Genome-wide identification of the HvSCAMP gene family in barley and functional characterization of the role of HvSCAMP1 in salt tolerance - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. 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Learn more: PMC Disclaimer | PMC Copyright Notice BMC Plant Biol . 2026 Mar 10;26:692. doi: 10.1186/s12870-026-08510-0 Search in PMC Search in PubMed View in NLM Catalog Add to search Genome-wide identification of the HvSCAMP gene family in barley and functional characterization of the role of HvSCAMP1 in salt tolerance Zhaoxia Shi Zhaoxia Shi 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China Find articles by Zhaoxia Shi 1, # , Jianbin Zeng Jianbin Zeng 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China Find articles by Jianbin Zeng 1, # , Xinyi Zhang Xinyi Zhang 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China Find articles by Xinyi Zhang 1 , Caiming Zhang Caiming Zhang 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China Find articles by Caiming Zhang 1 , Zhikui Wang Zhikui Wang 2 Jimo District Bureau of Agriculture and Rural Affairs of Qingdao, Qingdao, Shandong 266200 China Find articles by Zhikui Wang 2 , Jian Sun Jian Sun 3 Govement of Chunhua Town, Boxing County of Shandong Province, Binzhou, Shandong 256507 China Find articles by Jian Sun 3 , Huayan Yin Huayan Yin 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China Find articles by Huayan Yin 1 , Ping Mu Ping Mu 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China Find articles by Ping Mu 1, ✉ , Xiaoyan He Xiaoyan He 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China Find articles by Xiaoyan He 1, ✉ Author information Article notes Copyright and License information 1 College of Agronomy, Qingdao Agricultural University, Qingdao, Shandong 266109 China 2 Jimo District Bureau of Agriculture and Rural Affairs of Qingdao, Qingdao, Shandong 266200 China 3 Govement of Chunhua Town, Boxing County of Shandong Province, Binzhou, Shandong 256507 China ✉ Corresponding author. # Contributed equally. Received 2025 Sep 9; Accepted 2026 Mar 2; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13085525 PMID: 41803728 Abstract Background Secretory carrier membrane proteins (SCAMPs) play crucial roles in membrane trafficking and vesicle cycling in eukaryotes; however, their functions in plants remain inadequately characterized. Results Through a genome-wide bioinformatics analysis, we identified seven HvSCAMP genes in barley ( Hordeum vulgare L.). Phylogenetic analysis classified them into three distinct groups, with members within the same group sharing similar motif compositions and gene structures. Promoter analysis revealed that all HvSCAMP members contain methyl jasmonate-responsive elements, while the salicylic acid-responsive elements were exclusively present in HvSCAMP1 of Group 1. Collinearity analysis revealed evolutionary conservation between SCAMP genes in barley, rice, and Arabidopsis . The prediction of upstream regulators indicated that HvSCAMPs are modulated by stress-associated miRNAs and ERF family transcription factors. Expression profiling demonstrated that HvSCAMP1 was significantly induced under salt stress in a salt-tolerant barley genotype. Subcellular localization confirmed that HvSCAMP1 localizes to the plasma membrane. Heterologous expression of HvSCAMP1 enhanced salt stress tolerance in yeast. Furthermore, Arabidopsis lines overexpressing HvSCAMP1 exhibited superior growth under salt stress, which was associated with increased antioxidant enzyme activity, elevated accumulation of osmoprotectants, alleviated oxidative damage, and reduced leaf Na + accumulation. Conclusion This study provides the first systematic identification of the SCAMP gene family in barley and highlights the role of HvSCAMP1 in positively regulating salt tolerance, offering a valuable genetic resource for molecular breeding of stress-resistant barley. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-026-08510-0. Keywords: Barley, Secretory carrier membrane protein (SCAMP), Gene family, Salt tolerance, HvSCAMP1 Introduction Barley ( Hordeum vulgare L.) ranks as the fourth most important cereal crop worldwide and is important in global agricultural production. It not only serves as a major source of animal feed and raw material for malting and brewing but is also directly consumed by humans in certain regions [ 1 ]. Known for its broad adaptability, barley is cultivated across diverse ecological environments, including arid, semiarid, and saline marginal soils [ 2 ]. However, soil salinization has become a severe challenge for sustainable agricultural production worldwide, affecting an estimated 800 million hectares of arable land and posing a substantial threat to crop yield and food security [ 3 , 4 ]. As a relatively salt-tolerant cereal crop, barley represents a valuable genetic resource for studying salt adaptation in grasses. Analyzing its unique salt-tolerant gene repertoire and regulatory networks can provide insights into both conserved and species-specific tolerance mechanisms, thereby supporting the improvement of cereal crops under salt stress [ 5 ]. Therefore, mining salt-tolerant genes in barley and elucidating their molecular mechanisms hold considerable theoretical and practical importance for developing high-yielding, stable varieties suitable for saline environments. Secretory carrier membrane proteins (SCAMPs) are a group of evolutionarily conserved integral membrane proteins, widely distributed in eukaryotes [ 6 , 7 ]. They are characterized by a signature structural domain comprising a central core of four transmembrane segments, known as the SCAMP homology domain [ 6 ]. SCAMPs are predominantly localized to secretory membranes, including the trans-Golgi network, early endosomes, secretory vesicles, and the plasma membrane, positioning them at critical junctions of membrane trafficking pathways [ 7 , 8 ]. This strategic localization implicates SCAMPs in fundamental vesicle-mediated processes such as exocytosis, endocytosis, and membrane recycling. Functionally, they are believed to facilitate the sorting and targeted delivery of vesicular cargo, thereby contributing to essential cellular and physiological processes [ 7 ]. Plant responses to salt stress involve complex physiological and biochemical processes, primarily including osmotic stress, ionic toxicity (particularly Na⁺ accumulation in the cytosol), and subsequent secondary oxidative damage. Among these, the precise regulation of intracellular ion homeostasis represents a central adaptive strategy to mitigate the cytotoxic effects imposed by excessive Na + [ 9 – 11 ]. Two primary mechanisms underpin this regulation: the active extrusion of Na + from the cytosol across the plasma membrane, and its sequestration into the vacuole [ 12 ]. The salt overly sensitive 1 (SOS1) protein, a plasma membrane-localized Na + /H + antiporter, is a key mediator of cytosolic Na + efflux [ 13 , 14 ]. Concurrently, vacuolar Na + /H + antiporters (NHXs) compartmentalize excess Na + into the vacuole, thereby reducing its cytosolic concentration and also contributing to osmotic adjustment [ 15 , 16 ]. In plants, NHX proteins belong to an evolutionarily conserved gene family, phylogenetically related to the intracellular clade of mammalian Na + /H + exchangers (NHEs), and have been consistently shown to enhance salt tolerance when overexpressed [ 17 – 19 ]. Intriguingly, emerging evidence suggests that the membrane trafficking machinery, particularly through SCAMPs, may play a regulatory role in the function of key ion transporters under salt stress. In mammalian systems, specific NHE isoforms such as NHE5 and NHE7 have been shown to physically interact with SCAMP2, indicating that SCAMPs can influence the trafficking, localization, or activity of these transporters [ 20 , 21 ].This regulatory paradigm appears to extend to plants, where the expression of several AtSCAMP genes in Arabidopsis is upregulated under salt stress [ 22 ]. More directly, recent functional studies in soybean have demonstrated that GmSCAMP5 positively regulates ion homeostasis under salinity, with its overexpression altering Na + /H + content and the expression of ion transporter genes like SOS1 and NHX1 [ 23 ]. These findings position SCAMPs as potential regulators or functional partners in salt-tolerance-related ion transport pathways, possibly through the control of transporter vesicular trafficking, membrane stability, or complex assembly. However, the the direct evidence for a physical or functional interaction between plant SCAMPs and SOS1 and NHX1 remains lacking. To date, the SCAMP gene family has been identified and characterised in several plant species, including Arabidopsis [ 24 ] and soybean [ 23 ], and functional studies have been partially conducted in species such as Arabidopsis [ 24 ] and cotton [ 25 ]. However, a systematic genome-wide characterization of the SCAMP family in barley is still lacking: the copy number, phylogenetic classification, and structural features of HvSCAMP genes have not been elucidated. Moreover, the expression dynamics and functional relevance of barley SCAMPs under abiotic stress-especially salinity-remain completely unknown. To address these gaps, this study aimed to (1) identify all members of the SCAMP gene family in barley at the whole-genome level; (2) analyze their phylogenetic relationships, gene structures, promoter cis-acting elements, and expression patterns; and (3) investigate the subcellular localization of a key member, HvSCAMP1, and its role in salt stress tolerance through heterologous expression in yeast and Arabidopsis , thereby evaluating its conserved cellular functions. This study provides the first comprehensive analysis of the SCAMP gene family in barley and reveals the critical role of HvSCAMP1 in salt tolerance, offering a valuable candidate gene for molecular breeding of stress-resistant barley. Materials and methods Materials The reference genomes and annotations of barley ( Hordeum vulgare L.), Arabidopsis ( Arabidopsis thaliana L.), and rice ( Oryza sativa L.) were used for the genome-wide identification of SCAMP gene families. Transcriptomic data for barley were acquired from the Wheat Omics 1.0 database ( http://wheatomics.sdau.edu.cn/ ), including RNA-Seq datasets from the salt-tolerant genotype XZ113 (a Tibetan barley landrace) and salt-sensitive cultivar Gairdner (an Australian modern brewing barley) for expression profiling. The cultivated barley Golden Promise (a model barley originating from England) was employed for gene cloning and subcellular localization experiments. The yeast strain INVSC1, transformed with either the empty vector pYES2-NTB or the recombinant plasmid pYES2-NTB-HvSCAMP1, was utilized for heterologous salt tolerance assays. The model plant Arabidopsis thaliana ecotype Columbia-0 (Col-0) and transgenic Arabidopsis lines overexpressing HvSCAMP1 generated via Agrobacterium -mediated transformation were utilized for salt stress phenotyping. The barley and Arabidopsis seeds used in this study were collected in summer 2023 from the glass greenhouse at Qingdao Agricultural University, Qingdao, Shandong Province, China. Identification of barley SCAMP gene family members The reference genome sequences and annotation files of barley, Arabidopsis and rice were downloaded from Ensembl Plants ( http://plants.ensembl.org ). The Hidden Markov Model (HMM) profile of the SCAMP protein domain (PF04252) was obtained from Pfam ( http://pfam.xfam.org/ ). The hmmsearch command in HMMER 3.0 was used to query the barley proteome database with an E-value threshold of 1e − ⁵ to identify potential SCAMP members. Candidate sequences were further validated using SMART ( http://smart.embl-heidelberg.de/ ) and NCBI CDD ( https://www.ncbi.nlm.nih.gov/cdd/ ). Sequences lacking the core domain or containing incomplete regions were discarded, yielding the final set of HvSCAMP gene family members. The number of amino acids, molecular weight (MW), and theoretical isoelectric point (pI) of the identified HvSCAMPs were predicted using the ExPASy ProtParam tool ( http://web.expasy.org/protparam/ ). Phylogenetic analysis Multiple sequence alignment of the finalized HvSCAMP protein sequences along with SCAMP homologues from Arabidopsis and rice was conducted using ClustalW. A phylogenetic tree was constructed on the basis of the aligned sequences with the neighbour-joining (NJ) method in MEGA 11.0 software, with 1000 bootstrap replicates for assessing node support. The resulting tree was visualized and annotated using iTOL ( https://itol.embl.de/ ). Gene structure and conserved motif analysis The exon–intron structures of HvSCAMPs were analysed with GSDS 2.0 ( http://gsds.cbi.pku.edu.cn/ ) on the basis of the barley genome annotation file (GFF3). Conserved protein motifs were identified using MEME Suite 5.4.1 ( http://meme-suite.org/ ) with the following parameters: maximum number of motifs = 10 and width range = 6–50 amino acids. TBtools was used to integrate and visualize the gene structures and conserved motifs. Analysis of cis-acting elements in the promoter The 2000 bp promoter region upstream of the start codon (ATG) of each HvSCAMP gene was extracted using TBtools. These sequences were submitted to the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) for the prediction of cis-acting elements. The results were compiled and visualized using TBtools. The final figures were typeset and optimized using Adobe Illustrator CS6. Chromosomal localization and collinearity analysis Chromosomal locations, lengths, gene densities, and collinearity relationships of HvSCAMP genes were extracted from the barley genome database on the basis of the GFF3 file using TBtools. Chromosomal distribution and collinearity plots were generated, and genes were named according to their positions. The MCScanX algorithm was used to analyse interspecies collinearity between HvSCAMPs and SCAMP genes from Arabidopsis and rice. The results were visualized with TBtools. The Ka, Ks, and Ka/Ks values were calculated using KaKs_Calculator 2.0 with the Standard Genetic Code. For intraspecific comparisons, Yang–Nielsen (YN) method was applied, while the Model Averaging (MA) method was used for interspecific comparisons. miRNA target gene prediction The open reading frames (ORFs) of HvSCAMPs were submitted to the psRNATarget platform ( https://www.zhaolab.org/psRNATarget/ ), with barley set as the reference species to predict potential target miRNAs. The analysis was performed using Scoring Schema V2 (2017 release), with the number of top targets set to 200 and up to 2 mismatches allowed in the seed region. The miRNA–HvSCAMP regulatory network was constructed and visualized using Cytoscape 3.7.2. Prediction of upstream transcription factors The promoter sequences (2000 bp upstream of ATG) of HvSCAMPs were submitted to PlantTFDB ( http://plantregmap.gao-lab.org/ ), with barley selected as the reference species to predict transcription factors (TFs) potentially regulating HvSCAMP expression. Using the Network tool, the analysis was configured with Organism set to “All”, Prediction Method to “Motif”, and Running Mode to “Target (retrieve IFs)”. The TF–HvSCAMP regulatory network was constructed and visualized using Cytoscape 3.7.2. Expression profiling of HvSCAMPs Transcripts per million (TPM) expression data of HvSCAMPs across various barley tissues (young leaves, roots, stems, epidermises, inflorescences, and seeds) and developmental stages were downloaded from the Wheat Omics 1.0 database ( http://wheatomics.sdau.edu.cn/ ). A tissue-specific expression heatmap was generated on the basis of TPM values using TBtools. To analyze expression changes under salt stress, RNA-Seq data from a previous study were used to compare HvSCAMP transcript levels in the roots and shoots of salt-sensitive (cv. Gairdner) and salt-tolerant (genotype XZ113) barley plants. Genes were considered significantly upregulated if they met the thresholds of |log₂(fold change)| ≥ 1 and a false discovery rate (FDR) < 0.05. For salicylic acid responsiveness assays, leaves of barley (cv. Golden Promise) at the jointing stage were sprayed with 150 µM salicylic acid solution until runoff. Leaf samples were collected at 0 (control), 3, 6, 9, 12, 24, and 48 h post-treatment. Total RNA was extracted, and HvSCAMP1 transcript abundance was quantified by RT-qPCR. To validate tissue-specific expression patterns suggested by the TPM data, three key tissues (roots, stems, leaves) were collected from Golden Promise at the jointing stage. Total RNA was extracted, and the transcript abundance of two representative genes - HvSCAMP1 (low expression) and HvSCAMP4 (high expression) - was measured by RT-qPCR. Cloning of HvSCAMP1 Total RNA was extracted from leaves of the barley cultivar Golden Promise hydroponically grown to the two-leaf-one-heart stage. First-strand cDNA was synthesized using the Evo M-MLV Plus cDNA Synthesis Kit (Accurate Biology, Qingdao, China). The specific primers HvSCAMP1-F and HvSCAMP1-R (Table S1) were designed to amplify the ORF of HvSCAMP1 . The purified PCR product was subsequently cloned and inserted into the pEASY-Blunt cloning vector and transformed into E. coli DH5α competent cells. Positive clones were selected on LB plates supplemented with 100 mg/L kanamycin. After sequencing verification, the intermediate vector pEASY-Blunt-HvSCAMP1 containing the correct HvSCAMP1 ORF was obtained. Subcellular localization of HvSCAMP1 The primers yHvSCAMP1-F and yHvSCAMP1-R (Table S1) were designed to amplify the coding region of HvSCAMP1 (without the stop codon) from pEASY-Blunt-HvSCAMP1. The fragment was subsequently cloned and inserted into the SUPER1300-35 S-GFP vector to construct the SUPER1300-35 S-HvSCAMP1-GFP fusion expression vector. Correct construction and reading frames were confirmed. The fusion vector and a membrane localization marker were cotransformed into barley mesophyll protoplasts via PEG-mediated transformation: plasmid DNA, protoplasts, and 40% PEG4000 solution were mixed in equal volumes, incubated at 22.5 °C for 15–20 min, washed with W5 solution (154 mM NaCl, 125 mM CaCl₂, 2 mM KH₂PO₄, 2 mM MES, pH 5.7) to terminate transformation, and cultured under low light at 23 °C for 16–18 h. GFP and membrane marker fluorescence distributions were observed and recorded using a confocal laser scanning microscope (Olympus, Japan). Validation of salt tolerance via heterologous expression in yeast The primers pHvSCAMP1-F and pHvSCAMP1-R (Table S1) were used to clone the ORF of HvSCAMP1 into the yeast expression vector pYES2-NTB to construct pYES2-NTB-HvSCAMP1. The recombinant vector and empty vector control (pYES2-NTB) were transformed into the yeast strain INVSC1. Positive single colonies were cultured in SG-Ura liquid medium to OD₆₀₀ ≈ 0.6–0.8. The cultures were serially diluted (10⁰, 10 − ⁰.⁵, 10 − ¹, and 10 − ¹.⁵) and spotted onto SG-Ura solid plates containing different concentrations of NaCl (0, 0.5, 1.0, 1.3, 1.5, and 2.0 M). The plates were incubated at 30 °C for 3–4 days, after which colony growth was observed and recorded. Overexpression vector construction and Arabidopsis transformation The primers HvSCAMP1-OE-F and HvSCAMP1-OE-R (Table S1) were used to amplify the full ORF of HvSCAMP1 (with a stop codon) from pEASY-Blunt-HvSCAMP1. The product was subsequently cloned and inserted into the SUPER1300 vector (containing the 35 S promoter) to construct the plant overexpression vector SUPER1300-35 S-HvSCAMP1, and the correct construction was verified. The vector was subsequently transformed into Agrobacterium tumefaciens strain GV3101. Wild-type Arabidopsis (Columbia ecotype, Col-0) was transformed via the floral dip method: positive Agrobacterium clones were cultured, and cells in the logarithmic growth phase were collected and resuspended in infiltration medium to an OD₆₀₀ ≈ 1.0. Inflorescences were immersed in the suspension for 2 h. After infiltration, the plants were kept in the dark at high humidity for 24–48 h before they were returned to normal growth conditions (23 °C, 16 h light/8 h dark). T₁ seeds were harvested, surface-sterilized and sown on 1/2 MS medium supplemented with 25 mg/L hygromycin for selection. Resistant seedlings with well-developed roots were transferred to soil and grown to maturity. T₂ seeds were harvested individually and sown on 1/2 MS medium supplemented with 25 mg/L hygromycin, after which resistance/sensitivity segregation ratios were recorded. Lines showing a 3:1 segregation ratio were selected. Leaves from T₂ plants conforming to the ratio were used for RT‒qPCR analysis with the primers RT-HvSCAMP1-F/R and AtACTIN-F/R (Table S1) to identify lines with high HvSCAMP1 expression. T₃ seeds of these lines were harvested for subsequent phenotypic analysis. Salt tolerance phenotyping of transgenic Arabidopsis Seeds of wild-type (WT) and T₃ homozygous overexpression (OE) Arabidopsis lines were sterilized and sown on 1/2 MS medium, stratified at 4 °C for 3 days, and transferred to a growth chamber (23 °C, 16 h light/8 h dark) for 7 days. Seedlings of uniform size were transplanted into pots containing equal amounts of nutrient soil (with a consistent initial water content). After 15 days of growth, uniform seedlings were subjected to salt treatment. To simulate progressive stress and avoid acute osmotic shock, NaCl was applied in a stepwise gradient: the treatment group was first irrigated with 150 mM NaCl on day 1, followed by 200 mM on day 5, and finally 250 mM on day 10, maintaining this final concentration until the end of the experiment (total treatment duration of 14 days). The control group received normal watering (maintaining the soil water content at 80% ± 5%). The experimental design included three independent biological replicates for each genotype (WT and three OE lines) under each condition. A biological replicate consisted of one pot containing six seedlings derived from an independent sowing. After 14 days of salt treatment, the phenotypes were observed, and leaf samples were collected for physiological indicator measurements. Assay kits for malondialdehyde (MDA), superoxide anion (•O₂⁻), soluble protein, soluble sugar, and proline content, as well as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activities, were purchased from Grace Biotechnology Co., Ltd. (Suzhou, China). The Na + and K + contents in Arabidopsis leaves were quantified by flame photometry [ 26 ] using a flame photometer FP640 (Jingke, Shanghai, China). Data analysis All experimental data are presented as the mean ± standard deviation (SD) of three independent biological replicates ( n = 3). Statistical analysis was performed using IBM SPSS Statistics 22 software. One-way analysis of variance (ANOVA) was conducted, and significant differences among groups were determined by the Waller–Duncan post hoc test ( p < 0.05), with differences indicated by lowercase letters. Results Identification of seven SCAMP genes in the barley genome To comprehensively identify the SCAMP gene family in barley ( Hordeum vulgare L.), a search was performed against the barley protein database using the HMM profile of the SCAMP domain. After redundant sequences were removed and domain integrity was validated using SMART and NCBI-CDD, seven full-length SCAMP genes were obtained. These genes were designated HvSCAMP1 to HvSCAMP7 according to their physical positions on chromosomes 1 H to 7 H (Fig. 1 A; Table S2). Physicochemical analysis revealed that HvSCAMPs range from 263 to 309 amino acids (aa) in length, with a mean of 285 aa; their molecular weights vary between 29.7 and 34.9 kDa, with an average of 32 kDa; and their theoretical isoelectric points (pI) range from 6.39 to 9.26, with an average of 7.8. Notably, 57% of the members have pI values greater than 7, indicating that the family is generally alkaline (Table S2). Fig. 1. Open in a new tab Chromosomal distribution and phylogenetic analysis of HvSCAMPs in barley. A Chromosomal localization of HvSCAMP genes. Chromosome numbers are indicated beside each chromosome. The scale on the left indicates chromosomal length in megabases (Mb). B Phylogenetic tree of SCAMP proteins from barley, rice, and Arabidopsis . The tree was constructed using the neighbour-joining method with bootstrap values based on 1000 replicates. Barley, rice, and Arabidopsis SCAMPs are marked with solid green pentagrams, hollow red pentagrams, and hollow green pentagrams, respectively. The SCAMP family is divided into three subgroups (Groups 1–3), which are highlighted with green solid, blue dashed, and red solid arcs, respectively Phylogenetic analysis classified HvSCAMPs into three clades To elucidate the phylogenetic relationships of HvSCAMPs , multiple sequence alignment was conducted using the seven HvSCAMP barley protein sequences along with six from rice ( Oryza sativa L.) and five from Arabidopsis ( Arabidopsis thaliana L.) (Table S3). A phylogenetic tree was constructed using the neighbour-joining method (Fig. 1 B). The results of the analysis revealed that the seven HvSCAMPs can be classified into three clades (Groups 1–3) on the basis of their subfamily affiliations relative to those of Arabidopsis and rice. Group 1 is the largest, comprising three barley ( HvSCAMP1 , HvSCAMP2 , and HvSCAMP4 ), three rice, and four Arabidopsis members; Group 2 is the smallest, with only one barley ( HvSCAMP5 ) and one rice member; and Group 3 includes three barley ( HvSCAMP3 , HvSCAMP 6, and HvSCAMP7 ), two rice, and one Arabidopsis member. Within Group 1, members further separated into two distinct subclades: one consisting entirely of monocot (barley and rice) SCAMPs and the other entirely of dicot ( Arabidopsis ) SCAMPs (Fig. 1 B), suggesting specific divergence between monocots and dicots during evolution. HvSCAMPs within the same clade share similar motif compositions and gene structures To characterize the sequence features of HvSCAMPs , conserved motifs, domains, and gene structures were analysed in the context of phylogenetic grouping (Fig. 2 A). Using the MEME suite, 10 conserved motifs (motifs 1–10) ranging from 8 to 50 aa in length were identified (Fig. 2 B, E; Table S4). Members within the same clade exhibited highly similar motif compositions, numbers, and arrangements (Fig. 2 B). Specifically, all Group 1 members contained all 10 motifs; Group 2 lacked Motif 10; and two-thirds of Group 3 members lacked Motifs 6, 9, and 10. These results indicate that Group 1 members possess the most complete set of conserved motifs, suggesting potentially more complex or critical functions. All the HvSCAMPs contained the typical SCAMP domain (Fig. 2 C; Table S5), confirming their classification within the SCAMP family. All the genes contained 11 to 12 introns (Fig. 2 D). Genes within the same clade exhibited similar exon‒intron patterns, further supporting the phylogenetic grouping (Fig. 2 A, D). Fig. 2. Open in a new tab Analysis of conserved motifs, gene structure, and promoter cis-acting elements of HvSCAMPs in barley. A Phylogenetic tree of HvSCAMPs. B Distribution of conserved protein motifs in HvSCAMPs. C SCAMP domains of HvSCAMPs. D Exon‒intron structures of HvSCAMPs . E Sequence logos of the ten identified conserved motifs. F Schematic diagram of cis-acting elements in the promoters of HvSCAMPs Cis-acting elements in HvSCAMP promoters exhibit divergent profiles To investigate the potential roles of HvSCAMPs in stress response and signalling, a 2000 bp region upstream of the start codon (ATG) for each gene was analysed for cis-acting elements using PlantCARE. The composition and number of these elements varied among members (Table S6). Based on descriptive observation, almost all the HvSCAMP promoters contained ABA- and MeJA-responsive elements, suggesting possible involvement in the ABA and JA signalling pathways. Notably, compared with Groups 2 and 3, Group 1 members contained more MeJA-responsive elements. Stress-responsive elements were present in all the promoters except those of HvSCAMP6 and HvSCAMP7 in Group 3. Importantly, Group 1 promoters contained a greater number of cis-acting elements overall, and a salicylic acid-responsive element was identified only in the promoter of HvSCAMP1 in Group 1 (Fig. 2 F). To functionally validate the salicylic acid responsiveness suggested by this cis-element, exogenous salicylic acid was applied to barley leaves. HvSCAMP1 expression was strongly induced by salicylic acid and remained elevated from 9 to 24 h after treatment (Fig. S1), confirming that HvSCAMP1 is transcriptionally responsive to salicylic acid signaling. Segment duplication contributes to the expansion of the HvSCAMP gene family To understand the expansion mechanism of the HvSCAMP family, collinear relationships among the seven genes were analysed (Fig. 3 A; Table S7). The analysis revealed two segmentally duplicated gene pairs ( HvSCAMP1 / HvSCAMP4 and HvSCAMP3 / HvSCAMP6 ), each located on different chromosomes, indicating that segmental duplication contributed to the expansion of the HvSCAMP gene family. Notably, both duplication events occurred within the same group: HvSCAMP1 / HvSCAMP4 within Group 1 and HvSCAMP3 / HvSCAMP6 within Group 3, highlighting their role in expansion. Further selection pressure analysis (Ka/Ks) (Table S7) revealed that all Ka/Ks ratios were less than 1, indicating strong purifying selection during evolution and relative functional conservation. Fig. 3. Open in a new tab Intra- and interspecies collinearity analysis of HvSCAMPs . A Intraspecies synteny of HvSCAMPs in barley. The grey lines indicate all collinear gene pairs in the barley genome, whereas the coloured lines highlight collinear HvSCAMP gene pairs. Chromosome numbers are labelled adjacent to each chromosome. The colour scale represents gene density, with increasing red intensity indicating increased gene density. B Interspecies synteny of SCAMP genes among Arabidopsis , rice, and barley. The grey lines represent homologous gene pairs between barley–rice and barley– Arabidopsis , and the blue lines indicate homologous SCAMP gene pairs Collinearity analysis reveals higher homology between barley and rice SCAMP genes Collinearity analysis among barley, rice, and Arabidopsis was performed to assess homology (Fig. 3 B; Table S8). Nine homologous gene pairs were identified between barley and rice, whereas only three were identified between barley and Arabidopsis , suggesting increased evolutionary conservation of SCAMP genes between the two monocots. Certain barley genes showed cross-species conservation: HvSCAMP2 was homologous to rice OsSCAMP1 and Arabidopsis AtSCAMP4 ; similarly, HvSCAMP4 was homologous to rice OsSCAMP2 and Arabidopsis AtSCAMP4 (Table S8), highlighting the high function and sequence conservation of HvSCAMP2 and HvSCAMP4 between monocots and dicots. Further evolutionary analysis revealed that all interspecies Ka/Ks values were below 1 (ranging from 0.047 to 0.154), consistent with strong purifying selection acting on SCAMP genes in all three species (Table S8). Together, these findings support a high degree of functional conservation within the SCAMP gene family across divergent plant lineages. HvSCAMPs are putatively regulated by miRNAs and transcription factors, including ERFs To explore potential miRNA-HvSCAMP regulatory networks, five candidate miRNAs targeting the seven HvSCAMPs were predicted (Table S9). Three main regulatory patterns were revealed (Fig. 4 A): a single miRNA targeting a single gene (e.g., hvu-miR6177 targeting HvSCAMP5 ; hvu-miR6184 targeting HvSCAMP6 ; hvu-miR5049f targeting HvSCAMP3 ; hvu-miR6180 targeting HvSCAMP7 ); a single miRNA targeting multiple genes (e.g., hvu-miR6192 targeting HvSCAMP1 , HvSCAMP3 , and HvSCAMP 7); and multiple miRNAs cotargeting a single gene (e.g., hvu-miR6192 and hvu-miR5049f cotargeting HvSCAMP3 ; hvu-miR6192 and hvu-miR6180 cotargeting HvSCAMP7 ). Fig. 4. Open in a new tab Prediction of upstream regulators of HvSCAMPs . A Interaction network between miRNAs and HvSCAMPs . B Predicted transcription factors targeting HvSCAMPs . The size and color intensity of the circles represent the number of regulatory interactions, with larger size and darker red indicating a greater number Analysis of transcription factor (TF) binding sites in the promoters revealed 11 TF families (AP2, bHLH, C2H2, CPP, Dof, E2F, ERF, GATA, LBD, MYB, and Trihelix), involving 44 binding types (Table S10). Notably, the ERF family emerged as the predominant upstream regulator, with 27 binding types predicted to target six HvSCAMPs ( HvSCAMP1 to HvSCAMP5 , and HvSCAMP7 ) (Fig. 4 B; Table S10). These results point to a potential regulatory relationship between ERF transcription factors and the HvSCAMP family, suggesting that ERFs may mediate the expression of HvSCAMPs during barley development and stress responses. HvSCAMPs exhibit tissue-specific expression and differential salt stress responses The tissue-specific expression profiles of HvSCAMPs across various tissues and developmental stages were analysed using data from the WheatOmics 1.0 database (Fig. 5 ). The genes showed significant expression variation in the roots, stems, leaves, flowers, epidermis, and seeds at different stages. The expression levels of HvSCAMP2 , HvSCAMP3 , HvSCAMP4 , and HvSCAMP6 were generally higher than those of the other members (Fig. 5 A; Table S11), indicating broad basal expression. Fig. 5. Open in a new tab Tissue-specific and salt stress-responsive expression patterns of HvSCAMPs . A Heatmap of HvSCAMP expression across different tissues. A larger circle size and deeper red colour indicate higher expression levels. B Heatmap of HvSCAMP expression in response to salt stress in salt-tolerant (XZ113) and salt-sensitive (Gairdner) barley. An increasing red colour intensity denotes higher expression levels Experimental validation of tissue‑specific expression profiles confirmed that the database‑derived TPM trends were reliable. RT‑qPCR analysis of roots, stems, and leaves from barley (cv. Golden Promise) showed that HvSCAMP4 was highly expressed in roots, stems and leaves, while HvSCAMP1 maintained low basal levels across all tested tissues (Fig. S2). These results corroborate the public database profiles and support their use in assessing tissue‑preferential expression of HvSCAMP genes. Under salt stress, compared with the other members, HvSCAMP1 and HvSCAMP6 were significantly upregulated (|log₂(fold change)| ≥ 1). However, the responses were genotype specific: HvSCAMP1 was upregulated only in the shoots of the salt-tolerant genotype XZ113, whereas HvSCAMP6 was upregulated in the salt-sensitive cultivar Gairdner (Fig. 5 B; Table S12). Based on this distinct expression pattern-along with its complete set of conserved motifs, membership in the cis -element-enriched Group 1, and the unique salicylic acid-responsive element in its promoter- HvSCAMP1 was selected for subsequent functional validation. HvSCAMP1 is localized to the plasma membrane and enhances salt tolerance in yeast The ORF of HvSCAMP1 was subsequently cloned from the cDNA of the barley cultivar Golden Promise. Agarose gel electrophoresis revealed a single, specific band (Fig. S3A; Supplementary File 1). Sequencing confirmed an 840 bp ORF encoding a 280-aa protein (Fig. S3B). For subcellular localization, an HvSCAMP1-GFP fusion construct was transfected into barley protoplasts. Confocal microscopy revealed that the GFP fluorescence of HvSCAMP1 strongly overlapped with the YFP fluorescence of a plasma membrane marker (Fig. 6 A), confirming the plasma membrane localization of HvSCAMP1. Fig. 6. Open in a new tab Subcellular localization of HvSCAMP1 and functional validation of its salt tolerance in yeast. A Subcellular localization of HvSCAMP1 in barley mesophyll protoplasts. GFP: green fluorescent signal; CM marker: plasma membrane marker; Chlorophyll: chlorophyll autofluorescence indicating chloroplast positions; Bright field: bright field image; Merged: merged fluorescence and bright field images. B Heterologous expression of HvSCAMP1 enhances salt tolerance in yeast. The grey arrows indicate serial dilutions (10⁰, 10⁻⁰.⁵, 10⁻¹, and 10⁻¹.⁵) of the yeast cultures To assess function under salt stress, the ORF of HvSCAMP1 was cloned and inserted into the yeast expression vector pYES2-NTB, yielding pYES2-HvSCAMP1, which was subsequently transformed into yeast cells. Under nonstress conditions (SG-Ura + 0 M NaCl), the growth of HvSCAMP1 -expressing and empty vector (pYES2-NTB) cells was similar. On NaCl-containing plates, however, the salt tolerance of HvSCAMP1 -expressing yeast was much better than the empty vector-expressing cells. Notably, at 1.3 M NaCl, the growth of the HvSCAMP1 -expressing yeast was still high, whereas the growth of the empty vector-expressing cells was strongly inhibited (Fig. 6 B, S4). These results provide evidence that that heterologous expression of HvSCAMP1 enhances salt-stress tolerance in yeast, although the experimental conditions do not directly mirror those in plant cells. Overexpression of HvSCAMP1 enhances salt tolerance in Arabidopsis To further validate the salt tolerance function of HvSCAMP1 , the gene was transformed into Arabidopsis via the Agrobacterium-mediated floral dip method. Through RT‒qPCR analysis of the expression levels in positive transgenic lines, three homozygous T 3 generation lines exhibiting significantly elevated HvSCAMP1 expression were identified: OE1 (4 − 1), OE2 (6 − 1), and OE3 (7 − 3) (Fig. S5; Table S13). These lines were subsequently utilized for salt tolerance phenotype analysis. It should be noted that because Arabidopsis lacks an endogenous homolog of HvSCAMP1 , the transcript accumulation of the transgene in OE lines showed an extraordinarily high relative fold-increase compared with WT. Under normal growth conditions after 14 days of cultivation, no discernible difference in growth was detected between the wild-type (WT) and overexpression (OE) lines. However, after 14 days of exposure to salt stress, the WT plants exhibited severe leaf wilting and growth inhibition, whereas the OE lines maintained relatively normal growth (Fig. 7 A), visually demonstrating the enhanced salt tolerance conferred by HvSCAMP1 . Fig. 7. Open in a new tab Validation of salt tolerance in HvSCAMP1 -overexpressing Arabidopsis lines. A Growth phenotypes of the wild-type (WT) and overexpression lines (OE1, OE2 and OE3) under salt treatment. B–M Physiological parameters of the WT and OE lines after salt treatment: ( B ) MDA content; ( C ) O₂⁻ content; ( D ) SOD activity; ( E ) POD activity; ( F ) CAT activity; ( G ) APX activity; ( H ) proline content; ( I ) soluble sugar content; ( J ) soluble protein content. ( K ) Na + content; ( L ) K + content; ( M ) Na + /K + ratio. The values are presented as the means ± SDs of three biological replicates. Different lowercase letters indicate significant differences ( P < 0.05) To elucidate the mechanism underlying salt tolerance, key physiological parameters were compared between the OE lines and WT plants under salt stress. Oxidative damage was significantly attenuated in the OE lines: the malondialdehyde (MDA) contents of OE1, OE2, and OE3 were 73%, 44%, and 74% those of the WT, respectively (Fig. 7 B), while the superoxide anion (•O₂⁻) levels were 77%, 60%, and 92% those of the WT, respectively (Fig. 7 C). Concurrently, the antioxidant enzyme system was synergistically enhanced. The superoxide dismutase (SOD) activity increased by 47%, 30%, and 38% (Fig. 7 D); peroxidase (POD) activity increased by 167%, 182%, and 188% (Fig. 7 E); catalase (CAT) activity increased by 26%, 60%, and 59% (Fig. 7 F); and ascorbate peroxidase (APX) activity increased by 19%, 17%, and 32% (Fig. 7 G) in OE1, OE2, and OE3, respectively. Osmoregulatory capacity was also markedly improved. The proline content reached 1.2-, 1.5-, and 1.3-fold that of the WT (Fig. 7 H); the soluble sugar content increased to 1.7-, 2.1-, and 1.4-fold (Fig. 7 I); and the soluble protein content increased to 1.3-, 1.6-, and 1.5-fold (Fig. 7 J) in the three OE lines, respectively. Under salt stress, OE lines accumulated significantly less Na + (Fig. 7 K) but maintained higher K + content (Fig. 7 L) compared to WT, resulting in a substantially lower Na + /K + ratio (Fig. 7 M). In summary, the enhanced salt tolerance in HvSCAMP1- overexpressing Arabidopsis is associated with coordinated physiological improvements: increased activity of antioxidant enzymes to scavenge reactive oxygen species and reduce membrane lipid peroxidation, elevated accumulation of osmolytes to sustain cellular osmotic homeostasis, and critically, improved ion homeostasis due to reduced Na + accumulation and maintained K + levels (Fig. 8 ). Fig. 8. Open in a new tab Proposed model of the HvSCAMP1 -mediated salt tolerance mechanism in Arabidopsis Discussion Salt stress is among the key abiotic constraints limiting crop growth, yield, and quality [ 27 ]. Its detrimental effects primarily include ionic toxicity, osmotic imbalance, and oxidative stress [ 9 – 11 ]. Therefore, elucidating the molecular mechanisms by which plants maintain ion homeostasis and mitigate oxidative and osmotic stress under salt stress is highly important for improving crop salt tolerance. Secretory carrier membrane proteins (SCAMPs) are a class of membrane proteins that are conserved in eukaryotes and are involved in critical processes such as ion efflux, endocytic trafficking, and vesicle budding and fusion [ 6 , 7 ]. To date, the SCAMP gene family has been identified and characterized in various plant species, including Arabidopsis [ 24 ], soybean [ 23 ], cotton [ 25 ], and rubber tree [ 28 ]. However, systematic studies on SCAMP genes in barley ( Hordeum vulgare L.) remain scarce. In this study, we identified seven HvSCAMP genes from the barley genome (Table S2) and selected HvSCAMP1 , whose expression is significantly induced by salt stress, for further functional validation of its role in salt tolerance. Our findings provide an important foundation for in-depth analysis of the biological functions of barley SCAMP genes and their regulatory mechanisms in response to salt stress. Potential roles of HvSCAMPs in growth, development, and stress resistance In higher plants, gene promoters are key regulatory regions that control gene expression. The cis-acting elements within promoters bind specific transcription factors, thereby precisely regulating spatiotemporal expression patterns and responses to environmental signals [ 29 – 31 ]. Thus, analysis of promoter cis-elements helps elucidate gene function and regulatory mechanisms. In this study, we systematically analysed the promoter cis-elements of the HvSCAMP gene family. The results revealed that the promoter regions of these genes widely contain various hormone-responsive elements, including those for ABA, MeJA, auxin, and GA, as well as MYB binding sites and stress-responsive elements. These elements are known to mediate key stress-adaptive and developmental responses. For example, abscisic acid (ABA)-related transcription factors increase drought and salt tolerance by binding ABA-responsive elements in the promoters of downstream target genes such as rd29A and rd29B [ 32 ]. Methyl jasmonate (MeJA) alleviates salt, drought, heavy metal, and cold stress by inducing the expression of antioxidant enzymes, osmolytes, and secondary metabolites through the JA signalling pathway [ 33 ]. MeJA also enhances salt and drought tolerance via the COI1–JAZ–MYC2 pathway, where it releases JAZ-mediated repression of MYC2 transcription factors, thereby activating downstream targets such as P5CS and DREB1C [ 34 ]. ARF transcription factors improve salt tolerance by binding auxin-responsive elements and regulating downstream genes, including SIARF4 , SIARF8 , and SIARF10a [ 35 ]. Gibberellins (GAs) promote plant growth by inducing ubiquitin-mediated degradation of DELLA proteins, which relieves their repression of GA-responsive genes [ 36 ]. Additionally, MYB-type transcription factors (e.g., barley HvMYB1) increase drought resistance by binding MYB-responsive elements and regulating downstream genes such as HvDHN6 , HvDREB1 , HvABF2 , and HvABI5 [ 37 ]. Notably, the number of cis-elements in Group 1 members was greater than that in Group 2 and Group 3 (Fig. 2 F; Table S6). Salicylic acid-responsive elements were detected exclusively in the promoter of HvSCAMP1 in Group 1 (Fig. 2 F; Table S6). Consistent with this in silico prediction, exogenous salicylic acid treatment strongly induced HvSCAMP1 expression (Fig. S1), confirming its specific link to salicylic acid signaling and suggesting a specialized role in stress-related membrane trafficking. This divergent regulatory landscape indicates potential subfunctionalization, with Group 1 genes likely under more complex control and involved in broader physiological responses. HvSCAMPs are predicted to be targeted by miRNAs Beyond transcriptional control, post-transcriptional regulation by microRNAs (miRNAs) adds another layer of complexity to gene expression modulation [ 38 ], critically influencing growth, development, metabolic regulation, and stress responses [ 39 ]. Through predictive analysis of miRNA targets within the barley HvSCAMP family, we identified multiple regulatory relationships between miRNAs and HvSCAMPs , including one-to-one, one-to-many, and many-to-one modes (Fig. 4 A; Table S9). Specifically, hvu-miR6177 targets HvSCAMP5 . This miRNA is a low-copy sequence [ 40 ], located on the outer stem region of a hairpin structure [ 41 ], and is conserved in barley [ 42 ]. It is speculated that hvu-miR6177 may constitutively target HvSCAMP5 to maintain cellular homeostasis. hvu-miR6184 targets HvSCAMP6 and is known to regulate the stress-related genes HvPLATZ2 , HvPLATZ10 [ 43 ], and HvleckRLK96 [ 44 ]. With demonstrated DNA-binding transcription factor activity [ 45 ], hvu-miR6184 is suggested to participate in complex regulatory networks. hvu-miR5049f targets HvSCAMP3 and has been implicated in the response to powdery mildew [ 46 ] and drought [ 47 ]. Its sequence conservation in barley [ 42 ] suggests that HvSCAMP3 may be regulated by this miRNA in stress adaptation. hvu-miR6180 targets HvSCAMP7 and influences plant growth and development via the regulation of growth-regulating factors (GRFs) [ 48 ] and hormone-related targets [ 45 ], indicating a potential role for HvSCAMP7 in hormone signalling and developmental processes under the regulation of hvu-miR6180. Additionally, hvu-miR6192 simultaneously targets HvSCAMP1 , HvSCAMP3 , and HvSCAMP7 . This miRNA is upregulated under drought stress [ 49 ], suggesting its role in coordinating the response to adverse conditions through multigene regulation. Furthermore, instances of multiple miRNAs regulating the same HvSCAMP gene were observed: HvSCAMP3 is coregulated by hvu-miR6192 and hvu-miR5049f, and HvSCAMP7 is cotargeted by hvu-miR6192 and hvu-miR6180. These findings imply that these HvSCAMP genes may integrate signals from various miRNAs to participate in multiple biological processes. The results of the predictive analysis of regulatory interactions between HvSCAMPs and miRNAs presented in this study lay a theoretical foundation for further mechanistic investigations. ERF transcription factors as key upstream regulators of HvSCAMPs Transcription factors (TFs) specifically recognize and bind to cis-acting elements in the promoter regions of target genes, thereby positively or negatively regulating gene expression at the transcriptional level [ 50 ]. Ethylene response factors (ERFs), a major subfamily of the AP2/ERF superfamily, are widely involved in the regulation of various biological processes in plants, including growth and development, hormone signalling, and responses to abiotic stresses [ 51 – 53 ]. Previous studies have demonstrated that ERF TFs play pivotal roles in plant adaptations to multiple stresses, such as heavy metal toxicity [ 54 ], hypoxia [ 55 ], drought [ 56 ], low temperature [ 57 ], and salinity [ 58 ], while also participating in normal developmental processes [ 59 ]. For instance, the ERF transcription factor TaERF87 enhances drought resistance by binding to the promoter of its target gene TaABF2 [ 60 ]. In barley, the ERF transcription factor HvRAF improves salt tolerance through binding to the promoters of target genes such as PDF1.2 , KIN2 , and GSH1 [ 61 ]. In this study, a systematic analysis of the promoter regions of the seven HvSCAMP genes revealed 44 potential binding sites for TFs from 11 families. Among these, binding sites for the ERF family were the most frequently observed (Fig. 4 B; Table S10), suggesting that ERF TFs may regulate the expression of six HvSCAMP genes. On the basis of these findings, we propose that ERF transcription factors likely interact with specific cis-elements in HvSCAMP promoters via their conserved AP2/ERF domains, thereby modulating plant development and stress responses. Future work will focus on experimentally validating the interactions between ERF transcription factors and HvSCAMP promoters to further elucidate the precise molecular mechanisms governing HvSCAMP transcriptional regulation. HvSCAMP1 is associated with enhanced salt-stress tolerance The functional relevance of HvSCAMP1 in salt tolerance is supported by convergent evidence from expression profiling, heterologous systems, and transgenic plants. HvSCAMP1 exhibited a low basal expression but was significantly upregulated specifically in the salt-tolerant genotype XZ113 under salt stress (Fig. 5 ; Tables S11, S12). This genotype-specific induction pattern correlates with enhanced stress resilience and suggests that HvSCAMP1 is a candidate tolerance gene. Functional validation confirmed this role: heterologous expression of HvSCAMP1 improved yeast survival under salt stress (Fig. 6 B, S5), and its overexpression in Arabidopsis led to superior growth (Fig. 7 ). These results provide direct evidence for the role of HvSCAMP1 in conferring salt tolerance. In line with previous findings showing that silencing GhSCAMP2 and GhSCAMP4 compromises antioxidant capacity and salt tolerance in cotton [ 25 ]. Our study demonstrated that overexpression of HvSCAMP1 significantly reduced the accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS) under salt stress (Fig. 7 B-C). It also increased the activities of antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and promoted the synthesis of osmolytes, including proline, soluble sugars, and soluble proteins (Fig. 7 D-J). Since salt stress disrupts intracellular ROS homeostasis, leading to oxidative damage that simultaneously activates antioxidant defenses [ 62 ], and induces the accumulation of osmolytes to maintain cellular osmotic balance [ 63 ]. Our findings suggest that HvSCAMP1 may increase salt tolerance by synergistically increasing antioxidant enzyme activity and facilitating osmolyte biosynthesis, thereby alleviating ROS-induced oxidative damage and preserving membrane integrity. In addition to mitigating oxidative and osmotic stress, HvSCAMP1 likely influences the response of ion homeostasis. Plant SCAMP proteins, as key components of membrane trafficking, are primarily localized to membranes associated with the secretory and endocytic pathways, and may act as potential regulators or interactors of Na + /H + transporters such as SOS1 and NHX in ion homeostasis [ 6 , 7 ]. Consistent with reports of membrane localization in other species- including rice (OsSCAMP1), tobacco (NtSCAMP2), rubber tree (HbSCAMP3), soybean (GmSCAMP4), and cotton (GhSCAMP2 and GhSCAMP4) [ 8 , 25 , 28 , 64 – 67 ]-we confirmed that HvSCAMP1 is also mainly localized to the plasma membrane (Fig. 6 A). Notably, functional studies have shown that overexpression of GmSCAMP5 enhances salt tolerance in soybean by upregulating SOS1 and NHX1 expression and reducing Na + content [ 23 ], Similarly, in our study, HvSCAMP1 overexpression improved salt tolerance, which was associated with a significant reduction in leaf Na + accumulation, maintenance of higher K + levels, and a consequently lower Na + /K + ratio (Fig. 7 K-M). These findings further support the hypothesis that HvSCAMP1 may modulate salt stress responses by participating in plasma membrane-associated vesicle trafficking or by regulating the function of ion transporters. This study establishes HvSCAMP1 as a promising candidate gene associated for improving salt-stress resilience in barley. Integrating the observed improvements in ion balance, oxidative stress markers, and osmolyte accumulation, we propose a multi-layered working model for its function (Fig. 8 ). Building on this framework, future work should aim to validate HvSCAMP1 function in a barley genetic background, elucidate its mechanistic role-especially its potential interaction with ion transporters-and evaluate the performance of HvSCAMP1 -overexpressing lines under field-relevant saline conditions to assess its potential contribution to crop improvement strategies. Conclusion In this study, we conducted the first genome-wide identification of the SCAMP gene family in barley, revealing seven HvSCAMP members. Comprehensive analyses characterized their phylogenetic relationships, gene structures, cis-regulatory elements, collinearity, and expression patterns. HvSCAMP1 , a key member predominantly localized to the plasma membrane, was significantly induced by salt stress in salt-tolerant barley genotypes. Functional validation through heterologous expression in yeast and overexpression in Arabidopsis provided evidence that HvSCAMP1 substantially enhances salt tolerance. Physiological analyses indicated that HvSCAMP1 coordinates the upregulation of antioxidant enzyme activity, facilitates osmoprotectants accumulation, reduces membrane lipid peroxidation, and improves ion homeostasis under salt stress. This study not only expands the functional understanding of SCAMP genes in plants but also provides a valuable candidate gene and theoretical foundation for the genetic improvement of salt tolerance in barley. Future efforts should focus on barley genetic transformation and field-based evaluation to further assess the agronomic potential of HvSCAMP1. Supplementary Information 12870_2026_8510_MOESM1_ESM.xlsx (58.5KB, xlsx) Supplementary Material 1. Table S1 Primer information used in this study. Table S2 Characteristics of the SCAMP genes in Hordeum vulgare L. Table S3 The SCAMP genes in Arabidopsis thaliana L. and Oryza sativa L. Table S4 Conserved amino acid motifs of the SCAMP genes in Hordeum vulgare L. Table S5 The sequences of seven SCAMP genes identified in Hordeum vulgare L. Table S6 Promotion element analysis of HvSCAMP genes. Table S7 Syntenic blocks and Ka/Ks values of the SCAMP genes in Hordeum vulgare L. Table S8 One-to-one orthologous relationships and Ka/Ks values of the SCAMP genes between Hordeum vulgare L. and Arabidopsis thaliana L., and between Hordeum vulgare L. and Oryza sativa L. Table S9 miRNA and miRNA-acted HvSCAMPs. Table S10 Transcription factor prediction of HvSCAMP genes. Table S11 The expression profiles [log2(TPM+1)] of SCAMP genes in different tissues of Hordeum vulgare L. Table S12 Fold change in the expression of HvSCAMPs under salt-treated versus control (T/CK) conditions in salt-tolerant (XZ113) and salt-sensitive (Gairdner) barley. Table S13 Raw Ct values for HvSCAMP1 from quantitative RT-qPCR assays in wild-type (WT) and overexpression (OE) lines. Supplementary Material 2. (96.9KB, pdf) Supplementary Material 3. (1.2MB, docx) Acknowledgements We are deeply grateful to the editor and reviewers for their comments and suggestions on the manuscript. Research involving plants All experimental research and field studies on plants conducted in this work adhere to Chinese institutional, national, and international guidelines and legislation. The cultivation and management of experimental materials were carried out with permission from Qingdao Agricultural University (Qingdao, Shandong Province, China). Authors’ contributions X.H. and J.Z. conceived and designed the experiments. Z.S. performed the experiments and analysed the data. X.Z. and C.Z. harvested the Arabidopsis plants to obtain transgenic seeds. X.H., Z.S. and J.Z. wrote and revised the manuscript. Z.W., J. Sun., H.Y. and P.M. revised the manuscript. All the authors read and approved the final manuscript. Funding This work was supported by the Shandong Provincial Key Research and Development Plan (Agricultural Seed Improvement Project) (2023LZGC009, 2023LZGCQY011), the Special Projects of the Central Government Guiding Local Science and Technology Development (YDZX2021078), and the Wheat Innovation Team of Modern Agricultural Production Systems in Shandong Province (SDAIT01-06). Data availability Data is provided within the manuscript or supplementary information files. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Zhaoxia Shi and Jianbin Zeng contributed equally to this work. Contributor Information Ping Mu, Email: [email protected]. Xiaoyan He, Email: [email protected]. References 1. 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Overexpression of GmSCAMP4 enhances the tolerance of soybeans to low phosphorus stress. Plant Sci. 2025;359:112680. [ DOI ] [ PubMed ] [ Google Scholar ] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Supplementary Materials 12870_2026_8510_MOESM1_ESM.xlsx (58.5KB, xlsx) Supplementary Material 1. Table S1 Primer information used in this study. Table S2 Characteristics of the SCAMP genes in Hordeum vulgare L. Table S3 The SCAMP genes in Arabidopsis thaliana L. and Oryza sativa L. Table S4 Conserved amino acid motifs of the SCAMP genes in Hordeum vulgare L. Table S5 The sequences of seven SCAMP genes identified in Hordeum vulgare L. Table S6 Promotion element analysis of HvSCAMP genes. Table S7 Syntenic blocks and Ka/Ks values of the SCAMP genes in Hordeum vulgare L. Table S8 One-to-one orthologous relationships and Ka/Ks values of the SCAMP genes between Hordeum vulgare L. and Arabidopsis thaliana L., and between Hordeum vulgare L. and Oryza sativa L. Table S9 miRNA and miRNA-acted HvSCAMPs. Table S10 Transcription factor prediction of HvSCAMP genes. Table S11 The expression profiles [log2(TPM+1)] of SCAMP genes in different tissues of Hordeum vulgare L. Table S12 Fold change in the expression of HvSCAMPs under salt-treated versus control (T/CK) conditions in salt-tolerant (XZ113) and salt-sensitive (Gairdner) barley. Table S13 Raw Ct values for HvSCAMP1 from quantitative RT-qPCR assays in wild-type (WT) and overexpression (OE) lines. Supplementary Material 2. (96.9KB, pdf) Supplementary Material 3. (1.2MB, docx) Data Availability Statement Data is provided within the manuscript or supplementary information files. 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