 1154. https://doi.org/10.3390/horticulturae10111154

AMA Style

Zhang X, Ahmad B, Zeng S, Lan Y, Hu X, Fu L, Hu T, Li J, Zhang X, Pan Y, et al. Genome-Wide Characterization of Shi-Related Sequence Gene Family and Its Roles in Response to Zn2+ Stress in Cucumber. Horticulturae. 2024; 10(11):1154. https://doi.org/10.3390/horticulturae10111154

Chicago/Turabian Style

Zhang, Xinhui, Bilal Ahmad, Shuang Zeng, Yuhan Lan, Xin Hu, Lingling Fu, Tian Hu, Jinhua Li, Xingguo Zhang, Yu Pan, and et al. 2024. "Genome-Wide Characterization of Shi-Related Sequence Gene Family and Its Roles in Response to Zn2+ Stress in Cucumber" Horticulturae 10, no. 11: 1154. https://doi.org/10.3390/horticulturae10111154

APA Style

Zhang, X., Ahmad, B., Zeng, S., Lan, Y., Hu, X., Fu, L., Hu, T., Li, J., Zhang, X., Pan, Y., & Du, D. (2024). Genome-Wide Characterization of Shi-Related Sequence Gene Family and Its Roles in Response to Zn2+ Stress in Cucumber. Horticulturae, 10(11), 1154. https://doi.org/10.3390/horticulturae10111154

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