Genome-Wide Identification and Characterization of Stress-Responsive SNARE Genes in Quinoa (Chenopodium quinoa Willd.)

Simple Summary

SNARE proteins are crucial for plant development and stress response. Quinoa is a nutrient-rich crop with strong tolerance to harsh environmental conditions. In this study, 88 CqSNARE genes were identified in quinoa. A comprehensive analysis was conducted on the gene family members. Transcriptome analysis indicates that CqSNAP30a plays a crucial role in saline–alkaline stress response. When constitutively overexpressed in plants, CqSNAP30a enhances stress resistance by regulating ion balance, antioxidant capacity and photosynthesis efficiency. Our findings provide important insights into CqSNARE genes in quinoa and build a theoretical foundation for improving crop stress tolerance through genetic improvement.

Abstract

Plant SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) constitute a large superfamily and play pivotal roles in diverse biological processes and responses to various abiotic stresses. Quinoa (Chenopodium quinoa wild.) is a nutritionally superior crop endowed with robust tolerance to environmental stresses. In this study, we identified 88 CqSNARE genes in quinoa, which are unevenly distributed across 18 chromosomes and classified into 23 subfamilies. We systematically analyzed their physicochemical properties, phylogenetic relationships, gene and protein structures, and cis-acting elements. Furthermore, transcriptome analysis of quinoa leaves under saline–alkaline stress revealed that CqSNAP30a was the most significantly upregulated. This gene is predominantly expressed in leaves and localized on the plasma membrane. Constitutive expression of CqSNAP30a enhanced plant stress resistance by regulating ion homeostasis and antioxidant capacity. Our findings provide valuable insights into the SNARE genes of stress-tolerant crops and lays a theoretical foundation for the genetic improvement of stress resilience.

1. Introduction

Soil salinity and alkalinity stress represent major environmental constraints on global agricultural productivity, posing a profound threat to food security and impeding the advancement of sustainable agricultural systems [1]. As a major abiotic stress, saline–alkali stress exerts multifaceted adverse effects on plant growth and crop yield, primarily by triggering osmotic imbalance, inducing ion toxicity, and exacerbating oxidative damage in plants. Previous studies have elucidated that salt stress manifests in two sequential and interconnected phases [2,3]. The initial phase is an osmotic stress response, which is characterized by inhibition of plant growth, stomatal closure to reduce water loss, and a consequent decline in photosynthetic efficiency, all of which are direct physiological adaptations to the increase in soil osmolarity. The subsequent phase is a persistent ion toxicity, driven by the excessive accumulation of toxic ions (e.g., Na⁺ and Cl⁻) in plant cells. This ion overaccumulation disrupts the homeostasis of essential mineral elements (particularly K⁺, a critical cofactor for numerous enzymes), impairs the activity of key metabolic enzymes, and frequently provokes oxidative stress by disrupting the balance between reactive oxygen species (ROS) production and scavenging.

Eukaryotic cells possess an endomembrane system consisting of the nuclear envelope, PM (plasma membrane), and various membrane-bound organelles. Intracellular transport is typically mediated by membrane-enclosed vesicles, which shuttle cargo between different compartments via vesicular trafficking. Vesicular trafficking generally entails four core steps: vesicle budding, translocation, tethering, docking and membrane fusion. These steps rely on the coordinated participation and precise regulation of multiple protein factors, such as coat proteins, tethering factors, SNARE proteins, and SM (Sec1/Munc18) proteins [4,5,6,7,8,9]. The final membrane fusion step is driven by the assembly of SNARE complexes, which play a central role in directing the targeted delivery of cargo [10]. Based on their structural motifs and conserved amino acid residues, SNAREs are categorized into Q-SNAREs (Qa, Qb, and Qc), localized on target membranes, and R-SNAREs, localized on vesicles [11].

In plants, SNARE proteins play a pivotal regulatory role in adaptive responses to abiotic stress by dynamically modulating the localization, turnover, and activity of PM-localized transporters. With respect to ion homeostasis, vesicle-mediated trafficking and retrieval of ion channels are critical for maintaining cellular K⁺ and Na⁺ balance under salt stress. For instance, the R-SNARE VAMP721 directly interacts with the internalized K⁺ channels KAT1 and KC1 to fine-tune PM K⁺ conductance [12], whereas the Rab5 guanine nucleotide exchange factor (GEF) VPS9a is required to maintain K⁺ homeostasis during salt stress by regulating endosomal trafficking [13]. Accumulating evidence indicates that both R-SNAREs and Q-SNAREs in plants are implicated in abiotic stress responses. For example, NtSYP121 was identified as a key component to maintain homeostasis of PM ion channels in response to abscisic acid (ABA) [14]. It has been demonstrated that the AtSYP121–AtSNAP33–AtVAMP721 SNARE complex mediates the targeting of K⁺ channels to the PM [15,16]. The AtSYP4 protein family, comprising AtSYP41, AtSYP42, and AtSYP43, enhances plant tolerance to salt and osmotic stress [17]. Furthermore, deletion mutations in AtSYP61, a component of the AtSYP41 complex, result in altered osmotic stress tolerance and stomatal response phenotypes [18]. Additionally, specific plant SNARE and SNARE-like proteins regulate responses to salt, alkaline, and drought stress by maintaining ion and water balance, controlling reactive oxygen species (ROS) levels, and modulating stomatal behavior. For example, SbSLSP and SlSLSP6 enhance salt and drought tolerance by improving water retention capacity, promoting ROS scavenging, and facilitating Na⁺ compartmentalization [19,20], while GmSYP24 strengthens abscisic acid (ABA)-dependent stress responses [21]. In Arabidopsis, VAMP711 promotes ABA-induced stomatal closure, and SYP132 regulates aquaporin trafficking under salt stress [22,23,24].

Quinoa (Chenopodium quinoa Willd.) is a dicotyledonous annual herb with a cultivation history of approximately 7000 years [25]. Its seeds are the primary edible organ, characterized by high protein content, natural gluten-free properties, low glycemic index (GI), and abundant reserves of dietary fibers, minerals, and vitamins, as well as all essential amino acids [26]. Notably, quinoa seeds contain abundant secondary metabolites, including phenolic acids, flavonoids, and terpenoids, which exhibit diverse bioactivities such as anti-inflammatory, anti-diabetic, anti-bacterial, and anti-cancer properties [27]. Moreover, multi-variety analyses show that quinoa sprouts contain abundant minerals (e.g., K, Mg), vitamin C, reducing sugars, and moisture, and supply all essential amino acids (particularly high in leucine), along with significant levels of flavonoids, phenolics, carotenoids and unsaturated fatty acids [28]. Compared with seeds, germination boosts total amino acid content by ~7–14%, improving protein quality while elevating bioactive compound levels and antioxidant capacity [29,30,31]. These traits highlight quinoa sprouts as a functional vegetable with high-quality protein, micronutrients, and antioxidants, suitable for modern healthy diets [32]. In addition to its nutritional value, quinoa exhibits strong tolerance to abiotic stresses such as drought and salinity [33,34]. Recent physiological and molecular studies uncover the underlying tolerance mechanisms. Drought-tolerant genotypes maintain intact photosynthetic systems and upregulate antioxidant enzyme activities to preserve redox homeostasis and cellular integrity [35]. Genomic and transcriptomic analyses reveal differential regulation of drought-responsive genes (e.g., carbohydrate metabolism, osmotic adjustment, stress signaling) between tolerant and sensitive cultivars [36]. Under salt and alkali stress, transcriptome and small RNA sequencing demonstrated extensive re-programming of genes and miRNAs associated with reactive oxygen species (ROS) homeostasis, hormone signaling, cell wall synthesis and osmolyte metabolism [37,38]. And time-course RNA-seq combined with physiological assays confirms that quinoa seedlings mitigate salt-induced oxidative stress through enhanced antioxidant capacity and reduced membrane damage [39]. In summary, these findings demonstrate that quinoa employs multi-level adaptation strategies at the physiological, biochemical, and genetic levels. Therefore, identifying and characterizing stress-resistant candidate genes is critical for improving crop resilience. However, the role of SNARE-mediated vesicular trafficking in regulating quinoa’s salt–alkali stress response remains unexplored.

In this study, we identified the SNARE gene family in quinoa and performed comprehensive analyses, including phylogenetic reconstruction, gene structure and conserved motif characterization, cis-acting element prediction, and intra- and interspecific synteny assessments. Transcriptomic analysis of quinoa leaves under saline–alkali stress revealed the expression profiles of CqSNARE genes and pinpointed CqSNAP30a as a key differentially expressed candidate. Subsequently, we evaluated the drought and saline–alkali tolerance of CqSNAP30a-overexpressing Arabidopsis thaliana (Col-0) lines through phenotypic characterization, physiological and biochemical assays, and stress-responsive gene expression analysis s. Collectively, this work aims to elucidate the potential role and underlying molecular mechanisms of CqSNAP30a in quinoa’s adaptation to abiotic stresses, thereby providing theoretical insights for dissecting the stress resilience of this economically valuable crop.

2. Materials and Methods

2.1. Plant Materials, Growth Conditions, and Stress Treatments

Quinoa (Jiaqi#3) plants were grown at 22 °C under 16 h light/8 h dark conditions. Two-week-old quinoa seedlings were treated three times every five days with a 100 mM Na₂CO₃:NaHCO₃ solution (1:9) [28,38]. The control group was treated with water. Leaves were randomly harvested five days after the third treatment and subjected to RNA sequencing.

2.2. Identification of the SNARE Family in Quinoa

Genome sequences, functional annotations and coding sequence (CDS) predictions of Quinoa were downloaded from Ensemble Plants (https://ftp.ebi.ac.uk/ensemblgenomes/pub/release-62/plants/fasta/chenopodium_quinoa/, accessed on 10 January 2026). The Hidden Markov Model (HMM) files of the SNARE (PF05739), Syntaxin (PF00804), longin (PF13774), Synaptobrevin (PF00957), SEC20 (PF03908), v-SNARE-C (PF12352), v-SNARE (PF05008) and USE1 (PF09753) motifs were downloaded from the Pfam database to identify the CqSNARE proteins. Incomplete and redundant amino acid sequences were removed and the longest variant was retained for further analysis. The amino acid sequences of Arabidopsis AtSNARE proteins were obtained from the TAIR database (https://www.arabidopsis.org/, accessed on 10 January 2026). Furthermore, each protein was manually confirmed using BLASTp (version 2.15.0) in TBtools software (version 2.4.0) (https://github.com/CJ-Chen/TBtools-II/releases, accessed on 10 January 2026), the online NCBI batch Conserved Domain Database (CDD)-search tool. Physicochemical properties of the CqSNARE genes were obtained using tools from ExPASy website (https://www.expasy.org/, accessed on 10 January 2026). The subcellular localization of CqSNAREs was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 10 January 2026).

2.3. Chromosomal Location and Phylogenetic Analysis

Chromosomal location: Based on the quinoa genome database and its General Feature Format (GFF) annotation file, the number, length, and start and end positions of the chromosomes were obtained. The location of CqSNARE geneson chromosomes was mapped using an online website (http://mg2c.iask.in/mg2c_v2.1/, accessed on 10 January 2026).

The phylogenetic tree of Quinoa CqSNARE and Arabidopsis AtSNARE proteins was constructed based on multiple protein sequence alignment using MAGA 11 (version 11.0.13) software and the maximum-likelihood model (bootstrap is 1000). The final tree was refined and annotated using the Interactive Tree of Life (iTOL) website (https://itol.embl.de/, accessed on 12 January 2026).

2.4. Gene Collinearity in CqSNARE Family

The locations on the chromosomes of CqSNAREs were shown by Circos (version 0.69-10). Duplication events of CqSNAREs and synteny to different species (Arabidopsis thaliana, Oryza sativa) were analyzed using Multiple Collinearity Scan toolkit (MCScanX, version 1.0.0) and plotted using TBtools software with default parameters.

2.5. Gene Structure and Conserved Motif Analyses of CqSNARE Family Members

Conserved motifs in SNARE proteins were analyzed using searching multiple Em for Motif Elicitation (MEME) website (https://meme-suite.org/meme/tools/meme, accessed on 12 January 2026) allowing any number of repetitions with an optimum motif width of 6–50 residues and up to 20 motifs. The structural organization of the CqSNARE genes was characterized using TBtools, integrated with GFF3 annotation data to visualize exon–intron arrangements.

2.6. Promoter Analysis of CqSNARE Genes

The sequences of 2000 bp upstream of the start codon of CqSNARE genes were screened using TBtools and used as potential promoter sequences for analysis. The plant cis-acting regulatory elements (plantCARE) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi?cdsi, accessed on 10 January 2026) were used to screen the cis-acting regulatory elements in promoters. The distribution and abundance of these cis-acting elements were statistically analyzed and graphically represented via TBtools.

2.7. RNA Isolation, RT-qPCR and RT-PCR

Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. RNA concentration and quality were checked using NanoDrop 2000 (ThermoFisher Scientific, Beijing, China) and agarose gel electrophoresis, respectively. cDNA was then synthesized using a Prime Script RT reagent kit (Takara, Dalian, China). RT-qPCR was performed following the manufacturer’s instructions for Ultra SYBR Mixture (Low ROX) (CWBIO, Beijing, China) on the ABI7300 real-time PCR system. Elongation factor 1α (XM_021860126.1) was used as the reference gene for normalizing mRNA transcription. The relative expression level was calculated by the 2^−ΔΔCT method. For RT-qPCR reaction, three independent experiments were conducted per sample, with three technical replicates per experiment and three biological replicates per sample.

Semi-quantitative RT-PCR was performed to examine the ectopic expression of CqSNAP30a in transgenic Arabidopsis lines using gene-specific primers (the same primer pairs used for RT-qPCR), with AtACT2 as an endogenous control. The primers are listed in Supplemental Table S1.

2.8. RNA-Seq Data and Differential Expression of CqSNARE Genes

The original data of RNA sequencing was previously uploaded to the NCBI SRA database (accession number PRJNA972512) [40]. Fragments Per Kilobase of transcript per Million mapped fragments (FPKM) values of all the CqSNAREs were extracted and submitted to TBtools to generate heatmaps. The changes in expression of CqSNARE genes were represented by log₂FC (SAS vs. CK).

2.9. Subcellular Localization and Generation of Transgenic Plants

Subcellular localization: CqSNAP30a coding sequence was amplified using cDNA from total RNA of quinoa leaves and seamlessly cloned into binary vector pCAMBIA1300-eGFP using XbaI and KpnI restriction sites. The pCAMBIA1300-eGFP was previously modified by inserting a CaMV 35S promoter driving the eGFP coding sequence at the N-terminus of the interested gene. The chimeric and the empty (control) vectors were introduced into Agrobacterium tumefaciens strain GV3101, respectively, and the agrobacteria were infiltrated into the epidermal cells of Nicotiana benthamiana leaves. GFP fluorescence was examined by Leica TCS SP8 laser confocal microscope (Leica Microsystems, Wetzlar, Germany) at 48 h post-infiltration.

Generation of transgenic plants: The recombinant vector eGFP–CqSNAP30a/pCAMBIA1300 was transformed into Arabidopsis thaliana by agrobacterium GV3101 using the floral-dip method. The transgenic plants were screened by the antibiotic hygromycin at a concentration of 50 mg/L.

2.10. Analysis of Stress Tolerance of CqSNAP30a-Overexpressing Arabidopsis Lines

In total, nine CqSNAP30a-overexpressing (CqSNAP30a-OE) Arabidopsis transgenic lines were obtained, and eight of them had the same genotypes. Three CqSNAP30a-OE lines (OE4, OE7, and OE8) with higher expression levels were selected for salt and drought treatments. For root growth observation, seven-day-old seedlings vertically grown on 1/2 MS medium were transferred to 1/2 MS medium containing 100 or 120 mM NaCl, and 200 or 300 mM mannitol, and vertically cultivated for an additional seven days at 22 °C under a 16 h light/8 h dark cycle. The root lengths were measured by ImagJ (version 1.54).

For stress tolerance evaluation of CqSNAP30a-OE lines, three-week-old plants grown in soil were treated with or without 300 mM NaCl solution for an additional seven days. For drought tolerance evaluation, 20-day-old plants were treated with or without water for ten days.

2.11. Biochemical Analysis

The contents of total chlorophyll content were measured by an acetone solution (80% (v/v) extract) as described by Arnon [41]. The contents of free proline were measured by the sulphosalicylic acid method [42]. The malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) reaction method as described by Heath and Packer [43]. The activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were determined following the methods of Giannopolitis and Ries [44], Chance and Maehly [45], and Aebi [46], respectively. SOD activity was assayed by monitoring inhibition of NBT (Nitro blue tetrazolium chloride) reduction at 560 nm, POD activity by measuring guaiacol oxidation at 470 nm, and CAT activity by following H₂O₂ decomposition at 240 nm.

3. Results

3.1. Identification and Classification of SNAREs in Quinoa

To identify all members of the SNARE family in quinoa, three approaches were employed: HMM analysis, conserved domain search, and BLAST against orthologous sequences of Arabidopsis SNARE family genes. A total of 88 CqSNAREs were identified (Supplemental Table S2). Candidate SNAREs were named based on their homologs in Arabidopsis, with names prefixed by the species abbreviation. The length of CqSNARE proteins ranged from 73 (CqSFT11c) to 412 (CqSYP41a) amino acids. The predicted molecular weights (MW) of these proteins were between 0.80 kDa and 46.04 kDa; most CqSNAREs are acidic proteins with a theoretical isoelectric point (pI) < 7. Subcellular localization predictions revealed that quinoa SNAREs are primarily localized to the plasma membrane (PM), endoplasmic reticulum (ER)/Golgi apparatus, and trans-Golgi network (TGN)/vacuoles/endosomes (early endosomes/late endosomes, EE/LE).

3.2. Chromosomal Location and Phylogenetic Characterization of CqSNARE Family

Chromosomal mapping revealed that the 88 CqSNARE genes are distributed across all 18 chromosomes, albeit with a remarkably uneven distribution pattern. Specifically, several chromosomes, including Chr7, Chr12, and Chr18, harbor a relatively higher density of CqSNARE genes, implying potential local gene expansion or segmental duplication events in these genomic regions. In particular, Chr7 contains multiple members from different SNARE subfamilies (e.g., CqVAMP7, CqSYP11, CqSYP13, CqSEC20, CqVTI11), forming a distinct gene cluster that may reflect coordinated expansion or functional specialization. By contrast, Chr3, Chr5, Chr10, and Chr13 contain only a few CqSNARE genes, suggesting limited local duplication or stronger purifying selection (Figure 1A).

To analyze the evolutionary relationships of SNARE proteins in Chenopodium quinoa and Arabidopsis thaliana, the phylogenetic tree was constructed using 60 AtSNAREs (Arabidopsis) and 88 CqSNAREs (quinoa). Phylogenetic classification revealed 23 CqSNARE subfamilies, namely SYP11, SYP12, SYP13, SYP2, SYP3, SYP4, SYP8, GOS1, MEMB, NPSN1, SEC20, VTI1, SNAP, SFT1, BET1, USE1, SYP5, SYP6, SYP7, VAMP71, VAMP72, YKT6 and SEC22. By structural type, Qa, Qb, Qc, Qb + Qc and R-SNARE proteins account for 31 (35%), 17 (19%), 21 (24%), 3 (4%) and 16 (18%) of the total, respectively. The SYP12 subfamily contains the most members (eight), while MEMB1 had only one (Figure 1B). The phylogenetic relationships were generally consistent with the species’ evolutionary relationships, and CqSNAREs showed high similarity to AtSNAREs in many subfamilies.

3.3. Conserved Domain and Gene Structure Analysis

Analysis of conserved motifs and domains in CqSNARE proteins revealed that members within the same subfamily share highly similar motif and domain compositions (Figure 2A). Specifically, Motifs 7, 9, and 10 correspond to the longin domain, and Motifs 4 and 6 correspond to the synaptobrevin domain, the signature domains of R-SNAREs. In contrast, Motifs 2, 3, 5, and 11 encode the synaptophysin domain, which is typical of Qb-SNAREs, while Motif 8 is a characteristic domain of Qc-SNAREs. Additionally, SNAP subfamily members uniquely harbor both Motifs 8 and 19.

Moreover, Qa-SNAREs (syntaxins) exhibited the most conserved motif architecture, typically harboring multiple core motifs distributed throughout the protein sequence (Figure 2B). These motifs correspond to the evolutionarily conserved SNARE domain and its adjacent regulatory regions, a structural feature consistent with their canonical role as core components of the membrane fusion machinery in eukaryotic cells. In contrast, Qb- and Qc-SNAREs displayed considerably more variable motif profiles, characterized by fewer conserved motifs and greater positional heterogeneity across their sequences (Figure 2B); this structural plasticity implies functional diversification within these two subfamilies. R-SNAREs (VAMPs) featured relatively compact protein architectures, with a limited set of conserved motifs predominantly clustered in the C-terminal region (Figure 2B). This region encodes the conserved R-SNARE motif, which is indispensable for the assembly of trans-SNARE complexes and subsequent membrane fusion events. Notably, several members of the CqVAMP clade contained subgroup-specific motifs that are absent from other SNARE classes, indicating lineage-specific structural adaptations that may underpin specialized biological functions in quinoa. Conserved domain analysis further corroborated this motif-based classification (Figure 2B). All identified CqSNARE proteins possessed at least one signature SNARE domain, whereas auxiliary domains, including the Syntaxin_N domain and longin-like regions, were restricted to specific subfamilies. The presence and spatial arrangement of these accessory domains were highly conserved within each subfamily but diverged substantially between Qa-, Qb-, Qc-, and R-SNAREs. These observations collectively highlight a pattern of strong structural conservation within SNARE subfamilies, coupled with subfamily-specific divergence that likely shapes their functional specialization.

Gene structure analysis is a powerful tool for investigating intron gain/loss events during the evolution of gene families with well-characterized structures and functions, and it serves as an important approach for elucidating their evolutionary trajectories. The number and length of introns in CqSNARE genes showed a clear correlation with the phylogenetic clustering of the family. However, significant differences in gene size and intron distribution patterns were observed among distinct subfamilies. Specifically, the intron number of CqSNARE genes ranged from 0 to 15, and the majority of CqSNARE genes contained at least 5 introns (Figure S1).

3.4. Gene Collinearity Analysis

Gene duplication serves as a core evolutionary mechanism that drives the expansion and functional diversification of gene families, making it indispensable for deciphering gene family evolution and inferring gene functions. The tandem and segmental replication events of the CqSNARE genes were systematically characterized to elucidate their evolutionary trajectory. A total of 30 pairs of segmentally duplicated CqSNARE genes were detected across the quinoa genome, whereas no tandem duplication events were identified. These findings strongly suggest that segmental duplication has acted as the primary evolutionary force shaping the expansion and diversification of the CqSNARE gene family in quinoa (Figure 3A). To further explore the evolutionary dynamics and cross-species conservation of the CqSNARE gene family, interspecific collinearity analyses among quinoa, Arabidopsis thaliana (a dicot model plant), and Oryza sativa (a monocot model crop) were conducted. The results demonstrated a markedly higher degree of collinearity between quinoa and A. thaliana, indicating closer evolutionary affinities between these two dicot species. In contrast, the collinear relationships between quinoa and O. sativa were relatively limited (Figure 3B). Notably, chromosome 16 of quinoa contained the highest number of homologous CqSNARE gene pairs with A. thaliana, while quinoa chromosome 2 exhibits the most abundant collinear gene pairs with O. sativa. Collectively, these findings provide critical insights into the evolutionary origin, conservation, and divergence of the CqSNARE gene family across angiosperm lineages, laying a robust theoretical foundation for subsequent functional validation of CqSNARE genes and their potential utilization in enhancing abiotic-stress tolerance in quinoa and other crops.

3.5. Cis-Acting Elements of CqSNAREs

Cis-acting elements in gene promoter regions are critical regulatory modules for specific binding by transcription factors, thereby mediating the spatiotemporal expression of genes involved in plant growth, development, and stress adaptation. To decipher the transcriptional regulatory mechanisms underlying CqSNARE gene expression, the composition and distribution of cis-acting elements in the 2000 bp promoter sequences of CqSNARE genes were systematically analyzed. Two core cis-acting elements, the CAAT-box (a common enhancer element) and TATA-box (a basal promoter element essential for transcription initiation), were detected in the promoter regions of all CqSNARE genes (Figure 4A), indicating their conserved role in sustaining basic transcriptional activity.

The remaining identified cis-acting elements were classified into four functional categories based on their known regulatory roles: light-responsive, hormone-responsive, stress-responsive, and plant growth and development-related elements. The light-responsive category included multiple classic cis-elements, such as G-box, GT1-motif, GATA-motif, MRE (MYB recognition element), TCCC-motif, I-box, Box-4, and CHS-CMA1. The hormone-responsive category encompassed elements associated with diverse phytohormone signaling pathways: ABRE (ABA-responsive), G-Box (ABA-, JA-, SA-responsive) TGACG-motif and CGTCA-motif (JA-responsive), P-box, GARE-motif, TATC-box (gibberellin-responsive), TCA-element (salicylic acid (SA)-responsive), TGA-element and AuxRR-core (auxin-responsive), and ERE (ethylene-responsive). The stress-responsive category contained cis-elements involved in plant adaptation to various adverse environmental conditions, including ARE (anaerobic-responsive), MBS (MYB-binding site, drought-responsive), LTR (low-temperature-responsive), TC-rich repeats (pathogen- and stress-responsive), WUN-motif (wounding-responsive), and W-box (pathogen-responsive, recognized by WRKY transcription factors). The plant growth and development-related category included cis-elements regulating multiple biological processes: O2-site and HD-ZIP1 (metabolism regulation), CAT-box (meristematic-activity maintenance), GCN4-motif (endosperm development), Circadian element (circadian-rhythm control), and RY-elements (seed development) (Figure 4A). We further quantified the number of these cis-elements in the promoter region. Statistical analysis revealed a total of 1266 cis-acting elements, including 120 growth and development-related elements, 554 hormone-responsive elements, 479 light-responsive elements, and 113 environmental response elements. ABRE, G-Box, TGACG-motif, and CGTCA-motif, the ABA-, JA- and other hormone-responsive elements, appear most frequently in the promoter region (Figure 4B), suggesting that CqSNARE genes play important roles in plant development and environmental response.

3.6. Expression Profile of CqSNARE Genes in Quinoa Leaves Under Saline–Alkali Stress

To elucidate the functional responses of CqSNARE genes to saline–alkali stress, we analyzed the transcriptome profiles of quinoa leaves exposed to saline–alkali treatment. Transcriptome data revealed that two CqSNARE genes exhibited the most prominent transcriptional changes (Supplemental Table S3), with absolute log₂ fold change (|log₂FC|) values exceeding 1: CqSYP132a (log₂FC = 1.44439) and CqSNAP30a (log₂FC = 2.57991) (Figure 5A, highlighted by red lines). To validate the reliability of the transcriptome results, we performed reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays, and the results were consistent with the transcriptome data (Figure 5B,C). Collectively, these pronounced transcriptional alterations imply that the CqSYP132a and CqSNAP30a play a critical role in mediating the adaptive response of quinoa to saline–alkali stress. Then, we conducted an in-depth analysis of the regulatory role of CqSNAP30a in environmental stress adaptation.

3.7. Subcellular Localization and Tissue-Specific Expression Pattern of CqSNAP30a

To characterize the tissue-specific expression profile of CqSNAP30a, we performed RT-qPCR to quantify its transcript abundance in roots, stems and leaves. The results revealed that CqSNAP30a was most highly expressed in leaves, followed by stems, whereas its expression level in roots was comparatively low (Figure 6A). For subcellular-localization assays, the Chimeric gene eGFP-CqSNAP30a was transiently expressed in epidermal cells of Nicotiana benthamiana. Laser confocal microscopy observation revealed that the fluorescence signal of the eGFP-CqSNAP30a fusion protein was localized in the plasma membrane. In contrast, the fluorescence of the empty vector control (eGFP) was distributed in the nucleus and cytosol (Figure 6B). These results indicate that CqSNAP30a is a PM-localized SNARE protein and may regulate PM-associated vesicle trafficking.

3.8. CqSNAP30a Overexpression Enhanced Plant Stress Tolerance

To investigate the functional role of CqSNAP30a, a candidate gene implicated in plant abiotic-stress responses, we generated stable CqSNAP30a overexpression (CqSNAP30a-OE) lines in Arabidopsis Col-0 background. RT-PCR analysis confirmed that three independent transgenic lines (OE4, OE7, and OE8) exhibited substantially increased CqSNAP30a transcript abundance relative to wild-type (WT) plants (Figure 7A).

To assess how CqSNAP30a overexpression affects plant tolerance to abiotic stress, we conducted salt and drought treatments. First, phenotypic assays of seven-day-old seedlings grown on 1/2 MS medium supplemented with mannitol and NaCl were performed. Under normal conditions (1/2 MS medium), the primary root lengths of the three CqSNAP30a-OE lines were indistinguishable from WT, indicating that ectopic CqSNAP30a expression did not interfere with basal root development. In contrast, under osmotic stress (200 mM and 300 mM mannitol), all three transgenic lines displayed significantly longer primary roots compared with WT (Figure 7B,C). Consistently, upon exposure to salinity stress (100 mM and 120 mM NaCl), the CqSNAP30a-OE lines also showed a marked increase in primary root elongation relative to WT (Figure 7D,E). Collectively, these results demonstrate that constitutive CqSNAP30a overexpression enhances Arabidopsis tolerance to salinity and drought stress during the post-germinative growth phase.

To further confirm the putative role of CqSNAP30a in regulating plant responses to abiotic stress, soil-grown CqSNAP30a OE and WT plants were subjected to stress treatment. After 10-day water deprivation treatment on three-week-old plants, the mature rosette leaves of WT plants exhibited severe wilting, whereas those of CqSNAP30a-OE lines only showed incipient wilting symptoms (Figure 8A). Physiologically, CqSNAP30a-OE lines displayed markedly elevated activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), and accumulated significantly higher contents of chlorophyll and proline, while malondialdehyde (MDA) levels were significantly decreased compared to those in WT leaves (Figure 8B,C). Similarly, CqSNAP30a OE plants treated with 300 mM NaCl for 7 days also exhibited a resistance phenotype and stronger physiological indicators than WT (Figure 8D–F). Furthermore, the expression levels of stress-responsive marker genes associated with vesicle trafficking (Rab18) and ion transport (AHA1 and SOS1) were examined. Following drought and salt treatments, the transcriptional levels of these marker genes were markedly upregulated in CqSNAP30a OE plants compared to WT (Figure 8G). These results suggest that CqSNAP30a alleviates stress-induced plant growth inhibition by regulating ion homeostasis, adjusting osmotic pressure, protecting the photosynthesis system, and enhancing antioxidant capacity.

4. Discussion

The SNARE family represents a large and evolutionarily conserved superfamily, with hundreds of members identified across plant species. Accumulating evidence indicates that distinct SNARE isoforms, characterized by their subcellular compartmentalization and tissue-specific expression patterns, play pivotal roles in a wide range of biological processes. These include mediating interactions with ion channels, regulating cell wall synthesis and remodeling, facilitating auxin transport, and orchestrating adaptive responses to abiotic stresses [47,48,49,50]. Quinoa (Chenopodium quinoa Willd.), a nutrient-dense and nutritionally superior crop, is enriched in bioactive compounds (e.g., flavonoids, polyphenols, saponins, and polysaccharides) as well as vitamins and essential amino acids. These phytochemical and nutritional attributes endow quinoa with great potential health benefits, such as cancer prevention, free-radical scavenging, anti-inflammatory activity, and protective effects against cardiovascular diseases [28]. Despite the extensive characterization of the SNARE family in various model and crop plants, a systematic investigation of SNARE genes in quinoa remains elusive.

4.1. Characterization of CqSNARE Genes

In this study, a total of 88 CqSNARE genes were identified from the quinoa genome. A comparative analysis with other plant species revealed that the size of the quinoa CqSNARE gene family is comparable to that reported in Arabidopsis thaliana (64 members), rice (60 members) [51], tomato (63 members) [52], and Populus trichocarpa (69 members) [53]. However, it is smaller than those documented in rapeseed (237 members) [54], wheat (173 members) [55], and cultivated peanut (129 members) [56]. Variations in SNARE numbers across different plant species are primarily attributed to polyploidization (whole-genome duplication, WGD) events and the subsequent differential retention of duplicated genes [54]. Notably, plants harbor the largest number of SNARE proteins among eukaryotes. For instance, humans contain 38 SNAREs [57], Drosophila melanogaster has 26 [58], and Saccharomyces cerevisiae carries 21–25 members [59,60]. According to Sansebastiano and Piro [53], the expansion of the SNARE gene family in plants is mainly driven by the duplication of genes within conserved subfamilies, rather than the emergence of novel SNARE types.

Further analysis of the distribution of CqSNARE genes uncovered distinct genomic organization patterns. Specifically, the chromosomal distribution of Qa-SNAREs (CqSYP family) and R-SNARE CqVAMP members differs significantly, with these genes showing distinct enrichment on specific chromosomes. The presence of closely spaced CqVAMP and CqSYP gene pairs on these chromosomes suggests that segmental duplication, rather than tandem duplication, may have played a major role in the expansion of these subfamilies in quinoa. The limited number of tandemly arranged CqSNARE genes in the quinoa genome further supports this notion, indicating that WGD and large-scale segmental duplication events, well-documented features of quinoa’s allotetraploid evolutionary history, have been the dominant forces driving the expansion of the CqSNARE gene family in quinoa.

Notably, several chromosomes (Chr2, Chr8, Chr14, and Chr18) contain both homologous-like gene pairs and small CqSNARE gene clusters. This distribution pattern is consistent with quinoa’s hybrid A × B subgenome constitution [61,62,63], implying differential retention of duplicated CqSNARE genes following polyploidization, with certain chromosomal regions exhibiting a stronger bias toward gene retention. Such biased retention is a common phenomenon in membrane trafficking-related gene families. This is likely because duplicated CqSNARE genes can enhance functional robustness and facilitate the evolution of specialized roles in key biological processes, including vesicle fusion, stress signaling transduction, and membrane remodeling.

4.2. Structural Conservation and Divergence of CqSNAREs

Phylogenetic analysis of SNARE proteins from quinoa (Chenopodium quinoa Willd.) and Arabidopsis thaliana classified 88 CqSNARE proteins into 23 subgroups, a classification consistent with that previously reported for cultivated peanut. While the biological functions of most CqSNARE proteins remain uncharacterized, the majority of these genes cluster with functionally validated Arabidopsis AtSNARE orthologs, enabling reliable functional inference via homology. Functionally, the 23 subgroups exhibit distinct specialization profiles: members of the CqSYP12/13 subgroups are primarily associated with PM-localized secretion and membrane ion channel regulation; CqVAMP71/72 subgroups predominantly mediate endosome-to-vacuole trafficking and vacuolar membrane fusion; and the CqBET1, CqSFT1, CqSEC22, CqUSE1, and CqGOS1 subgroups are involved in the ER-Golgi-TGN/early secretory pathway. Additionally, the CqSNAP33, CqNPSN, and cytokinesis-related CqSYP11 subgroups show strong conservation in regulating cell plate formation and cell division. Collectively, the CqSNARE family retains the core functional architecture of Arabidopsis AtSNARE family, while copy-number expansion in specific CqSYP and CqVAMP clades suggests potential functional diversification or subfunctionalization events subsequent to genome duplication.

Within the quinoa genome, CqSNARE proteins in the same subgroup share highly conserved motif alignment and domain architectures, indicating the retention of structural features essential for membrane fusion activity. This conservation aligns with other plant species (e.g., Brassica napus), where CqSNARE subfamilies maintain structural homogeneity as core components of the vesicle fusion machinery. Notably, substantial variation in intron number and exon-intron organization was observed across different CqSNARE subgroups, whereas intrasubgroup members displayed relatively stable structural patterns. Such intersubgroup divergence likely reflects distinct evolutionary trajectories following gene family expansion, driven by differential selective constraints and functional requirements.

Synteny analyses integrating sequence similarity and genomic collinearity revealed extensive conserved homologous linkages among quinoa, Arabidopsis, and rice (Oryza sativa). Orthologous relationships frequently exhibited one-to-many or many-to-one patterns, indicative of gene duplication followed by differential retention in quinoa. Despite deep evolutionary divergence among these angiosperm lineages, locally conserved syntenic blocks were detectable, underscoring the evolutionary conservation of CqSNARE genes across flowering plants [64]. Intra-genomic synteny analysis of quinoa further identified widespread CqSNARE gene correspondences across chromosomes, with these linkages predominantly arising from segmental rather than tandem duplication events. As an allotetraploid species, quinoa has undergone ancient hybridization and subsequent genome reorganization, providing the evolutionary framework for the observed patterns of gene retention, loss, rearrangement, and diversification [61]. Moreover, the persistence of syntenic SNARE loci between Arabidopsis (Brassicaceae) and rice (Poaceae), two distantly related taxa, supports prior reports of angiosperm-wide micro-synteny. Although large-scale gene order has been extensively reshaped during evolution, subsets of functionally constrained SNARE gene clusters remain conserved, reflecting strong selective pressure to maintain core components of vesicle trafficking and membrane fusion pathways across diverse plant lineages [65].

Our cis-element analysis of quinoa CqSNARE gene promoters provides key insights into their transcriptional regulation and potential functional divergence. First, hormone-responsive elements including ABRE, AuxRR-core/TGA, and CGTCA/TGACG motifs are widely distributed across the promoters, implying that these genes are likely involved in ABA-, auxin-, JA-, and ethylene-mediated developmental processes and stress responses. This observation aligns with reports on SNARE families in banana [66] and cotton [67], where promoters are similarly enriched with hormone- and stress-related elements and show transcriptional induction under stress treatments, thereby validating our findings. Second, light-responsive elements (e.g., G-box, GATA-motif) and development-associated elements (e.g., CAT-box, RY-element) are broadly represented in CqSNARE promoters. This is consistent with the conserved core functions of SNARE proteins in membrane trafficking, organelle biogenesis, and cell differentiation [68], and further indicates that certain CqSNARE genes not only sustain basal vesicle transport activities but also coordinate transcriptional responses to environmental and developmental cues [69].

4.3. Functions of CqSNAP30a

Profiling gene expression across diverse tissues and under environmental stress is a fundamental approach for deciphering gene function [70,71]. Prior studies have established that multiple environmental stresses, including salt, ABA exposure, low temperature, and osmotic stress, can induce the transcription of SNARE genes [14,72,73]. In this study, we found that CqSNAP30a exhibited the most pronounced induction under saline–alkali stress. Tissue-specific expression pattern and subcellular localization of CqSNAP30a were consistent with that of AtSNAP33, a member of the same subfamily in Arabidopsis thaliana [74]. Furthermore, GFP-CqSNAP30a PM-localization aligns with SNAP25-type SNARE proteins in Arabidopsis [75], wild soybean [76], tomato [77] and rice [78].

In recent years, plasma membrane (PM)-localized SNAP25-type SNARE proteins have been shown to mediate diverse biological processes, including pathogen defense, cytokinesis, vesicle trafficking, and mechanical stimulus perception [75,79,80]. Among these, Arabidopsis AtSNAP33 is the most thoroughly characterized member. It assembles a functional SNARE complex with the cytokinesis-specific Qa-SNARE KNOLLE and R-SNAREs VAMP721/722 to drive membrane fusion during cell plate formation, and also plays a pivotal role in pathogen-triggered immunity-related exocytosis [74]. Beyond biotic interactions, mounting evidence indicates that SNAP25-type SNAREs contribute to plant adaptation to abiotic stresses. For instance, heterologous expression of Glycine soja GsSNAP33 enhances salt and drought tolerance in Arabidopsis [76], while overexpression of its tomato homolog improves salt tolerance, presumably by regulating endocytic pathways [77].

In this study, we demonstrated that CqSNAP30a constitutive overexpression markedly improved plant stress tolerance. This phenotypic response mirrors that of GsSNAP33; heterologous expression of the wild soybean gene in Arabidopsis similarly enhances primary root growth under drought and salt stress [76]. Recent studies have identified multiple SNARE proteins, including OsSYP71 [81], AtSYP61 [18], AtVAMP71/AtVAM7C [82], and OsSNAP32 [78], as key regulators of stress responses. Building on these findings, we further characterized the function of CqSNAP30a in stress tolerance during the adult stage of Arabidopsis. Consistent with our seedling data, transgenic plants overexpressing CqSNAP30a exhibited significantly higher peroxidase activity and greater biomass accumulation under both salt and drought stress compared with wild-type controls. Phenotypic analyses suggest that CqSNAP30a enhances salt and drought tolerance primarily via an ion regulatory mechanism. Given that vesicle trafficking is a conserved core function of all SNARE proteins, we hypothesize that CqSNAP30a may maintain PM stability by modulating membrane-localized transporters or ion channels, thereby improving plant performance under abiotic stress.

Transcriptional upregulation of stress-responsive genes represents a major protective strategy in plants exposed to adverse environments [83]. Numerous well-characterized abiotic-stress marker genes, such as Rab18 and RD29A, are known to participate in multiple stress response pathways [84,85]. In addition, AHA1 (a key member of the PM H⁺-ATPase family) acts as the central proton pump of the PM and serves as a sensitive indicator of membrane transport activity, proton pump function, and salt/drought stress responses [86,87]. SOS1, a PM Na⁺/H⁺ antiporter, is another core marker gene that plays an essential role in Na⁺ extrusion under salt stress [88,89,90]. In this study, we found that CqSNAP30a overexpression significantly upregulated the transcription of several stress-responsive and ion homeostasis-related marker genes (including Rab18, AHA1, and SOS1) under salt and osmotic stress conditions. Based on these observations, we propose that distinct abiotic stresses activate specific defense pathways in Arabidopsis, and CqSNAP30a facilitates enhanced expression of key stress-responsive and ion transport-related genes, thereby conferring improved tolerance to salt and drought stresses.

5. Conclusions

In this study, we performed a genome-wide identification of the CqSNARE gene family in quinoa and systematically characterized the protein and gene structural features, phylogenetic relationships, and cis-acting regulatory elements in the promoters. Transcriptome analysis revealed that the expression level of CqSNAP30a in leaves was significantly upregulated under abiotic-stress induction. The CqSNAP30a OE Arabidopsis plants exhibited markedly enhanced tolerance to salt and drought stresses, which is primarily attributed to the regulation of ion homeostasis, adjustment of osmotic pressure, protection of the photosynthetic system, and enhancement of antioxidant capacity. Overall, our study provides novel insights into the CqSNARE genes in quinoa and lays a theoretical foundation for the genetic improvement of stress tolerance in quinoa (Figure 9).