VaTPS9 from Vitis amurensis Encodes a Trehalose-6-Phosphate Synthase Correlated with Cold Tolerance

Abstract

Vitis amurensis is a cold-hardy wild grape species and represents valuable germplasm for breeding cold-tolerant grapevines. In this study, we identified a highly expressed gene (VaTPS9) in one-year-old shoots of V. amurensis ‘Shuangfeng’ during overwintering, but its biological function remained unclear. Temporal and spatial expression analyses revealed distinct expression patterns of VaTPS9 among different tissues from June to November, with the highest transcript abundance detected in one-year-old shoots in November. Gene cloning and sequence alignment showed that VaTPS9 encoded a type II trehalose-6-phosphate synthase (TPS) and was designated as VaTPS9. Functional analyses demonstrated that overexpression of VaTPS9 enhanced cold tolerance in yeast, Arabidopsis thaliana, and V. amurensis callus tissues. Conversely, virus-induced gene silencing (VIGS) of VaTPS9 in grapevine plantlets markedly increased cold sensitivity under low-temperature stress. These reciprocal gain- and loss-of-function phenotypes indicate that VaTPS9 positively regulates cold tolerance, likely by modulating trehalose metabolism and associated physiological responses, including reactive oxygen species (ROS) homeostasis. Collectively, our findings provide new insights into the molecular basis of cold adaptation in wild grape species and highlight VaTPS9 as a promising candidate gene for improving cold tolerance in cultivated grapevine.

1. Introduction

Low temperature is a major environmental factor limiting grapevine growth, productivity, and geographical distribution, particularly in cold and temperate regions [1]. Exposure to freezing temperatures can cause severe cellular damage, including membrane disruption, metabolic imbalance, and oxidative stress [2], ultimately leading to reduced survival and yield. Although winter protection practices such as soil burial are commonly used in cold regions, they are labor-intensive and costly [3], highlighting the need to improve intrinsic cold tolerance in grapevine.

Cold acclimation is a crucial adaptive process that enhances plant tolerance to subsequent freezing stress through extensive physiological and metabolic reprogramming [4]. Among the metabolic adjustments, carbohydrate metabolism plays a central role in the cold stress response [5]. The accumulation of soluble sugars and trehalose has been shown to stabilize cellular membranes, protect proteins, and mitigate oxidative damage under low-temperature conditions [6]. Trehalose metabolism is primarily regulated by trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), which together control trehalose biosynthesis and its key regulatory intermediate, trehalose-6-phosphate (T6P) [7].

In grapevine, several members of the TPS gene family have been identified and implicated in abiotic stress responses [8]. However, the roles of specific TPS genes in cold acclimation and freezing tolerance remain poorly understood, particularly in cold-tolerant wild grape species. V. amurensis, a wild grape native to northeastern Asia, exhibits exceptional cold hardiness and represents a valuable genetic resource for elucidating the molecular mechanisms underlying cold adaptation [9]. Although advances have been made in characterizing cold tolerance mechanisms in cultivated grapevine (Vitis vinifera) [10], the genetic and molecular basis of cold adaptation in V. amurensis is still incompletely defined. Our initial screening pointed to VaTPS9 as a putative cold-responsive gene, leading us to conduct the present functional investigation [3].

Virus-induced gene silencing (VIGS) has emerged as an efficient reverse genetics tool for the functional characterization of stress-responsive genes in horticultural crops without stable transformation [11]. In this study, we identified a TPS gene, VaTPS9, from V. amurensis that was strongly upregulated during cold acclimation based on transcriptome analysis. We systematically investigated its function through heterologous expression in yeast and Arabidopsis, along with physiological and biochemical analyses of VaTPS9-overexpressing grape calli under cold stress. This study aims to elucidate the role of VaTPS9 in cold adaptation, providing new insights into TPS-mediated pathways that confer cold tolerance in grapevine.

2. Results

2.1. Analysis of the Temporal and Spatial Expression Patterns of VaTPS9

In this study, qRT-PCR was used to analyze the temporal and spatial expression patterns of VaTPS9 in V. amurensis ‘Shuangfeng’ (Figure 1). The gene exhibited distinct expression patterns across various tissues from June to November. Before August, VaTPS9 expression was relatively higher in the roots and tendrils. As temperatures gradually decreased, its expression in the shoots began to increase from September, peaking in November when the plants entered dormancy.

2.2. Cloning and Sequence Characterization of VaTPS9

Following the observation of peak VaTPS9 expression in shoots in November, we cloned the 2589 bp full-length CDS of the candidate gene VaTPS9 from one-year-old shoots collected at that time (Figure 2). We then constructed a phylogenetic tree by aligning its deduced protein sequence with homologs from the NCBI database (Figure 3). This analysis confirmed its classification within the TPS family, leading to its designation as VaTPS9.

Nucleotide sequence alignment identified six base differences between VaTPS9 (from cold-tolerant V. amurensis) and its ortholog VvTPS9 (from cold-sensitive V. vinifera) (Figure S1). At the protein level, however, only a single amino acid substitution was identified: methionine (Met) at position 856 in VaTPS9 replacing isoleucine (Ile) in VvTPS9 (Figure S2). VaTPS9 encodes an 862-amino-acid protein with a predicted molecular weight of 97.63 kDa and an isoelectric point (pI) of 5.78.

2.3. Subcellular Localization of VaTPS9

We used VAMP711-RFP, the Arabidopsis SNARE protein AtVAMP711 that has been frequently adopted as a tonoplast marker, and co-expressed it with VaTPS9-GFP in Arabidopsis protoplasts to determine whether VaTPS9 localizes to the vacuolar membrane [12]. Under control conditions (25 °C), Arabidopsis protoplasts were co-transformed with 35S-GFP, VaTPS9-GFP and the tonoplast marker VAMP711-RFP, and 35S-GFP was used as a fluorescence control. The VaTPS9-GFP signal predominantly localized to the tonoplast and overlapped with the VAMP711-RFP signal (Figure 4) [13].

To test whether cold stress affects VaTPS9 localization, transfected protoplasts were incubated at 4 °C and imaged after 4 d and 6 d of treatment. Under cold conditions, the VAMP711-RFP signal was not stably detectable in our imaging assays; therefore, the chlorophyll autofluorescence channel was used as an auxiliary reference (because chloroplasts are often compressed and cluster along the vacuolar boundary in protoplasts). After cold treatment, VaTPS9-GFP appeared more broadly distributed within the vacuolar region (Figure 5), suggesting that low temperature alters the fluorescence distribution pattern of VaTPS9 in Arabidopsis protoplasts.

2.4. Verification of the Cold-Tolerant Phenotype of Yeast Overexpressing VaTPS9

To verify the involvement of VaTPS9 in cold tolerance, heterologous expression was performed in yeast. In this study, yeast cells transformed with pYES2-NTB-VaTPS9 were defined as the VaTPS9-expressing (overexpression) group, whereas cells harboring the empty vector pYES2-NTB served as the control. Yeast cells from both groups were exposed to 4 °C, 0 °C, −10 °C, and −20 °C for 24, 48, 72, and 96 h. A clear difference in cold tolerance was observed at −10 °C after 72 h (Figure 6A): the VaTPS9-expressing yeast cells still formed colonies, whereas the control cells showed markedly reduced colony formation (Figure 6B). These results indicate that VaTPS9 expression enhances yeast survival and growth under low-temperature stress.

2.5. Analysis of Cold Tolerance and Physiological Responses in Arabidopsis VaTPS9 Overexpression

Arabidopsis mutants overexpressing VaTPS9 were generated to confirm the involvement of this gene in cold tolerance. Homozygous T3 Arabidopsis lines overexpressing VaTPS9 (OE2, OE4 and OE5) were confirmed by PCR (Figure 7) and used to carry out the low temperature treatment. Prior to cold treatment, VaTPS9-OE lines and wild-type (WT) plants displayed comparable growth. After freezing treatment followed by recovery, VaTPS9-OE plants exhibited less visible injury than WT, as assessed at 7 d and 14 d of recovery (Figure 8A). Survival rate analysis at 14 d showed that the three VaTPS9-OE lines exhibited markedly higher survival (66.7%, 58.3%, and 54.2%) than WT plants (29.2%) (Figure 8B).

To examine whether VaTPS9 overexpression affected endogenous TPS-family gene expression during cold stress, the transcript levels of AtTPS1, AtTPS2, AtTPS3, and AtTPS9 were quantified by qRT-PCR in WT and VaTPS9-OE plants after low-temperature treatment. Compared with WT, VaTPS9-OE lines showed significantly higher expression of AtTPS9 and increased transcript levels of AtTPS1 and AtTPS2, whereas AtTPS3 exhibited no significant change (Figure 9). Collectively, these results indicate that VaTPS9 overexpression is associated with coordinated changes in the expression of endogenous TPS-family genes during cold stress.

To assess cold-induced physiological changes, we measured membrane damage and reactive oxygen species (ROS)-related parameters. Compared with the wild type (WT), the VaTPS9-OE lines exhibited significantly higher superoxide dismutase (SOD) activity (Figure 10A), which corresponded to markedly lower levels of hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻) (Figure 10B,C), collectively indicating an enhanced ROS-scavenging capacity. Accordingly, the malondialdehyde (MDA) content, an indicator of lipid peroxidation, was significantly lower in VaTPS9-OE lines than in WT (Figure 10D). Consistent with these findings, histochemical staining with DAB and NBT revealed weaker signals in VaTPS9-OE leaves than in WT leaves after cold treatment (Figure 11 and Figure 12), and staining intensity was quantified by ImageJ-based image analysis as described in Materials and Methods, further confirming the reduced accumulation of H₂O₂ and O₂⁻. Furthermore, analysis of trehalose metabolism showed that cold-treated VaTPS9-OE lines had significantly higher TPS activity, slightly increased TPP activity, and elevated trehalose content compared with WT (Figure 13).

2.6. VaTPS9 Overexpression Enhances Cold Tolerance of V. amurensis Calli

To investigate the role of VaTPS9 in cold tolerance of V. amurensis, we overexpressed this gene in V. amurensis callus tissues. The overexpression of VaTPS9 in V. amurensis calli was first confirmed by PCR and qRT-PCR (Figure 14). Programmed cooling assays revealed distinct exothermic patterns between VaTPS9-overexpressing and control (CK) calli before and after cold stress, suggesting that overexpression altered freezing behavior following acclimation (Figure 15 and Figure 16). Compared with CK, the overexpression lines exhibited reduced browning after cold stress, indicating alleviated cold-induced injury (Figure 17A). Physiological measurements further supported the enhanced cold tolerance of VaTPS9-overexpressing calli. Relative electrolyte leakage and malondialdehyde (MDA) content were generally lower in VaTPS9-overexpressing calli than in CK after cold stress (Figure 17B,F), indicating improved membrane integrity and reduced lipid peroxidation. Meanwhile, soluble sugar and trehalose contents were higher in VaTPS9-overexpressing calli before and after cold stress (Figure 17C,I). Antioxidant enzyme activities, including peroxidase (POD) and catalase (CAT), were also elevated in VaTPS9-overexpressing calli after cold stress (Figure 17D,E). Moreover, activities of trehalose metabolism–related enzymes, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), were consistently higher in VaTPS9-overexpressing calli during cold acclimation (Figure 17G,H), consistent with enhanced trehalose accumulation.

2.7. Cold Tolerance of V. amurensis Plantlets with VIGS-Mediated Silencing of VaTPS9

To further explore the function of VaTPS9, we subjected tissue-cultured plantlets to virus-induced gene silencing (VIGS). To identify effective VIGS materials, Tobacco rattle virus (TRV) infection was first confirmed by PCR amplification of TRV1 and TRV2 viral sequences in inoculated plantlets (Figure 18A). qRT-PCR further showed that VaTPS9 transcript levels were markedly reduced in three independent TRV1:TRV2(VaTPS9) lines compared with WT and the TRV empty-vector control (Figure 18B), confirming successful silencing. These VaTPS9-silenced plantlets were then used to assess cold tolerance.

Following cold stress, VaTPS9-silenced plantlets exhibited more severe injury than WT and the TRV empty-vector control, whereas no obvious differences were observed among genotypes before stress (Figure 19A). Consistently, membrane damage was increased in VaTPS9-silenced plantlets under cold stress, as indicated by higher MDA content and relative electrolyte leakage (Figure 19B,C). By contrast, soluble sugar and trehalose contents, as well as TPS and TPP activities, were significantly lower in VaTPS9-silenced plantlets after cold stress (Figure 19D–G). Overall, these results support a role for VaTPS9 in cold tolerance of V. amurensis, likely through maintaining trehalose metabolism and cellular integrity during cold stress.

3. Discussion

Vitis amurensis is widely recognized as one of the most cold-hardy species within the genus Vitis and represents an important genetic resource for improving cold tolerance in cultivated grapevine [14]. Through transcriptome-based screening, we identified VaTPS9 as a candidate gene potentially associated with overwintering cold hardiness in V. amurensis shoots. Notably, VaTPS9 expression was strongly induced during the November dormancy period (Figure 1), which coincides with the stage when grapevine tissues undergo metabolic adjustments that support overwintering survival. Previous studies have shown that dormancy establishment in grapevine is closely accompanied by reprogramming of carbohydrate metabolism, which contributes to cold acclimation and freezing tolerance [15,16].

Sequence comparison between VaTPS9 and its homolog from the cold-sensitive Eurasian grape (V. vinifera) revealed six nucleotide differences, including one nonsynonymous substitution resulting in a single amino-acid change (Figure S1). Although the functional significance of this substitution remains to be determined, even minor sequence variation can potentially influence protein properties and stress-related functions. Therefore, divergence between the TPS9 homologs may contribute to the contrasting cold hardiness observed between V. amurensis and V. vinifera [10,14]. Future studies such as allelic replacement or site-directed mutagenesis will be required to directly test the functional importance of this variation.

Members of the trehalose-6-phosphate synthase (TPS) family are generally divided into class I (TPS1–TPS4) and class II (TPS5–TPS11) groups based on sequence features and catalytic properties [17,18]. Class I TPS proteins possess catalytic activity responsible for trehalose-6-phosphate synthesis, whereas class II TPS proteins typically show limited enzymatic activity and are thought to function mainly as regulatory components involved in carbohydrate metabolism and stress signaling [7,18,19]. Increasing evidence suggests that class II TPS proteins participate in regulatory networks linking trehalose metabolism with broader carbon signaling pathways in plants.

In this study, phylogenetic analysis placed VaTPS9 within the class II TPS group (Figure 2 and Figure 3). Functional analyses further demonstrated that VaTPS9 positively regulates cold tolerance. Overexpression of VaTPS9 enhanced cold tolerance in yeast (Figure 6A,B) and improved freezing survival in transgenic Arabidopsis plants (Figure 8A,B). In addition, overexpression reduced browning and membrane damage in V. amurensis calli under cold stress (Figure 15, Figure 16 and Figure 17). Conversely, VIGS-mediated silencing of VaTPS9 significantly increased cold injury and electrolyte leakage (Figure 19). Together, these gain- and loss-of-function results consistently indicate that VaTPS9 plays a positive role in cold stress tolerance.

Physiological analyses suggest that this effect is closely associated with trehalose metabolism. In Arabidopsis, low-temperature treatment of VaTPS9-overexpressing lines induced the expression of AtTPS9, as well as the catalytically active AtTPS1 and AtTPS2, and was accompanied by significantly increased TPS/TPP enzyme activities and trehalose accumulation (Figure 13). Similar increases in soluble sugar content, trehalose levels, and trehalose metabolism enzyme activities were also observed in VaTPS9-overexpressing calli, whereas these parameters were reduced following VaTPS9 silencing (Figure 17 and Figure 19). Trehalose and its precursor trehalose-6-phosphate (T6P) are well-established signaling molecules that integrate carbon status with plant stress responses [7,19]. In this context, the coordinated induction of TPS genes together with increased TPS/TPP activities and trehalose accumulation observed in our study suggests that VaTPS9 may function as a regulatory component that indirectly promotes trehalose biosynthesis under cold stress. Nevertheless, whether this regulatory effect involves altered T6P dynamics or SnRK1-mediated carbon signaling remains to be investigated.

Our findings are consistent with emerging evidence that class II TPS members can contribute to cold tolerance through regulatory roles rather than strong intrinsic catalytic activity [18]. Notably, a grapevine class II TPS, VvTPS10, was recently reported to enhance cold tolerance via engagement with the CBF pathway and ubiquitination-related regulation [20]. Although VaTPS9 and VvTPS10 are both class II TPS proteins, differences in species background (V. amurensis vs. V. vinifera), tissue context (overwintering shoots/calli vs. leaves or other organs), and stress regimes may lead to divergent downstream outputs across studies [15,20]. These consistencies and potential discrepancies highlight the need to test whether VaTPS9 also interfaces with canonical cold transcriptional modules (e.g., CBF/COR) in grape-derived tissues.

In addition to carbohydrate metabolism, VaTPS9 also appears to influence antioxidant defense mechanisms. Under cold stress, VaTPS9-overexpressing Arabidopsis lines exhibited higher SOD activity, reduced ROS accumulation, and lower levels of membrane damage indicators (Figure 10, Figure 11 and Figure 12). Trehalose metabolism has also been associated with improved stress tolerance in plants [21,22]. The enhanced antioxidant response observed in VaTPS9-overexpressing plants therefore provides a plausible explanation for their improved freezing tolerance and reduced cellular damage. Consistent with this interpretation, comparative proteomics in grapevine has shown that cold-resistant genotypes display stronger adjustments in stress-defense and metabolism-related proteins under freezing exposure [23].

Subcellular localization analysis further provides clues to the cellular context of VaTPS9 function. The VaTPS9–GFP fusion protein localized predominantly to the tonoplast (Figure 4) and displayed a broader vacuolar distribution under cold treatment (Figure 5). Because the vacuole plays a central role in osmotic regulation, metabolite compartmentation, and cellular homeostasis during abiotic stress responses [2,24], the altered vacuolar association of VaTPS9 under low temperature may be related to metabolic adjustments occurring during cold adaptation. However, the precise functional relationship between VaTPS9 localization and trehalose metabolism or osmotic regulation remains to be clarified.

Taken together, our results reveal a previously uncharacterized function of VaTPS9, a grapevine class II TPS protein, in enhancing cold tolerance. Based on our findings, we propose a model in which VaTPS9 promotes cold hardiness through coordinated regulation of trehalose metabolism and antioxidant defense. Although class II TPS proteins generally exhibit limited catalytic activity, VaTPS9 may enhance trehalose accumulation by inducing the expression of catalytically active TPS genes under low temperature, leading to increased TPS/TPP enzyme activities and trehalose levels (Figure 13, Figure 17 and Figure 19) [7,18,19]. At the same time, VaTPS9 enhances antioxidant capacity by increasing SOD activity and reducing ROS accumulation, thereby protecting cellular membranes from cold-induced oxidative damage (Figure 10, Figure 11 and Figure 12) [22]. The cold-induced redistribution of VaTPS9 within the vacuolar region (Figure 5) may further reflect adjustments in vacuole-associated metabolic or osmotic processes during cold stress.

Overall, this study identifies VaTPS9 as an important regulator of cold tolerance in grapevine and provides new insights into the functional roles of class II TPS proteins in abiotic stress adaptation. These findings not only contribute to a better understanding of the molecular mechanisms underlying cold hardiness in V. amurensis, but also highlight VaTPS9 as a promising genetic target for improving cold tolerance in grapevine breeding. Future work should investigate the relationship between VaTPS9, T6P signaling, and SnRK1-mediated carbon regulation, as well as explore potential interactions with established cold-response pathways in grapevine.

4. Materials and Methods

4.1. Plant Materials

The cold-tolerant V. amurensis cultivar ‘Shuangfeng’ was used in this study. Plants were grown in the National Field Gene Bank for Amur Grapevine located in Zuojia Town, Jilin Province, China (44°04′ N, 126°05′ E; 190 m above sea level). Leaves, shoots, tendrils, fruits, and roots were sampled at mid-month from June to November 2023 to analyze the temporal and spatial expression patterns of VaTPS9. In addition, one-year-old shoots collected in November from the south-facing side of the espalier at a height of 1.0–1.8 m were used for cloning of VaTPS9.

For virus-induced gene silencing (VIGS) experiments, one-year-old cutting-derived ‘Shuangfeng’ plantlets were prepared. Shoot segments were collected from field-grown plants, soaked in rooting solution, and then planted in pots with regular watering. After the emergence of new shoots, plantlets were subjected to tissue culture propagation and subsequently used for VIGS inoculation experiments. The same tissue culture–derived materials were also used for the generation of transgenic calli overexpressing VaTPS9 and for subsequent physiological analyses.

4.2. Gene Expression Analysis Using Quantitative Real-Time PCR

The gene expression levels were analyzed by quantitative real-time PCR (qRT-PCR). The qRT-PCR assays were performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using SYBR Green Master Mix. Relative expression levels were calculated using the 2^−ΔΔCT method [25,26]. Detailed information on the target genes and their specific primers is provided in Table S1 (Supplementary Material). All qRT-PCR reactions were performed in triplicate for each biological replicate. For statistical analysis, data were analyzed using ordinary one-way ANOVA (Gaussian distribution assumed) in GraphPad Prism 10. Homogeneity of variances was assessed using the Brown–Forsythe test. When ANOVA indicated significant differences, Tukey’s multiple comparison test was applied. Data are presented as means ± SD, and different lowercase letters indicate significant differences at p < 0.05.

4.3. Cloning and Sequence Analysis of the Target Gene VaTPS9

Total RNA was extracted from one-year-old shoots of V. amurensis ‘Shuangfeng’ collected in November and reverse-transcribed into cDNA [27] The coding sequence (CDS) of VaTPS9 was amplified using cDNA as the template. PCR products were verified by agarose gel electrophoresis and purified prior to cloning. The VaTPS9 CDS was inserted into the plant expression vector pRI101-GFP to generate the recombinant construct. The resulting plasmid was transformed into Escherichia coli DH5α, and positive clones were confirmed by antibiotic selection and sequencing [20]. Primer sequences used in this study are listed in the Supplementary Materials.

The VaTPS9 sequence and its orthologs were retrieved from the NCBI database. The cloned nucleotide sequence of VaTPS9 was translated into its corresponding amino acid sequence, and protein physicochemical properties were predicted using online tools [28]. Nucleotide and amino acid sequence alignments were performed using SnapGene 7.1.2 software. Phylogenetic analysis of the TPS gene family was conducted using MEGA 11 software [29]. Multiple sequence alignment was carried out with the MUSCLE method, and a phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with 1000 bootstrap replications to assess branch reliability.

4.4. Subcellular Localization Analysis of VaTPS9

Arabidopsis protoplasts were prepared according to previously described methods and transiently transformed with the VaTPS9-GFP fusion construct using a polyethylene glycol (PEG)-mediated method [30]. Fluorescence signals were observed and recorded using a confocal laser scanning microscope. To examine the effect of cold treatment on the subcellular localization of VaTPS9, Arabidopsis seedlings were subjected to cold treatment at 4 °C for 4 and 6 days prior to protoplast isolation.

4.5. Functional Analysis of VaTPS9 in Yeast Under Cold Stress

The VaTPS9 coding sequence was cloned into the yeast expression vector pYES2-NTB and transformed into the yeast strain BY4741. Positive transformants and empty-vector controls were cultured under induction conditions, then yeast cells harboring pYES2-NTB-VaTPS9 and the empty vector (pYES2-NTB) were exposed to 4 °C, 0 °C, −10 °C, and −20 °C for 24, 48, 72, and 96 h.

Cold tolerance was evaluated using a serial dilution spot assay [31]. After treatment, cultures were serially diluted (10⁰, 10⁻¹, and 10⁻²) and spotted onto SG-Ura plates. Plates were incubated at 30 °C for 3 days, Colonies exhibiting distinct differences were selected for image analysis. The analytical method is described in Section 4.9.

4.6. Generation of Transgenic Arabidopsis Lines and Freezing Tolerance Assays

The VaTPS9 gene was introduced into Arabidopsis using the floral dip method [32]. Homozygous T3 transgenic lines were obtained through antibiotic selection. Twenty-five-day-old potted transgenic plants and wild-type Arabidopsis were used for freezing tolerance assays. Prior to freezing treatment, plants were cold-acclimated at 4 °C for 12 h, followed by exposure to −4 °C for 15 h. After freezing treatment, plants were allowed to recover at 4 °C in darkness for 12 h and then transferred back to normal growth conditions. Plant phenotypes were recorded at 7 and 14 days after recovery, and survival rates were counted at 14 days [33]. For each treatment, 24 seedlings per line were used, and the survival rate of Arabidopsis lines was manually counted.

4.7. Overexpression of VaTPS9 in Grapevine and Cold Treatment

To investigate the role of VaTPS9 in its native species, the coding sequence of VaTPS9 was cloned into a plant overexpression vector driven by a constitutive promoter and introduced into V. amurensis callus tissues via an established Agrobacterium tumefaciens–mediated transformation system [34]. Transgenic calli were screened and verified by PCR and quantitative real-time PCR (qRT-PCR).

Following transformation, callus tissues were maintained under normal growth conditions for 3 d to allow recovery from transformation-associated stress and stabilization of transgene expression. Samples collected at this stage, immediately before the initiation of cold treatment, were defined as the pre-stress control. At least three independent VaTPS9-overexpressing callus lines showing stable transgene expression were selected for subsequent analyses, while non-transformed calli were used as the control (CK).

For cold stress treatment, calli were first exposed to 0 °C for 6 h and subsequently returned to normal growth conditions (24 °C) for 12 h to allow recovery. In addition, for cold acclimation treatment, calli were incubated at 4 °C for 9 d. After the respective treatments, samples were collected for phenotypic observation, physiological and biochemical analyses, and freezing behavior measurements [35].

Grapevine callus tissues were used as a controlled experimental system that allows uniform cold treatment and precise assessment of cellular responses to cold stress and cold acclimation.

4.8. Virus-Induced Gene Silencing (VIGS) of VaTPS9 in V. amurensis

Virus-induced gene silencing (VIGS) was employed to suppress VaTPS9 expression in V. amurensis plantlets. A specific fragment of VaTPS9 was amplified and inserted into the pTRV2 vector to generate the TRV2–VaTPS9 construct. The pTRV1 plasmid and the empty pTRV2 vector were used as controls. TRV (Tobacco rattle virus) vectors were obtained from Beijing Huayueyang Biotechnology Co., Ltd. (Beijing, China). The silencing fragment was designed using SnapGene software, and primers used for vector construction are listed in Supplementary Table S1. The resulting constructs were introduced into Agrobacterium tumefaciens strain GV3101 and co-infiltrated into grapevine plantlets following a Tobacco rattle virus (TRV)–based VIGS protocol as previously described [36].

After VIGS inoculation, plantlets were maintained under normal growth conditions for 3 d to allow establishment of viral infection and effective gene silencing while minimizing acute stress responses. Samples collected at this stage, immediately prior to cold stress treatment, were designated as the pre-stress control. Successful viral infection was confirmed by PCR amplification of TRV1 and TRV2 sequences, and the silencing efficiency of VaTPS9 was further verified by quantitative real-time PCR (qRT-PCR). Plantlets exhibiting a significant reduction in VaTPS9 transcript levels were selected for subsequent experiments, with at least three independent silenced plantlets analyzed for each treatment.

For cold stress treatment, VIGS-treated and control plantlets were exposed to chilling conditions at 4 °C for 7 d, while plants maintained at 24 °C served as normal growth controls. The duration of cold treatment was selected to accommodate the reduced stress tolerance of VIGS-treated plantlets caused by viral infection, allowing reliable evaluation of cold stress responses without excessive lethality. After treatment, phenotypic assessment and physiological measurements were conducted.

4.9. Measurement of Physiological Indexes

Reactive oxygen species (ROS) accumulation was examined by histochemical staining with 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) following the method described in [37] with minor modifications. Briefly, leaves were vacuum-infiltrated, incubated in the dark, destained, and photographed. For DAB staining, leaves were immersed in freshly prepared DAB solution (Solutions A and B mixed 1:1, 20×), vacuum-infiltrated, incubated for 8 h, and destained in boiling 70% ethanol; the brown precipitate indicates H₂O₂ accumulation. For NBT staining, leaves were immersed in NBT solution (1 mg mL⁻¹ NBT in 10 mM phosphate buffer, pH 7.8), vacuum-infiltrated, incubated for 8 h, and destained in boiling 70% ethanol; the blue formazan precipitate indicates O₂⁻ accumulation. Images were acquired under identical settings, and staining intensity was quantified in ImageJ (Fiji) from 8-bit grayscale images as mean gray value within leaf Region of Interest (ROI) after background subtraction (≥3 biological replicates per genotype and treatment) [38], As described in Section 4.5, yeast colonies treated at −10 °C for 72 h were processed using the same image analysis method.

Physiological and biochemical parameters were measured in Arabidopsis and grapevine samples subjected to cold treatment. Soluble sugar, malondialdehyde (MDA), and trehalose contents, as well as the activities of superoxide dismutase (SOD), Hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻), catalase (CAT), peroxidase (POD),trehalose-6-phosphate synthase (TPS), and trehalose-6-phosphate phosphatase (TPP), were determined using commercial assay kits according to the manufacturer’s instructions (BOXBIO, Beijing, China).

For grape callus tissues, a programmable cooling system was used to analyze freezing-induced exothermic characteristics. Samples were cooled at a constant rate of 10 °C h⁻¹, and tissue temperature was continuously recorded. Exothermic events were identified based on temperature–time curves [39]. Membrane stability under cold stress was assessed by measuring relative electrolyte leakage, which was determined by calculating (C1/C2) × 100% according to established methods [40,41].

4.10. Statistical Analysis

All data are presented as means ± standard deviation (SD). Statistical analyses and figure generation were performed using GraphPad Prism 10, while temperature–time data obtained from programmed cooling experiments were processed and visualized using Origin 2021 (OriginLab Corporation, Northamption, MA, USA). For experiments involving two independent factors, such as genotype and temperature or treatment duration, differences among groups were analyzed by two-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison test. For comparisons involving a single factor, one-way ANOVA followed by Tukey’s multiple comparison test or Student’s t-test was applied as appropriate.

For qRT-PCR analysis, all reactions were performed in triplicate for each biological replicate. Statistical analysis of qRT-PCR data was conducted using ordinary one-way ANOVA (Gaussian distribution assumed) in GraphPad Prism 10. Homogeneity of variances was assessed using the Brown–Forsythe test. When ANOVA indicated significant differences, Tukey’s multiple comparison test was applied. All experiments were performed with at least three independent biological replicates. Differences were considered statistically significant at p < 0.05, and different lowercase letters indicate significant differences among groups.

5. Conclusions

This study demonstrates that VaTPS9 plays a crucial role in cold tolerance in V. amurensis. Overexpression of VaTPS9 enhanced cold tolerance in yeast, Arabidopsis, and grapevine calli, while silencing VaTPS9 compromised cold stress resistance. Mechanistically, VaTPS9 enhances cold tolerance by promoting trehalose biosynthesis, maintaining membrane integrity, and reducing oxidative damage during cold stress. These findings highlight VaTPS9 as a potential candidate gene for improving cold resilience in grapevine breeding programs aimed at expanding grapevine cultivation to colder regions. Future research should focus on understanding the molecular mechanisms underlying VaTPS9 regulation of trehalose metabolism and its interactions with other cold-responsive genes.