The beetroot (Beta vulgaris L. ssp. vulgaris) seeds used in this study were of the commercial cultivar ‘AK3018’. The seeds were purchased from Jinyangpu Agricultural Co., LTD, located in Beijing, China. The seeds were obtained as a commercial product for agricultural use and did not require any specific permission for collection. Formal identification of the plant material was conducted by the seed supplier based on the cultivar characteristics described in the product catalog. As the material is a widely available commercial cultivar, no voucher specimen was deposited. This study complied with relevant institutional, national, and international guidelines and legislation for the use of cultivated plant material in experimental research.

Seeds were sown in seed trays and placed in a growth chamber with a light/dark cycle of 16 hours at 26 °C and 8 hours at 22 °C. Seedlings with 6–8 leaves and consistent growth vigor were selected for NaCl root irrigation treatment at concentrations of 0, 200, 400, 600, and 800 mM. After 7 days, roots and leaves were harvested, immediately frozen in liquid nitrogen, and stored at −80 °C for later use. Arabidopsis seeds of the Columbia WT (Col-0) were used. Prior to sowing, the seeds were disinfected and then sown on MS medium. The 15-day-old seedlings were transferred to seedling trays and placed in a growth chamber for continued cultivation.

Total RNA was extracted from salt-tolerant beet leaves using the TransZol Plant reagent kit (Kangwei Century) and then reverse transcribed to synthesize complementary DNA (cDNA). Specific primers BvKUP13-F/R, designed by NCBI (accession number: XM010671674.3), were used for PCR amplification, with the cDNA sequence from salt-tolerant beet leaves serving as the template. The target band was obtained, purified using gel electrophoresis, extracted and ligated into the T vector pEASY-T1. Following identification through colony PCR, the construct was submitted to Beijing Huada Biotechnology Co., Ltd. for sequencing, and the correctly sequenced bacterial cultures were preserved. The conserved domains of the BvKUP13 protein were analyzed using NCBI’s Conserved Domain Database (CDD). The predicted secondary structure of the protein was analyzed using SWISS-MODEL, and its transmembrane domains were assessed using TMHMM. The sequence alignment and conservation of BvKUP13 and homologs were visualizedusing Espript. The accession numbers of KUP homologs from other species were: Chenopodium quinoa (CAN99589.XP021774497.1; Cq), Triticum aestivum (XP044424600.1; Ta), Hordeum vulgare (KAE8804611.1; Hv), Arabidopsis thaliana (OAPO8615.1; At), and Malus domestica (XP008346085.2; Md). The phylogenetic tree was constructed using the Neighbor-Joining method in MEGA orrelevant software.

Target gene amplification was performedusing gfpBvKUP13-R/F primers, and the full-length BvKUP13 cDNA was amplified viaPCR. The resulting product was ligated into the pAN580 expression vector, which had been pre-digested with BamHI and XbaI restriction enzymes, yielding the recombinant construct BvKUP13-pAN580. Nicotiana benthamiana plants were grown under controlled conditions at 25 °C for 15-20 days. Leaf-derived protoplasts were isolated via enzymatic digestion of young leaf tissue using standard protocols. A total of 100 µL of the prepared protoplast suspension was mixed with 10 µL of plasmid DNA and 100 µL of PEG4000 solution (w/v). After incubation, the mixture was washed to remove residual PEG, and the supernatant was discarded, and approximately 100 µL of protoplasts were retained. The samples were then observed using either fluorescence or laser scanning confocal microscopy. Excitation wavelengths of 488 nm and 561 nm were applied to detect GFP and the endoplasmic reticulum marker, respectively.

The full-length BvKUP13 was amplified by PCR using the GFPBvKUP13-R/F primers. The amplified product was recovered from the gel and recombined with the linearized pBWA(V)H2S-ccdB-egfp vector, which had been digested with BsaI/Eco31I enzymes, to obtain the recombinant vector pBWA(V)H2S-BvKUP13-GFP. The endoplasmic reticulum (ER) marker plasmid was transformed into Agrobacterium, and then co-suspended with Agrobacterium harboring the target gene. Prior to injection, the two Agrobacterium cultures were mixed at a 1:1 ratio and infiltrated into one-month-old tobacco leaves. Observations were performed after two days of cultivation under low-light conditions.

Real-time PCR was used to measure the relative expression levels of BvKUP13 in the roots, petioles, and leaves of the salt-tolerant beet variety AK3018, as well as in the roots and leaves of seedlings after 7 days of NaCl stress at concentrations of 0, 200, 400, 600, and 800 mM. Changes in the expression levels of BvKUP13 in the roots and leaves were also measured after 48 hours of treatment with 600 mM NaCl. Specific primers for quantitative real-time PCR were designed based on the cloned BvKUP13 sequence: qBvKUP13-F/R. The beet Actin gene served as the internal reference, with primers designed as BvActin-F/R. Total RNA was extracted from both aerial and root tissues using the TransZol Plant reagent kit, followed by reverse transcription into cDNA. The cDNA was subsequently diluted 5-fold and used as the template for real-time quantitative PCR. Each treatment was performed with three biological replicates and three technical replicates. Relative gene expression was calculated using the 2^−ΔΔCt method.

The full-length BvKUP13 cDNA was amplified using pBI101-BvKUP13-F/R primers, which target the gene. The PCR products were then inserted into the pBI101-km-35S::Gus-Hm vector, previously linearized by BamHI restriction enzymes, to generate the recombinant plasmid. This plasmid was subsequently transformed into the Agrobacterium tumefaciens strain GV3101. The Agrobacterium-mediated floral dip method was used to transform WT, generating T₀ generation seeds. All T₀ generation seeds were sown on MS solid medium supplemented with Kanamycin (50 mg/L) to generate T1 generation seeds. DNA was extracted from the target lines, and PCR was performed using kanamycin resistance primers Kan-F/R to identify and screen positive lines. These positive lines were subsequently self-pollinated to obtain T₃ generation Arabidopsis lines overexpressing BvKUP13. RNA was extracted and reverse transcribed, utilizing WT Arabidopsis as a control and AtActin as the reference gene, to assess the expression of BvKUP13 in three homozygous transgenic lines. All special primer sequences are shown in Table (S1).

The concentrations of NaCl used in the experiments were determined through preliminary trials to reflect the specific salt sensitivity of each developmental stage under investigation. Homozygous transgenic Arabidopsis seeds were sown on MS medium supplemented with 0, 50, or 100 mM NaCl for germination and survival assays. Germination rates were recorded after 7 days, and survival rates after 14 days. For root development analysis, 15-day-old seedlings grown on standard MS medium were transferred to vertical MS plates containing 0, 75, or 150 mM NaCl and cultivated for an additional 7 days, after which root lengths were measured. Following 20 days of plate growth, seedlings were transplanted into soil and grown for another 20 days. Subsequently, these soil-grown plants were irrigated with 0, 100, or 200 mM NaCl solutions for 7 days prior to phenotypic evaluation. Fresh and dry weights were measured using an electronic balance. Leaf discs (1 cm in diameter) were collected for senescence assays under 300 mM NaCl treatment. Chlorophyll content was assessed using a SPAD-502 meter, and the maximum photochemical efficiency of PSII (Fv/Fm) was measured using a chlorophyll fluorescence imaging system. For ion content analysis, plant tissues from the 200 mM NaCl treatment were dried at 80 °C to constant weight, ground into powder, and digested in 10% nitric acid. Na⁺ and K⁺ contents were quantified using a flame photometer. Ion-specific fluorescence probes were obtained from Shanghai Maokang Biotechnology Co., Ltd., and staining was performed according to the manufacturer’s instructions. Fluorescence signals were observed using a stereo fluorescence microscope. Additionally, fresh leaves were stained with NBT, DAB, and Evans blue to assess reactive ROS accumulation and cell death. Activities of superoxide dismutase SOD and POD, as well as MDA and Pro contents, were measured using commercial assay kits (Greysun Biotech Co., Ltd.) in plants treated with 100 mM and 200 mM NaCl.

All statistical analyses were performed using SPSS 26.0 software, and graphs were generated using Microsoft Excel. Data are expressed as the mean ± standard deviation (SD). Statistical differences were determinedusing one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (MRT). Differences were considered statistically significant at P<0.05. Significant differences between groups are indicated by different lowercase letters (e.g., a, b, c).

In the transcriptomic dataset under salt stress (AK3018), BvKUP13 expression was significantly increased. The full-length cDNA of BvKUP13 was obtained using sequencing and RT-PCR. The CDS of BvKUP13 was 2199 bp long and encoded a 733-amino acid protein, with a predicted molecular weight of 81.76 kDa and an isoelectric point (pI) of 8.96. Sequence alignment with the NCBI database revealed that BvKUP13 contained conserved domains of the K-trans and Kup superfamilies (Figure 1A), classifying it as a member of the high-affinity K⁺ transporter family. The predicted secondary structure of the protein consists of 56.81% α-helix, 3.93% β-sheet, 26.31% random coil, and 12.96% extended strand. Transmembrane prediction indicated that the protein possesses 12 transmembrane domains (TM1–TM12). BvKUP13 showed high sequence homology with CqKUP13 from quinoa (90.74%), TaKUP13 from wheat (86.45%), HvKUP13 from barley (86.20%), AtKUP13 from Arabidopsis (82.13%), and MdKUP13 from apple (88.40%) (Figure 1B). Phylogenetic analysis of amino acid sequences from BvKUP13 in sugar beet and KUP proteins from 16 species, including wheat and Arabidopsis, revealed that BvKUP13 clusters with spinach and quinoa proteins, both belonging to the Chenopodiaceae family, indicating close evolutionary relationships (Figure 1C).

To investigate the subcellular localization of BvKUP13, transient co-expression assays were performed in two independent Nicotiana systems. First, tobacco protoplasts were co-transformed with the pan580-BvKUP13-GFP recombinant vector and an ER red fluorescent marker, using the pan580-GFP empty vector as a negative control. Confocal imaging revealed that the green fluorescence of BvKUP13-GFP co-localized with the red fluorescence of the ER marker, indicating that BvKUP13 is localized to the ER in protoplasts (Figure 2A). To further validate this result, the fusion construct pBWA(V)H2S-BvKUP13-GFP and the ER marker were introduced into tobacco leaf epidermal cells via Agrobacterium-mediated infiltration. Confocallaser scanning microscopy showed that the green fluorescence of BvKUP13-GFP displayed a typical reticulate distribution pattern, which overlapped precisely with the red ER marker signal (Figure 2B). This co-localization was evident from the merged images, where a strong yellow fluorescence was observed. These findings provide compelling evidence that BvKUP13 is localized to the endoplasmic reticulum.

To investigate the expression pattern of the BvKUP13 under salt stress, real-time PCR was used to analyze its expression in the leaves and roots of sugar beet at various NaCl concentrations and time points. Under normal conditions, the BvKUP13 was mainly expressed in the roots, petiole and leaves, with the highest expression in the leaves and lowest in the roots (Figure 3A). Under different NaCl concentrations, the expression of BvKUP13 in both leaves (Figure 3B) and roots (Figure 3C) initially increased and then decreased, and expression peaked at 600 mM NaCl. After 48 hours of treatment at this concentration, peak expression in leaves occurred at 16 hours (Figure 3D), and in the roots at 20 hours (Figure 3E). These results suggested that the BvKUP13 can be significantly induced by salt stress.

The pBI101-BvKUP13 overexpression vector was constructed, and WT was genetically transformed using the Agrobacterium-mediated floral dip method. Homozygous transgenic lines were obtained through self-pollination. Three resistant lines (OE3, OE5,and OE9) were randomly selected and screened for kanamycin resistance (50 mg/L) through the T₃ generation (Figure 4A). Genomic PCR analysis confirmed the insertion of the foreign gene into the genomes of these plants (Figure 4B). Real-time PCR analysis revealed that the expression of BvKUP13 in transgenic Arabidopsis was significantly higher than in WT plants, with varying expression levels among different lines (Figure 4C), confirming the successful generation of transgenic Arabidopsis.

To examine the impact of salt stress on the growth of transgenic Arabidopsis, seeds were planted in MS medium containing varying NaCl concentrations, and the plant phenotypes were observed. The results indicated no significant difference between WT and transgenic Arabidopsis under normal conditions. Under salt stress, however, the seed germination rate and seedling survival rate of transgenic Arabidopsis overexpressing BvKUP13 were significantly higher than those of WT (Figures 5A, C, D). After 7 days of vertical growth, no significant difference in root length was observed between the WT and transgenic Arabidopsis. However, under different NaCl concentration conditions, the root length of the transgenic Arabidopsis was significantly greater than that of the WT (Figures 5B, E). This suggested that BvKUP13 may be involved in significantly improving the germination rate, survival rate and root development of seedlings under salt stress.

Transgenic Arabidopsis and WT plants grown in soil for 30 d were subjected to various NaCl concentrations. No significant difference was observed in the growth between WT and transgenic plants under normal conditions. However, under salt stresses, both WT and overexpressing lines exhibited growth inhibition and leaf chlorosis, though the inhibition was more pronounced in the WT (Figure 6A). The dry and fresh weights of WT plants were significantly lower than those of the overexpressing lines (Figures 6B, C). These results suggested that overexpression of BvKUP13 significantly enhanced the salt tolerance of transgenic Arabidopsis.

To investigate the role of BvKUP13 in regulating the photosynthetic system under salt stress, we analyzed leaf disc senescence, chlorophyll content, and photosystem II (PSII) activity in both transgenic and WT plants. The results indicated that in the control group, both transgenic Arabidopsis and WT plants exhibited similar growth, with no significant differences. Under salt stress, the overexpressing transgenic Arabidopsis displayed lower leaf disc senescence and significantly higher chlorophyll content compared to WT (Figures 7A, B). The overexpressing lines exhibited stronger chlorophyll fluorescence under 200 mM NaCl stress compared to WT (Figure 7C), which was consistent with the Fv/Fm measurement results (Figure 7D). Chlorophyll content is positively correlated with photosynthetic efficiency within a certain range, suggesting that BvKUP13 enhances photosynthetic activity by increasing chlorophyll content under salt stress.

To examine the role of BvKUP13 in regulating ion balance under salt stress in transgenic Arabidopsis, the contents of Na⁺ and K⁺, along with their distribution in the roots and leaves of transgenic lines and WT, were analyzed. Under normal conditions, Na⁺ fluorescence signals in both the roots and leaves of all lines were weak, with no significant differences. Under salt stress conditions, the Na⁺ fluorescence signal was significantly enhanced in the root apex and elongation zone, as well as in mesophyll cells and around the leaf veins of both WT and BvKUP13-overexpressing Arabidopsis (Figure 8A), the contents of Na⁺ were significantly higher in both the leaves (Figure 8C) and roots (Figure 8F), but Na⁺ fluorescence intensity and content in transgenic plants were significantly lower than in WT. Under normal conditions, both WT and overexpressing lines exhibited strong, evenly distributed K⁺ fluorescence signals in their roots and leaves. However, K⁺ fluorescence intensity in WT significantly decreased compared to overexpressing lines under salt stress conditions (Figure 8B). Additionally, K⁺ content in both the leaves (Figure 8D) and roots (Figure 8G) was significantly reduced, with the roots exhibiting a more sensitive response. Under salt stress, the Na⁺ content in the leaves of transgenic plants was significantly lower than in WT, while the K⁺ content remained unchanged. This led to a substantial reduction in the Na⁺/K⁺ ratio in transgenic plants compared to WT (Figure 8E). In contrast, both Na⁺ and K⁺ contents in the roots of transgenic plants were significantly higher than in WT, resulting in a markedly lower Na⁺/K⁺ ratio (Figure 8H). These findings suggested that BvKUP13 enhanced potassium ion uptake, reduced sodium ion accumulation, and maintained a low Na⁺/K⁺ ratio, thereby improving the salt tolerance of Arabidopsis. Furthermore, BvKUP13 may play a more prominent role in ion transport within the roots, limiting the transport of sodium ions to the shoot.

To further investigate how the BvKUP13 enhanced plant tolerance to salt stress, the activity of the ROS scavenging system and the accumulation of osmotic regulators were examined under stress conditions. Under normal conditions, no significant differences were observed in the levels of antioxidants and osmotic regulators between transgenic plants and WT. As NaCl concentration increased, the activities of SOD, POD, and CAT (Figures 9A–C) in transgenic plants were significantly higher than in WT, while H₂O₂ and O₂^- (Figures 9E, F) levels were significantly lower in the transgenic plants. These findings were consistent with the NBT and DAB (Figure 9D) staining results. After salt stress, Evans Blue staining was more intense in WT, indicating greater cell damage (Figure 9D). MDA accumulation was significantly higher in the WT, indicating greater lipid peroxidation of the membrane (Figure 9H). Proline (Figure 9G), soluble sugars (Figure 9I), and soluble proteins (Figure 9J), which serve as osmotic regulators, were significantly more abundant in the overexpressing lines under high-salt conditions than in the WT. These results suggested that BvKUP13 overexpression enhanced the efficiency of the ROS scavenging system in Arabidopsis under salt stress conditions, promoted osmotic regulator accumulation, and improved salt tolerance.