De Novo Transcriptome Profiling of Salt Stress Responses in the Crop Wild Legume Vicia hirsuta (L.) Gray

TL;DR

This study explores how the wild legume Vicia hirsuta responds to salt stress at the genetic level, offering insights into improving crop resilience to salty soils.

Contribution

The study provides a de novo transcriptomic resource for Vicia hirsuta, revealing key genes and pathways involved in salt stress adaptation.

Findings

Salt stress in Vicia hirsuta activates genes related to signaling, transcription factors, and ion transporters.

Genes involved in Na+/K+ balance are consistently up-regulated under salt stress.

Transcription factor families like bHLH, MYB, bZIP, NAC, and WRKY are key regulators during stress adaptation.

Abstract

Soil salinity is an increasing agricultural challenge that limits crop growth and yield worldwide. As salinity stress becomes more widespread, crop wild relatives are gaining attention as genetic resources for improving stress tolerance, but molecular information for many wild species remains limited. In this study, we analyzed genome-wide gene expression in Vicia hirsuta (L.) Gray (hereafter referred to as V. hirsuta), a wild legume relative, after seven days of salt stress. The results revealed coordinated changes in stress-related signaling, transcription factor activity, and ion transporter genes, particularly those involved in Na+ and K+ balance, while antioxidant enzyme responses indicated that plants experienced stress. This study provides a foundational transcriptomic resource for V. hirsuta and highlights the potential value of wild legumes for improving salt stress tolerance in cultivated crops.

Salt stress is a major environmental constraint affecting plant growth and productivity. Crop wild relatives provide valuable genetic resources for stress tolerance, yet transcriptomic information for forest-derived wild species remains limited. In this study, we analyzed transcriptional responses of V. hirsuta, a crop wild relative (CWR) of legumes, after seven days of salt stress using de novo transcriptome sequencing. Seedlings were exposed to salt stress, and differentially expressed genes (DEGs) were identified between control (Vh_S0) and salt-treated (Vh_S7) plants using an FDR-adjusted threshold (q < 0.05). Gene Ontology and KEGG enrichment analyses revealed that salt-responsive DEGs were mainly involved in regulatory signaling, metabolic adjustment, redox-related processes, and macromolecular organization. Up- and down-regulated DEGs showed distinct yet overlapping enrichment patterns, indicating complex transcriptional reprogramming under salt stress. Transcription factor analysis identified bHLH, MYB, bZIP, NAC, and WRKY families as major regulators, with many families containing both up- and down-regulated members. Notably, genes associated with Na+/K+ homeostasis were consistently up-regulated and validated by qRT-PCR. These results suggest that continuous seven days salt stress adaptation in V. hirsuta involves coordinated regulation of signaling pathways, transcriptional networks, and transporter-mediated ion homeostasis, providing a valuable transcriptomic resource for crop wild relatives.

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1. Introduction

The genus Vicia (hereafter referred to as V.), a member of the Fabaceae family, encompasses a wide diversity of species, including both annuals and perennials [1,2]. These plants are found worldwide, mainly in temperate regions, and they show great adaptability to different environmental conditions [1,2]. Vicia species commonly grow in grasslands, roadsides, and agricultural fields [3]. Vicia species have shown strong resilience, competing well with other plants for survival [4,5,6]. The genus includes over 140 species, some of which play important agricultural roles [1]. Representative species of the genus Vicia, such as V. faba, V. Sativa, and V. villosa, are agriculturally significant [1,2]. For example, V. faba, known for its high protein content, is widely grown and valued as a staple crop, especially in the Mediterranean and Middle East regions [1]. V. Sativa and V. villosa are mainly used as green manure crops, helping increase soil nitrogen content and improve the structure of the soil [2,4]. Another species, V. hirsute, is commonly found in the wild and can adapt to various environmental conditions [5,6]. Like other Vicia species, V. hirsuta grows in grasslands, roadsides, and agricultural areas, demonstrating strong survival abilities even when competing with other plants [4]. Although several studies have focused on V. hirsuta, relatively less is known about this species compared to other major crops and legumes [5,6].

Globally, environmental stress factors profoundly impact agricultural productivity due to climate change, persistent landuse changes, degradation, and the over-exploitation of natural resources [7,8,9,10]. In particular, alongside natural saltwater exposure, excessive chemical fertilizers, soil amendments, and inappropriate irrigation practices have intensified soil salinization, severely restricting plant growth and threatening agricultural production worldwide [7,8,11,12]. Under salt stress conditions, plants initiate various physiological and biochemical responses to adapt [8,13,14,15]. Osmotic adjustments enable cellular water retention to prevent dehydration by accumulating compatible solutes such as proline and glycine betaine, helping plants maintain cell turgor [7,8,9,10]. Concurrently, redox regulation mechanisms control reactive oxygen species (ROS) levels, allowing ROS to function as signaling molecules without reaching toxic levels that could damage cells [10,16]. Additionally, Na^+^/H^+^ antiporters and K^+^ retention systems regulate intracellular Na^+^ concentrations, mitigating ion toxicity and supporting normal metabolism [8]. Hormonal signaling pathways, including those involving abscisic acid (ABA) and brassinosteroids (BR), further regulate these processes, enhancing the tolerance of plants to salt stress [15,17]. However, when these adaptive processes do not function effectively, plants experience physiological issues [7,8]. Osmotic adjustment failures lead to cellular dehydration, causing wilting and reduced growth [8]. Ineffective ROS regulation results in oxidative damage to cellular membranes and proteins, potentially leading to cell death [10,16]. Disruption of the ionic balance due to improper Na^+^/K^+^ regulation interferes with enzyme function and metabolism through Na^+^ toxicity, while deficient K^+^ levels impair essential cellular activities [8]. Hormonal signaling abnormalities can lead to uncontrolled stomatal closure, exacerbating dehydration stress and inhibiting growth and development [15,17]. Consequently, plants unable to adapt to salt stress will show stunted growth, developmental delays, damage to reproductive organs, and reduced yields, significantly impacting agricultural productivity and crop quality [7,8]. Over 20% of cultivated land worldwide has been salinized, and crops in arid and semiarid regions already show significant damage [7,8,12]. The negative impacts of salt stress on crops are becoming increasingly evident worldwide.

The need for crop research addressing salt stress is becoming increasingly important. This involves developing salt-tolerant crop varieties capable of maintaining high productivity in saline soils through various approaches, including traditional breeding techniques, gene editing technologies, and utilizing resistance genes discovered in CWRs [18,19,20,21]. Such research can enhance crop tolerance under salt stress, enable sustainable agricultural practices, and bolster food security [20,22]. CWRs are the wild relatives of cultivated crops, representing a treasure trove of genetic diversity [18,23]. Naturally adapted to various environmental stress conditions, they are likely to contain genes for resistance to drought, salinity, pests, and diseases [18,19,20]. These traits can be invaluable resources for crop improvement, especially with regard to enhancing resistance to stress conditions related to climate change. Recently, CWRs have been recognized as a crucial store of wealth capable of expanding the genetic base of cultivated crops and improving crop varieties in response to climate change and sustainable agriculture challenges [18,23]. The identification and application of functional genes from CWRs is considered an attractive research area.

However, lacking a reference genome is a bottleneck in research on CWRs [24,25]. Reference genomes are applied to precise mapping and annotation with the DNA sequences of specific species and are necessary to identify and understand genetic variations [26]. The lack of such reference genomes poses considerable challenges in analyzing genomic data and identifying genes. De novo assembly, the process of assembling a genome from high-throughput sequencing data without a reference genome, is invaluable for uncovering the structure of unknown genomes and exploring genetic diversity [26]. This approach is particularly suitable for research on plants such as CWRs, where genomic information is limited, enabling the identification of new genes, gene families, and pathways related to stress responses [21,24,27].

Common vetch (V. Sativa L.), a relative of V. hirsuta, is commonly cultivated as animal feed due to its high leaf crude protein content and high digestibility [28]. Common vetch seeds contain relatively high amounts of starch and crude protein, making them a valuable and sustainable food resource [29,30]. Research comparing 54 common vetch populations found that under salt stress conditions, the salt-tolerant populations had higher transcript levels of Na^+^ and K^+^ transporter genes, as well as higher levels of proline and antioxidants, compared to the salt-sensitive populations. Additionally, the transcript levels of genes associated with the salt stress pathway, in particular, NHX7, HKT1, AKT2, and HAC17, were induced in both the leaves and roots after salt stress [5]. However, lacking a reference genome for vetch populations limits our understanding of the genetic mechanisms underlying salt stress. At the same time, de novo transcriptome analysis remains analytically challenging due to the complexity of transcript reconstruction and the reliance on homology-based annotation, which may affect transcript-level resolution in non-model wild species. Despite these challenges, de novo transcriptomics can yield biologically meaningful insights into stress-responsive pathways when carefully applied to wild plants without reference genomes, particularly at the level of gene families and functional categories.

This study examines transcriptomic responses in normally grown four-week-old V. hirsuta, a CWR of leguminous crops, plants subjected to 200 mM NaCl treatment for seven days, using de novo transcriptome sequencing. Genome-wide gene expression was analyzed at a single sampling time point to identify salt-responsive genes and associated biological processes in this species. Particular attention was given to transcription factor families potentially involved in regulating stress-responsive gene networks, as well as to ion transporter genes related to cellular sodium and potassium balance. By integrating differential expression analysis, functional enrichment, and qRT-PCR validation, this work provides a transcriptomic resource for V. hirsuta and identifies candidate regulatory and transporter genes relevant to salt stress responses in legumes.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The V. hirsuta seeds used for germination and salt treatment experiments were obtained from the National Baekdu Mountain Arboretum Seed Bank (Korea Forest Service, Republic of Korea). To promote uniform germination, seeds were surface-sterilized and imbibed in distilled water for 24 h at 4 °C, germinated on Petri dishes at 25 °C for 3 days, and uniformly germinated seedlings were transplanted into plastic pots (15 cm × 15 cm × 17 cm) filled with 250 g of commercial horticultural soil per pot. Plants were grown in a controlled greenhouse for 4 weeks under a 16 h light/8 h dark photoperiod at 25 °C. During the growth period, plants were watered every two days with a baseline volume of 100 mL of distilled water per pot, and soil moisture was maintained at approximately 60~65% using a soil moisture sensor (HY Systems, Seoul, Republic of Korea). At 4 weeks after transplanting, soil moisture content was reduced to 40% prior to salt treatment to standardize water conditions and minimize confounding effects of water availability. Salt stress was imposed by applying 100 mL of 200 mM NaCl solution per pot once per day for seven days, following protocols modified from previous studies [31,32,33]. Control plants received the same volume of water without NaCl. Shoot tissues (aboveground tissues excluding roots) were harvested immediately before salt treatment (day 0) and after seven days of treatment (day 7) for antioxidant enzyme activity measurements, frozen in liquid nitrogen, and stored at −80 °C until analysis (Figure 1).

2.2. Antioxidant Activity Measurement

The antioxidant enzyme activities of peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) were assessed in V. hirsuta samples designated as Salt + 0 (S + 0) and Salt + 7 (S + 7). For each treatment, 100 mg of fresh tissue was immediately frozen in liquid nitrogen and finely ground using a pre-chilled mortar and pestle. The powdered tissue was homogenized in an ice-cold protein extraction buffer containing 0.2 M potassium phosphate and 0.1 mM EDTA (pH 7.0). The homogenate was centrifuged at 12,000× g for 15 min at 4 °C to remove insoluble cell debris, and the resulting clarified supernatant, free of insoluble cell debris, was used for total protein quantification and subsequent antioxidant enzyme activity assays. Total protein concentration was determined using a SMART™ BCA Protein Assay Kit (iNtRON Biotech., Seongnam, Gyeonggi Province, Republic of Korea) according to the manufacturer’s instructions. Enzyme activity assays were performed following the methods described by Ko et al. (2018) and Kim et al. (2024) [34,35]. Statistical differences in antioxidant enzyme activities between S + 0 and S + 7 treatments were evaluated using a t-test, and asterisks indicate statistically significant differences (* p < 0.05, ** p < 0.01).

2.3. Library Construction and RNA-Sequencing

RNA was extracted from salt-treated V. hirsuta using a Ribospin™ Plant kit (GeneAll Biotechnology, Seoul, Republic of Korea) for RNA sequencing. Three independent biological replicates were prepared for each condition, with each replicate consisting of pooled tissues from three individual plants. RNA quantity and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Detailed RNA quality metrics, including RIN values and A260/A280 ratios, are summarized in Table S1. RNA libraries were prepared for 151 bp paired-end sequencing using a TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA). Library quality was assessed using an Agilent 2100 Bio-Analyzer (Agilent, Santa Clara, CA, USA), and sequencing was performed on the Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA).

2.4. Transcriptome De Novo Assembly and Filtering

Quality filtering removed low-quality reads and adapter sequences, leaving only high-quality reads for the de novo transcriptome assembly. We used Trinity software v2.15.0, which is well regarded for its effective transcript assembly [36,37]. During the assembly process, Trinity merged overlapping reads into longer sequences referred to as contigs. These contigs were then processed to identify and extend non-terminal sequences, assembling unique genes representing the distinct transcript sequences in the sample.

2.5. Differentially Expressed Gene (DEG) Analysis

A differential gene expression analysis was conducted to identify genes with expression changes in response to salinity stress. RNA-seq count data were analyzed using the TCC package with the DESeq method, which models read counts with a negative binomial distribution and normalizes library size [38]. In this exploratory analysis, genes with a p-value < 0.05 and FPKM > 1 were considered differentially expressed genes (DEGs). We also calculated the q-value adjusted by the false discovery rate (FDR), and used a more conservative criterion of q-value <0.05 for DEGs.To reduce false-positive detection in the absence of a reference genome, differentially expressed genes used for the main analyses were defined using a conservative false discovery rate threshold (q < 0.05). Genes meeting a nominal significance threshold (p < 0.05) were considered for exploratory analyses. For each gene, the log2 fold-change (log2FC) between the Salinity + 7 (S7) and Salinity + 0 (S0) conditions was calculated, and DEGs with positive log2FC values were classified as up-regulated, whereas those with negative log2FC values were classified as down-regulated. In addition, q-values were calculated for all genes, and the number of DEGs that meet different combinations of p-value and q-value thresholds is summarized in Table S2. Functional annotation of these DEGs was then carried out by Gene Ontology (GO) enrichment analysis, grouping genes into biological process, cellular component, and molecular function categories. To explore biological pathways associated with these DEGs, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using KOBAS (http://bioinfo.org/kobas/, accessed on 22 April 2024). Additionally, transcription factors among the DEGs were profiled using PlantTFDB v5.0 (https://planttfdb.gao-lab.org/, accessed on 14 April 2024).

2.6. Expression Pattern Analysis of Candidate DEGs

Selected DEGs identified from RNA-seq data were validated using quantitative real-time PCR (qRT-PCR). All qRT-PCR analyses were performed using three independent biological replicates, with each replicate consisting of pooled tissues from three individual plants. cDNA was synthesized from RNA samples using a Power cDNA synthesis kit (iNtRON Biotechnology, Seoul, Republic of Korea). Specific primers for each selected gene were designed using Primer3 Plus software version 3.3.0 (https://www.primer3plus.com/index.html, accessed on 4 March 2024). The qRT-PCR reactions were performed on a Rotor-Gene Q real-time PCR machine using the SYBR Green PCR Master Mix (QIAGEN, Hilden, Germany). Relative expression levels of target genes were calculated using the 2^−ΔΔCt^ method [39] and normalized against a V. hirsuta Actin homolog, which was identified from the de novo assembled transcriptome using Pisum sativum Actin1 as a reference sequence for homology search [40] and subsequently used as the internal reference gene. Primer sequences for the Actin homolog and all target genes, together with primer amplification efficiency values, are provided (Tables S3 and S4).

3. Results

3.1. Antioxidant Enzyme Responses to Salt Stress in V. hirsuta

Antioxidant enzyme activities were examined to evaluate oxidative stress-related responses of V. hirsuta to salt treatment. Enzyme activities were measured in plant samples collected before salt treatment (S + 0) and after seven days of salt treatment (S + 7) (Figure 2). Peroxidase (POD) activity did not differ significantly between treatments (p = 0.0561; Figure 2a). In contrast, superoxide dismutase (SOD) activity was significantly higher in S + 7 plants than in the control (p = 0.0375; Figure 2b). Ascorbate peroxidase (APX) activity showed a significant reduction under salt stress (p = 0.0446; Figure 2c), whereas catalase (CAT) activity increased significantly after seven days of salt treatment (p = 0.0148; Figure 2d). Overall, distinct and enzyme-specific changes in antioxidant activities were observed in response to salt stress.

3.2. Unigene Profiling of V. hirsuta Under Salt Stress Using De Novo Assembly

De novo assembly processes were undertaken to generate unigenes with which to evaluate raw transcriptome data under salt-treated V. hirsuta and non-treated control. Table 1 shows a statistical summary of the number of unigenes in V. hirsuta under salt stress conditions. Unigenes refer to sequences identified among all gene sequences obtained under specific conditions or experiments; such sequences can provide essential data when studying the diversity and expression of genes.

RNA-seq generated on average 64,112,323 raw reads per control library (Salinity + 0) and 53,195,609 raw reads per salt-treated library (Salinity + 7). These corresponded to 9680.96 Mb and 8032.54 Mb of raw bases, respectively (Table 1). Of these bases, 8874.50 Mb in the control libraries and 7425.68 Mb in the treated libraries had Phred quality scores ≥ Q30. After quality trimming and filtering, the control libraries retained on average 57,564,545 clean reads (8405.48 Mb), whereas the treated libraries retained 48,782,712 clean reads (7112.20 Mb) (Table 1). These results indicate a slight decrease in the number of clean reads and base pairs in the treated group and a reduction in the average read quality following salt stress. Table 2 presents the results of the expression analysis and the expression patterns of unigenes constructed by de novo assembly for the Vh_S0 vs. Vh_S7 groups. The genes detected in both groups were categorized as expressed genes (Expressed), known genes (Known), newly discovered or predicted genes (Novel), and unexpressed genes (Unexpressed). The expression of all genes was quantitatively assessed by counting the number of genes with FPKM (fragments per kilobase of transcript per million mapped reads) values greater than 1.0. In the Vh_S0 (control) group, the average number of expressed genes was 60,567, which included 34,148 known genes, 26,420 novel genes, and 69,130 unexpressed genes. The Vh_S7 group showed a slight increase in the number of expressed genes, with an average of 62,030 expressed genes, including 35,346 known genes and 26,684 novel genes, while the number of unexpressed genes was 67,667. These results indicate that the overall gene expression patterns of the two groups were similar, with only a slight increase in the number of genes detected in the treated group. The data support the suitability of the sequencing data quality and the de novo assembly process for further analysis.

3.3. Exploring DEGs Under Salinity Stress Conditions in V. hirsuta

Differentially expressed genes (DEGs) between the Salinity + 0 (S0) and Salinity + 7 (S7) conditions were identified to characterize transcriptional responses to salt stress (Table 3). The RNA-seq analysis detected a total of 122,128 genes. Before applying statistical thresholds, 58,571 genes showed higher expression and 63,557 genes showed lower expression in S7 compared with S0. DEGs between the Salinity + 0 (S0) and Salinity + 7 (S7) conditions were identified to characterize transcriptional responses to salt stress (Table 3). The RNA-seq analysis detected a total of 122,128 genes. Before statistical filtering, 58,571 genes showed higher expression and 63,557 genes showed lower expression in S7 compared with S0. DEGs were primarily defined using a false discovery rate threshold of q-value < 0.05 to ensure robust statistical significance. Based on this criterion, a total of 9839 DEGs were identified, including 4044 up-regulated and 5795 down-regulated genes under the S7 condition (Table 3). This DEG set was used for downstream analyses, including GO and KEGG enrichment analyses and transcription factor profiling (Table S2). In addition, for exploratory purposes, genes meeting a nominal significance threshold (p-value < 0.05) and an expression level of FPKM > 1 were also examined to provide a broader overview of transcriptional changes under salinity stress. Using this criterion, 11,971 genes were identified as differentially expressed, comprising 5002 up-regulated and 6969 down-regulated genes under the S7 condition. GO and KEGG results based on this nominal p-value threshold are presented in Figure S1.

Figure 3a presents a scatter plot of log expression, x-axis for Vh_S0 and y-axis for Vh_S7, enabling direct comparison between groups. This type of plot allowed for a visual comparison of the overall gene expression changes between the two groups, with red and blue points highlighting up-regulated and down-regulated genes, respectively, and black points representing genes with no significant expression changes. A volcano plot (Figure 3b) was used to visualize DEGs by plotting the log2 fold change on the x-axis and the negative log10 of the p-value on the y-axis, allowing for the identification of genes with both large fold changes and statistically significant differences. Red points in the plot represent significantly up-regulated genes, blue points represent significantly down-regulated genes, and black points correspond to genes that were not significantly different between the two groups. This plot highlights the magnitude and significance of gene expression differences across the conditions. A heatmap (Figure 3c) was generated to visualize gene expression patterns and the relationships between genes in the Vh_S0 and Vh_S7 groups. It showed the expression levels of selected genes across all samples, with the color gradient indicating the expression level. Hierarchical clustering of genes and samples revealed groups of genes with similar expression patterns, providing a more comprehensive view of gene expression changes across the conditions. This heatmap enabled a deeper analysis of specific gene expression profiles and their relationships within the Vh_S0 vs. Vh_S7 groups.

3.4. Functional Analysis of DEGs in V. hirsuta Under Salt Stress

Gene Ontology (GO) enrichment analysis was conducted to classify the differentially expressed genes (DEGs) identified between the Salt 0 and Salt 7 treatments into three functional categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). The distribution of enriched GO terms reflects the major functional characteristics of salt stress-responsive genes (Figure 4a–c). Within the Biological Process (BP) category, DEGs were predominantly associated with regulatory and signaling-related processes. The most highly represented terms were ‘protein phosphorylation’, ‘regulation of DNA-templated transcription’, and ‘intracellular signal transduction’. In addition to these major categories, several other regulatory and post-translational processes were enriched, including ‘protein ubiquitination’, ‘intra-cellular protein transport’, ‘ubiquitin-dependent protein catabolic process’, ‘protein folding’, and ‘protein autophosphorylation’ (Figure 4a). In the Cellular Component (CC) category, enriched DEGs were mainly localized to intracellular compartments and membrane-associated structures. The most abundant annotations corresponded to ‘nucleus’ and ‘cytoplasm’, followed by ‘plasma membrane’ and ‘cytosol’. Other frequently enriched components included ‘membrane’, ‘chloroplast’, ‘mitochondrion’, ‘Golgi apparatus’, ‘endoplasmic reticulum’, and ‘intracellular membrane-bounded organelle’, indicating broad subcellular distribution of salt stress-responsive genes (Figure 4b). The Molecular Function (MF) category was characterized by enrichment of genes involved in nucleic acid- and protein-related molecular activities. The most frequently observed terms were ‘RNA binding’ and ‘structural constituent of ribosome’, followed by ‘protein serine/threonine kinase activity’ and ‘oxidoreductase activity’. Additional enriched functions included ‘DNA-binding transcription factor activity’, ‘ubiquitin protein ligase activity’, ‘ATPase activity’, ‘unfolded protein binding’, ‘GTPase activity’, and ‘mRNA binding’ (Figure 4c).

To further examine pathways related to salt stress, KEGG pathway enrichment analysis was performed separately for up-regulated and down-regulated DEGs (Figure 4d,e; Table S6). Both DEG groups were enriched in pathways involved in metabolism, macromolecular processing, and cellular organization. However, the enriched pathways and their gene ratio patterns differed between the up- and down-regulated DEGs. Among the up-regulated DEGs, enriched pathways were mainly related to metabolic processes and signal transduction. Pathways with relatively high gene ratios included ‘Carbon metabolism’, ‘Glycolysis/Gluconeogenesis’, and ‘Biosynthesis of amino acids’. In addition, signaling-related pathways such as the ‘MAPK signaling pathway’ and ‘Plant hormone signal transduction’ were enriched. Pathways involved in redox and energy metabolism, including ‘Glutathione metabolism’, ‘Cysteine and methionine metabolism’, and ‘Oxidative phosphorylation’, were also detected. Furthermore, pathways related to gene expression and protein processing, such as ‘Ribosome’, ‘RNA transport’, ‘Spliceosome’, and ‘Protein processing in endoplasmic reticulum’, were present in the up-regulated DEG set (Figure 4d). In contrast, the down-regulated DEGs were enriched mainly in pathways related to lipid and carbohydrate metabolism. These included ‘Fatty acid metabolism’, ‘Pyruvate metabolism’, ‘Fructose and mannose metabolism’, and ‘Purine metabolism’, as well as ‘Biosynthesis of amino acids’. Several lipid-related pathways, such as ‘Glycerolipid metabolism’, ‘Glycerophospholipid metabolism’, and ‘Starch and sucrose metabolism’, were also identified among down-regulated DEGs (Figure 4e). Pathways related to general metabolic functions and cellular organization including ‘Metabolic pathways’, ‘Ribosome’, ‘Endocytosis’, ‘RNA transport’, ‘Spliceosome’, and ‘Protein processing in endoplasmic reticulum’ were shared with the up-regulated DEG group, although their gene ratios differed between the two sets.

The bar chart presents the transcription factor ratios between Vh_S0 and Vh_S7 (Figure 5a). The data indicate that the transcription factor bHLH has the highest ratio, with a value of 45, followed by bZIP and C2H2 with ratios of 32 and 25, respectively. Other notable transcription factors include MYB and MYB-related transcription factor, NAC and WRKY, with corresponding ratios of 22, 22, 20, and 19. The chart shows a clear predominance of specific transcription factors, particularly bHLH, which was the most represented transcription factor family. The Venn diagram compares the up-regulated and down-regulated genes between Vh_S0 vs. Vh_S7 (Figure 5b). Of the total transcription factors, 28 (59.6%) were shared between the up-regulated and down-regulated DEG sets, indicating that these transcription factor families contained members showing both up- and down-regulated expression patterns. In addition, 7 transcription factors (14.9%) were uniquely up-regulated, while 12 transcription factors (25.5%) were uniquely down-regulated (Figure 5b).

3.5. Expression Pattern of DEGs Related to Salinity Response Mechanisms

Salt stress involves various mechanisms and pathways, with different genes activated depending on the timing and duration of exposure. Understanding these dynamic genetic responses is crucial for a proper examination of plant adaptation to salt stress. Analyzing differentially expressed genes (DEGs) at each stage can identify the key regulatory patterns and mechanisms involved in stress adaptation. DEGs identified from the Vh_S0 and Vh_S7 groups were profiled based on the four stages of salinity stress mechanisms proposed by Zelm et al. (2020) [41]. The identified DEGs were analyzed by categorizing them into four stages: perception, early signaling, down-stream signaling, and adaptive response. The analysis identified 53 DEGs in the perception stage, 65 in the early signaling stage, 95 in the downstream signaling stage, and 129 in the adaptive response stage. Among the DEGs categorized into the four stages, the heat shock protein (HSP) family was most prevalent, with 45 genes identified. Additionally, the transcription factors MYB (25 DEGs), MAPK (16 DEGs), and ERF (11 DEGs) were detected (Table S5). DEG profiling based on gene expression in the Vh_S0 and Vh_S7 groups enabled the selection of gene sets associated with salt stress. The selected DEGs were subsequently validated by quantitative RT-PCR (Figure 6 and Figure S3). Notably, genes involved in NaK homeostasis and in the maintenance of intracellular ion transport and balance showed distinct expression patterns between the Vh_S0 and Vh_S7 groups. DEGs related to genes known to regulate the balance of Na^+^ and K^+^ ions, including NHX (Na^+^/H^+^ exchanger), KEA (K^+^ efflux antiporter), HAK (high-affinity potassium transporters), TPK (two-pore K^+^ channel), and AKT (Arabidopsis potassium channel), were up-regulated under continuous salt stress (Figure 6).

4. Discussion

Soil salinization is considered one of the major factors affecting global agricultural productivity. Numerous studies have reported that improper irrigation practices and excessive fertilizer application exacerbate soil salinization in various types of agricultural land [7,8,41]. The resilience of plants to salt stress in high-salinity soils is essential to maintain food security. In this study, we subjected V. hirsuta, a wild relative of leguminous crops, to continuous salt stress for seven days to simulate field-relevant conditions (Figure 1). Differentially expressed genes (DEGs) were identified using a de novo transcriptome assembly approach to investigate transcriptional responses under sustained salinity. Stress responses are often described as a sequence of physiological and molecular changes, including perception, signaling, and downstream regulation [8,41]. However, under prolonged salt stress, these stages may overlap or occur concurrently, complicating temporal separation. Gene expression patterns were analyzed based on functional response phases rather than fixed time points. This approach enabled a more comprehensive interpretation of the adaptation-related transcriptional patterns in V. hirsuta under continuous salt stress.

Salt stress causes extensive changes in plants, such as metabolic disturbances, while also affecting cellular functions and various physiological processes, as reflected in the gene expression patterns and metabolic pathway regulation activities of plants [41]. Gene Ontology and KEGG pathway analyses were conducted to examine the response of V. hirsuta under continuous salt stress. In the Biological Process (BP) category, genes associated with cellular and metabolic processes were highly expressed (Figure 4a), indicating a putative adaptive response of the plant to regulate fundamental cellular functions and metabolic pathways under salt stress conditions. In addition, KEGG enrichment analysis indicated the involvement of plant hormone-related signaling pathways, including abscisic acid (ABA)-associated processes, suggesting a possible association between metabolic adjustment and hormonal signaling under continuous salt stress (Figure 4d). These findings indicate that hormonal modulation may contribute to metabolic regulation and support the maintenance of physiological stability under salinity stress [10,17,42].

In the Molecular Function (MF) analysis, binding and catalytic-related activities were highly enriched under salt stress (Figure 4c) [43,44]. The most frequent terms included ‘RNA binding’ and ‘structural constituent of ribosome’, indicating sustained regulation of transcriptional and translational processes under stress conditions [45,46]. In addition, several enzymatic activities, such as ‘protein serine/threonine kinase activity’ and ‘oxidoreductase activity’, were enriched, suggesting active redox-related regulation and signaling under salt stress [10]. The enrichment of ‘ubiquitin protein ligase activity’ and ‘unfolded protein binding’ further indicates enhanced protein turnover and quality control mechanisms, which are known to contribute to stress adaptation [47,48].

These findings from the MF analysis are closely linked to the KEGG pathway results. Among the enriched pathways, the ‘MAPK signaling pathway’ and ‘Glutathione metabolism’ were particularly prominent under salt stress (Figure 4d). In the context of V. hirsuta, enrichment of these pathways indicates the involvement of conserved stress-related signaling and redox regulatory processes under the applied salt stress conditions.

The MAPK signaling pathway is a well-known regulator of Na^+^/K^+^ homeostasis, ion channel activity, and reactive oxygen species (ROS) production and scavenging in plants [10,16,49]. Previous studies in model and crop species have shown that modulation of MAPK cascade components, including MPK3, MPK6, and MAPKKs, can influence salt stress sensitivity and tolerance [50,51,52,53]. However, the direction and magnitude of these effects appear to be context-dependent, varying with species, genetic background, developmental stage, and stress conditions. In particular, contrasting outcomes observed in gain- and loss-of-function studies of MAPK components, such as increased salt sensitivity upon overexpression and enhanced tolerance in certain knockout backgrounds, suggest that additional regulatory mechanisms may modulate MAPK-associated stress responses. Therefore, while MAPK signaling is likely involved in salinity responses, its specific regulatory role under continuous stress in V. hirsuta cannot be directly inferred from studies in other plant systems.

The enzyme-specific changes observed in antioxidant activities may reflect the activation of distinct physiological responses under the applied salt stress conditions. In this study, catalase (CAT) activity was markedly increased after seven days of salt treatment, whereas ascorbate peroxidase (APX) activity showed a significant decrease. Similar contrasting response patterns among antioxidant enzymes have been reported in other plant species exposed to salinity stress and have been discussed in the context of functional differentiation within the antioxidant defense system [10,41]. CAT has been reported to function as a high-capacity enzyme involved in hydrogen peroxide detoxification, whereas APX is known to be more tightly regulated and closely associated with cellular redox homeostasis [10,16]. In line with this observation, the differential regulation of these enzymes appears to be broadly aligned with the enrichment of glutathione metabolism and MAPK signaling pathways observed in this study, suggesting a potential association between antioxidant enzyme regulation and broader ROS-related signaling pathways under salt stress conditions [15,16,49,50].

Gene expression analysis conducted after seven days of continuous salt stress revealed complex transcriptional profiles across diverse functional categories (Figure 6 and Figure S2, Table S5), indicating that multiple stress-responsive pathways are transcriptionally engaged under sustained salinity. Although salt stress responses are often conceptually divided into perception, early signaling, and downstream signaling stages [8], transcriptomic profiling at a single sampling point reflects the concurrent detection of response-associated transcriptional signatures rather than a resolved temporal sequence.

In addition, genes annotated to downstream signaling and adaptive response categories showed elevated expression levels at the seven-day time point compared with their basal expression (Figure S2). These observations suggest sustained transcriptional engagement of stress-related pathways under continuous salt stress, while the extent to which these transcriptional changes contribute to effective physiological adaptation requires further investigation [15,51].

A differential expression analysis between the control (S0) and salt-stressed (S7) groups revealed that continuous salt stress in V. hirsuta is associated with transcriptional changes across multiple functional categories related to salt stress responses (Figure 6). Among these categories, transporter-related genes represented a prominent and consistently up-regulated group. Functional annotation indicated enrichment of ion transporter gene families, including NHX, KEA, HAK, TPK, and AKT, which are transcriptionally associated with Na^+^ extrusion and K^+^ uptake or redistribution processes (Figure 6). This transporter-enriched expression profile highlights the strong representation of ion homeostasis-related genes at the transcriptional level in V. hirsuta under sustained salinity.

Rather than directly reflecting physiological ion fluxes, these results are consistent with transcriptional changes involving ion transporter genes during salt stress in this study. The presence of transporter-related transcripts across multiple response-associated functional categories suggests that genes associated with ion homeostasis-related pathways are transcriptionally responsive after seven days of salt treatment. Similar patterns of transcriptional enrichment of transporter gene families under salinity have been reported in other plant species, including wheat, where salt-tolerant genotypes show elevated expression of ion transport-related genes [54]. In addition, Long and Huang (2020) reported the widespread transcriptional involvement of membrane transporter genes in K^+^/Na^+^ homeostasis across diverse plant species [55]. Collectively, these observations suggest that ion transporter genes represent a prominent transcriptional feature of the salt stress response in V. hirsuta under continuous salinity stress.

Transcription factors are well known to play central roles in plant salt stress responses by integrating stress perception, signaling, and downstream regulatory processes. In this study, transcriptomic analysis of V. hirsuta identified multiple transcription factor families, including ERF, bHLH, MYB, bZIP, NAC, and WRKY, among salt-responsive genes (Figure 5a), consistent with previous reports in other plant species [14,56,57,58,59,60,61,62,63]. ERF family members, which are known to interact with hormonal signaling pathways such as jasmonic acid and salicylic acid, were transcriptionally responsive under salt stress, and several ERF genes were also associated with growth- and development-related functional categories (Table S5), in line with their reported roles in sustained stress adjustment [56,57,58,59]. Members of the bHLH and MYB families were among the most abundant transcription factors detected, a pattern that has been widely observed in salt stress studies and is often linked to downstream regulatory processes including ABA signaling, ROS homeostasis, circadian regulation, and cell wall modification [14,60,61,62,63]. Although precise stage-specific roles cannot be resolved based on a single sampling point, the transcriptional representation of these transcription factor families suggests broad engagement of conserved regulatory networks during continuous salt stress in V. hirsuta. Consistent with this interpretation, bZIP and NAC transcription factors, which have been implicated in ABA-responsive gene regulation and long-term stress tolerance in diverse plant species, were also detected among salt-responsive transcripts [64,65,66,67].

Overall, the TFs identified in V. hirsuta were not only functionally diverse but also spanned multiple stages of the stress response. A comparative analysis of S0 vs. S7 samples revealed that 59.6% of differentially expressed TFs were commonly regulated between the control and seven days salt stress conditions (Figure 5b), suggesting a sustained and stage-overlapping transcriptional network. Furthermore, the functional annotations and KEGG pathways of these TFs showed high consistency with those found in V. faba under salt stress [68], indicating conserved regulatory strategies across Vicia species.

These findings collectively suggest that V. hirsuta employs a temporarily changing yet interconnected transcription factor network to handle both immediate and continuous responses to salt stress. Categorizing TFs by functional stage offers clearer insights into their roles and highlights potential targets for improving stress resilience through stage-specific genetic interventions.

Taken together, these findings indicate that multiple transcription factor families are transcriptionally responsive to salt stress in V. hirsuta. Grouping transcription factors by functional categories provides a useful framework for organizing salt-responsive regulatory components at the transcriptomic level, while further studies will be required to resolve their temporal dynamics and functional contributions under salt stress conditions.

In this first transcriptomic study of salt stress in the forest-derived crop wild relative V. hirsuta, all primary downstream analyses were conducted using DEGs defined by a stringent false discovery rate threshold (q < 0.05), ensuring statistical robustness of the results presented in this study. However, because genomic and transcriptomic information for V. hirsuta is still limited, we additionally provide DEG information based on a nominal p-value threshold (p < 0.05) to offer a broader descriptive view of salt-responsive transcriptional changes (Table S2). This dual presentation is intended to facilitate data interpretation and reuse by readers, particularly for exploratory analyses and comparative studies in crop wild relatives and legume species. Table S2 summarizes DEG counts obtained under different p-value and FDR-adjusted q-value thresholds, allowing transparent assessment of how multiple-testing correction influences candidate gene selection.

A key survival strategy for plants under salt stress is to maintain the cellular ion balance [41]. In our analysis of V. hirsuta, the genes NHX (Na^+^/H^+^ exchanger), KEA (K^+^ efflux antiporter), HAK (high-affinity K^+^ transporter), TPK (two-pore K^+^ channel), and AKT (K^+^ channel) were significantly up-regulated under continuous salt stress (Figure 6). This observation is consistent with previous findings in Vicia species [69]. In particular, NHX plays a key role in intracellular Na^+^ sequestration [70,71,72]. While earlier studies mainly focused on Na^+^ exclusion mechanisms [8,41], our results also suggest an active transcriptional response related to K^+^ transport, with elevated expression of HAK, KEA, and TPK genes, which are involved in K^+^ uptake, efflux, and vacuolar redistribution. Hence, V. hirsuta may maintain Na^+^/K^+^ homeostasis not only through Na^+^ extrusion but also via the regulation of K^+^ dynamics. These findings imply that genes related to K^+^ transport could serve as useful markers for breeding salt-tolerant crops. Zhang et al. (2021) [66] showed that overexpression of DREB1A in wheat improved salt tolerance, probably through pathways related to ion transport.

This study provides transcriptomic insight into salt stress responses in the crop wild relative V. hirsuta based on genome-wide gene expression patterns observed after seven days of salt treatment. The results highlight transporter-mediated ion homeostasis, particularly the regulation of K^+^ uptake and distribution, as a major component of the stress response, together with transcriptional regulation involving multiple transcription factor families. The consistent enrichment of ion transporters and regulatory genes across different functional response phases underscores the importance of integrated transcriptional control in maintaining cellular stability under salinity stress.

Comparative studies in the Vicia genus provide useful context for interpreting the salt stress responses observed in V. hirsuta. In common vetch (V. sativa), substantial variation in salt tolerance has been reported among germplasms, with physiological performance under salinity reflecting genotype-dependent stress sensitivity [5]. Transcriptome-level analyses in V. sativa further revealed that salt stress induces widespread transcriptional reprogramming involving stress-responsive regulatory pathways and multiple transcription factor families, indicating that adaptive responses in this genus are mediated by coordinated gene expression rather than by isolated stress-related genes [6]. Consistent with these observations, the transcriptional patterns identified in V. hirsuta suggest that salt stress responses in Vicia species share common regulatory features at the pathway and gene-family levels, while the specific composition and magnitude of responsive genes may vary among species. In addition, accurate characterization of such transcriptional responses requires careful methodological consideration, as demonstrated in V. faba, where the stability of reference genes differed across experimental conditions, emphasizing the importance of species- and stress-specific validation in gene expression studies [40]. Taken together, these comparative findings support the interpretation that salt stress responses in V. hirsuta reflect both conserved legume-associated regulatory mechanisms and species-specific transcriptional features shaped by its wild genetic background.

Given these characteristics, the transcriptional signatures identified in V. hirsuta indicate that this species represents a potentially valuable genetic resource for pre-breeding efforts aimed at improving salt stress responses in cultivated legumes. As a crop wild relative of soybean, V. hirsuta harbors stress-responsive transcription factors and ion transporter genes that may be explored as candidate loci for enhancing salinity tolerance in Glycine max through comparative genomics and molecular breeding approaches. Notably, conserved regulatory components associated with Na^+^/K^+^ homeostasis and stress-related signaling pathways suggest that wild Vicia species may provide alleles that are underrepresented or absent in modern cultivated varieties, thereby reinforcing the value of CWRs as reservoirs of adaptive variation under salinity stress.

This study is based on a de novo transcriptome assembly, which inherently involves analytical challenges, including fragmented contigs, imperfect isoform reconstruction, and reliance on homology-based annotation in non-model wild species lacking reference genomes. In light of these limitations, interpretation was focused on consistently enriched pathways, gene families, and shared transcriptional patterns rather than on individual transcript isoforms. The expression patterns of selected salt-responsive genes were further examined by qRT-PCR, showing trends generally consistent with the RNA-seq results. Although a strong linear correlation was not observed, qRT-PCR analysis confirmed the direction of expression changes for the majority of selected genes (Table S3).

Viewed in this context, the present transcriptomic dataset provides a reliable framework for capturing major regulatory features of salt stress responses in V. hirsuta. Overall, this study lays a foundation for future research aimed at clarifying regulatory mechanisms underlying salt stress responses and translating these insights into pre-breeding strategies for legume improvement, particularly in saline soils and reclaimed coastal environments.

5. Conclusions

This study provides transcriptome-level insights into the salt stress response in V. hirsuta, a crop wild relative within the legume family, based on gene expression patterns observed after seven days of salt treatment. The results highlight coordinated transcriptional responses involving ion transport, stress-related signaling, and regulatory gene networks, rather than isolated pathway activation. In particular, the consistent upregulation of ion transporter genes associated with Na^+^ efflux and K^+^ uptake and redistribution suggests that transcriptional regulation of Na^+^/K^+^ homeostasis is a key component of salt stress responsiveness in this species.

In addition, multiple transcription factor families, including ERF, bHLH, MYB, NAC, and bZIP, were identified among salt-responsive genes, together with enrichment of MAPK signaling and glutathione metabolism, pointing to integrated redox- and signaling-related transcriptional responses under salinity stress. Collectively, these findings establish a valuable transcriptomic resource for the relatively understudied wild Vicia species and provide a foundation for future functional characterization and pre-breeding efforts aimed at improving salt stress responses in cultivated legumes.

Building on this resource, future studies may prioritize selected ion transporter genes, such as NHX and HAK family members, as candidates for functional validation or marker-assisted selection. Stress-responsive transcription factors, particularly those belonging to the ERF and NAC families, may also represent promising targets for regulatory studies to facilitate the translation of transcriptomic insights from V. hirsuta into legume improvement programs.