VsAPX1 Is Up-Regulated by ABA and Heat Stress in Common Vetch (Vicia sativa)

Abstract

Ascorbate peroxidase (APX) is a heme-containing enzyme involved in hydrogen peroxide (H₂O₂) detoxification within the ascorbate–glutathione (AsA–GSH) cycle. In this study, the full-length genomic DNA and cDNA of an APX1 gene (VsAPX1) were cloned and characterized from Vicia sativa. The genomic sequence of VsAPX1 is 2425 bp in length and comprises 10 exons separated by nine introns, with the first intron located within the 5′ untranslated region (5′UTR). The corresponding cDNA is 1010 bp long and includes a 61 bp 5′UTR, a 753 bp open reading frame, and a 196 bp 3′UTR. VsAPX1 encodes a predicted cytosolic APX protein of 250 amino acids, with a molecular weight of 27.1 kDa and a theoretical isoelectric point (pI) of 5.60. Bioinformatics analysis revealed that the deduced VsAPX1 protein shares high sequence similarity with cytosolic APX1 proteins from other plant species, contains conserved APX domains, and clusters within the cytosolic APX clade in phylogenetic analysis. Quantitative real-time PCR analysis showed that VsAPX1 expression exhibits transient and moderate changes in response to abiotic stress and phytohormone treatments. Transcript levels increased at early time points following heat stress (42 °C), abscisic acid, and salicylic acid treatments, and after 4 h of jasmonic acid exposure, whereas hydrogen peroxide treatment resulted in a gradual down-regulation of expression. Overall, this study provides the first molecular and expression characterization of a cytosolic APX1 gene from Vicia sativa and establishes a foundation for future functional analyses of antioxidant genes in this species.

1. Introduction

In nature, plants are exposed to various environmental stresses during their lifetime [1,2]. Global climatic change increases plants’ vulnerability to various abiotic stresses, including temperature, salinity, drought, flooding, high and low light intensity, nutrient deficiency, and chemical factors (e.g., pH and heavy metals) [3]. Plants also suffer from biotic stresses (e.g., pests and diseases) [4]. All these stresses cause morphological, physiological, and molecular changes [5,6] that adversely impact agricultural yield, production, and global food security. Furthermore, stress conditions induce the overproduction and accumulation of reactive oxygen species (ROS) in plant cells, including superoxide anion (O₂^•−), hydroxyl radical (^•OH), hydrogen peroxide (H₂O₂), and singlet oxygen (₁O²) [7,8]. ROS are highly reactive and harmful by-products generated from different organelles (e.g., chloroplasts, peroxisomes, and mitochondria) that impose oxidative damage [9,10] to different cellular components, including lipids, proteins, carbohydrates, and nucleic acid, and may ultimately lead to apoptosis and senescence [7,11]. On the other hand, ROS, especially H₂O₂ at the steady-state level, acts as an essential cell signaling molecule and regulates many plant processes such as development, growth, reproduction, and responses to biotic and abiotic stresses [12].

In response to ROS overproduction, plants evolve a specific and flexible combination of signaling molecules and defense systems. These signaling molecules and defense systems are kinases/phosphatase signaling cascade, plant bioregulators (e.g., salicylic acid (SA), ethylene (ET), jasmonic acid (JA), abscisic acid (ABA), and auxin), defense, and antioxidant genes [3]. Therefore, plants can avoid and tolerate the adverse effects of oxidative stresses through the antioxidant defense system (ROS scavengers), which represents the first line of defense [13], including non-enzymatic antioxidants (e.g., flavonoids, carotenoids, ascorbic acid (AsA), glutathione (GSH), proline, and α-tocopherol) (3), and enzymatic antioxidants (e.g., superoxide dismutase (SOD), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX)) [11]. Together, enzymatic and non-enzymatic antioxidants minimize oxidative stress injuries and maintain ROS homeostasis by rapidly detoxifying the excess ROS [12,14].

Ascorbate peroxidase (APX, EC: 1.11.1.11) is a class I heme peroxidase, and an important ROS-scavenging enzyme [3]. It has a high affinity to H₂O₂ and regulates and maintains cellular H₂O₂ content at a steady-state level [15,16]. Therefore, APX is considered as a key enzyme in the ascorbate–glutathione (AsA-GSH) cycle, reducing H₂O₂ to water (H₂O) and oxygen (O₂) by utilizing ascorbate as an electron donor [4]. Plants’ APXs are encoded by multigenic families and found as isoenzymes that are classified according to their subcellular localization as cytosolic, peroxisomal/glyoxysomal, mitochondrial, or chloroplastic (stromal sAPX and thylakoid tAPX) [16]. They have been cloned from different plant species [15,16]. Furthermore, APX activity and expression were reported to increase under different stresses [11], such as high light intensity, high and low temperature, drought, heavy metals, wounding, and pathogen infection [15]. Moreover, the overexpression of APXs improves transgenic plants’ tolerance to drought, heat, high light, and salinity [17].

Common vetch (Vicia sativa L.) is an annual leguminous herb that belongs to the Fabaceae family [18]; it is self-pollinated with 2n = 12 chromosomes [19]. It originated in arid regions of the Middle East [20], and is now widely distributed in Central Asia, North Asia, Europe, and North America [18]. Common vetch can grow in a wide range of soil and climatic conditions and can tolerate drought and cold conditions [21]. Common vetch is suitable for crop rotation, cultivated either as a monocrop or as an intercrop with cereals, which enhances forage harvesting, and yield [21]. Moreover, it is used as a cover crop not only for cereals but also for vegetables such as tomato and pepper [22]. Moreover, this plant reduces herbicides application by suppressing weed growth [22], due to its high content of polyphenols and flavonoids [23]. These chemicals have allelopathic properties that are released in the soil via leachates and root exudates [24]. In addition, V. sativa has a high capacity for nitrogen fixation [18], and therefore, it decreases utilization of fertilizer and pesticides [21], enhancing microbial community, and soil enzyme activities such as sucrase, urease, phosphatase, and catalase [18]. Furthermore, common vetch is easy to cultivate, has a high protein content, and grows rapidly; therefore, it can be used as a green manure, livestock feed as grain, and for silage and hay production [25].

Heat stress represents an increasingly important abiotic stress on cool-season legumes, including common vetch, particularly in the Mediterranean regions where rising temperatures negatively affect germination, vegetative growth, and forage yield. In addition, they lead to ROS production, reducing photosynthesis efficiency and cellular homeostasis, which requires efficient antioxidant mechanisms. Therefore, investigating the expression of VsAPX1 under heat stress may provide important insights into the thermotolerance in common vetch.

Recent studies characterized some of the antioxidant genes in V. sativa, such as catalase (VsCAT) and chloroplastic copper/zinc superoxide dismutase (VsCu/Zn-SOD) [26,27]. APX plays a central and isoform-specific role in ROS detoxification, stress signaling, and redox homeostasis. Although APX genes have been characterized in several species [28], APX from Vicia sativa has not been functionally examined, and its regulatory behavior under specific stresses remains unknown. Species-specific APX isoenzymes often differ in expression patterns, subcellular localization, kinetic properties, and stress responsiveness; thus, cloning and characterizing APX from V. sativa provides a first functional characterization. Moreover, our study integrates expression profiling, structural/sequence analysis, and stress-responsive induction patterns, which together would provide a comprehensive characterization.

Despite extensive studies on APX1 genes in several legumes, no molecular or expression-level characterization has been reported for V. sativa, limiting comparative and applied research in this species. Therefore, this study aims to characterize the APX gene from V. sativa, and perform bioinformatic analyses of the APX protein and investigate its structure and expression pattern in response to abiotic stresses.

2. Materials and Methods

2.1. Plant Growth and Treatments

Common vetch (V. sativa, Mahali) seeds were received from the National Agricultural Research Center (NARC), Jordan. Seeds were grown in peatmoss and perlite mix (2:1) in plastic pots (14 × 14 cm, three seeds per pot) in a greenhouse (16 h photoperiod and 55 μmol m⁻² s⁻¹ from white fluorescent lamps, around 60% relative humidity) and were irrigated on alternate days with tap water (100 mL). For gene expression purposes, one-month-old seedlings were subjected to different treatments for 6 h, including: heat stress (seedlings were exposed to 42 °C in incubator for 6 h), phytohormone treatment (seedlings were sprayed until dripping with 1 mM salicylic acid (SA), 100 μM abscisic acid (ABA), or 100 μM jasmonic acid (JA) one time), and hydrogen peroxide (H₂O₂) (seedlings were sprayed with 10 mM H₂O₂ one time). Leaflet tissues were collected after 0, 2, 4, and 6 h of treatment, from each treated seedling and control (at each time point). The harvested tissue samples were directly dipped in liquid nitrogen and stored at −20 °C for RNA extraction.

2.2. Gene Cloning

For gene cloning, total RNA was extracted from one-month-old vegetative tissues treated by 12% PEG (6000) by using IQeasy_TM Plus Plant RNA Extraction Mini Kit (iNtRON Biotechnology, Gyeonggi-do, Republic of Korea) according to the manufacturer’s protocol, RNA integrity was verified by gel electrophoresis (Figure S5). A reverse transcription (RT) reaction was performed to synthesize the first-strand cDNA from 2 μg of total RNA by using the SCRIPT cDNA Synthesis Kit (Jena Bioscience, Jena, Germany), with oligo (dT)₂₀ as a primer in a reaction volume of 20 μL. cDNA samples’ concentration and purity were measured by spectrophotometer at 260 and 280 nm (Biochrom, Cambridge, UK), and stored at −20 °C.

For identifying APX gene structure, genomic DNA was isolated from vegetative tissues of three-week-old seedlings. Collected tissues were harvested and dipped immediately in liquid nitrogen and the tissues were ground into a fine powder in liquid nitrogen using a micro pestle. Total genomic DNA was isolated by using GenElute^TM Plant Genomic DNA Miniprep Kit (Sigma, Steinheim, Germany) according to the manufacturer’s protocol. Genomic DNA quality was detected using agarose gel, and their concentration and purity were estimated by spectrophotometer based on absorbance at 260 and 280 nm (Biochrom, Cambridge, UK), and stored at −20 °C.

In silico sequence of VsAPX was retrieved from V. sativa RNAseq SRA database “https://www.ncbi.nlm.nih.gov/sra (accessed on 1 January 2025)” using APX orthologs from related legumes. Thereafter, specific primers spanning the entire cDNA (VsAPX-F: 5′-CTCGTGTCACTAGGGTTTATCT-3′) and (VsAPX-R: 5′-CAAATTAGCTGGGCATTACCAC-3′) were designed on Primer-BLAST “https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi (accessed on 1 January 2025)” and utilized in PCR amplification. The PCR reaction was performed using iNtRON i-MAX II system (iNtRON, Gyeonggi-do, Republic of Korea). cDNA PCR amplification was carried out as follows: Initial denaturation at 95 °C for 5 min, then 35 cycles of: 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1.5 min, and then final extension at 72 °C for 10 min. In case of using gDNA as a template, the following PCR amplification program was as follows: Initial denaturation at 95 °C for 5 min and then 35 cycles of: 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 2.5 min, and then final extension at 72 °C for 10 min. PCR products were separated on 1% agarose gel and visualized under UV light. The single specific PCR product bands (around 1 kb in case of cDNA as a template and 2.5 kb in case of gDNA as a template) were cut from the gel by a razor blade and recovered as stated by Wizard^® SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The purified single specific PCR product was ligated into the pGEM^®-T Easy Vector (Promega, Madison, WI, USA), and then transformed into Escherichia coli JM109 competent cells (Promega, Madison, WI, USA) by the heat shock method. The recombinant plasmid DNA was extracted by using E.Z.N.A. ^TM Plasmid Miniprep Kit I (Omega Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer’s instructions. Positive clones were verified using PCR (either using VsAPX-specific primers or universal primers (T7p and SP6)) and sequenced (Macrogen, Seoul, Republic of Korea).

2.3. VsAPX Sequence Analysis and Gene Structure

The result of bidirectional sequencing of cDNA-APX was assembled in a contig by using BioEdit software, and the vector backbone was removed by VecSecreen “https://www.ncbi.nlm.nih.gov/tools/vecscreen/ (accessed on 1 January 2025)”. The open reading frame was detected by using the ORF finder of NCBI “https://www.ncbi.nlm.nih.gov/orffinder/ (accessed on 1 January 2025)” and BLAST with SRA data (https://blast.ncbi.nlm.nih.gov/). For gDNA-APX, the received sequence results were used as a template to design a pair of internal primers (VsAPX-Fc: 5′-TAACAGCGGTCTTGATATTGC-3′) and (VsAPX-Rc: 5′-AATGGTGTGACCACCAGATAG-3′), to cover the whole internal region of the gene. All gDNA sequences were assembled in one contig, and the genomic structure (exon/intron organization) of VsAPX gene was detected by pairwise alignment with cDNA using the BLAST algorithm, Splign-NCBI “https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi (accessed on 1 January 2025)”, and Gene Structure Display Server 2.0 “http://gsds.gao-lab.org/ (accessed on 1 January 2025)”. Gene structure was constructed by using BioRender “https://biorender.com/ (accessed on 1 January 2025)”. The VsAPX1 promoter (1500 bps upstream) was retrieved from chromosome 1 of the reference genome sequence for V. sativa at the Genbank. PlantPAN 4.0 was used to find promoter TFBS and related TFs “https://plantpan.itps.ncku.edu.tw/plantpan4/index.html (accessed on 1 January 2025)”.

2.4. Bioinformatic Analysis

The full-length nucleotide sequence was translated by ExPASY Translate tool “https://web.expasy.org/translate/ (accessed on 1 January 2025)”, and the physical and chemical parameters of the protein were obtained by using the ProtParam tool “https://web.expasy.org/protparam/ (accessed on 1 January 2025)”. Subcellular localization of VsAPX was predicted by TargetP 2.0 “https://services.healthtech.dtu.dk/services/TargetP-2.0/ (accessed on 1 January 2025)”, ProtComp 9.0 online tool http://www.softberry.com/cgi-bin/programs/proloc/protcomppl.pl (accessed on 1 January 2025)”, and DeepLoc 2.0 https://services.healthtech.dtu.dk/services/DeepLoc-2.0 (accessed on 1 January 2025)”. The conserved protein domains were identified by NCBI Conserved Domain Database https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi (accessed on 1 January 2025)” and InterPro 91.0 https://www.ebi.ac.uk/interpro/ (accessed on 1 January 2025)”. Clustal Omega was performed for multiple sequence alignment https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 1 January 2025)”. Protein 3D structure was predicted using SWISS-MODEL https://swissmodel.expasy.org/ (accessed on 1 January 2025)”. Phylogenetic analysis of APX proteins was carried out by a neighbor-joining algorithm using the MEGA 11 program after bootstrap re-sampling analysis with 1000 replicates to assess branch support.

2.5. VsAPX Gene Expression Analysis

Expression of VsAPX gene was quantified in V. sativa seedlings under different treatment conditions at different time points by using quantitative real-time PCR (qRT-PCR). Total RNA was isolated from the treated leaflet tissues and their control by using Hybrid-R_TM (GeneALL, Seoul, Republic of Korea), according to the manufacturer’s protocol. One hundred nanograms of total RNA were used to synthesize the first strand of cDNA using EasyScript^® First-Strand cDNA synthesis Super Mix Kit (TransGen Biotech, Beijing, China). The relative expression pattern of the VsAPX gene was assayed using a pair of specific primers designed by Primer3 input online tool, (VsAPXq-F: 5′-AGCAGTTCCCTATTGTGAGCT-3′) and (VsAPXq-R: 5′-GCCCCATAGCTTTTCCAAACA-3′), that results in 195 bp amplicon, whereas the V. sativa Actin gene was used as an internal control (GenBank accession No. GU946218) and amplified using specific primers (VsActinqF: 5′-CAATCCAGGCCGTCTTGTCTC-3′; VsActinqR: 5′-TCTGTTAAATCACGCCCAGCA-3′), resulting in 157 bp amplicon [26]. The qRT-PCR reaction mix contained 10 μL of 2xTransScript^® Green qPCR SuperMix, 1 μL of each specific primer (10 μM), 3 μL of cDNA template, and 5 μL of nuclease-free water to make up a total volume of 20 μL. The amplification reaction was carried out using a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) with two steps, initial denaturation at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 s then annealing and extension at 60 °C for 30 s at which florescent was acquired. Following qRT-PCR, to confirm primer stringency, melting curve from 65 °C to 95 °C was used; increment 0.5 °C each 5 s at which florescent was acquired. The fold change in transcript level (stresses compared to control) was achieved using the 2^−ΔΔC_T method [29] according to the following equation: fold change in expression = 2^−ΔΔC_T, while ΔΔC_T = ((C_{T VsAPX} − C_{T VsAct})_Treated − (C_{T VsAPX} − C_{T VsAct})_Control). All qRT-PCR experiments were performed with three biological replicates. The gene expression results are expressed as fold change, and the experiment was a completely randomized design, and subjected to one-way analysis of variance (ANOVA) using SAS 9.4. Differences between interval times of treatment means were determined by least significant difference (LSD) test at 95% confidence interval, and a significance level less than 0.05 (p ≤ 0.05).

3. Results

3.1. Cloning and Sequence Analysis of V. sativa APX Gene

As the GenBank was lacking any entry related to APX under V. sativa, the SRA database was searched for RNAseq data. V. sativa RNAseq (SRX6435314) was selected and assembled using CLC Genomic Workbench (Version 9.1). Assembly was BLAST-searched using APX orthologous sequences with complete cDNA entries of related legumes (Pisum sativum). Based on BLAST results, a pair of gene-specific primers (VsAPX-F and VsAPX-R) were designed to amplify the APX gene from V. sativa. The cDNA was generated from total RNA extracted from one-month-old V. sativa seedlings subjected to 12% PEG for 24 h. The amplified PCR product (around 1 kb) was cloned into the pGEM^®-T Easy Vector then sequenced. The full-length APX cDNA was 1010 bp, including 753 bp of ORF flanked by 61 bp of 5′-UTR and 196 bp of 3′-UTR. The ORF of V. sativa APX encodes 250 amino acid residues, with a molecular weight of 27.09862 kDa and a theoretical isoelectric point (pI) of 5.60. The V. sativa APX gene was designated as VsAPX1, and its nucleotide sequence was submitted to GenBank under the accession number OR842549.

The subcellular localization of the VsAPX1 protein was predicted using online bioinformatic tools: TargetP 2.0, ProtComp 9.0, and DeepLoc 2.0 (Figure S1). TargetP 2.0 excludes the probability of VsAPX1 being localized in the mitochondria or chloroplast, while the highest value was recorded for “other” cellular locations. Therefore, ProtComp 9.0 was checked, and its result showed that VsAPX1 is most likely predicted to be cytoplasmic. On the other hand, DeepLoc 2.0 confirms the previous results and showed the lack of localization signals in the VsAPX1 protein.

BLASTp search against protein sequence database showed that the VsAPX1 sequence shared high similarity to known APX homologs from other plant species (Figure S2), including Lens culinaris subsp. culinaris AXI69835.1 (97%), Pisum sativum XP_050898393.1 (98%), Medicago truncatula XP_003606510.1 (97%), Cicer arietinum XP_004505943.1 (95%), Medicago sativa AIY27528.1 (94%), Trifolium pratense PNX98620.1 (96%), Glycine max NP_001237785.1 (96%), Vigna unguiculata AAB03844.1 (96%), Solanum lycopersicum NP_001234782.1 (92%), Zea mays NP_001152746.1 (92%), Arabidopsis thaliana BAA03334.1 (91%), and Hordeum vulgare subsp. vulgare CAA06996.1 (89%).

Multiple sequence alignment (Figure 1) was performed by using Clustal Omega between the VsAPX1 protein and other legumes cytosolic APX homologs: Pisum sativum (PsAPX1, AAA33645.1), Lens culinaris subsp. culinaris (LcAPX1, AXI69835.1), Trifolium pratense (TpAPX, PNX98620.1) Medicago sativa (MsAPX2, AIY27528.1) and Arabidopsis thaliana (AtAPX1, BAA03334.1). The alignment showed the presence of conserved amino acid residues. The conserved domains of the VsAPX1 protein were predicted using NCBI Conserved Domains (CDDs) and InterPro. InterPro analysis revealed conserved APX active site (APLILRLAWHSA, 33-44 aa), proximal heme-ligand motif (DIVALSGGHTI, 155-165 aa) and other conserved sites including six K⁺ binding sites (T164, T180, N182, I185, D187, S189), eight substrate binding sites (P111, H163, I165, G166, A168, E193, L202, L203), and 24 heme binding sites (P34, L35, R38, W41, P132, D133, A134, F145, L159, S160, G162, H163, I165, G166, A167, A168, H169, R172, S173, W179, L205, S207, Y235, H239) (Figure 1). Furthermore, CDD results confirmed the presence of previously conserved sites and demonstrated that the VsAPX1 protein belongs to a plant-peroxidase-like superfamily (cl00196), member (PLN02364).

Homology modeling of the VsAPX1 protein was performed using SWISS-MODEL (Figure 2). The best structural template identified was the cytosolic ascorbate peroxidase 1APX (PDB: 1apx.1.A), an X-ray structure of 2.2 Å resolution, showing 95.18% sequence identity, 0.98 coverage, and a GMQE score of 0.98. The predicted quaternary structure was supported by a QSQE value of 0.66, consistent with the dimeric organization of the template.

The resulting 3D model correctly positioned the heme prosthetic group and the nearby potassium-binding region (Figure 3). The amino acids shown surrounding these sites represent predicted spatial proximities from the homology model, not experimentally verified contacts. Therefore, these features should be interpreted as structural hypotheses. Experimental validation, such as mutagenesis or biochemical assays, will be required to confirm the contribution of individual residues.

To study the phylogeny of VsAPX1 and plant homologs, APX proteins were retrieved from GenBank and aligned by ClustalW. Based on sequence similarity, a phylogenetic tree was constructed by a neighbor-joining method using the MEGA 11 program with bootstrap re-sampling analysis with 1000 replicates. The phylogenetic tree showed that APX proteins clearly clustered into three clades according to ascorbate peroxidase subcellular localization: cytosolic, peroxisomal, and chloroplast. The VsAPX1 protein clustered with the cytosolic APX clade and was closely related to other legumes like pea and lentil (Figure 4).

3.2. Ascorbate Peroxidase Gene Structure

To determine VsAPX1 gene structure, APX gDNA was amplified by PCR using the same specific primers (VsAPX-F and VsAPX-R). The amplified PCR resulted in around 2.5 kb of product, which was cloned into pGEM^®-T Easy Vector and was sequenced. To complete the waking primer sequencing and cover the entire internal region of the gene, a pair of internal primers (VsAPX-Fc and VsAPX-Rc) designed based on the received sequence were used as a template. The VsAPX1 gene structure was determined by comparison of the APX cDNA with the gDNA sequence, using the BLAST algorithm, Splign-NCBI, and Gene Structure Display Server 2.0. The full-length VsAPX1 gDNA nucleotide sequence was 2425 bp, and was submitted to GenBank under the accession number OR756531, and the comparison in Figure S3 shows that the VsAPX1 gene contains 10 exons separated by nine introns. All introns contain the GT-AG dinucleotide at both ends, and the first intron was localized within the 5′UTR closer to the start codon (ATG). Introns of the VsAPX1 gene were determined according to the intron’s position relative to the reading frame of the gene. Intron 1 is located in the 5′UTR, introns 3, 4, 6, and 8 are located between two codons (phase 0 intron), intron 5 is located between the first and second nucleotide of a codon (phase 1 intron), and introns 2, 7, and 9 are located between the second and third nucleotide of a codon (phase 2 intron).

A comparison between the VsAPX1 genomic structure (exon/intron organization) and cytosolic APX of other plants (P. sativum APX1 (M93051.1), A. thaliana APX1a (D14442.1), A. thaliana APX1b (X80036.1), and F. ananassa APX (AF158654.1)) was performed to study structural changes (Figure S4). The results revealed that the VsAPX1 gene structure is similar to that of PsAPX1 and FaAPX and has a conserved exon/intron order, including exons with the same length as well as conserved intron phases, while VsAPX1 is different from AtAPX1b as it lacks the 5′UTR intron. In addition, exons 5 and 6 of VsAPX1 may have been merged to form exon 5 in AtAPX1a. However, this hypothesis needs further investigation using synteny analysis.

The promoter region (1500 bps) of VsAPX1 revealed multiple transcription factor binding sites (TFBS) for major TFs including MYB/SANT, WRKY and NAC NAM (Figure 5).

3.3. Expression Pattern Analysis of VsAPX1 Gene

The differential expression of the VsAPX1 gene was studied in one-month-old V. sativa seedlings by using qRT-PCR in response to heat stress, hydrogen peroxide, and phytohormonal (ABA, SA, and JA) stimuli at different time points (0, 2, 4, and 6 h).

To examine VsAPX1 transcript level under abiotic stresses, seedlings were exposed to heat stress (42 °C); the expression of VsAPX1 compared to the control was highly up-regulated by 8.4-fold after 2 h then decreased sharply to 4.8-fold after 4 h and to 1.36-fold after 6 h, which reflects a transient stress shock response (Figure 6A). The VsAPX1 transcript was down-regulated gradually over time by 0.86, 0.60, and 0.39-fold after 2 h, 4 h, and 6 h, respectively, in response to hydrogen peroxide (10 mM) compared to the control (Figure 6B). Although the 10 mM H₂O₂ treatment exceeds endogenous physiological concentrations, this level is widely used in plant molecular studies to induce reproducible oxidative stress and trigger antioxidant gene responses (e.g., 5–20 mM in leaf and callus assays). During the exposure period used here, the tissues remained visually healthy, with no necrosis or collapse observed. While electrolyte leakage or additional physiological viability indicators were not measured, the transcriptional responses and absence of tissue damage suggest that the treatment elicited oxidative signaling rather than extensive toxicity.

The VsAPX1 transcript levels showed modest responses to phytohormone treatments. Following ABA (100 μM) treatment, transcript abundance increased slightly to 2.7-fold after 2 h, before decreasing to 1.4- and 1.3-fold at 4 h and 6 h, respectively (Figure 6C). SA (1 mM) treatment resulted in a small increase to 1.38-fold after 2 h, which then decreased slightly to 1.27-fold at 4 h and further declined to 0.65-fold at 6 h (Figure 6D). JA (100 μM) treatment caused a modest rise to 1.18-fold at 2 h, peaking at 1.2-fold at 4 h, and subsequently decreasing to 0.34-fold at 6 h compared to the control (Figure 6E). These results indicate that VsAPX1 transcript levels exhibit only slight and transient changes in response to the tested phytohormones.

4. Discussion

4.1. Sequence Analysis of VsAPX1 Gene and Protein

APXs are important H₂O₂ scavenger antioxidant enzymes, which maintain H₂O₂ homeostasis under normal and stressful conditions. The cytosolic APX isoenzyme is the most studied among other isoforms, due it being moderately responsive and induced under different stresses [30], as well as its role in different defense processes, and in plant homeostasis redox regulation [31]. Guo et al. (2020) proved the role of cytosolic APX1 in maintaining redox balance to regulate cotton photosynthetic rate and yield [32]. Also, in Arabidopsis thaliana, APX1 protected chloroplast under high light intensity [30]. Therefore, the present study characterized the full-length gDNA and cDNA cytosolic ascorbate peroxidase gene from the V. sativa plant. The VsAPX1 cDNA sequence contains an in-frame start site (ATG, 62 bp) and stop site (TAG, 812 bp); these signals suggest that the VsAPX1 cDNA is full length. Furthermore, a putative polyadenylation signal site (AAATAA) was detected in the VsAPX1 sequence at a 159 bp downstream stop codon; this signal site was previously reported in other legumes [28]. Different studies have cloned and characterized cytosolic APX cDNA from different plant species such as pea [33], sweet potato [34], potato [35], and spinach [36].

The ORF of VsAPX1 encodes 250 amino acid residues (Figure 1), with a molecular weight of 27.1 kDa. APX isoenzymes are different in molecular weight, for example, in rice chloroplast (thylakoidal APX (~51 kDa), stromal APX (~33-38 kDa)), peroxisomal APX (~32 kDa), and cytosolic APX (~27 kDa) [37], while in potato mitochondrial APX they are ~31 kDa [38]. VsAPX1 amino acid residues lack localization signals, an N-terminal transit peptide sequence and C-terminal transmembrane domains, according to the protein targeting analysis. This confirms the bioinformatic results that the VsAPX1 protein is cytosolic ascorbate peroxidase. APX can be localized to different cellular compartments which perform distinct antioxidant functions. However, the cytosolic VsAPX1 acts as a central hub for maintaining whole-cell redox balance and integrating stress and hormone signaling pathways. Therefore, the predicted cytosolic localization suggests a broader regulatory role in coordinating cellular antioxidant responses rather than organelle-specific protection.

Sequence similarity analysis of VsAPX1 with closely related proteins from different plant species was investigated using BLASTp (Figure S2). The results showed that the deduced VsAPX1 amino acid sequence was highly similar to other cytosolic APX proteins; this could indicate that the cloned gene encodes cytosolic APX.

Ascorbate peroxidase protein, in its active form, is a dimer (containing two identical subunits) distinguished by two domains, C-terminal and N-terminal, surrounded by the heme, and specified by its residues that are essential for the activity [39]. Ascorbate, heme (iron), and potassium ions are critical for APX activity [40]. In the present study, the multiple sequence analysis of the VsAPX1 primary structure (Figure 1) revealed fundamental and functional conserved motifs, domains and residues that are essential for APX protein structure and function. These include the APX active site, proximal heme-ligand motif, K⁺ binding site, substrate binding sites, and heme binding sites. Several studies have identified the proximal His-163 in the heme binding site and Arg-38 which are essential for heme binding. The distal His-42 and Arg-38 in the active site are responsible for the heterolytic cleavage of H₂O₂. The hydrogen bonding between His-163, Asp-208, and Trp-179 forms the active site structure [41]. The substrate ascorbate binds to the active site by four hydrogen bonds with Lys-30, Arg-172, Cys-32, and the heme moiety. At the proximal domain, K⁺ ions bind to K⁺ binding sites (Thr-164, Thr-180, Asp-187) that are required for APX activity [39]. Phosphorylation of Thr-59 and Thr-164 residues was reported to increase tomato APX enzyme activity, and S-nitrosylation of Cys-32 enhances APX enzyme catalytic activity [42]. APX class I is distinguished from other classes by Trp-41 and Trp-179 instead of Phe-41 and Phe-179 [39]. Moreover, cytosolic APX is differentiated by Phe-175 instead of Trp-175 found in the chloroplast, and Ser-43, Phe-57, and Thr-59 are replaced by Asp-43, Asn-57, and Ser-59 in other APX isoforms [40]. These data suggest that VsAPX1 belongs to the class I cytosolic ascorbate peroxidase.

Molecular phylogenetics deals with evolutionary relationships based on different macromolecules (DNA, RNA, and protein) [43]. This study clarifies the phylogenetic relationship of VsAPX1 with APX orthologs from other plant species; for this purpose, a neighbor-joining phylogenetic tree was constructed (Figure 4). The resulting tree showed that APX proteins were clearly separated into three groups based on their subcellular localization: cytosolic, peroxisomal, and chloroplastic, and the VsAPX1 protein was clustered within the cytosolic clade which is closely related to the leguminous species Pisum sativum and Lens culinaris, indicating a close relationship between these APX proteins. This result is consistent with the phylogenetic tree generated in the study of 44. This apparent divergence between orthologs was revealed in previous studies indicating that cytosolic and peroxisomal APX isoenzymes were generated by a duplication event of a non-chloroplastic ancestral gene [37]. According to Qu et al. (2020), results of APX genes’ phylogenetic tree and exons’ structure revealed that the ancestors of monocots and dicots underwent genome duplication [16]. Ozyigit et al.’s (2016) APX phylogenetic tree of 18 plant species showed segmental and tandem duplications in some APX genes [15].

4.2. Ascorbate Peroxidase Gene Structure

The identification of the VsAPX1 gene structure provides insights into the organization of exons and introns in this gene, which can aid in further genetic studies and manipulation of the gene. To investigate the structure of VsAPX1, the exon/intron organization was constructed and compared with other plant cytosolic APX genes. In the present study, the VsAPX1 gene contains 10 exons interspaced by nine introns (Figure S3). Comparing the VsAPX1 gene structure with other plant species (Figure S4), the length of VsAPX1 exons of ORF 9 exons is highly identical to pea APX1 (which is the only gene available with details for APX in legumes) [28], and strawberry [44]. Moreover, similar results were found in other plant APX genes such as tomato [45], rice [37], maize [16,46], and wild watermelon [47]. Whereas there are differences in intron length and nucleotide sequences, the intron number and intron phases are conserved [48]. This suggests a conserved cytosolic APX1 gene architecture in higher plants [16,37]. Likewise, APX genes’ promoter cis-elements, exon–intron organization, and number were studied in Populus trichocarpa [49]. On the other hand, Arabidopsis thaliana APX1a, APX1b had nine exons and eight introns [50]. A major difference appears in exon 5 of AtAPX1a, which is separated by an 86 bp intron to exon 5 and exon 6 in the VsAPX1 gene. The first intron was located within 5′UTR. A similar observation was made in those of other plant species except for the A. thaliana APX1b (Supplementary Figure S4), which lacks the 5′UTR intron, the gene encoding a second family of cytosolic APX; and a similar observation was made in Dancy’ tangerine (Citrus reticulata Blanco) [50,51]. Mittler and Zilinskas (1992) detected a part of the GPEI enhancer (TGATTCAG) sequence in the 5′UTR intron, which is a regulator element for glutathione transferase P that regulates gene transcription by interaction with transcription factors and RNA II polymerase [28]. Some studies discussed the effect of the 5′UTR intron, which may interact with other elements located in the promoter that regulate APX1 gene expression; in fact, the leader intron of the APX20 gene in tomato increased expression of a reporter gene in leaves but it was absent in roots [46]. The gain of an intron in the 5′-untranslated region and in exon number five of VsAPX1 causes exon fission but does not cause a shift in reading frame, unlike the exon loss/gain that can cause a shift in reading frame [52]. The intron insertion at this location may be due to the presence of proto-splice sites (G|G, and MAG|R; M: A or C, R: A or G) that increase the chance of intron insertion at this position [53].

4.3. Expression Pattern Analysis of VsAPX1 Gene in Response to Stresses

Plants have complex defense systems that protect themselves from environmental changes. These defense systems are augmented and activated by the perception of stress signaling molecules (phytohormones and ROS (H₂O₂)), which induce signal transduction cascades and then regulate gene expression of the stress-responsive genes [11]. Antioxidant enzymes scavenge the excess ROS and maintain cellular homeostasis from oxidative damage [13]. APX is one of the crucial enzymes in the AsA-GSH cycle and can scavenge excess H₂O₂ [54]. Recent studies showed up-regulation of APX gene expression under different abiotic stresses and stress-response chemicals, which could be due to the presence of stress-response cis-acting elements in the promoter that activate APX gene expression [49]. These elements include phytohormone-responsive, abiotic stress-responsive, and growth and development-responsive elements. Furthermore, several TFs were found to bind TFBS on the VsAPX1 promoter (Figure 5), including major abiotic stress TFs including WRKY and NAC.

Generally, the cAPX 5′ regulatory region has a heat shock-responsive element and anti-peroxidative element (ARE) that might aid in H₂O₂ scavenging. The present study investigated the expression pattern of the APX gene in V. sativa under phytohormone treatments (ABA, SA, and JA) and abiotic stresses (42 °C and H₂O₂) using qRT-PCR, to understand and define the possible involvement of the VsAPX1 gene to stresses.

The stress-induced expression patterns observed for VsAPX1 are consistent with reports from other legumes, supporting the conserved role of cytosolic APX1 in ROS detoxification. While no novel functional properties were identified, the present data establish VsAPX1 as a canonical APX1 homolog in V. sativa, thereby extending the comparative framework of antioxidant gene regulation within legumes.

The increased global temperature is a critical climate-change problem, resulting in an increasing rate of evaporation and dehydration in plants and soil [55]. Heat stress resulted in the deactivation of enzymes, protein misfolding, disturbance of cell metabolism, increased fluidity of the membrane lipid, and increased production and accumulation of ROS (e.g., oxidative damage) [56]. Recent studies demonstrated that some APX genes are heat inducible [1]. Under heat stress, the increasing level of cellular H₂O₂ acts as a signaling molecule that induces heat stress signal transduction components, including the heat stress transcription factor, which binds to the heat shock element (HSE) in the promoter of the APX gene and controls its expression [57]. This explains the quick response of the APX gene to heat stress [58]. HSE has been determined in the APX promoter of pea [28] and strawberry [45]. In the present study, after exposing seedlings to 42 °C, the expression of VsAPX1 scored an early increase after 2 h (Figure 6A). Other studies found that heat stress increased APX gene expression in a variety of plant species, including pea [28], rice [59], alfalfa [60], Arabidopsis thaliana APX2 [61], and sweet potato APX1 [34]. Moreover, heat stress increased the activity of the cucumber cAPX enzyme [62]. The overexpression of pea cAPX enhanced heat tolerance in transgenic tomato [63]. Furthermore, the heat tolerance of Arabidopsis thaliana was increased by the overexpression of the cabbage APX gene [56].

H₂O₂ is an important non-radical ROS, generated from normal cellular metabolism as a harmful by-product that causes damage and inactivation for cellular components [64,65]. At basal levels, H₂O₂ acts as a regulatory signal for different physiological processes including photosynthesis, photorespiration, stomatal closure, growth, cell cycle, development, and senescence [66]. Furthermore, under stress conditions, H₂O₂ acts as a signaling transduction molecule, due to being highly stable and diffusible, and can pass through the plasma membrane via aquaporins and diffuse from different organelles and transport the signal to the nucleus through redox reactions, integrated with the MAPK pathway [67], which are involved in the regulation of nuclear gene expression of many transcription factors, and the up-regulation of antioxidative enzymes [62].

Interestingly, exogenous H₂O₂ treatment did not result in sustained induction of VsAPX1 expression (Figure 6B), but instead a reduction in expression was observed over time. In fact, similar responses have been reported for several antioxidant genes and may reflect the complex dynamics of ROS signaling. APX genes are often rapidly and transiently induced during the early phase of oxidative stress, with expression peaks occurring within minutes to 1 h, followed by feedback repression once cellular redox homeostasis is restored. Moreover, high concentrations of exogenously applied H₂O₂ can impose oxidative damage on cellular components and transiently inhibit transcriptional processes. Moskova et al. (2009) reported that exogenous application of H₂O₂ decreased APX enzyme activity in pea [68]. In contrast to other studies, the exogenous application of H₂O₂ increased gene expression in sweet potato [34], and cucumber cAPX enzyme activity [62]. In addition, H₂O₂ treatment was reported to induce APX gene expression in cultured soybean cells [69]. This is possibly due to the presence of an anti-peroxidative element (ARE) in the APX promoter, which could be involved in the regulation of the APX gene and responsible for the H₂O₂-induced response [28,69]. It is also possible that regulation occurs predominantly at post-transcriptional or enzymatic levels, or that other APX isoforms and antioxidant systems compensate for ROS detoxification. Therefore, the observed decline in VsAPX1 transcript levels likely reflects tight redox-dependent feedback control rather than reduced involvement in oxidative stress defense.

4.4. Expression Analysis of VsAPX1 Gene in Response to Phytohormones

Plant bioregulators are chemicals found in small quantities that have a large influence on regulating plant growth, development, and yield under normal and stressful conditions [70,71]. The plant bioregulators SA, ABA, and JA were selected because they are key regulators in stress signaling. ABA mediates abiotic stress responses such as drought, salinity, and heat, while JA and SA are central components of defense and stress-adaptation interacting with ROS signaling [72]. Therefore, evaluating VsAPX1 expression under these treatments enables assessment of its potential involvement in hormone-mediated stress responses and signaling crosstalk. According to previous studies, the cis-elements were detected in APX genes that contribute to the phytohormones’ stress responses, mainly the abscisic acid-responsive elements, MeJA-responsive element and salicylic acid-responsive element [73]. These elements were detected in different plant species’ APX promotor such as peanut AhAPX genes [73], Ammopiptanthus nanus [74], Triticum aestivum [75] and Brassica spp [76]. Furthermore, the exogenous application of phytohormones at a suitable dose enhanced plant tolerance to abiotic stress conditions [77].

ABA acts as a stress-responsive hormone that plays a crucial role in plant adaptation [78]. Its biosynthesis increases under stress conditions and regulates the expression of different stress-responsive genes and protective proteins like antioxidant enzymes, late embryogenesis abundant proteins, and dehydrins [79]. Therefore, exogenous ABA treatment was reported to increase the expression and activity of ascorbate peroxidase in different plant species, which is possibly due to the presence of the APX promoter cis-elements antioxidant-responsive element and ABA-responsive element [30]. In the peanut AhAPX1 promoter, an abscisic acid-responsive element (ABRE) was recorded, which was supported by up-regulation of AhAPX1 expression when peanut tissues were exposed to ABA [73]. In this study, the foliar application of ABA markedly increased the VsAPX1 expression after 2 h; this indicates that ABA has a signaling effect on VsAPX1 expression. Similar observations were reported after ABA treatment in pea [28], maize [47], and sweet potato [34].

The distinct temporal expression profiles of VsAPX1 observed under different phytohormone treatments suggest that this gene is regulated through hormone-specific signaling pathways rather than through a generalized stress response. In particular, the earlier transcriptional peak detected after ABA treatment (2 h) compared with the delayed induction under JA (2–4 h) likely reflects differences in the kinetics of their respective signaling cascades. ABA signaling is typically mediated through rapid activation of ABRE-dependent transcription factors, including AREB/ABF and ABI5 [80], which directly bind ABA-responsive elements in promoters of stress-associated genes and promote early transcriptional activation of antioxidant defenses. Such fast ABA-mediated responses are consistent with the role of ABA as a primary regulator of drought, heat, and oxidative stress adaptation. In contrast, JA signaling proceeds through the COI1–JAZ–MYC2 pathway [81], where degradation of JAZ repressors is required prior to activation of MYC2-dependent transcription, a multistep process that may account for the relatively delayed VsAPX1 induction observed following JA application. Therefore, the different response kinetics likely reflect intrinsic differences in hormone perception, signal transduction, and transcriptional regulation.

SA acts as a growth-regulating and protector molecule that improves crop plant tolerance under stress conditions [70], and induces the gene expression of antioxidants, HSPs, chaperones, and genes involved in the biosynthesis of secondary metabolites [79]. Foliar application of SA under abiotic stresses enhances the activity of antioxidant enzymes and reduces oxidative stress impacts such as lipid peroxidation and membrane injury [82]. The current study showed a slight rise in the VsAPX1 transcript level after 2 h of foliar SA treatment. Some studies reported that SA application increased endogenous H₂O₂ accumulation and increased the expression and activity of antioxidant enzymes [83]. The SA application increases the transcript level of sweet potato cytosolic ascorbate peroxidase [34], and pea APX enzyme activity [84].

JA aids in plant reproduction, development, tendril coiling, fruit ripening, chlorosis, allelopathy, production of secondary metabolites, flower and seed development, wounding and herbivory, and leaf senescence [77]. Whereas, under a stressful environment, JA acts as a signaling molecule and stress-responsive hormone [85]. JA was reported to interact with other plant hormones, transcription factors, and enhance the expression of JA-associated genes and stress-responsive genes and increase the activity of the antioxidant defense system [72]. VsAPX1 transcript level was highly accumulated after 4 h of JA application. A similar observation was recorded in rice OsAPX1 [86], and sweet potato [34]. The foliar application of JA alone or in combination with salinity on bitter melon seedlings decreased the activity of the APX enzyme [85].

In the present study, the expression profiling of VsAPX1 was conducted primarily in leaf tissues, as leaves represent the major site of photosynthesis and consequently the principal source of reactive oxygen species (ROS) production under both normal and stress conditions. Chloroplast electron transport, photorespiration, and environmental stresses such as heat and exogenous hormone treatments are known to markedly enhance H₂O₂ generation in photosynthetically active tissues, necessitating strong antioxidant defenses. Cytosolic APX isoforms have been widely reported to exhibit higher activity and functional relevance in leaves [87], where they cooperate with chloroplastic antioxidant systems to maintain cellular redox homeostasis. Therefore, focusing on leaf tissues allowed us to capture the most physiologically relevant and dynamic transcriptional responses of VsAPX1 to oxidative and hormonal stimuli. Nevertheless, antioxidant genes often display tissue-specific regulation depending on metabolic activity and stress perception. Future investigations examining roots and stems under similar stress conditions will provide a more comprehensive understanding of the spatial regulation and whole-plant physiological roles of VsAPX1.

It should be noted that we presented VsAPX1 expression, which may not correlate with enzymatic activity. APX proteins are frequently regulated at post-transcriptional and post-translational levels, including protein stability, redox modifications, and cofactor availability. Therefore, measurements of enzyme activity and protein accumulation will be required in future studies to fully elucidate the functional contribution of VsAPX1 to oxidative stress tolerance.

4.5. Redox Reaction and VsAPX1

Plant APX is known to have the strongest affinity for H₂O₂ scavenging within the AsA–GSH cycle compared to other peroxidases in plant cells, which is critical for maintaining cellular homeostasis under stress conditions [88]. Furthermore, stress-related genes interact extensively through crosstalk during stress perception and response, ultimately inducing the expression of tolerance and resistance pathways.

In cotton, silencing the cytosolic GhAPX1 gene led to reduced photosynthesis, impaired growth and development, and increased water loss, whereas its overexpression enhanced tolerance of cotton fibers to oxidative stress [32]. Similarly, overexpression of the Chinese cabbage APX gene in Arabidopsis thaliana increased heat tolerance in transgenic lines, resulting in higher chlorophyll content, improved germination rates, and lower MDA and H₂O₂ levels compared to non-transgenic plants [56].

We focused specifically on APX1 rather than the entire gene family due to its relative importance. Chloroplastic and mitochondrial APXs are highly sensitive to ascorbate deficiency and have short half-inactivation times (less than 30 s), in contrast to cytosolic and peroxisomal isoenzymes, which remain active for about an hour or more [16]. Among the APX isoforms, cytosolic APX is the most extensively studied because it is strongly induced under diverse stresses, is more resistant to ascorbate depletion, and is involved in multiple defense processes and redox homeostasis regulation [31]. It is also considered the most responsive isoform to environmental constraints [30]. In addition, overexpression or suppression of APX1 regulates redox balance and thereby influences cotton photosynthetic rate and yield [32].

A recent genome-wide study in common bean (Phaseolus vulgaris) identified 101 APX family members and demonstrated strong tissue-specific and stress-inducible expression patterns under drought and salinity, highlighting the central role of APX genes in oxidative stress mitigation and abiotic stress tolerance. These findings further support the conserved involvement of cytosolic APX isoforms in regulating H₂O₂ homeostasis across legumes [89]. Similarly, a cytosolic APX gene (LcAPX1) cloned from lentil (Lens culinaris) showed conserved structural features and stress-responsive expression, with significant induction under salinity, H₂O₂, ABA, and JA treatments. These findings further support the conserved role of legume APX1 isoforms in hormone-mediated antioxidant defense and oxidative stress regulation [90]. Transcriptomic analysis of common vetch under salt stress also revealed enhanced activation of ROS-scavenging pathways, including significant up-regulation of APX4, with stronger induction in the salt-tolerant genotype. These findings highlight the importance of APX-mediated antioxidant defenses in stress adaptation and further support the conserved role of APX genes in oxidative stress regulation in Vicia species [91].

5. Conclusions

In this study, we cloned and characterized the full-length VsAPX1 gene from the forage legume Vicia sativa. Sequence and bioinformatic analyses indicate that VsAPX1 encodes a cytosolic ascorbate peroxidase and is closely related to APX1 homologs from other legumes. Expression analyses under heat and phytohormone treatments revealed transient and moderate changes in transcript abundance, indicating that VsAPX1 transcription is responsive to environmental and hormonal cues.

While these expression patterns are consistent with the conserved involvement of APX1 genes in stress-related processes, the present data do not allow direct conclusions regarding the functional contribution of VsAPX1 to stress tolerance or adaptation. Rather, this work provides a species-specific molecular and expression framework for VsAPX1 in Vicia sativa. Further studies, including promoter analyses and functional characterization, will be required to more precisely elucidate the physiological role and regulatory control of VsAPX1 under stress conditions.