Molecular Characterization of Peroxidase (PRX) Gene Family in Cucumber

Abstract

Background: The Peroxidase (PRX) gene family is essential for plant growth and significantly contributes to defense against stresses. However, information about PRX genes in cucumber (Cucumis sativus L.) remains limited. Methods: In this present study, CsPRX genes were identified and characterized using bioinformatics analysis. The expression pattern analysis of CsPRX genes were examined utilizing the RNA-seq data of cucumber from public databases and real-time quantitative PCR (qRT-PCR) analysis. Results: Here, we identified 60 CsPRX genes and mapped them onto seven chromosomes of cucumber. The CsPRX proteins exhibited the presence of 10 conserved motifs, with motif 8, motif 2, motif 5, and motif 3 consistently appearing across all 60 CsPRX protein sequences, indicating the conservation of CsPRX proteins. Furthermore, RNA-seq analysis revealed that differential expression of CsPRX genes in various tissues. Notably, a majority of the CsPRX genes exhibited significantly higher expression levels in the root compared to the other plant tissues, suggesting a potential specialization of these genes in root function. In addition, qRT-PCR analysis for four selected CsPRX genes under different stress conditions indicated that these selected CsPRX genes demonstrated diverse expression levels when subjected to NaCl, CdCl₂, and PEG treatments, and the CsPRX17 gene was significantly induced by NaCl, CdCl₂, and PEG stresses, suggesting a vital role of the CsPRX17 gene in response to environmental stresses. Conclusions: These findings will contribute valuable insights for future research into the functions and regulatory mechanisms associated with CsPRX genes in cucumber.

1. Introduction

Peroxidases (EC 1.11.1.X) belong to a significant class of isomerases that utilize hydrogen peroxide (H₂O₂) as an oxidizing agent to facilitate the oxidation of a variety of substrates. These enzymes are prevalent across a diverse range of living organisms, including plants, animals, and microorganisms. Peroxidases can be categorized into two primary types: hemoglobin peroxidases and non-hemoglobin peroxidases, based on their structural and catalytic properties [1]. Furthermore, the non-animal hemoglobin peroxidases can be divided into three categories: class I, class II, and class III [2]. Specifically, class III peroxidases (EC 1.11.1.7) consist of those peroxidases that are exclusive to plants [2]. In various previous studies, these peroxidases have been referred to by a multitude of abbreviations, including PRX, POX, POD, Px, and PER. In the present study, we will adopt the abbreviation “PRX” to represent class III peroxidases. These peroxidases (PRXs) are glycoproteins known for their thermal stability and can be found in the cell walls and vacuoles of plant tissues [3]. The structure of the PRX protein is characterized by highly conserved amino acid residues and consists of two components: a single peptide chain and a protoporphyrin IX domain [4]. Furthermore, two histidine residues engage with a heme group and eight cysteine residues within its internal framework to establish disulfide bridges, which are crucial for enzymatic catalytic activity [5].

Some studies have demonstrated that PRX proteins play crucial roles in various physiological processes in plants, such as lignin and liposome formation, cell wall protein cross-linking [6], auxin metabolism [7], cell growth, and elongation [8]. Furthermore, PRX genes can also enhance stress resistance by modulating the levels of reactive oxygen species (ROS) in plants [9]. For instance, the overexpression of the OsPRX38 gene in Arabidopsis increased SOD, POD, and GST activities; lowered H₂O₂ levels; decreased electrolyte leakage and malondialdehyde contents; and thus improved arsenic stress tolerance [10]. Similarly, the overexpression of the TaPRX-2A gene in wheat enhanced SOD, POD, and CAT activities; lowered ROS levels; and improved salt and drought stress resistance [11,12]. The overexpression of the IbPRX17 gene in sweet potatoes enhanced tolerance to salt and drought stresses by effectively scavenging ROS [13]. Furthermore, the overexpression of the OsPRX30 gene in rice reduced the the bacterial blight resistance by reducing H₂O₂ contents [14]. Taken together, PRX genes are crucial for plant biological functions and responses to multiple biotic and abiotic stresses; therefore, a comprehensive analysis of these genes is essential to further explore their roles in plants.

PRX genes were first reported in Arabidopsis by Intapruk et al. [15]. Thereafter, numerous PRX genes have been extensively characterized across a variety of plant species due to the results of transcriptomic data. Currently, a total of 73 PRX genes have been identified in Arabidopsis [16]. Other significant discoveries included 138 PRX genes in rice [5], 124 PRX genes in soybean [17], and 102 PRX genes in potatoes [18]. Additional studies revealed the presence of 47 PRX genes in grapes [19], 82 PRX genes in sugarcane [20], and 75 PRX genes in carrot [21]. A comprehensive bioinformatics analysis of the PRX gene family will significantly enhance our understanding of their physiological functions and characteristics. Nevertheless, to date, there has been no genome-wide analysis performed on the PRX gene family in cucumber.

Cucumber is one of the most extensively cultivated vegetable crops globally and has important economic and social value. In 2022, the global cucumber planting area was more than 2.17 million hectares, resulting in a remarkable total production of approximately 94.72 million tons. In China, the cucumber planting area was approximately 1.31 million hectares, yielding more than 77.3 million tons, which constituted 60.37% of the world’s cucumber cultivation area and 81.61% of its total output [22]. Nevertheless, various adverse environmental factors, including extreme weather, low temperature, insufficient light, drought, salinity, and pollution from heavy metals, have severely hindered cucumber growth, resulting in a decline in yield and quality. Consequently, it is of significant importance to understand the mechanisms of stress response in cucumbers.

In this present study, we comprehensively investigated the PRX gene family in cucumbers using bioinformatics methods, including gene structure, chromosomal location, and phylogenetic relationship. Moreover, we examined the tissue-specific expression profiles of CsPRX genes and their expression patterns when subjected to various abiotic stresses, including treatments with NaCl, CdCl₂, and PEG. Our findings will undoubtedly be helpful for in-depth research on the biological roles of CsPRX genes in cucumber.

2. Materials and Methods

2.1. Characterization of PRX Gene Family in Cucumber

The cucumber genome annotation files, coding sequences (CDS), and protein sequence files were obtained from the Cucurbitaceae database (CuCgenDB: the Index of /FTP/genome/cucumber/Chinese_long/v3, accessed on 11 April 2024). The reference sequences of the PRX gene family in Arabidopsis were obtained from the TAIR database (TAIR: https://www.arabidopsis.org/browse/genefamily/index.jsp, accessed on 11 April 2024) and preliminarily screened by Blast comparison. The HMM file of the conserved domain of the PRX gene family (PF00141) was obtained from the Pfam online website (https://www.ebi.ac.uk/interpro/, accessed on 11 April 2024), and then the hmmsearch program of the TBtools software v2.056 was used to search for the proteins containing the conserved domain in cucumber. NCBI CDD (CDD, http://www.ncbi.nlm.nih.gov/cdd, accessed on 12 April 2024) was used to verify the proteins. The physical and chemical properties of CsPRX proteins were conducted using ExPASy (https://web.expasy.org/protparam/, accessed on 12 April 2024). The subcellular localization of CsPRX proteins was predicted through the Wolfpsort online tool (https://wolfpsort.hgc.jp/, accessed on 13 April 2024).

2.2. Gene Structure and Phylogenetic Relationship of PRX Proteins

MEME (https://meme-suite.org/meme/, accessed on 13 April 2024) was utilized for the identification of conserved motifs in all the CsPRX proteins. TBtools was employed to display the conserved domain, along with the positions of introns and exons, as well as the chromosomal location of CsPRX genes. The phylogenetic tree of CsPRX proteins was generated through multiple sequence alignments, applying the Neighbor-Joining (NJ) method. For the syntenic analysis of PRX genes between Arabidopsis and cucumber, the One StepMC ScanX-Superfast program within TBtools was used. Cis-regulatory elements (CREs) were predicted using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 13 April 2024).

2.3. Plant Materials and Treatments

In this study, a cucumber ‘Jinyou 1’ variety was utilized as plant material. The cucumber seeds were soaked in sterilized water at 55 °C for 15 min and then germinated at 28 ± 1 °C in sterile culture dishes with filter paper. The 5-day-old cucumber seedlings were transferred to 1/2 MS liquid medium with 100 mmol·L⁻¹ NaCl solutions (salt stress), 125 mg·L⁻¹ CdCl₂ solutions (cadmium stress), and 10% PEG-6000 solutions (drought stress), respectively. Cucumber roots were harvested after 0, 6, 12, 24, and 48 h following stresses for RNA isolation.

2.4. Gene Expression Analysis

Transcriptome data for CsPRX genes in various tissues (PRJNA80167) were sourced from the Cucurbitaceae database. In this study, the read counts were characterized as fragments per kilobase of transcript per million fragments (FPKMs) for CsPRX genes. A heatmap of expression was generated using log₂FPKM values. The expression patterns of four CsPRX genes subjected to NaCl, CdCl₂, and PEG treatments were examined using real-time quantitative PCR (qRT-PCR) analysis. The total RNA of cucumber was isolated using the DNase I plant RNA extraction kit (Kangweishiji, Beijing, China, CW2598), and complementary DNA (cDNA) was synthesized using PrimeScript™ RTMaster Mix (Takara, Kusatsu, Japan, RR036A). The qRT-PCR analysis was conducted using TB Green^®Premix Ex Taq™ Ⅱ (Takara, RR820A). The primer sequences for the four CsPRX genes and the reference gene (CsActin) are detailed in Supplementary Table S1. The relative expression levels of the four CsPRX genes were quantified employing the 2^{−(∆∆Ct)} method.

3. Results

3.1. Identification of PRX Genes in Cucumber

A total of 73 PRX candidate genes were identified by Blast and Pfam in cucumber, and 60 CsPRX proteins were obtained by CDD verification, which were renamed as CsPRX01-CsPRX60 according to their distribution on chromosomes. The number of amino acids (aas) encoded for the CsPRX proteins in cucumber ranged from 255 aa to 742 aa. The relative molecular weight (MW) was between 27.94 kD and 80.80 kD, and the theoretical isoelectric point (pI) was from 4.61 to 9.40. The Grand average of Hydropathicity (GRAVY) analysis showed that 6 proteins (CsPRX14, CsPRX27, CsPRX30, CsPRX32, CsPRX36, and CsPRX52) were hydrophobic, while the remaining 54 CsPRX proteins were hydrophilic. In addition, the instability index (II) of 29 CsPRX proteins was less than 40, ranging from 26.49 to 39.8, showing the stable characteristics. While, the II of 31 CsPRX proteins was greater than 40, ranging from 40.02 to 52.01, showing the unstable characteristics. Subcellular localization revealed that CsPRX proteins in cucumber were located in the chloroplasts (23), extracellular (14), vacuole membrane (10), endoplasmic reticulum (5), mitochondria (3), cytoplasm (2), plasma membrane (2), and cell membrane (1). Detailed information on the CsPRX proteins in cucumber is shown in

Table 1. Physical and chemical properties of CsPRX proteins in cucumber.

Name	Gene ID	AA	MW (Da)	pI	II	GRAVY	Subcellular Localization
CsPRX1	CsaV3_1G001790.1	409	44,782.37	6.57	36.19	−0.106	endoplasmic reticulum
CsPRX2	CsaV3_1G008260.1	332	36,902.27	6.36	30.43	−0.096	extracellular
CsPRX3	CsaV3_1G009980.1	343	38,125.62	5.84	40.75	−0.173	extracellular
CsPRX4	CsaV3_1G010780.1	331	37,686.25	8.47	41.32	−0.352	vacuole membrane
CsPRX5	CsaV3_1G012650.1	347	37,950.81	9.30	39.59	−0.224	chloroplasts
CsPRX6	CsaV3_1G014970.1	255	35,741.98	6.53	40.29	−0.029	chloroplasts
CsPRX7	CsaV3_1G030170.1	323	35,722.78	5.63	32.33	−0..155	extracellular
CsPRX8	CsaV3_1G040180.1	255	27,942.32	5.15	35.64	−0.314	cytoplasm
CsPRX9	CsaV3_2G014160.1	329	36,569.78	4.96	30.07	−0.047	extracellular
CsPRX10	CsaV3_2G016300.1	335	36,566.83	8.86	34.73	−0.092	vacuole membrane
CsPRX11	CsaV3_2G024340.1	317	34,683.64	9.09	49.80	−0.095	extracellular
CsPRX12	CsaV3_2G034090.1	331	35,690.26	4.74	36.49	−0.001	vacuole membrane
CsPRX13	CsaV3_2G034100.1	330	35,735.71	8.74	37.72	−0.137	cytoplasm
CsPRX14	CsaV3_2G034110.1	326	34,863.86	6.93	26.49	0.080	endoplasmic reticulum
CsPRX15	CsaV3_2G035150.1	328	36,082.20	8.10	40.23	−0.139	vacuole membrane
CsPRX16	CsaV3_3G016670.1	316	34,565.47	7.56	38.95	−0.053	cell membrane
CsPRX17	CsaV3_3G035690.1	323	36,388.92	8.31	34.08	−0.181	chloroplasts
CsPRX18	CsaV3_3G042040.1	326	35,890.94	8.63	34.12	−0.196	chloroplasts
CsPRX19	CsaV3_3G047080.1	323	36,036.27	9.06	27.15	−0.170	extracellular
CsPRX20	CsaV3_4G004610.1	273	30,455.90	5.40	41.04	−0.041	chloroplasts
CsPRX21	CsaV3_4G005430.1	342	37,447.46	9.18	49.20	−0.294	extracellular
CsPRX22	CsaV3_4G023590.1	322	34,297.21	4.94	42.45	−0.153	vacuole membrane
CsPRX23	CsaV3_4G023610.1	318	34,844.31	6.43	52.01	−0.196	vacuole membrane
CsPRX24	CsaV3_4G023620.1	742	80,803.84	6.17	40.02	−0.229	plasma membrane
CsPRX25	CsaV3_4G023630.1	329	36,469.33	6.24	46.57	−0.264	extracellular
CsPRX26	CsaV3_4G023640.1	351	38,281.28	9.11	43.07	−0.006	chloroplasts
CsPRX27	CsaV3_4G023650.1	330	35,502.55	5.51	37.98	0.102	extracellular
CsPRX28	CsaV3_4G023660.1	336	36,453.37	8.34	41.41	−0.224	chloroplasts
CsPRX29	CsaV3_4G023670.1	334	35,554.70	4.61	43.64	−0.082	vacuole membrane
CsPRX30	CsaV3_4G023680.1	334	36,476.81	5.63	42.78	0.043	vacuole membrane
CsPRX31	CsaV3_4G029960.1	318	35,080.10	8.10	44.63	−0.092	chloroplasts
CsPRX32	CsaV3_4G036360.1	327	34,873.18	5.10	37.76	0.006	plasma membrane
CsPRX33	CsaV3_5G005000.1	365	39,538.89	5.16	42.23	−0.038	chloroplasts
CsPRX34	CsaV3_5G012680.1	322	35,243.14	8.16	38.21	−0.012	endoplasmic reticulum
CsPRX35	CsaV3_5G012840.1	320	34,594.32	7.57	43.66	−0.099	extracellular
CsPRX36	CsaV3_5G033660.1	341	37,746.19	5.24	30.42	0.024	extracellular
CsPRX37	CsaV3_5G037450.1	319	35,013.07	8.36	42.21	−0.002	chloroplasts
CsPRX38	CsaV3_6G002170.1	322	34,128.24	8.63	48.52	−0.097	chloroplasts
CsPRX39	CsaV3_6G005630.1	326	36,159.26	9.28	49.78	−0.187	chloroplasts
CsPRX40	CsaV3_6G006890.1	337	37,933.75	9.14	47.17	−0.461	vacuole membrane
CsPRX41	CsaV3_6G013360.1	345	38,494.56	9.34	33.68	−0.045	vacuole membrane
CsPRX42	CsaV3_6G018990.1	320	34,795.30	7.58	27.51	−0.160	chloroplasts
CsPRX43	CsaV3_6G019010.1	326	35,456.53	9.26	40.02	−0.196	extracellular
CsPRX44	CsaV3_6G019020.1	313	34,396.09	6.99	32.03	−0.236	chloroplasts
CsPRX45	CsaV3_6G019040.1	325	35,404.37	9.12	42.38	−0.123	chloroplasts
CsPRX46	CsaV3_6G043090.1	327	36,656.70	6.43	33.14	−0.165	extracellular
CsPRX47	CsaV3_6G043930.1	329	36,083.28	9.36	36.70	−0.077	chloroplasts
CsPRX48	CsaV3_6G046710.1	332	36,387.16	9.04	46.80	−0.319	chloroplasts
CsPRX49	CsaV3_6G047430.1	318	34,499.38	9.06	39.26	−0.084	endoplasmic reticulum
CsPRX50	CsaV3_7G003750.1	329	35,669.05	9.20	36.63	−0.030	chloroplasts
CsPRX51	CsaV3_7G005720.1	314	34,009.52	8.60	29.85	−0.181	chloroplasts
CsPRX52	CsaV3_7G006200.1	324	35,012.99	6.11	41.44	0.013	chloroplasts
CsPRX53	CsaV3_7G022880.1	386	43,158.60	5.79	41.10	−0.294	chloroplasts
CsPRX54	CsaV3_7G030360.1	284	30,755.02	6.83	36.75	−0.134	extracellular
CsPRX55	CsaV3_7G030370.1	279	30,498.63	5.20	38.27	−0.200	endoplasmic reticulum
CsPRX56	CsaV3_7G030380.1	338	36,567.71	9.40	42.64	−0.148	chloroplasts
CsPRX57	CsaV3_7G030390.1	339	36,860.73	8.04	41.24	−0.180	mitochondria
CsPRX58	CsaV3_7G030400.1	339	36,852.71	8.32	39.80	−0.183	mitochondria
CsPRX59	CsaV3_7G030410.1	339	36,896.89	8.03	42.37	−0.158	chloroplasts
CsPRX60	CsaV3_7G030420.1	337	36,908.94	8.62	43.11	−0.236	mitochondria

.

3.2. Chromosomal Location of CsPRX Genes

To reveal the distribution of CsPRX genes across different chromosomes in cucumber, a chromosomal graph was constructed. A total of 60 members of the CsPRX family were located on seven distinct chromosomes (Figure 1). Specifically, chromosome 1 housed 8 CsPRX genes, chromosome 2 contained 7 CsPRX genes, chromosome 3 had 4 CsPRX genes, chromosome 4 comprised 13 CsPRX genes, chromosome 5 included 5 CsPRX genes, chromosome 6 featured 12 CsPRX genes, and chromosome 7 held 11 CsPRX genes.

3.3. Phylogenetic Analysis of CsPRX Genes

To elucidate the evolutionary relationships within the CsPRX gene family, a phylogenetic tree was generated utilizing the Neighbor-Joining (NJ) method (Figure 2). The findings revealed that 60 CsPRX genes were divided into 8 groups: groups I and II each contained 2 CsPRX genes, groups III and IV included 3 CsPRX genes, group V had 4 CsPRX genes, group VI contained 7 CsPRX genes, group VII included 14 CsPRX genes, and group VIII had 25 CsPRX genes. Furthermore, the syntenic relationships among the PRX family genes from cucumber and Arabidopsis were examined to further understand the evolution of PRX genes (Figure 3). The results indicated that a total of 36 syntenic gene pairs of PRX genes were identified between cucumber and Arabidopsis, suggesting a high level of conservation among the PRX family members throughout evolution.

3.4. Conserved Motifs and Gene Structures of CsPRX Genes

MEME was utilized to identify the conserved motifs of the CsPRX proteins in cucumber, resulting in the identification of 10 divergent motifs (Figure 4). Among the 60 members of the CsPRX family, 37 CsPRX proteins contained 10 motifs, 19 proteins had 9 motifs, and 4 proteins included 7–8 motifs. Notably, among the ten motifs, motifs 8, motif 2, motif 5, and motif 3 were present in all 60 CsPRX protein sequences and appeared in the same order, indicating their conservation. The gene structure analysis of the CsPRX genes revealed that the number of introns varied from 0 to 7, with most genes containing between 1 and 4 introns. CsPRX24 had the highest number of introns (7), while both CsPRX1 and CsPRX37 lacked introns (Figure 5). Furthermore, the structural analysis of the CsPRX family genes indicated that most CsPRX genes contained both 5’ UTR and 3’ UTR; however, CsPRX24 contained only a 5’ UTR, while CsPRX14, CsPRX33, and CsPRX57 contained only a 3′ UTR (Figure 5).

3.5. Cis-Regulatory Elements of CsPRX Genes in Cucumber

Transcription factors have crucial roles in the regulation of gene expression by binding to CREs located within the promoter regions of genes. To investigate the CREs of the CsPRX family genes in cucumber, we scanned the 2000 bp promoter regions of CsPRX genes by utilizing the online PlantCARE database. As illustrated in Figure 6, 175 CREs were categorized into light response elements, plant hormone response elements, stress response elements, and growth and development elements, according to their putative functions. The light response elements included the CTT-motif, chs-CMA1a, GATA-motif, L-box, 3-AF1 binding site, chs-CMA2b, G-box, GT1-motif, GCN4-motif, chs-CMA2a, CAG-motif, LAMP-element, MRE, and TCC-motif, among others. The plant hormone response elements comprised ABRE, MBSI, CGTCA-motif, TGACG-motif, TATC-box, P-box, and ARE-motif. The stress response elements included TC-rich repeats, STRE, MBS, and LTR. The growth and development elements encompassed O2-site, GCN4-motif, TGA-element, AuxRE, and TGA-box. Additionally, a substantial number of promoter/enhancer elements like the TATA-box and CAAT-box, as well as binding elements like the W-box, MYB recognition site, and MYC, were also identified. Thus, the variety of CREs present within the CsPRX genes suggested their varied roles in biological processes, including responses to light, plant hormones, and environmental stress.

3.6. Tissue Specificity of CsPRX Genes in Cucumber

To further investigate the tissue specificity of the CsPRX genes, transcript data from various cucumber tissues were sourced from an accessible genome database. The tissues examined included the root, stem, leaf, ovary, unfertilized ovary, and fertilized ovary. Based on the FPKM values, the CsPRX genes exhibited varying levels of expression across these six tissues, with CsPRX04, CsPRX49, CsPRX26, CsPRX21, and CsPRX25 generally showing higher expression levels in all the tissues. Furthermore, we observed that the majority of the CsPRX genes displayed higher expression in the root compared to the other tissues. The expression abundances of CsPRX25 and CsPRX37 were significantly greater in the stem than in the other three tissues. CsPRX16 demonstrated relatively high expression in the leaf. Additionally, CsPRX05, CsPRX45, and CsPRX53 exhibited higher expression levels in the ovary, unfertilized ovary, and fertilized ovary than in the other three tissues (Figure 7). These results suggested that the expression of CsPRX genes was regulated in a tissue-specific manner within cucumber, and the unique expression patterns observed across the different tissues reflected their various functions in cucumber.

3.7. Expression Profiles of CsPRX Genes under Abiotic Stresses

CsPRX genes are not only involved in plant developmental processes but also in response to multiple abiotic stresses. The CRE analysis of promoter sequences discovered the presence of several stress response elements within the CsPRX genes. To further explore how the CsPRX genes respond to NaCl stress, CdCl₂ stress, and PEG stress, qRT-PCR experiments were conducted to measure the relative expression levels. The expression profiles of these four genes (CsPRX04, CsPRX17, CsPRX30, and CsPRX36) demonstrated considerable variability across the various treatments (Figure 8). Specifically, the expression levels of CsPRX04 and CsPRX30 were down-regulated in response to NaCl stress. Conversely, CsPRX17 was significantly induced by NaCl stress, exhibiting higher expression levels from 12 h to 24 h after treatment. Unlike the other three genes, CsPRX36 displayed an initial increase followed by a decrease during NaCl stress. Additionally, the expression level of CsPRX04 was suppressed by CdCl₂ stress. The transcript levels of CsPRX17 and CsPRX36 were remarkably induced at both 24 h and 48 h of CdCl₂ stress. CsPRX30 was induced after 6 h of CdCl₂ stress, followed by a subsequent decrease and then a rise. Under PEG stress, the expression level of CsPRX04 showed a significant decrease initially, followed by an increase, while CsPRX17 exhibited a notable upward trend. CsPRX30 and CsPRX36, however, were not significantly affected by PEG stress. Collectively, these observations implied functional divergence among the CsPRX genes in their response to abiotic stresses. The CsPRX17 gene was significantly induced by NaCl, CdCl₂, and PEG stresses, exhibiting higher expression levels from 12 h to 24 h after treatment, suggesting a vital role of the CsPRX17 gene in response to environmental stresses.

4. Discussion

Peroxidases are enzymes found specifically in plants that are crucial for multiple stress responses during plant development. Although extensive studies have been carried out on the PRX gene family across various plant species, there is still a deficiency in research concerning the CsPRX genes in cucumber. In this research, we identified 60 CsPRX genes in cucumber, designated CsPRX1-60 (Table 1). This number is slightly less than that found in Arabidopsis (73) and carrot (75), but greater than that in grape (47). Furthermore, our analysis revealed that motifs 8, 2, 5, and, 3 were present in all 60 CsPRX protein sequences in the same order, indicating a level of conservation among CsPRX proteins. It is known that variations in the intron/exon structures contribute to gene diversity, driving the evolution of multi-gene families and resulting in diverse functional outcomes in gene evolution [18]. In this study, we observed considerable variability in the number of introns among CsPRX genes, ranging from 0 to 7. Of the 60 CsPRX genes, 29 genes contained three introns and four exons. This pattern was also noted as a significant proportion in Arabidopsis [16], rice [5], and potato [18], suggesting that this represented an ancestral intronic model for PRX genes. In addition, the syntenic analysis of PRX genes between Arabidopsis and cucumber also implied the high level of conservation among the PRX family members throughout evolution.

The patterns of gene expression and the presence of CREs in gene family members provide significant insights into gene functions and regulatory mechanisms [20]. It was reported that the ShPRX family genes were involved in regulating plant hormones, responding to abiotic stresses, managing tissue-specific cell cycles, and controlling circadian rhythms through the analysis of CREs. In this study, the CREs of CsPRX genes were classified into four groups, suggesting that these genes served as regulators actively participating in cucumber development, and multiple stress responses.

The expression patterns of CsPRX genes were examined, revealing that these genes were present in various tissues, such as the root, stem, leaf, ovary, unfertilized ovary, and fertilized ovary. This indicated that CsPRX genes exhibited extensive expression in cucumber, although variations in expression patterns were observed. In the case of sugarcane, of the 44 ShPRX genes with high expression levels, 26 and 43 were detected in the leaf and stem, respectively, suggesting that most of these genes might play significant roles in these tissues [20]. In the current study, the expression profiles of CsPRX genes demonstrated variation across various tissues. For instance, CsPRX25 and CsPRX37 were significantly higher in the stem, while CsPRX16 exhibited relatively higher expression in the leaf. Additionally, CsPRX05, CsPRX45, and CsPRX53 showed elevated expression levels in the ovary. These findings suggested the unique functions of CsPRX genes in cucumber development. Notably, a majority of CsPRX genes exhibiting higher expression levels were identified in the root of cucumber. Similar observations have been documented in Arabidopsis [16], rice [5], maize [23], and potato [18]. For example, the overexpression of AtPRX01, AtPRX44, and AtPRX73 has been shown to promote root hair elongation in Arabidopsis [24]. Furthermore, a decrease in the expression of PRX2/ATPRX1, PRX8, PRX35, and PRX73 negatively affected cell elongation, vegetative growth, and the development of vascular structures in Arabidopsis [25]. These findings provide basic information about the role of CsPRX genes in cucumber root functionality, and the gene expression patterns and functions need further validation.

Abiotic stresses significantly hinder plant growth and productivity, leading to economic losses. Previous studies have reported that PRX genes can be markedly influenced by abiotic stresses, exhibiting distinct expression patterns in soybean [17], grapevine [19], and potato [18]. Plant peroxidase is involved in various cell growth and development processes, responding to both abiotic and biotic stresses through plant hormone signaling pathways [26]. Numerous researchers have indicated that plants produce ROS under stresses from salt and Cd, with the PRX family genes playing crucial roles in mitigating these stresses by scavenging ROS levels [12,27,28]. In wheat, the overexpression of the TaPRX-2A gene has been shown to trigger the abscisic acid (ABA) signaling pathway and enhance antioxidant enzyme activity, leading to a decrease in ROS levels and an increase in osmotic metabolites, thereby improving salt tolerance [12]. Furthermore, in sugarcane, ShPRX genes enhanced tolerance to Cd and salt stresses by activating the antioxidant defense mechanism and reducing ROS levels [20]. Previous researches has also indicated that drought stress enhanced the POD activity [29], and higher expression levels of POD genes have been shown to be associated with enhanced tolerance to drought and osmotic stresses in plants [30,31]. The GsPOD40 gene promoted drought resistance in soybean by modulating critical physiological and biochemical pathways involved in defense responses [17]. To verify the response of CsPRX genes under various environmental stimuli and cucumber development, the qRT-PCR analysis of four CsPRX genes in response to NaCl, CdCl₂, and PEG stresses demonstrated a range of differential expression, and the CsPRX17 gene was significantly induced by NaCl, CdCl₂, and PEG stresses, suggesting a vital role of the CsPRX17 gene in response to environmental stresses. Further studies are needed to elucidate the functions of the CsPRX17 gene through either gain-of-function or loss-of-function experiments in cucumber.

5. Conclusions

In this study, 60 CsPRX genes were identified in cucumber by comprehensive investigation. Motif 8, motif 2, motif 5, and motif 3 consistently appeared across all 60 CsPRX protein sequences, indicating the conservation of CsPRX proteins. Furthermore, we analyzed the expression patterns of the CsPRX genes across various tissues and their responses to abiotic stresses in cucumber, indicating that a majority of the CsPRX genes might be essential for root development, and the CsPRX17 gene might be linked to environmental stresses. Our results provided a systematic insight into the characterization of CsPRX genes and highlighted the possible functions of the CsPRX17 gene in cucumber development and stress responses. This work lays the foundation for future investigations into the roles and regulatory mechanisms of CsPRX genes in particular environments.