The plant material used in this study was the potato cultivar Désirée provided by our laboratory. The tubers were planted in a climate chamber, in a vermiculite substrate mixed with soil, in a 1:1 ratio, v/v, for 4 weeks at a temperature of 23°C ± 1°C, 16h/8h(day/night). The seeds of WT Arabidopsis thaliana (Columbia (Col-0) ecotype) and 3 T3 transgenic lines (L1-L3) were vernalized for 3 days in 1/2MS medium, transferred to an artificial climate chamber at 24°C and a relative humidity of 80% for ten days, and then transplanted to a vermiculite medium with small square pots (7x7cm). Next, plants were grown for 3 weeks (Kim and Hwang, 2015). Samples were taken before and after salt stress treatment and the collected fresh leaves were quickly placed in −80°C for storage (Zhang et al., 2003). At the same time, chlorophyll content, soluble sugar, SOD, MDA, and other physiological indexes and enzyme activities were measured before and after salt stress treatment.

The StVQ31 gene was amplified through PCR using primers specific to the gene ( Table 1 ). The CDS of StVQ31 was ligated to the pCAMBIA1302 overexpression vector within the KpnI and BstbI restriction sites. The recombinant plasmid was verified by sequencing and was then cloned in E. coli DH5α. Subsequently, it was introduced into Agrobacterium GV3101, which was for the genetic transformation of Arabidopsis plants by the floral-dip method (Zhao et al., 2018). When Arabidopsis seeds matured, the T0 generation seeds were harvested. The T0-generation seeds were then sowed on 1/2 MS medium containing 50μg/ml kanamycin, and 16 lines were identified through PCR verification ( Figure 1A ). The above steps were repeated until T3 transgenic overexpression lines were obtained.

We obtained the properties of StVQ31 from the ExPASy website, including its physical and chemical characteristics (http://web.expasy.org/protparam/) (Pedrini et al., 2015). Then, the secondary structure of StVQ31 was predicted using the PHD software (http://www.predictProtein.org/) (Sajid et al., 2018). In addition, the conserved motifs of StVQ31 were determined by MEME (version 4.12.0) (Bailey et al., 2009).

The CDS region of StVQ31 (after removal of the stop codon) was ligated to the pCAMBIA1302 vector carrying a green fluorescent protein sequence to produce pCAMBIA1302-StVQ31: GFP. The recombinant plasmid was transferred to Agrobacterium and then infiltrated into tobacco leaves, with the nuclear maker AtWRKY25-mCherry used as a control. All transformed tobacco plants grew in the dark at 22°C for 16 hours and then returned to normal conditions. The GFP and RFP fluorescence signals were observed by IX83-FV1200 confocal laser-scanning microscopy two days after tobacco inoculation.

Seeds from the WT and transgenic plants were cultivated in a growth medium called 1/2 MS, supplemented with 150 mM NaCl. The seeds were incubated at 4°C for 3 days, then transferred to 22°C, with 16 hours of light/8 hours of darkness, and incubated for 10 days. Radicle appearance from the seed coat was used as the criterion for seed germination (Stacey et al., 2016). Meanwhile, seeds of WT and transgenic plants (homozygous lines) were placed in 1/2MS medium for the subsequent experiments. After 7 days, seedlings with uniform appearance and growth were selected, transplanted into 1/2 MS medium containing 150mM NaCl, and grown vertically for 7 days. Subsequently, the changes in root length were recorded. In addition, the salt tolerance of 4-week-old Arabidopsis thaliana transgenic plants was evaluated by irrigation with 150 mM NaCl solution every 2 days for 10 days. The phenotypes of the plants were photographed after 7 days of salt treatment.

Ten physiological indexes in WT and transgenic plants were measured before and after the application of salt treatment (150 mM NaCl). Approximately 0.5 g of leaf tissue, avoiding the midvein of the leaves, was sampled and quickly placed in liquid nitrogen. The samples were fully ground and 9 folds volumes of pH 7.4 PBS buffer were added. After centrifugation at 12,000 ×g at 4°C for 30 min, the supernatant was taken, and the protein concentration was determined using the BCA Protein Assay Kit, following the manufacturer’s instructions (Stanley et al., 2020). Chlorophyll was determined based on the protocol by Guo et al., with slight modifications (Guo et al., 2022). The measurement of soluble sugars was slightly modified based on the Dubois et al. (1951) method, and the content of MDA was determined by the thiobarbituric acid (TBA) method (Basit et al., 2022). The SOD activity was determined using a total SOD assay kit (the wst-8 technique; Beyotime, China) (Chen et al., 2019). Furthermore, the CAT, POD, and Pro antioxidant enzyme activities, as well as the H₂O₂ and O₂ ^- content, were determined using the Solarbio Kit (Beijing, China) following the guidelines provided by the manufacturer (Ren et al., 2021).

To investigate the regulatory mechanisms of StVQ31, we further examined the expression changes of genes associated with salt stress. Four-week-old transgenic Arabidopsis were moved to MS liquid medium with 150 mM NaCl for 8 hours, while the remainder seedlings were kept in MS liquid medium as a control. Total RNA from the transgenic Arabidopsis leaves was extracted using the RNAprep Pure Plant Total RNA Extraction Kit (DP432), and cDNA was synthesized using the FastKing cDNA First Strand Synthesis Kit (Degenomics)(KR116) (Tiangen, Beijing, China). The template cDNA was then diluted according to experimental requirements. The NCBI primer design suite was used to design gene-specific primers ( Table 1 ) with Actin as the internal reference. qPCR was performed using the ABI QuantStudio7Flex system (Applied Biosystems, USA). The reaction mixture consisted of 2 μl of cDNA, 10 μl of 2× Super Real Color PreMix SYBR (Tiangen, China), 0.6 μl of gene-specific primers, and ddH₂O was added up to 20 μl. The reaction conditions were set to: 95 °C 15 min; 95 °C 10 s, 60 °C 32s, 72 °C 32 s, 95 °C 15 s, 60 °C 60 s, 95 °C 15 s for a total of 40 cycles. The expression levels of the evaluated genes were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). Three biological replicates were performed for each sample.

All data were expressed as the mean ± standard errors of three replicates. Prism 7.0 was used for image rendering. The mean differences between groups were statistically analyzed by independent sample t-test, analysis of variance (ANOVA), and post hoc LSD test. SPSS 27.0 was used for correlation analysis.

A gene-specific amplification primer StVQ31-F/R was designed based on the gene sequence ( Table 1 ), and total RNA was extracted from potato leaves for reverse transcription for the isolation of the complete cDNA of StVQ31 ( Supplementary File 1A ). DNA Sequencing revealed that the StVQ31 gene fragment amplified by PCR had an open reading frame of 672bp, encoding a protein consisting of 223 amino acids and contained a conserved VQ domain ( Supplementary File 1C ; Figure 2A ). StVQ31 contained only one exon. Based on the ExPASy ProtParam prediction, the theoretical MW of the encoded protein was 23.697 kDa, and the theoretical PI was 8.58. Ser (13%), Gly (10.3%), Val (7.6%), and Thr (7.6%) residues were the most abundant in the StVQ31 protein amino acid sequence. The overall mean hydrophilicity of StVQ31 was -0.295, indicating that the protein was hydrophilic. The secondary structure analysis showed that StVQ31 protein was composed by α helixes covering 19.73%, β fold covering 1.79%, and random coils covering 78.48% of the protein, respectively ( Figure 2B ). The motif analysis revealed that motif1 depicted in Figure 2C , served as the central conserved region of the protein and encompassed the functional domain ( Figure 2A ). In addition, the StVQ31 gene promoter contained TGA-element cis-elements, which means it may be involved in the regulation of plant growth and development.

In order to confirm the subcellular location of StVQ31, we removed the termination codon and fused StVQ31 to a GFP vector expressed by the 35s promoter of the tobacco mosaic virus (TMV). Individual cells carrying the control GFP vector exhibited detectable green fluorescence, while the cells expressing STVQ31-GFP emitted a green fluorescence signal from within the nucleus. Thus, the green fluorescence signal emitted by STVQ31-GFP could only be detected in the nucleus, and it overlapped with the red fluorescent nuclear marker AtWRKY25-mCherry ( Figure 3 ), thus indicating that StVQ31 is localized in the nucleus.

The transgenic plants were identified by PCR amplification after DNA extraction. Distinct bands were amplified in the 16 transgenic lines identified. In contrast, no corresponding fragment was amplified in the WT plants ( Figure 1A ). These results were subsequently validated by RT-PCR analysis ( Figure 1B ), confirming the successful overexpression of StVQ31 in transgenic Arabidopsis lines. In the early growth stages of Arabidopsis plants, we observed that the StVQ31 and wild-type plants exhibited certain differences, with StVQ31 overexpressing plants being bigger than wild-type Arabidopsis ( Figure 4B ). This discrepancy may be attributed to the elevated expression of StVQ31 protein in the stem, suggesting that it is involved in the regulation of plant development. This result is consistent with the phenotypes exhibited in AtVQ29 expressing plants (Cheng et al., 2012). Furthermore, we conducted a statistical analysis of the phenological stages and flowering dates of transgenic and wild-type Arabidopsis thaliana lines. We found that the flowering in transgenic plants occurred at about 30 days after germination, showing a significant increase in flowering time compared with wild-type Arabidopsis ( Figure 4A ). The StVQ31 gene not only affected the flowering time of Arabidopsis but also affected its growth and development. This implies that it potentially has pivotal regulatory functions that control Arabidopsis growth. Based on this, we observed the phenotypes of transgenic and wild plants under salt stress. We found that in contrast to the wild type, the leaves of the transgenic plants exhibited a vibrant green color, while the wild type plants had yellow leaves, short plants, and were in a wilted state ( Supplementary File 2 ). These results indicated that the transgenic plants exhibited an increased tolerance to salt stress.

Seed germination serves as the foundation for plant growth and overall plant vigor. Hence, examining the impact of salt stress on seed germination is critical to assess the resistance of the plants. Under 0mM NaCl treatment, the germination rates of transgenic lines were consistent with that of WT ( Figure 5A ; Supplementary File 3 ). After salt treatment, the germination rates of the three transgenic lines evaluated were 89.29%, 78.57%, and 82.14%, respectively ( Figure 5D ; Supplementary File 3 ), increased by 66.67%, 46.67%, and 53.33%, respectively, compared to the WT (germination rate of 53.57%). At the same time, the transgenic plants had an increased root length. Specifically, compared to WT plant (3.53cm), the root length of the three transgenic lines was 4.27cm, 4.37cm, and 4.76cm, respectively ( Figure 5C ; Supplementary File 3 ), increased by20.96%, 23.80%, and 34.84%, respectively. While under 0mM NaCl treatment, the root length of transgenic lines were consistent with that of WT ( Figure 5B ; Supplementary File 3 ).These findings suggest that StVQ31overexpression reduced salinity-mediated inhibition of seed germination, conferring a stronger capacity to the seedlings to reduce salt stress damage and maintain longer root length.

Under abiotic stress conditions, considerable amounts of excessive ROS, such as H₂O₂ and O₂ ⁻ are generated within plant cells. This leads to a rise in lipid peroxidation and subsequent cellular oxidative stress ( Figures 6A, B ). The contents of H₂O₂ and O₂ ⁻ in Arabidopsis leaves were determined before and after salt treatment. The H₂O₂ and O₂ ⁻ contents in WT under salt stress were 1.26 and 1.53 times higher than in the control conditions. The H₂O₂ and O₂ ⁻contents increased by 3.45%~5.76% and 17.89%~24.33%, respectively, while the WT increased even more. The increase of transgenic plants was significantly lower than that of WT ( Figures 6A, B ). The WT plants, overall, did not exhibit any notable variation. Next, we measured the activities of CAT, POD, and SOD. CAT catalyzes H₂O₂ breakdown, while SOD and POD are responsible for the reduction of stress-induced H₂O₂ and O₂ ⁻, respectively. Under salinity stress treatment, the enzyme activities increased in both transgenic plants and WT in different amplitudes. Compared with WT, CAT, SOD, and POD in transgenic plants were 1.66-1.68-fold, 1.37-1.38-fold, and 1.69-1.76-fold higher than WT, respectively ( Figures 7A–C ). These results are consistent with the H₂O₂ and O₂ ⁻ contents, suggesting that transgenic plants could reduce cell oxidative damage under stress conditions by increasing ROS clearance capacity.

We examined the alterations in proline levels, a significant stress indicator, before and after subjecting plants to salt stress. The results found that The Pro content in WT increased 1.31-fold after salt stress, while the Pro content in transgenic plants was 1.5-1.54-fold increased ( Figure 8D ). Thus, the increase of Pro content in transgenic plants was considerably higher than that in WT, by 1.19-1.21-fold ( Figure 8D ). MDA content analysis was conducted to evaluate the oxidative damage caused by salt stress in both WT and transgenic plants. The results showed that all StVQ31 overexpression lines produced significantly less MDA (0.216 to 0.277µmol g⁻¹ FW) than WT plants (0.36µmol g−1 FW) ( Figure 8B ). These results suggest that overexpression of StVQ31 may contribute to maintaining membrane permeability under salt stress by enhancing their antioxidant metabolism. The accumulation of soluble sugars was similar to that of proline. After being subjected to salinity stress ( Figure 8A ), the soluble sugar content in WT exhibited a substantial increase of 39.17%. In transgenic plants, soluble sugar content showed an increase ranging from 56.22% to 62.07%. The total protein content decreased in WT decreased under salt stress by 25.82% and in transgenic plants by 3.66%-5.58%. The trend was similar to that of MDA, albeit with a different amplitude of decrease ( Figure 8C ). Thus, compared with WT, the reduction was lower in transgenic plants. The chlorophyll content is directly related to the light-harvesting process in plants. It can be used as an indicator of plant photosynthesis to determine the plant’s physiological status and salt tolerance. Under salt stress, chlorophyll content decreased, affecting plant growth and development. The contents of chlorophyll a and b and the a/b ratio decreased notably under 150 mM NaCl treatment, but the total content of chlorophyll, chlorophyll a, and chlorophyll b in the transgenic plants was significantly higher than that in the WT plants ( Figures 9A–D ). These results indicate that the photosynthetic capacity of transgenic plants after salt treatment is even higher than that of WT plants.

The involvement of the StVQ31 gene in the adaptive response of plants to salt stress was determined by analyzing the expression of four genes that function as stress markers. Under high salinity conditions, the expression of the genes encoding LEA 4-5, NCED-3, ERD, and At2g38905 was notably upregulated in transgenic plants, as shown in Figures 10A–D . While under non-stress conditions, their expression in transgenic plants and wild-type plants remained unaltered. Taken together, these findings suggest that the enhanced salt tolerance in plants that overexpress StVQ31 genes is also associated with increased expression levels of LEA 4-5, NCED-3, ERD, and At2g38905genes.

Based on the results, 61.99% of the measured indicators were correlated, of which 47.95% were strongly correlated and 39.77% were weakly correlated ( Figure 11 ). Root length was found to have a positive correlation with SOD and POD activities (P<0.01), while the levels of superoxide anion and MDA were significantly negatively correlated. This suggests that reactive oxygen species play a role in regulating root growth. Similarly, the germination rate showed a positive correlation with SOD and CAT, and a negative correlation with superoxide anion and MDA levels. These findings indicate that superoxide anion and MDA have varying degrees of negative regulatory effects on both germination rate and root length. Moreover, total protein, SOD, POD, CAT, and Pro showed positive correlations with other indexes, whereas MDA and H2O2 were predominantly negatively correlated with other indexes. Specifically, CAT was highly correlated with SOD and POD. Root length was highly correlated with the expression of At2g38905 genes and NCED-3. Moreover, certain indexes, such as H2O2 and Chlorohyll b, were not correlated. The findings suggest a possible correlation between the indexes, which could collectively impact the salt tolerance of the StVQ31 overexpressing plants.