Novel Ascorbate Peroxidase from Common Vetch (Vicia sativa)

Novel Ascorbate Peroxidase from Common Vetch (Vicia sativa) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Novel Ascorbate Peroxidase from Common Vetch (Vicia sativa) Farah Abu Siam, Saeid Abu-Romman, Monther T. Sadder This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5468156/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Ascorbate peroxidase (APX) is a heme containing enzyme that acts as a key scavenging enzyme in the Ascorbate-Glutathione (AsA-GSH) cycle to scavenge hydrogen peroxide (H 2 O 2 ). APX expression in plants can enhance their tolerance to environmental stresses, potentially increasing crop yield. In this study, the full-length genomic DNA and cDNA of the APX gene were successfully cloned and characterized from Vicia sativa . The full-length gDNA- APX was 2425 bp with 10 exons that is enterspaced by nine introns. The first intron is located within the 5’-untranslated region (5’UTR). The transcribed cDNA (1010 bp) covers 5’UTR (61 bp), 3’UTR (196 bp) and open reading frame (ORF) (753 bp). It encodes the cytosolic APX protein (250 amino acids) with a molecular weight of 27.1 kDa and a theoretical isoelectric point (pI) of 5.60. Bioinformatic analysis of the deduced VsAPX1 amino acid sequence displays a high similarity with other plant species cytosolic APX. The main conserved domains were also determined, and the phylogenetic analysis showed that VsAPX1 clustered with the cytosolic APX clade. The expression pattern of VsAPX1 was determined using qRT-PCR in response to different stresses. Compared to the control, the level of VsAPX1 transcript showed an early increase after 2 h in response to heat stress (42 o C), abscisic acid, and salicylic acid, while being upregulated after 4 h of jasmonic acid treatment. While hydrogen peroxide treatment, caused a downregulation of VsAPX1 expression over time. The overall results suggest that VsAPX1 plays a role major in common vetch response to heat and plant bioregulators. Ascorbate Peroxidase. Abiotic stress. Cloning. Gene structure. Gene expression. Vicia sativa. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction In nature, plants are exposed to various environmental stresses during their lifetime (Sadder et al. 2014 ; 2021 ). 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) (Sachdev et al. 2021 ). Plants also suffer from biotic stresses (e.g., pests and diseases) (Saini et al. 2018 ). All these stresses cause morphological, physiological, and molecular changes (Alsadon et al. 2015 ; Al-Kiyyam et al. 2024 ), 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 (O 2 • − ), hydroxyl radical (OH•), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ) (Kumari et al. 2021 ; Tahtamouni et al. 2024 ). ROS are highly reactive and harmful by-products generated from different organelles (e.g., chloroplasts, peroxisomes, and mitochondria) that impose oxidative damage (Abu-Romman 2016a ; Huang et al. 2019 ) to different cellular components, including lipids, proteins, carbohydrates, and nucleic acid, and may ultimately lead to apoptosis and senescence (Nadarajah 2020 ; Kumari et al. 2021 ). On the other hand, ROS especially H 2 O 2 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 (Kumar et al. 2021 ). 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 (Sachdev et al. 2021 ). 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 (Rajput et al. 2021 ), including non-enzymatic antioxidants (e.g., flavonoids, carotenoids, ascorbic acid (AsA), glutathione (GSH), proline, and α-tocopherol) (Sachdev et al. 2021 ), 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)) (Nadarajah 2020 ). Together, enzymatic and non-enzymatic antioxidants minimize oxidative stress injuries and maintain ROS homeostasis by rapidly detoxifying the excess ROS (Abu-Romman and Shatnawi 2011 ; Kumar et al. 2021 ). Ascorbate peroxidase (APX, EC: 1.11.1.11) is a class I heme peroxidase, and an important ROS-scavenging enzyme (Sachdev et al. 2021 ). APX has a high affinity to H 2 O 2 and regulates and maintains cellular H 2 O 2 content at a steady-state level (Ozyigit et al. 2016 ; Qu et al. 2020 ). Therefore, APX is considered as a key enzyme in the ascorbate-glutathione (AsA-GSH) cycle, reducing H 2 O 2 to water (H 2 O) and oxygen (O 2 ) by utilizing ascorbate as an electron donor (Saini et al. 2018 ). Plants APXs are encoded by multigenic families and found as isoenzymes that are classified according to their subcellular localization to cytosolic, peroxisomal/glyoxysomal, mitochondrial, or chloroplastic (stromal sAPX and thylakoid tAPX) (Qu et al. 2020 ). APX has been cloned from different plant species (Ozyigit et al. 2016 ; Qu et al. 2020 ). Furthermore, APX activity and expression were reported to increase under different stresses (Nadarajah, 2020 ), such as high light intensity, high and low temperature, drought, heavy metals, wounding, and pathogen infection (Ozyigit et al. 2016 ). Moreover, the overexpression of APXs improves transgenic plants tolerance to drought, heat, high light, and salinity (He et al. 2018 ). Common vetch ( Vicia sativa L.) is an annual leguminous herb, that belongs to the Fabaceae family (Wang et al. 2021 ), it is self-pollinated with 2n = 12 chromosomes (Mikić et al. 2013 ). It originated in arid regions of the Middle East (Huang et al. 2017 ), and is now widely distributed in Central Asia, North Asia, Europe, and North America (Wang et al. 2021 ). Common vetch can grow in a wide range of soil and climatic conditions and can tolerate drought and cold conditions (Córdoba et al. 2021 ). Common vetch is suitable for crop rotation, cultivated either as a monocrop or as an intercrop with cereals, which enhances forage harvesting, and yield (Córdoba et al. 2021 ). Moreover, it is used as a cover crop not only for cereals but also for vegetables such as tomato and pepper (Mennan et al. 2020 ). Moreover, this plant reduces herbicides application by suppressing weed growth (Mennan et al. 2020 ), due to its high content of polyphenols and flavonoids (Magalhaes et al. 2017 ). These chemicals have allelopathic properties, that are released in the soil via leachates and root exudates (Ch et al. 2016 ). In addition, V . sativa has a high capacity for nitrogen fixation (Wang et al. 2021 ), and therefore, it decreases utilization of fertilizer and pesticides (Córdoba et al. 2021 ), enhancing microbial community, and soil enzyme activities such as sucrase, urease, phosphatase, and catalase (Wang et al. 2021 ). 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 (Tigka et al. 2021 ). Recent studies characterized some of the antioxidant genes in V . sativa , such as catalase ( VsCAT ) and chloroplastic copper/zinc superoxide dismutase ( VsCu/Zn - SOD ) (Abu-Romman 2016b ; 2019 ). Whereas the VsAPX gene has not been identified or characterized, while other studies have identified and characterized the APX gene in different legumes (Mittler and Zilinskas 1992 ). Therefore, this study aims to characterize APX gene from V . sativa , perform bioinformatic analyses of APX protein and investigate its structure and expression pattern in response to abiotic stresses. Materials and methods 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 x 14 cm, three seeds per pot) in a greenhouse 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 ο 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) for one time), and hydrogen peroxide (H 2 O 2 ) (seedlings were sprayed with 10 mM H 2 O 2 for one time). Leaflet tissues were collected after 0, 2, 4, and 6 h of treatment, from each treated seedlings and control (at each time point). The harvested tissue samples were directly dipped in liquid nitrogen and stored at -20 ο C for RNA extraction. 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, Korea) according to the manufacturer's protocol. 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, 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), and stored at -20 ο C. For identifying APX gene structure, genomic DNA was isolated from vegetative tissues of three-weeks 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™ Plant Genomic DNA Miniprep Kit (Sigma, 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), and stored at -20 ο C. In silico sequence of VsAPX was retrieved from V . sativa RNAseq SRA database ( https://www.ncbi.nlm.nih.gov/sra ) using APX orthologs form 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 ) and utilized in PCR amplification. The PCR reaction was performed using iNtRON i -MAX II system (iNtRON, Korea). cDNA PCR amplification was carried out as follows: Initial denaturation at 95 ο C for 5 min, then 35 cycles of: 95 ο C for 30 sec, 56 ο C for 30 sec, and 72 ο C for 1.5 min, and then final extension at 72 ο C for 10 min. In case of using gDNA as a template, the following PCR amplification program was followed: Initial denaturation at 95 ο C for 5 min and then 35 cycles of: 95 ο C for 30 sec, 56 ο C for 30 sec, and 72 ο C for 2.5 min, and then final extension at 72 ο 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, USA). The purified single specific PCR product was ligated into the pGEM ® -T Easy Vector (Promega, USA), and then transformed into Escherichia coli JM109 competent cells (Promega, 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. US) 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, Korea). 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/ ). The open reading frame was detected by using the ORF finder of NCBI ( https://www.ncbi.nlm.nih.gov/orffinder/ ) 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 ), and Gene Structure Display Server 2.0 ( http://gsds.gao-lab.org/ ). Gene structure was constructed by using BioRender ( https://biorender.com/ ). Bioinformatic analysis The full-length nucleotide sequence was translated by ExPASY Translate tool ( https://web.expasy.org/translate/ ), and the physical and chemical parameters of the protein were obtained by using the ProtParam tool ( https://web.expasy.org/protparam/ ). Subcellular localization of VsAPX was predicted by TargetP 2.0 ( https://services.healthtech.dtu.dk/services/TargetP-2.0/ ), ProtComp 9.0 online tool ( http://www.softberry.com/cgi-bin/programs/proloc/protcomppl.pl ), and DeepLoc 2.0 ( https://services.healthtech.dtu.dk/services/DeepLoc-2.0 ). The conserved protein domains were identified by NCBI Conserved Domain Database ( https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi ) and InterPro 91.0 ( https://www.ebi.ac.uk/interpro/ ). Clustal Omega was performed for multiple sequence alignment ( https://www.ebi.ac.uk/Tools/msa/clustalo/ ). Protein 3D structure was predicted using SWISS-MODEL ( https://swissmodel.expasy.org/ ). 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. VsAPX gene expression analysis Expression of VsAPX g ene 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, 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). 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’) that results in 157 bp amplicon (Abu-Romman 2016b ). The qRT-PCR reaction mix was containing 10 µl of 2x TransScript ® 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, U.S.A) with two steps, initial denaturation at 95 ο C for 10 minutes, followed by 45 cycles of denaturation at 95 ο C for 10 seconds then annealing and extension at 60 ο C for 30 seconds at which florescent was acquired. Following qRT-PCR, to confirm primer stringency melting curve was used 65 ο C to 95 ο C; increment 0.5 ο C each 5 sec at which florescent was acquired. The fold change in transcript level (stresses compared to control) was achieved using the 2 − ΔΔC T method (Livak and Schmittgen 2001 ) 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 expressed as fold change, and experiments design as 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). Results Cloning and sequence analysis of V. sativa APX gene As the GenBank was missing any entry related to APX under V . sativa (NCBI 2024), 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 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 VsAPX1 protein was predicted using online bioinformatic tools: TargetP 2.0, ProtComp 9.0, and DeepLoc 2.0 (Supplementary Fig. 1). TargetP 2.0 excludes the probability of VsAPX1 to be 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 VsAPX1 protein. BLASTp search against protein sequence database showed that VsAPX1 sequence shared high similarity to known APX homologs from other plant species (Supplementary Fig. 2), 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 (Fig. 1 ) was performed by using Clustal Omega between 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 (CDD) 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) (Fig. 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). The predicted three-dimensional structure of the VsAPX1 protein was built using SWISS-MODEL with homology-based modeling. The search template on SWISS-MODEL found that the highest identity template (98.18%) was the 1apx.1.A cytosolic ascorbate peroxidase crystal structure of recombinant ascorbate peroxidase, with a resolution of X-RAY DIFFRACTION 2.20 Å, which indicates the target sequence was well compatible with the template (Patterson and Poulos, 1995). The structure describes the binding and interaction of the potassium ion and heme (Fig. 2 ). A zoom in view reveals major interacting amino acids with both the heme group (Fig. 3 A) and the potassium ion (Fig. 3 B) To study the phylogeny of VsAPX1 and plant homologs, APX proteins were retrieved from GenBank and aligned by ClustalW. Based on sequence similarity 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 (Fig. 4 ). Ascorbate peroxidase gene structure (dup: abstract ?) 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 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, was submitted to GenBank under the accession number OR756531 and the comparison in (Supplementary Fig. 3) showed that VsAPX1 gene contains 10 exons separated by 9 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 VsAPX1 gene were determined according to the introns 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 VsAPX1 genomic structure (exon/intron organization) and cytosolic APX of other plants was performed to study structural changes; P . sativum APX1 (M93051.1), A . thaliana APX1a (D14442.1), A . thaliana APX1b (X80036.1), and F . ananassa APX (AF158654.1) (Supplementary Fig. 4). The results revealed that 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 appear to have been merged to form exon 5 in AtAPX1a . 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 ο C), the expression of VsAPX1 compared to the control was highly upregulated 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 (Fig. 5 A). Whereas VsAPX1 transcript was downregulated 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 (Fig. 5 B). The VsAPX1 was stimulated by phytohormone treatments. In response to ABA (100 µM) treatment, transcript level compared to control was increased to reach 2.7-fold after 2 h then decreased to 1.4-fold and 1.3-fold after 4 and 6 h, respectively (Fig. 5 C). SA (1 mM) treatment resulted in an increase of VsAPX1 expression by about 1.38-fold after 2 h while after 4 h decreased slightly to 1.27-fold, then highly downregulated to 0.65-fold after 6 h (Fig. 5 D). However, the treated seedlings with JA (100 µM), resulted in an increase of VsAPX1 expression to 1.18-fold after 2 h, then peaked to 1.2-fold after 4 h, while decreased to 0.34-fold after 6 h compared to the control (Fig. 5 E). Discussion Sequence analysis of VsAPX1 gene and protein APXs are important H 2 O 2 scavenger antioxidant enzymes, which maintain H 2 O 2 homeostasis under normal and stressful conditions. The cytosolic APX isoenzyme is the most studied among other isoforms, due it is highly responsive and induced under different stresses (Pandey et al. 2017 ; Saxena et al. 2020 ), as well as its role in different defense processes, and in plant homeostasis redox regulation (Caverzan et al. 2019 ). Guo et al. ( 2020 ), proved the role of cytosolic APX1 in maintaining redox balance to regulate cotton photosynthetic rate and yield. Also, in Arabidopsis thaliana APX1 protected chloroplast under high light intensity (Saxena et al. 2020 ). 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 (Mittler and Zilinskas 1992 ). Different studies have cloned and characterized cytosolic APX cDNA from different plant species such as pea (Mittler and Zilinskas 1991 ), sweet potato (Park et al. 2004 ), potato (Kawakami et al. 2002 ), and spinach (Ishikawa et al. 1995 ). The ORF of VsAPX1 encodes 250 amino acid residues (Fig. 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) (Teixeira et al. 2004 ), while in potato mitochondrial APX (~ 31 kDa) (De Leonardis et al. 2000 ). VsAPX1 amino acid residues lack localization signals; an N-terminal transit peptide sequence and a C-terminal transmembrane domains, according to the protein targeting analysis. This confirms the bioinformatic results that the VsAPX1 protein is cytosolic ascorbate peroxidase. Sequence similarity analysis of VsAPX1 with closely related proteins from different plant species was investigated using BLASTp (Supplementary Fig. 2). 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 (Dąbrowska et al. 2007 ; Pandey et al. 2017 ). Ascorbate, heme (iron), and potassium ions are critical for APX activity (Jespersen et al. 1997 ). In the present study, the multiple sequence analysis of the VsAPX1 primary structure (Fig. 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 H 2 O 2 . The hydrogen bonding between His-163, Asp-208, and Trp-179 form the active site structure (Lazzarotto et al. 2021 ). The substrate ascorbate binds to the active site by four hydrogen bonds with Lys-30, Arg-172, Cys-32, and the heme moiety (Pandey et al. 2017 ). At the proximal domain, K + ions bind to K + binding sites (Thr-164, Thr-180, Asp-187) that are required for APX activity (Dąbrowska et al. 2007 ). Phosphorylation of Thr-59 and Thr-164 residues was reported to increase tomato APX enzyme activity, and S-nitrosylation of Cys-32 enhance APX enzyme catalytic activity (Ravi et al. 2023 ). APX class I is distinguished from other classes by Trp-41 and Trp-179 instead of Phe-41 and Phe-179 (Dąbrowska et al. 2007 ). 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 (Jespersen et al. 1997 ). These data suggest that VsAPX1 belongs to the class I cytosolic ascorbate peroxidase. Some studies reported a dual function for APX protein under abiotic stresses, which means functional switching; under salinity stress act as peroxidase, and under heat stress tend to be molecular chaperones. The dual function of APX resulted from structural conformation, which leads to the association of oligomer to high molecular weight (molecular chaperone) under heat stress, or dissociation of oligomers to low molecular weight (APX enzyme activity) under salinity stress. Molecular phylogenetics deals with evolutionary relationship based on different macromolecules (DNA, RNA, and protein) (Bogusz and Whelan 2017 ). This study clarifies the phylogenetic relationship of VsAPX1 with APXs orthologs from other plant species, for this purpose a neighbor-joining phylogenetic tree was constructed (Fig. 4 ). The resulting tree showed that APX proteins were clearly separated into three groups based on their subcellular localization: cytosolic, peroxisomal, and chloroplastic, and 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 Malambane et al. ( 2018 ). This apparent divergence between orthologs, revealed in previous studies indicating that cytosolic and peroxisomal APX isoenzymes were generated by a duplication event of a non-chloroplastic ancestral gene (Teixeira et al. 2004 ). 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. While Ozyigit et al. ( 2016 ) APX phylogenetic tree of 18 plant species showed segmental and tandem duplications in some APX genes. 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 enterspaced by 9 introns (Supplementary Fig. 3). Comparing VsAPX1 gene structure with other plant species (Supplementary Fig. 4), the length of VsAPX1 exons of ORF 9 exons is highly identical to pea APX1 (which is the only gene available with detailed for APX in legumes) (Mittler and Zilinskas 1992 ), and strawberry (Kim and Chung 1998 ). Moreover, similar result was found in other plant APX genes such as tomato (Gadea et al. 1999 ) rice (Teixeira et al. 2004 ), maize (Liu et al. 2012 ; Qu et al. 2020 ), and wild watermelon (Malambane et al. 2018 ). Whereas there are differences in intron length, nucleotide sequences, while the intron number, and intron phases are conserved (Patthy 1987 ). This suggests a conserved cytosolic APX1 gene architecture in higher plants (Teixeira et al. 2004 ; Qu et al. 2020 ). Likewise, APX genes promoter cis -elements, exon-intron organization, and number were studied in Populus trichocarpa (Leng et al. 2021 ). On the other hand, Arabidopsis thaliana APX1a , APX1b had 9 exons and 8 introns (Santos et al. 1996 ). A major difference appears in exon5 of AtAPX1a which is separated by 86 bp intron to exon 5 and exon 6 in VsAPX1 gene. The first intron was located within 5’UTR. Similar observation was made in those of other plant species except for the A . thaliana APX1b (Supplementary Fig. 4) which lacks the 5’UTR intron, the gene encoding a second family of cytosolic APX and similar observation in Dancy’ tangerine ( Citrus reticulata Blanco ) (Santos et al. 1996 ; Kunta et al. 2010 ). Mittler and Zilinskas ( 1992 ) detected a part of the GPEI enhancer (TGATTCAG) sequence in 5’UTR intron, which is a regulator element for glutathione transferase P that regulates gene transcription by the interaction with transcription factors and RNA II polymerase (Mittler and Zilinskas 1992 ). Some studies discussed the effect of 5’UTR intron, which may interact with other elements located in the promoter that regulate APX1 gene expression, in fact, the leader intron of APX20 gene in tomato increased expression of a reporter gene in leaves but it was absent in roots (Gadea et al. 1999 ). The gain of an intron in 5’-untranslated region and in exon number five of VsAPX1 causes exon fission but did not cause a shift in reading frame unlike the exon loss/gain that can cause a shift in reading frame (Xu et al. 2012 ). 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 chance of intron insertion at this position (Nguyen et al. 2006 ). 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 (H 2 O 2 )), which induce signal transduction cascades and then regulate gene expression of the stress responsive genes (Nadarajah 2020 ). Antioxidant enzymes scavenge the excess ROS and maintain cellular homeostasis from oxidative damage (Rajput et al. 2021 ). APX is one of the crucial enzymes in the AsA-GSH cycle and can scavenge excess H 2 O 2 (Hasanuzzaman et al. 2019 ). Recent studies showed the 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 (Leng et al. 2021 ). These elements include phytohormone-responsive, abiotic stress-responsive, and growth and development-responsive elements. Generally, cAPX 5' regulatory region has a heat shock responsive element and anti-peroxidative element (ARE) that might aid in H 2 O 2 -scavenging (Pandey et al. 2017 ). The present study investigated the expression pattern of APX gene in V . sativa under phytohormone treatments (ABA, SA, and JA) and abiotic stresses (42 ο C and H 2 O 2 ) using qRT-PCR, to understand and define the possible involvement of the VsAPX1 gene to stresses. The increased global temperature is a critical climate-change problem, resulting in an increasing rate of evaporation and dehydration in plants and soil (Hassan et al. 2020 ). Heat stress resulted in the deactivation of enzymes, protein misfolding, disturbs cell metabolism, increased the fluidity of membrane lipid, and increased production and accumulation of ROS (e.g., oxidative damage) (Chiang et al. 2015 ). Recent studies demonstrated that some APX genes are heat inducible (Sadder et al. 2014 ). Under heat stress, the increasing level of cellular H 2 O 2 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 control its expression (Panchuk et al. 2002 ). This explains the quick response of APX gene to heat stress (Banerjee and Roychoudhury 2019 ). HSE has been determined in the APX promoter of pea (Mittler and Zilinskas 1992 ), and strawberry (Kim and Chung 1998 ). In the present study, after exposing seedlings to 42ºC, the expression of VsAPX1 scored an early increase after 2 h (Fig. 5 A). Other studies found that heat stress increased APX gene expression in a variety of plant species, including pea (Mittler and Zilinskas 1992 ), rice (Sato et al. 2001 ), alfalfa (Li et al. 2013 ), Arabidopsis thaliana APX2 (Wang et al. 2020 b), and sweet potato APX1 (Park et al. 2004 ). Moreover, heat stress increased the activity of the cucumber cAPX enzyme (Song et al. 2005 ). The overexpression of pea cAPX enhanced heat tolerance in transgenic tomato (Wang et al. 2006 ). Furthermore, the heat tolerance of Arabidopsis thaliana was increased by the overexpression of cabbage APX gene (Chiang et al. 2015 ). H 2 O 2 is an important non-radical ROS, generated from normal cellular metabolism as a harmful by-product that cause damage and inactivation for cellular components (Das and Roychoudhury 2014 ; Mittler 2017 ). At basal levels, H 2 O 2 acts as a regulatory signal for different physiological processes including photosynthesis, photorespiration, stomatal closure, growth, cell cycle, development, and senescence (Hasanuzzaman et al. 2020 ). Furthermore, under stress conditions, H 2 O 2 acts as a signaling transduction molecule, due to being highly stable and diffusible, 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 (Singh et al. 2019 ), which involved in the regulation of nuclear gene expression of many transcription factors, and upregulation of antioxidative enzymes (Song et al. 2005 ). The current study reports a gradual decline in the VsAPX1 gene expression over time under foliar application of H 2 O 2 , this may be due to the high concentration of H 2 O 2 (Fig. 5 B). Moskova et al. ( 2009 ) reported that exogenous application of H 2 O 2 decreased APX enzyme activity in pea. In contrast to other studies, the exogenous application of H 2 O 2 increased gene expression in sweet potato (Park et al. 2004 ), and cucumber cAPX enzyme activity (Song et al. 2005 ). In addition, H 2 O 2 treatment was reported to induce APX gene expression in cultured soybean cells (Lee et al. 1999 ). This is possibly due to the presence of an Antiperoxidative element (ARE) in the APX promoter, which could be involved in the regulation of the APX gene and responsible for the H 2 O 2 -induced response (Mittler and Zilinskas 1992 ; Lee et al. 1999 ). 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 (Alhaithloul et al. 2020 ; Hasanuzzaman et al. 2021 ). Whereas, under unfavorable environmental conditions, plant hormones (ABA, JA, SA, and ethylene) play a role in plant adaptation and regulate the balance between plant development and stress responses (Wang et al. 2020 a). Furthermore, the exogenous application of phytohormones at a suitable dose enhanced plant tolerance to abiotic stress conditions (Awan et al. 2021 ). ABA acts as a stress-responsive hormone that plays a crucial role in plant adaptation (Gong et al. 2020 ). 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 (Wani et al. 2016 ). 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 APX promoter cis -elements antioxidant responsive element and ABA responsive element (Saxena et al. 2020 ). 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 (Mittler and Zilinskas 1992 ), maize (Liu et al. 2012 ), and sweet potato (Park et al. 2004 ). SA acts as a growth regulating and protector molecule that improves crop plant tolerance under stress conditions (Alhaithloul et al. 2020 ), and induces the gene expression of antioxidants, HSPs, chaperones, and genes involved in the biosynthesis of secondary metabolites (Wani et al. 2016 ). 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 (Pourghasemian et al. 2020 ). 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 H 2 O 2 accumulation and increased the expression and activity of antioxidant enzymes (Dikilitas et al. 2020 ). The SA application increases the transcript level of sweet potato cytosolic ascorbate peroxidase (Park et al. 2004 ), and pea APX enzyme activity (Yadu et al. 2017 ). 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 (Awan et al. 2021 ). Whereas, under a stressful environment, JA act as a signaling molecule and stress-responsive hormone (Alisofi et al. 2020 ). JA was reported to interact with other plant hormones, transcription factors, and enhanced the expression of JA-associated genes and stress-responsive genes and increased the activity of the antioxidant defense system (Wang et al. 2020 a). VsAPX1 transcript level was highly accumulated after 4 h of JA application. Similar observation was recorded in rice OsAPX1 (Wang et al. 2015 ), and sweet potato (Park et al. 2004 ). The foliar application of JA alone or in combination with salinity on bitter melon seedlings decreased the activity of APX enzyme (Alisofi et al. 2020 ). Conclusions The identification and characterization of VsAPX1 is the first step to understand function of APX1 in plants that are related to stress response, plant adaptation, and development under stress condition. In this study we have cloned and characterized the full-length VsAPX1 gene from the forage legume V . sativa . The bioinformatic analysis confirm that the cloned VsAPX1 gene encode cytosolic APX. Gene expression analysis suggested that VsAPX1 is possibly involved in response to heat and phytohormones treatments. According to the results of this study, to explore the regulatory mechanisms that control VsAPX1 gene expression in response to different stresses and signaling pathways, additional research are recommended to analyze the regulatory cis -elements in the promoter region of VsAPX1 and studying the transcription factors that involved in its regulation. Furthermore, there is a need for additional studies to investigate the functional role of the first intron on VsAPX1 gene expression in different types of tissues of V . sativa under different stress conditions. Moreover, knockout or overexpression of APX in V . sativa is recommended for more clarification of the APX functional role on forage plant growth and development, the differential response and tolerance under environmental stresses, as well as the detection of APX protein activity and their efficiency in scavenging H 2 O 2 under these stress conditions. Declarations Author contribution: Farah Abu Siam: Conducted plant growth and treatments and gene cloning besides contributing to writing. Acknowledgment The authors are grateful for Ahliyya Amman University, Al-Balqa Applied University, and the University of Jordan for their support to conduct the research. References Abu-Romman S (2016a) Genotypic response to heat stress in durum wheat and the expression of small HSP genes. 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Proceedings of the National Academy of Sciences, 109 (4), 1187–1192. https://doi.org/10.1073/pnas.1109047109 Yadu S, Dewangan TL, Chandrakar V, Keshavkant S (2017) Imperative roles of salicylic acid and nitric oxide in improving salinity tolerance in Pisum sativum L. Physiol Mol Biology Plants 23(1):43–58. https://doi.org/10.1007/s12298-016-0394-7 Supplementary Files SupplementaryFigure1.jpg SupplementaryFigure2.png SupplementaryFigure3.jpg SupplementaryFigure4.jpg Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 18 Jul, 2025 Reviewers invited by journal 17 Jul, 2025 Editor assigned by journal 19 Nov, 2024 First submitted to journal 16 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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07:00:37","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":226367,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/53f457bff486130886c7d55e.html"},{"id":94888195,"identity":"cf1c2b05-b18b-44d1-b8cf-c5496f40babf","added_by":"auto","created_at":"2025-10-31 19:18:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":884163,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of \u003cem\u003eV. sativa\u003c/em\u003e APX protein sequence with related cytosolic APX proteins from different plant species. Red box indicates conserved APX active site, yellow box is a proximal heme-ligand motif, and gray box is K\u003csup\u003e+\u003c/sup\u003e binding site, arrowheads indicate substrate binding sites, and green boxes heme binding sites. Conserved amino acid residues are indicated by asterisks below the sequence, and numbers to the right indicate the amino acid positions.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/008ea9df28469ee1d1b07453.jpg"},{"id":94888193,"identity":"44535d88-c9f5-411b-b7b0-a0b6dbb2d4d3","added_by":"auto","created_at":"2025-10-31 19:18:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":173251,"visible":true,"origin":"","legend":"\u003cp\u003eTheoretical three-dimensional structure modeling of the VsAPX1 protein using SWISS-MODEL online tool. Predicted VsAPX1 protein structure based on the crystal structure of pea APX protein.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/cb8b7aa84efd721ea3905575.jpg"},{"id":94888196,"identity":"422fa33f-9c9c-4b89-a854-cf63ea62bc21","added_by":"auto","created_at":"2025-10-31 19:18:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":309634,"visible":true,"origin":"","legend":"\u003cp\u003eA) Amino acid residues of ascorbate peroxidase interacting with heme are represented with stick model, ChainA Heme site P34, R.38, W.41, P.132, D.133, A.134, F.145, L.159, S.160, G.162, H.163, I.165, G.166, A.167, A.168, H.169, R.172, S.173, W.179, L.205, S.207, Y.235. Interaction between residues include: Hydrophobic interactions in gray color (A:W.41, A:W.41, A:W.41, A:P.132, A:P.132, A:F.145, A:L.159, A:A.168, A:W.179, A:L.205. Hydrogen bonds in blue color: A:A.167, A:A.167, A:H.169, A:R.172, A:S.173. Salt bridges in yellow color: A:R.38, A:H.169, A:H.169, pi-Stacking in green color: A:W.41, Metal complexes purple color: A:H.163. B) Amino acid residues of ascorbate peroxidase interacting with K\u003csup\u003e+ \u003c/sup\u003ebinding Chain A: T.164, T.180, N.182, I.185, D.187, S.189. Four interactions with chain A Metal complexes: A:T.164, A:T.180 , A:N.182, A:I.185.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/b4568d5c357fe4e081f50ef3.jpg"},{"id":94987183,"identity":"82a48063-ebe2-4c7b-9816-84a8bc63a15c","added_by":"auto","created_at":"2025-11-03 07:01:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":505827,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of V. sativa APX protein with other plant species. APX proteins were aligned by ClustalW, and the phylogenetic tree was constructed using the Neighbor-Joining method with 1000 bootstrap replicates by MEGA11. GenBank accession numbers are indicated in parentheses.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/a68371d43e43aa38442b369a.jpg"},{"id":94987221,"identity":"40272128-0fe6-483a-9120-ea5e17804355","added_by":"auto","created_at":"2025-11-03 07:01:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":283564,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression pattern of VsAPX1 in V. sativa seedlings exposed to (A) heat stress, (B) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, (C) ABA, (D) SA, and (E) JA as measured by qRT-PCR. The transcript level of VsAPX1 was normalized to VsActin (GU946218) as a housekeeping gene and expressed as a ratio relative to the control which was set at 1. Each value represents the average of three biological replicates, the error bars indicate the standard error (±SE). One-way ANOVA test LSD values that are indicated by different letters above the bars at P\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/6e8609f44f6c2c3e21ccfa58.jpg"},{"id":95221042,"identity":"863882d0-7f8c-4bec-ab05-01dd8bfcf567","added_by":"auto","created_at":"2025-11-05 16:18:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3039178,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/001997df-ddef-49a0-b2b2-44c6c1e261f3.pdf"},{"id":94888200,"identity":"eb471ece-3bdb-468d-bc3e-1a4c31eed6ea","added_by":"auto","created_at":"2025-10-31 19:18:15","extension":"jpg","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":65000,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/9a5387da720c11641f7ad279.jpg"},{"id":94888211,"identity":"b18d7bd2-5a4d-4a30-8dcb-13b1644b4ea6","added_by":"auto","created_at":"2025-10-31 19:18:16","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":138550,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/d73114643e3afcdcd89ffc04.png"},{"id":94888205,"identity":"a1f52b8b-af09-4163-a7d7-563529607dc7","added_by":"auto","created_at":"2025-10-31 19:18:15","extension":"jpg","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":141636,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/cc20614dac5463fc0d026b6e.jpg"},{"id":94987222,"identity":"59c873d8-a0f8-4279-8c85-aaa460c74a45","added_by":"auto","created_at":"2025-11-03 07:01:30","extension":"jpg","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":130197,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5468156/v1/bedd9739181956bf0cc82903.jpg"}],"financialInterests":"","formattedTitle":"Novel Ascorbate Peroxidase from Common Vetch (Vicia sativa)","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn nature, plants are exposed to various environmental stresses during their lifetime (Sadder et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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) (Sachdev et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Plants also suffer from biotic stresses (e.g., pests and diseases) (Saini et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). All these stresses cause morphological, physiological, and molecular changes (Alsadon et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Al-Kiyyam et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), 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 (O\u003csub\u003e2\u003c/sub\u003e\u0026bull;\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e), hydroxyl radical (OH\u0026bull;), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), and singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) (Kumari et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tahtamouni et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). ROS are highly reactive and harmful by-products generated from different organelles (e.g., chloroplasts, peroxisomes, and mitochondria) that impose oxidative damage (Abu-Romman \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) to different cellular components, including lipids, proteins, carbohydrates, and nucleic acid, and may ultimately lead to apoptosis and senescence (Nadarajah \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumari et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, ROS especially H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 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 (Kumar et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn 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 (Sachdev et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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 (Rajput et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), including non-enzymatic antioxidants (e.g., flavonoids, carotenoids, ascorbic acid (AsA), glutathione (GSH), proline, and α-tocopherol) (Sachdev et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and enzymatic antioxidants (e.g., superoxide dismutase (SOD), glutathione reductase (GR), glutathione \u003cem\u003eS\u003c/em\u003e-transferase (GST), glutathione peroxidase (GPX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX)) (Nadarajah \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Together, enzymatic and non-enzymatic antioxidants minimize oxidative stress injuries and maintain ROS homeostasis by rapidly detoxifying the excess ROS (Abu-Romman and Shatnawi \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAscorbate peroxidase (APX, EC: 1.11.1.11) is a class I heme peroxidase, and an important ROS-scavenging enzyme (Sachdev et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). APX has a high affinity to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and regulates and maintains cellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content at a steady-state level (Ozyigit et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Qu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, APX is considered as a key enzyme in the ascorbate-glutathione (AsA-GSH) cycle, reducing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to water (H\u003csub\u003e2\u003c/sub\u003eO) and oxygen (O\u003csub\u003e2\u003c/sub\u003e) by utilizing ascorbate as an electron donor (Saini et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Plants APXs are encoded by multigenic families and found as isoenzymes that are classified according to their subcellular localization to cytosolic, peroxisomal/glyoxysomal, mitochondrial, or chloroplastic (stromal sAPX and thylakoid tAPX) (Qu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eAPX\u003c/em\u003e has been cloned from different plant species (Ozyigit et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Qu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, APX activity and expression were reported to increase under different stresses (Nadarajah, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), such as high light intensity, high and low temperature, drought, heavy metals, wounding, and pathogen infection (Ozyigit et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, the overexpression of APXs improves transgenic plants tolerance to drought, heat, high light, and salinity (He et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCommon vetch (\u003cem\u003eVicia sativa\u003c/em\u003e L.) is an annual leguminous herb, that belongs to the Fabaceae family (Wang et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), it is self-pollinated with 2n\u0026thinsp;=\u0026thinsp;12 chromosomes (Mikić et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It originated in arid regions of the Middle East (Huang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and is now widely distributed in Central Asia, North Asia, Europe, and North America (Wang et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Common vetch can grow in a wide range of soil and climatic conditions and can tolerate drought and cold conditions (C\u0026oacute;rdoba et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Common vetch is suitable for crop rotation, cultivated either as a monocrop or as an intercrop with cereals, which enhances forage harvesting, and yield (C\u0026oacute;rdoba et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, it is used as a cover crop not only for cereals but also for vegetables such as tomato and pepper (Mennan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, this plant reduces herbicides application by suppressing weed growth (Mennan et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), due to its high content of polyphenols and flavonoids (Magalhaes et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These chemicals have allelopathic properties, that are released in the soil via leachates and root exudates (Ch et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e has a high capacity for nitrogen fixation (Wang et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and therefore, it decreases utilization of fertilizer and pesticides (C\u0026oacute;rdoba et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), enhancing microbial community, and soil enzyme activities such as sucrase, urease, phosphatase, and catalase (Wang et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 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 (Tigka et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRecent studies characterized some of the antioxidant genes in \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e, such as catalase (\u003cem\u003eVsCAT\u003c/em\u003e) and chloroplastic copper/zinc superoxide dismutase (\u003cem\u003eVsCu/Zn\u003c/em\u003e-\u003cem\u003eSOD\u003c/em\u003e) (Abu-Romman \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e; \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Whereas the \u003cem\u003eVsAPX\u003c/em\u003e gene has not been identified or characterized, while other studies have identified and characterized the \u003cem\u003eAPX\u003c/em\u003e gene in different legumes (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Therefore, this study aims to characterize \u003cem\u003eAPX\u003c/em\u003e gene from \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e, perform bioinformatic analyses of APX protein and investigate its structure and expression pattern in response to abiotic stresses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant growth and treatments\u003c/h2\u003e\u003cp\u003eCommon vetch (\u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e, 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 x 14 cm, three seeds per pot) in a greenhouse 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\u003csup\u003eο\u003c/sup\u003eC in incubator for 6 h), phytohormone treatment (seedlings were sprayed until dripping with 1 mM salicylic acid (SA), 100 \u0026micro;M abscisic acid (ABA), or 100 \u0026micro;M jasmonic acid (JA) for one time), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (seedlings were sprayed with 10 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for one time). Leaflet tissues were collected after 0, 2, 4, and 6 h of treatment, from each treated seedlings and control (at each time point). The harvested tissue samples were directly dipped in liquid nitrogen and stored at -20\u003csub\u003eο\u003c/sub\u003eC for RNA extraction.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGene cloning\u003c/h3\u003e\n\u003cp\u003eFor gene cloning, total RNA was extracted from one-month old vegetative tissues treated by 12% PEG (6000) by using IQeasy\u003csub\u003eTM\u003c/sub\u003e Plus Plant RNA Extraction Mini Kit (iNtRON Biotechnology, Korea) according to the manufacturer's protocol. A reverse transcription (RT) reaction was performed to synthesize the first-strand cDNA from 2 \u0026micro;g of total RNA by using the SCRIPT cDNA Synthesis Kit (Jena Bioscience, Germany), with oligo (dT)\u003csub\u003e20\u003c/sub\u003e as a primer in a reaction volume of 20 \u0026micro;l. cDNA samples concentration and purity were measured by spectrophotometer at 260 and 280 nm (Biochrom, Cambridge), and stored at -20\u003csub\u003eο\u003c/sub\u003eC.\u003c/p\u003e\u003cp\u003eFor identifying \u003cem\u003eAPX\u003c/em\u003e gene structure, genomic DNA was isolated from vegetative tissues of three-weeks 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\u0026trade; Plant Genomic DNA Miniprep Kit (Sigma, 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), and stored at -20\u003csup\u003eο\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn silico\u003c/em\u003e sequence of \u003cem\u003eVsAPX\u003c/em\u003e was retrieved from \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e RNAseq SRA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/sra\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using \u003cem\u003eAPX\u003c/em\u003e orthologs form related legumes. Thereafter, specific primers spanning the entire cDNA (VsAPX-F: 5\u0026rsquo;-CTCGTGTCACTAGGGTTTATCT-3') and (VsAPX-R: 5\u0026rsquo;-CAAATTAGCTGGGCATTACCAC-3') were designed on Primer-BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and utilized in PCR amplification. The PCR reaction was performed using iNtRON \u003cem\u003ei\u003c/em\u003e-MAX II system (iNtRON, Korea). cDNA PCR amplification was carried out as follows: Initial denaturation at 95\u003csub\u003eο\u003c/sub\u003eC for 5 min, then 35 cycles of: 95\u003csub\u003eο\u003c/sub\u003eC for 30 sec, 56\u003csub\u003eο\u003c/sub\u003eC for 30 sec, and 72\u003csub\u003eο\u003c/sub\u003eC for 1.5 min, and then final extension at 72\u003csub\u003eο\u003c/sub\u003eC for 10 min. In case of using gDNA as a template, the following PCR amplification program was followed: Initial denaturation at 95\u003csub\u003eο\u003c/sub\u003eC for 5 min and then 35 cycles of: 95\u003csub\u003eο\u003c/sub\u003eC for 30 sec, 56\u003csub\u003eο\u003c/sub\u003eC for 30 sec, and 72\u003csub\u003eο\u003c/sub\u003eC for 2.5 min, and then final extension at 72\u003csub\u003eο\u003c/sub\u003eC 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\u0026reg; SV Gel and PCR Clean-Up System (Promega, USA). The purified single specific PCR product was ligated into the pGEM\u003csup\u003e\u0026reg;\u003c/sup\u003e-T Easy Vector (Promega, USA), and then transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e JM109 competent cells (Promega, USA) by the heat shock method. The recombinant plasmid DNA was extracted by using E.Z.N.A. \u003csup\u003eTM\u003c/sup\u003e Plasmid Miniprep Kit I (Omega Bio-tek, Inc. US) according to the manufacturer\u0026rsquo;s instructions. Positive clones were verified using PCR (either using VsAPX-specific primers or universal primers (T7p and SP6)) and sequenced (Macrogen, Seoul, Korea).\u003c/p\u003e\u003cp\u003e\u003cb\u003eVsAPX\u003c/b\u003e \u003cb\u003esequence analysis and gene structure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe result of bidirectional sequencing of cDNA-APX was assembled in a contig by using BioEdit software, and the vector backbone was removed by VecSecreen (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/tools/vecscreen/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/tools/vecscreen/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The open reading frame was detected by using the ORF finder of NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/orffinder/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/orffinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and BLAST with SRA data (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). 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 \u003cem\u003eVsAPX\u003c/em\u003e gene was detected by pairwise alignment with cDNA using the BLAST algorithm, Splign-NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Gene Structure Display Server 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gao-lab.org/\u003c/span\u003e\u003cspan address=\"http://gsds.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene structure was constructed by using BioRender (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biorender.com/\u003c/span\u003e\u003cspan address=\"https://biorender.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBioinformatic analysis\u003c/h3\u003e\n\u003cp\u003eThe full-length nucleotide sequence was translated by ExPASY Translate tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/translate/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/translate/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the physical and chemical parameters of the protein were obtained by using the ProtParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subcellular localization of VsAPX was predicted by TargetP 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/TargetP-2.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/TargetP-2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ProtComp 9.0 online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.softberry.com/cgi-bin/programs/proloc/protcomppl.pl\u003c/span\u003e\u003cspan address=\"http://www.softberry.com/cgi-bin/programs/proloc/protcomppl.pl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and DeepLoc 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/DeepLoc-2.0\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/DeepLoc-2.0\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The conserved protein domains were identified by NCBI Conserved Domain Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and InterPro 91.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Clustal Omega was performed for multiple sequence alignment (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/msa/clustalo/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein 3D structure was predicted using SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). 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.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVsAPX\u003c/b\u003e \u003cb\u003egene expression analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExpression of \u003cem\u003eVsAPX g\u003c/em\u003eene was quantified in \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e 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\u003csub\u003eTM\u003c/sub\u003e (GeneALL, Korea), according to the manufacturer's protocol. One hundred nanograms of total RNA were used to synthesize the first strand of cDNA using \u003cem\u003eEasyScript\u003c/em\u003e\u0026reg; First-Strand cDNA synthesis Super Mix Kit (TransGen Biotech). The relative expression pattern of the \u003cem\u003eVsAPX\u003c/em\u003e 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 \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa Actin\u003c/em\u003e gene was used as an internal control (GenBank accession No. GU946218) and amplified using specific primers (VsActinqF: 5\u0026rsquo;-CAATCCAGGCCGTCTTGTCTC-3\u0026rsquo;; VsActinqR: 5\u0026rsquo;- TCTGTTAAATCACGCCCAGCA-3\u0026rsquo;) that results in 157 bp amplicon (Abu-Romman \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e). The qRT-PCR reaction mix was containing 10 \u0026micro;l of 2x\u003cem\u003eTransScript\u003c/em\u003e\u0026reg; Green qPCR SuperMix, 1 \u0026micro;l of each specific primer (10 \u0026micro;M), 3 \u0026micro;l of cDNA template, and 5 \u0026micro;l of nuclease- free water to make up a total volume of 20 \u0026micro;l. The amplification reaction was carried out using a CFX96 Real-Time PCR Detection System (Bio-Rad, U.S.A) with two steps, initial denaturation at 95\u003csub\u003eο\u003c/sub\u003eC for 10 minutes, followed by 45 cycles of denaturation at 95\u003csub\u003eο\u003c/sub\u003eC for 10 seconds then annealing and extension at 60\u003csub\u003eο\u003c/sub\u003eC for 30 seconds at which florescent was acquired. Following qRT-PCR, to confirm primer stringency melting curve was used 65\u003csub\u003eο\u003c/sub\u003eC to 95\u003csub\u003eο\u003c/sub\u003eC; increment 0.5\u003csub\u003eο\u003c/sub\u003eC each 5 sec at which florescent was acquired. The fold change in transcript level (stresses compared to control) was achieved using the 2\u003csup\u003e\u0026minus;\u0026thinsp;ΔΔC\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e method (Livak and Schmittgen \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) according to the following equation: Fold change in expression\u0026thinsp;=\u0026thinsp;2\u003csup\u003e\u0026minus;\u0026thinsp;ΔΔC\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e, while ΔΔC\u003csub\u003eT\u003c/sub\u003e = ((C\u003csub\u003eT VsAPX\u003c/sub\u003e - C\u003csub\u003eT VsAct\u003c/sub\u003e)\u003csub\u003eTreated\u003c/sub\u003e - (C\u003csub\u003eT VsAPX\u003c/sub\u003e-C\u003csub\u003eT VsAct\u003c/sub\u003e)\u003csub\u003eControl\u003c/sub\u003e). All qRT-PCR experiments were performed with three biological replicates. The gene expression results expressed as fold change, and experiments design as 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 (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.05).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCloning and sequence analysis of\u003c/b\u003e \u003cb\u003eV. sativa APX\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs the GenBank was missing any entry related to \u003cem\u003eAPX\u003c/em\u003e under \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e (NCBI 2024), SRA database was searched for RNAseq data. \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e RNAseq (SRX6435314) was selected and assembled using CLC Genomic Workbench (Version 9.1). Assembly was BLAST-searched using \u003cem\u003eAPX\u003c/em\u003e orthologous sequences with complete cDNA entries of related legumes (\u003cem\u003ePisum sativum\u003c/em\u003e). Based on BLAST results, a pair of gene-specific primers (VsAPX-F and VsAPX-R) were designed to amplify \u003cem\u003eAPX\u003c/em\u003e gene from \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e. The cDNA was generated from total RNA extracted from one-month old \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e seedlings subjected to 12% PEG for 24 h. The amplified PCR product (around 1 kb) was cloned into the pGEM\u003csup\u003e\u0026reg;\u003c/sup\u003e-T Easy Vector then sequenced. The full-length \u003cem\u003eAPX\u003c/em\u003e cDNA was 1010 bp, including 753 bp of ORF flanked by 61 bp of 5\u0026rsquo;-UTR and 196 bp of 3\u0026rsquo;-UTR. The ORF of \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa APX\u003c/em\u003e encodes 250 amino acid residues, with a molecular weight of 27.09862 kDa and a theoretical isoelectric point (pI) of 5.60. The \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa APX\u003c/em\u003e gene was designated as \u003cem\u003eVsAPX1\u003c/em\u003e, and its nucleotide sequence was submitted to GenBank under the accession number OR842549.\u003c/p\u003e\u003cp\u003eThe subcellular localization of VsAPX1 protein was predicted using online bioinformatic tools: TargetP 2.0, ProtComp 9.0, and DeepLoc 2.0 (Supplementary Fig.\u0026nbsp;1). TargetP 2.0 excludes the probability of VsAPX1 to be localized in the mitochondria or chloroplast, while the highest value was recorded for \u0026ldquo;other\u0026rdquo; 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 VsAPX1 protein.\u003c/p\u003e\u003cp\u003eBLASTp search against protein sequence database showed that VsAPX1 sequence shared high similarity to known APX homologs from other plant species (Supplementary Fig.\u0026nbsp;2), including \u003cem\u003eLens culinaris\u003c/em\u003e subsp. culinaris AXI69835.1 (97%), \u003cem\u003ePisum sativum\u003c/em\u003e XP_050898393.1 (98%), \u003cem\u003eMedicago truncatula\u003c/em\u003e XP_003606510.1 (97%), \u003cem\u003eCicer arietinum\u003c/em\u003e XP_004505943.1 (95%), \u003cem\u003eMedicago sativa\u003c/em\u003e AIY27528.1 (94%), \u003cem\u003eTrifolium pratense\u003c/em\u003e PNX98620.1 (96%), \u003cem\u003eGlycine max\u003c/em\u003e NP_001237785.1 (96%), \u003cem\u003eVigna unguiculata\u003c/em\u003e AAB03844.1 (96%), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e NP_001234782.1 (92%), \u003cem\u003eZea mays\u003c/em\u003e NP_001152746.1 (92%), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e BAA03334.1 (91%), and \u003cem\u003eHordeum vulgare\u003c/em\u003e subsp. vulgare CAA06996.1 (89%).\u003c/p\u003e\u003cp\u003eMultiple sequence alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) was performed by using Clustal Omega between VsAPX1 protein and other legumes cytosolic APX homologs: \u003cem\u003ePisum sativum\u003c/em\u003e (PsAPX1, AAA33645.1), \u003cem\u003eLens culinaris\u003c/em\u003e subsp. \u003cem\u003eculinaris\u003c/em\u003e (LcAPX1, AXI69835.1), \u003cem\u003eTrifolium pratense\u003c/em\u003e (TpAPX, PNX98620.1) \u003cem\u003eMedicago sativa\u003c/em\u003e (MsAPX2, AIY27528.1) and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (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 (CDD) and InterPro. InterPro analysis revealed, conserved APX active site (APLILRLAWHSA, 33\u0026ndash;44 aa), proximal heme-ligand motif (DIVALSGGHTI, 155\u0026ndash;165 aa) and other conserved sites including six K\u003csup\u003e+\u003c/sup\u003e 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) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). 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).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe predicted three-dimensional structure of the VsAPX1 protein was built using SWISS-MODEL with homology-based modeling. The search template on SWISS-MODEL found that the highest identity template (98.18%) was the 1apx.1.A cytosolic ascorbate peroxidase crystal structure of recombinant ascorbate peroxidase, with a resolution of X-RAY DIFFRACTION 2.20 \u0026Aring;, which indicates the target sequence was well compatible with the template (Patterson and Poulos, 1995). The structure describes the binding and interaction of the potassium ion and heme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A zoom in view reveals major interacting amino acids with both the heme group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and the potassium ion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo study the phylogeny of VsAPX1 and plant homologs, APX proteins were retrieved from GenBank and aligned by ClustalW. Based on sequence similarity 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eAscorbate peroxidase gene structure (dup: abstract ?)\u003c/h3\u003e\n\u003cp\u003eTo determine \u003cem\u003eVsAPX1\u003c/em\u003e gene structure, \u003cem\u003eAPX\u003c/em\u003e gDNA was amplified by PCR using the same specific primers (VsAPX-F and VsAPX-R). The amplified PCR resulted in around 2.5 kb product, which was cloned into pGEM\u003csup\u003e\u0026reg;\u003c/sup\u003e-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 \u003cem\u003eVsAPX1\u003c/em\u003e 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 \u003cem\u003eVsAPX1\u003c/em\u003e gDNA nucleotide sequence was 2425 bp, was submitted to GenBank under the accession number OR756531 and the comparison in (Supplementary Fig.\u0026nbsp;3) showed that \u003cem\u003eVsAPX1\u003c/em\u003e gene contains 10 exons separated by 9 introns. All introns contain the GT-AG dinucleotide at both ends, and the first intron was localized within the 5\u0026rsquo;UTR closer to the start codon (ATG). Introns of \u003cem\u003eVsAPX1\u003c/em\u003e gene were determined according to the introns position relative to the reading frame of the gene. Intron 1 is located in the 5\u0026rsquo;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).\u003c/p\u003e\u003cp\u003eA comparison between \u003cem\u003eVsAPX1\u003c/em\u003e genomic structure (exon/intron organization) and cytosolic \u003cem\u003eAPX\u003c/em\u003e of other plants was performed to study structural changes; \u003cem\u003eP\u003c/em\u003e. \u003cem\u003esativum APX1\u003c/em\u003e (M93051.1), \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ethaliana APX1a\u003c/em\u003e (D14442.1), \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ethaliana APX1b\u003c/em\u003e (X80036.1), and \u003cem\u003eF\u003c/em\u003e. \u003cem\u003eananassa APX\u003c/em\u003e (AF158654.1) (Supplementary Fig.\u0026nbsp;4). The results revealed that \u003cem\u003eVsAPX1\u003c/em\u003e gene structure is similar to that of \u003cem\u003ePsAPX1\u003c/em\u003e and \u003cem\u003eFaAPX\u003c/em\u003e and has a conserved exon/intron order, including exons with the same length as well as conserved intron phases, while \u003cem\u003eVsAPX1\u003c/em\u003e is different from \u003cem\u003eAtAPX1b\u003c/em\u003e as it lacks the 5\u0026rsquo;UTR intron. In addition, exons 5 and 6 of \u003cem\u003eVsAPX1\u003c/em\u003e appear to have been merged to form exon 5 in \u003cem\u003eAtAPX1a\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression pattern analysis of\u003c/b\u003e \u003cb\u003eVsAPX1\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe differential expression of the \u003cem\u003eVsAPX1\u003c/em\u003e gene was studied in one-month-old \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e 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).\u003c/p\u003e\u003cp\u003eTo examine \u003cem\u003eVsAPX1\u003c/em\u003e transcript level under abiotic stresses, seedlings were exposed to heat stress (42\u003csup\u003eο\u003c/sup\u003eC), the expression of \u003cem\u003eVsAPX1\u003c/em\u003e compared to the control was highly upregulated 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Whereas \u003cem\u003eVsAPX1\u003c/em\u003e transcript was downregulated 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 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eVsAPX1\u003c/em\u003e was stimulated by phytohormone treatments. In response to ABA (100 \u0026micro;M) treatment, transcript level compared to control was increased to reach 2.7-fold after 2 h then decreased to 1.4-fold and 1.3-fold after 4 and 6 h, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). SA (1 mM) treatment resulted in an increase of \u003cem\u003eVsAPX1\u003c/em\u003e expression by about 1.38-fold after 2 h while after 4 h decreased slightly to 1.27-fold, then highly downregulated to 0.65-fold after 6 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). However, the treated seedlings with JA (100 \u0026micro;M), resulted in an increase of \u003cem\u003eVsAPX1\u003c/em\u003e expression to 1.18-fold after 2 h, then peaked to 1.2-fold after 4 h, while decreased to 0.34-fold after 6 h compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eSequence analysis of\u003c/b\u003e \u003cb\u003eVsAPX1\u003c/b\u003e \u003cb\u003egene and protein\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAPXs are important H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenger antioxidant enzymes, which maintain H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e homeostasis under normal and stressful conditions. The cytosolic APX isoenzyme is the most studied among other isoforms, due it is highly responsive and induced under different stresses (Pandey et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Saxena et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as well as its role in different defense processes, and in plant homeostasis redox regulation (Caverzan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Guo et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), proved the role of cytosolic \u003cem\u003eAPX1\u003c/em\u003e in maintaining redox balance to regulate cotton photosynthetic rate and yield. Also, in \u003cem\u003eArabidopsis thaliana APX1\u003c/em\u003e protected chloroplast under high light intensity (Saxena et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, the present study characterized the full-length gDNA and cDNA cytosolic ascorbate peroxidase gene from the \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e plant. The \u003cem\u003eVsAPX1\u003c/em\u003e cDNA sequence contains an in-frame start site (ATG, 62 bp) and stop site (TAG, 812 bp), these signals suggest that the \u003cem\u003eVsAPX1\u003c/em\u003e cDNA is full length. Furthermore, a putative polyadenylation signal site (AAATAA) was detected in the \u003cem\u003eVsAPX1\u003c/em\u003e sequence at a 159 bp downstream stop codon, this signal site was previously reported in other legumes (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Different studies have cloned and characterized cytosolic \u003cem\u003eAPX\u003c/em\u003e cDNA from different plant species such as pea (Mittler and Zilinskas \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), sweet potato (Park et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), potato (Kawakami et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and spinach (Ishikawa et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe ORF of \u003cem\u003eVsAPX1\u003c/em\u003e encodes 250 amino acid residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), with a molecular weight of 27.1 kDa. APX isoenzymes are different in molecular weight for example, in rice chloroplast (thylakoidal APX (~\u0026thinsp;51 kDa), stromal APX (~\u0026thinsp;33\u0026ndash;38 kDa)), peroxisomal APX (~\u0026thinsp;32 kDa), and cytosolic APX (~\u0026thinsp;27 kDa) (Teixeira et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), while in potato mitochondrial APX (~\u0026thinsp;31 kDa) (De Leonardis et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). VsAPX1 amino acid residues lack localization signals; an N-terminal transit peptide sequence and a C-terminal transmembrane domains, according to the protein targeting analysis. This confirms the bioinformatic results that the VsAPX1 protein is cytosolic ascorbate peroxidase.\u003c/p\u003e\u003cp\u003eSequence similarity analysis of VsAPX1 with closely related proteins from different plant species was investigated using BLASTp (Supplementary Fig.\u0026nbsp;2). 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.\u003c/p\u003e\u003cp\u003eAscorbate 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 (Dąbrowska et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Pandey et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ascorbate, heme (iron), and potassium ions are critical for APX activity (Jespersen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). In the present study, the multiple sequence analysis of the VsAPX1 primary structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) 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\u003csup\u003e+\u003c/sup\u003e 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 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The hydrogen bonding between His-163, Asp-208, and Trp-179 form the active site structure (Lazzarotto et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The substrate ascorbate binds to the active site by four hydrogen bonds with Lys-30, Arg-172, Cys-32, and the heme moiety (Pandey et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At the proximal domain, K\u003csup\u003e+\u003c/sup\u003e ions bind to K\u003csup\u003e+\u003c/sup\u003e binding sites (Thr-164, Thr-180, Asp-187) that are required for APX activity (Dąbrowska et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Phosphorylation of Thr-59 and Thr-164 residues was reported to increase tomato APX enzyme activity, and S-nitrosylation of Cys-32 enhance APX enzyme catalytic activity (Ravi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). APX class I is distinguished from other classes by Trp-41 and Trp-179 instead of Phe-41 and Phe-179 (Dąbrowska et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). 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 (Jespersen et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). These data suggest that VsAPX1 belongs to the class I cytosolic ascorbate peroxidase.\u003c/p\u003e\u003cp\u003eSome studies reported a dual function for APX protein under abiotic stresses, which means functional switching; under salinity stress act as peroxidase, and under heat stress tend to be molecular chaperones. The dual function of APX resulted from structural conformation, which leads to the association of oligomer to high molecular weight (molecular chaperone) under heat stress, or dissociation of oligomers to low molecular weight (APX enzyme activity) under salinity stress.\u003c/p\u003e\u003cp\u003eMolecular phylogenetics deals with evolutionary relationship based on different macromolecules (DNA, RNA, and protein) (Bogusz and Whelan \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This study clarifies the phylogenetic relationship of VsAPX1 with APXs orthologs from other plant species, for this purpose a neighbor-joining phylogenetic tree was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The resulting tree showed that APX proteins were clearly separated into three groups based on their subcellular localization: cytosolic, peroxisomal, and chloroplastic, and VsAPX1 protein was clustered within the cytosolic clade which is closely related to the leguminous species \u003cem\u003ePisum sativum\u003c/em\u003e and \u003cem\u003eLens culinaris\u003c/em\u003e, indicating a close relationship between these APX proteins. This result is consistent with the phylogenetic tree generated in the study of Malambane et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This apparent divergence between orthologs, revealed in previous studies indicating that cytosolic and peroxisomal APX isoenzymes were generated by a duplication event of a non-chloroplastic ancestral gene (Teixeira et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). According to Qu et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), results of APX genes phylogenetic tree and exons structure revealed that the ancestors of monocots and dicots underwent genome duplication. While Ozyigit et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) APX phylogenetic tree of 18 plant species showed segmental and tandem duplications in some \u003cem\u003eAPX\u003c/em\u003e genes.\u003c/p\u003e\n\u003ch3\u003eAscorbate peroxidase gene structure\u003c/h3\u003e\n\u003cp\u003eThe identification of the \u003cem\u003eVsAPX1\u003c/em\u003e 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 \u003cem\u003eVsAPX1\u003c/em\u003e, the exon/intron organization was constructed and compared with other plant cytosolic \u003cem\u003eAPX\u003c/em\u003e genes. In the present study, the \u003cem\u003eVsAPX1\u003c/em\u003e gene contains 10 exons enterspaced by 9 introns (Supplementary Fig.\u0026nbsp;3). Comparing \u003cem\u003eVsAPX1\u003c/em\u003e gene structure with other plant species (Supplementary Fig.\u0026nbsp;4), the length of \u003cem\u003eVsAPX1\u003c/em\u003e exons of ORF 9 exons is highly identical to pea \u003cem\u003eAPX1\u003c/em\u003e (which is the only gene available with detailed for \u003cem\u003eAPX\u003c/em\u003e in legumes) (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), and strawberry (Kim and Chung \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Moreover, similar result was found in other plant \u003cem\u003eAPX\u003c/em\u003e genes such as tomato (Gadea et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) rice (Teixeira et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), maize (Liu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Qu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and wild watermelon (Malambane et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Whereas there are differences in intron length, nucleotide sequences, while the intron number, and intron phases are conserved (Patthy \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). This suggests a conserved cytosolic \u003cem\u003eAPX1\u003c/em\u003e gene architecture in higher plants (Teixeira et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Qu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Likewise, \u003cem\u003eAPX\u003c/em\u003e genes promoter \u003cem\u003ecis\u003c/em\u003e-elements, exon-intron organization, and number were studied in \u003cem\u003ePopulus trichocarpa\u003c/em\u003e (Leng et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). On the other hand, \u003cem\u003eArabidopsis thaliana APX1a\u003c/em\u003e, \u003cem\u003eAPX1b\u003c/em\u003e had 9 exons and 8 introns (Santos et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). A major difference appears in exon5 of \u003cem\u003eAtAPX1a\u003c/em\u003e which is separated by 86 bp intron to exon 5 and exon 6 in \u003cem\u003eVsAPX1\u003c/em\u003egene. The first intron was located within 5\u0026rsquo;UTR. Similar observation was made in those of other plant species except for the \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ethaliana APX1b\u003c/em\u003e (Supplementary Fig.\u0026nbsp;4) which lacks the 5\u0026rsquo;UTR intron, the gene encoding a second family of cytosolic \u003cem\u003eAPX\u003c/em\u003e and similar observation in \u003cem\u003eDancy\u0026rsquo; tangerine\u003c/em\u003e (\u003cem\u003eCitrus reticulata Blanco\u003c/em\u003e) (Santos et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Kunta et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Mittler and Zilinskas (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) detected a part of the GPEI enhancer (TGATTCAG) sequence in 5\u0026rsquo;UTR intron, which is a regulator element for glutathione transferase P that regulates gene transcription by the interaction with transcription factors and RNA II polymerase (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Some studies discussed the effect of 5\u0026rsquo;UTR intron, which may interact with other elements located in the promoter that regulate \u003cem\u003eAPX1\u003c/em\u003e gene expression, in fact, the leader intron of \u003cem\u003eAPX20\u003c/em\u003e gene in tomato increased expression of a reporter gene in leaves but it was absent in roots (Gadea et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The gain of an intron in 5\u0026rsquo;-untranslated region and in exon number five of \u003cem\u003eVsAPX1\u003c/em\u003e causes exon fission but did not cause a shift in reading frame unlike the exon loss/gain that can cause a shift in reading frame (Xu et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). 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 chance of intron insertion at this position (Nguyen et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression pattern analysis of\u003c/b\u003e \u003cb\u003eVsAPX1\u003c/b\u003e \u003cb\u003egene in response to stresses\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlants 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 (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)), which induce signal transduction cascades and then regulate gene expression of the stress responsive genes (Nadarajah \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Antioxidant enzymes scavenge the excess ROS and maintain cellular homeostasis from oxidative damage (Rajput et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). APX is one of the crucial enzymes in the AsA-GSH cycle and can scavenge excess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Hasanuzzaman et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Recent studies showed the up-regulation of \u003cem\u003eAPX\u003c/em\u003e gene expression under different abiotic stresses and stress-response chemicals, which could be due to the presence of stress-response \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoter that activate \u003cem\u003eAPX\u003c/em\u003e gene expression (Leng et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These elements include phytohormone-responsive, abiotic stress-responsive, and growth and development-responsive elements. Generally, cAPX 5' regulatory region has a heat shock responsive element and anti-peroxidative element (ARE) that might aid in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging (Pandey et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The present study investigated the expression pattern of \u003cem\u003eAPX\u003c/em\u003e gene in \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e under phytohormone treatments (ABA, SA, and JA) and abiotic stresses (42\u003csup\u003eο\u003c/sup\u003eC and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) using qRT-PCR, to understand and define the possible involvement of the \u003cem\u003eVsAPX1\u003c/em\u003e gene to stresses.\u003c/p\u003e\u003cp\u003eThe increased global temperature is a critical climate-change problem, resulting in an increasing rate of evaporation and dehydration in plants and soil (Hassan et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Heat stress resulted in the deactivation of enzymes, protein misfolding, disturbs cell metabolism, increased the fluidity of membrane lipid, and increased production and accumulation of ROS (e.g., oxidative damage) (Chiang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Recent studies demonstrated that some \u003cem\u003eAPX\u003c/em\u003e genes are heat inducible (Sadder et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Under heat stress, the increasing level of cellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 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 \u003cem\u003eAPX\u003c/em\u003e gene and control its expression (Panchuk et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). This explains the quick response of APX gene to heat stress (Banerjee and Roychoudhury \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). HSE has been determined in the \u003cem\u003eAPX\u003c/em\u003e promoter of pea (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), and strawberry (Kim and Chung \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In the present study, after exposing seedlings to 42\u0026ordm;C, the expression of \u003cem\u003eVsAPX1\u003c/em\u003e scored an early increase after 2 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Other studies found that heat stress increased \u003cem\u003eAPX\u003c/em\u003e gene expression in a variety of plant species, including pea (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), rice (Sato et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), alfalfa (Li et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eArabidopsis thaliana APX2\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003eb), and sweet potato \u003cem\u003eAPX1\u003c/em\u003e (Park et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Moreover, heat stress increased the activity of the cucumber cAPX enzyme (Song et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The overexpression of pea cAPX enhanced heat tolerance in transgenic tomato (Wang et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, the heat tolerance of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was increased by the overexpression of cabbage \u003cem\u003eAPX\u003c/em\u003e gene (Chiang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is an important non-radical ROS, generated from normal cellular metabolism as a harmful by-product that cause damage and inactivation for cellular components (Das and Roychoudhury \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mittler \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At basal levels, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e acts as a regulatory signal for different physiological processes including photosynthesis, photorespiration, stomatal closure, growth, cell cycle, development, and senescence (Hasanuzzaman et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, under stress conditions, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e acts as a signaling transduction molecule, due to being highly stable and diffusible, 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 (Singh et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which involved in the regulation of nuclear gene expression of many transcription factors, and upregulation of antioxidative enzymes (Song et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The current study reports a gradual decline in the \u003cem\u003eVsAPX1\u003c/em\u003e gene expression over time under foliar application of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, this may be due to the high concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moskova et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported that exogenous application of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decreased APX enzyme activity in pea. In contrast to other studies, the exogenous application of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e increased gene expression in sweet potato (Park et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and cucumber cAPX enzyme activity (Song et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In addition, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment was reported to induce \u003cem\u003eAPX\u003c/em\u003e gene expression in cultured soybean cells (Lee et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). This is possibly due to the presence of an Antiperoxidative element (ARE) in the \u003cem\u003eAPX\u003c/em\u003e promoter, which could be involved in the regulation of the \u003cem\u003eAPX\u003c/em\u003e gene and responsible for the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced response (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression Analysis of\u003c/b\u003e \u003cb\u003eVsAPX1\u003c/b\u003e \u003cb\u003eGene in Response to Phytohormones\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlant bioregulators are chemicals found in small quantities that have a large influence on regulating plant growth, development, and yield under normal and stressful conditions (Alhaithloul et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hasanuzzaman et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Whereas, under unfavorable environmental conditions, plant hormones (ABA, JA, SA, and ethylene) play a role in plant adaptation and regulate the balance between plant development and stress responses (Wang et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003ea). Furthermore, the exogenous application of phytohormones at a suitable dose enhanced plant tolerance to abiotic stress conditions (Awan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eABA acts as a stress-responsive hormone that plays a crucial role in plant adaptation (Gong et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 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 (Wani et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). 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 \u003cem\u003eAPX\u003c/em\u003e promoter \u003cem\u003ecis\u003c/em\u003e-elements antioxidant responsive element and ABA responsive element (Saxena et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, the foliar application of ABA markedly increased the \u003cem\u003eVsAPX1\u003c/em\u003e expression after 2 h, this indicates that ABA has a signaling effect on \u003cem\u003eVsAPX1\u003c/em\u003e expression. Similar observations were reported after ABA treatment in pea (Mittler and Zilinskas \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), maize (Liu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and sweet potato (Park et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSA acts as a growth regulating and protector molecule that improves crop plant tolerance under stress conditions (Alhaithloul et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and induces the gene expression of antioxidants, HSPs, chaperones, and genes involved in the biosynthesis of secondary metabolites (Wani et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). 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 (Pourghasemian et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The current study showed a slight rise in the \u003cem\u003eVsAPX1\u003c/em\u003e transcript level after 2 h of foliar SA treatment. Some studies reported that SA application increased endogenous H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation and increased the expression and activity of antioxidant enzymes (Dikilitas et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The SA application increases the transcript level of sweet potato cytosolic ascorbate peroxidase (Park et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and pea APX enzyme activity (Yadu et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eJA 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 (Awan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Whereas, under a stressful environment, JA act as a signaling molecule and stress-responsive hormone (Alisofi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). JA was reported to interact with other plant hormones, transcription factors, and enhanced the expression of JA-associated genes and stress-responsive genes and increased the activity of the antioxidant defense system (Wang et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003ea). \u003cem\u003eVsAPX1\u003c/em\u003e transcript level was highly accumulated after 4 h of JA application. Similar observation was recorded in rice \u003cem\u003eOsAPX1\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and sweet potato (Park et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The foliar application of JA alone or in combination with salinity on bitter melon seedlings decreased the activity of APX enzyme (Alisofi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe identification and characterization of \u003cem\u003eVsAPX1\u003c/em\u003e is the first step to understand function of \u003cem\u003eAPX1\u003c/em\u003e in plants that are related to stress response, plant adaptation, and development under stress condition. In this study we have cloned and characterized the full-length \u003cem\u003eVsAPX1\u003c/em\u003e gene from the forage legume \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e. The bioinformatic analysis confirm that the cloned \u003cem\u003eVsAPX1\u003c/em\u003e gene encode cytosolic APX. Gene expression analysis suggested that \u003cem\u003eVsAPX1\u003c/em\u003e is possibly involved in response to heat and phytohormones treatments.\u003c/p\u003e\u003cp\u003eAccording to the results of this study, to explore the regulatory mechanisms that control \u003cem\u003eVsAPX1\u003c/em\u003e gene expression in response to different stresses and signaling pathways, additional research are recommended to analyze the regulatory \u003cem\u003ecis\u003c/em\u003e-elements in the promoter region of \u003cem\u003eVsAPX1\u003c/em\u003e and studying the transcription factors that involved in its regulation. Furthermore, there is a need for additional studies to investigate the functional role of the first intron on \u003cem\u003eVsAPX1\u003c/em\u003e gene expression in different types of tissues of \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e under different stress conditions. Moreover, knockout or overexpression of \u003cem\u003eAPX\u003c/em\u003e in \u003cem\u003eV\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e is recommended for more clarification of the \u003cem\u003eAPX\u003c/em\u003e functional role on forage plant growth and development, the differential response and tolerance under environmental stresses, as well as the detection of APX protein activity and their efficiency in scavenging H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e under these stress conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor contribution:\u003c/h2\u003e\u003cp\u003eFarah Abu Siam: Conducted plant growth and treatments and gene cloning besides contributing to writing.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e\u003cp\u003eThe authors are grateful for Ahliyya Amman University, Al-Balqa Applied University, and the University of Jordan for their support to conduct the research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbu-Romman S (2016a) Genotypic response to heat stress in durum wheat and the expression of small HSP genes. 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Abiotic stress. Cloning. Gene structure. Gene expression. Vicia sativa.","lastPublishedDoi":"10.21203/rs.3.rs-5468156/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5468156/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAscorbate peroxidase (APX) is a heme containing enzyme that acts as a key scavenging enzyme in the Ascorbate-Glutathione (AsA-GSH) cycle to scavenge hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). \u003cem\u003eAPX\u003c/em\u003e expression in plants can enhance their tolerance to environmental stresses, potentially increasing crop yield. In this study, the full-length genomic DNA and cDNA of the \u003cem\u003eAPX\u003c/em\u003e gene were successfully cloned and characterized from \u003cem\u003eVicia sativa\u003c/em\u003e. The full-length gDNA-\u003cem\u003eAPX\u003c/em\u003e was 2425 bp with 10 exons that is enterspaced by nine introns. The first intron is located within the 5\u0026rsquo;-untranslated region (5\u0026rsquo;UTR). The transcribed cDNA (1010 bp) covers 5\u0026rsquo;UTR (61 bp), 3\u0026rsquo;UTR (196 bp) and open reading frame (ORF) (753 bp). It encodes the cytosolic APX protein (250 amino acids) with a molecular weight of 27.1 kDa and a theoretical isoelectric point (pI) of 5.60. Bioinformatic analysis of the deduced VsAPX1 amino acid sequence displays a high similarity with other plant species cytosolic APX. The main conserved domains were also determined, and the phylogenetic analysis showed that VsAPX1 clustered with the cytosolic APX clade. The expression pattern of \u003cem\u003eVsAPX1\u003c/em\u003e was determined using qRT-PCR in response to different stresses. Compared to the control, the level of \u003cem\u003eVsAPX1\u003c/em\u003e transcript showed an early increase after 2 h in response to heat stress (42\u003csup\u003eo\u003c/sup\u003eC), abscisic acid, and salicylic acid, while being upregulated after 4 h of jasmonic acid treatment. While hydrogen peroxide treatment, caused a downregulation of \u003cem\u003eVsAPX1\u003c/em\u003e expression over time. The overall results suggest that \u003cem\u003eVsAPX1\u003c/em\u003e plays a role major in common vetch response to heat and plant bioregulators.\u003c/p\u003e","manuscriptTitle":"Novel Ascorbate Peroxidase from Common Vetch (Vicia sativa)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 19:18:11","doi":"10.21203/rs.3.rs-5468156/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-07-18T11:52:16+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-17T20:30:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-19T08:31:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Physiologiae Plantarum","date":"2024-11-16T23:18:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-physiologiae-plantarum","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"acpp","sideBox":"Learn more about [Acta Physiologiae Plantarum](http://link.springer.com/journal/11738)","snPcode":"11738","submissionUrl":"https://www.editorialmanager.com/acpp/default2.aspx","title":"Acta Physiologiae Plantarum","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e0c0dd3d-aea4-48b5-9faa-8ddcf5335e79","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-10-31T19:18:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-31 19:18:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5468156","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5468156","identity":"rs-5468156","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}