Protein sequences for the nine Arabidopsis thaliana Shaker family members were obtained from the TAIR database (https://www.arabidopsis.org/).These sequences were then used as queries for a subsequent BLAST search in the NCBI database (https://www.ncbi.nlm.nih.gov/) to identify Shaker K⁺ channel family in tobacco (N. tabacum), using an E-value threshold of 1e^-5. Redundant sequences were manually removed. The redundant entries caused by alternative splicing were removed manually, and the identified sequences were then used as new queries to do a second BLASTP search. The sequences without Shaker K⁺ channel family-specific motifs were removed after sequence alignment and SMART analysis (http://smart.embl-heidelberg.de/). The molecular weight, isoelectric point, and amino acid number of proteins were calculated with the ExPASy tool (https://web.expasy.org/protparam/).

A phylogenetic tree of Shaker K⁺ channel family members from N. tabacum, Arabidopsis thaliana and Oryza sativa was constructed using the Neighbor-Joining method in MEGA6, under the default parameters (bootstrap replicates=1000) (Tamura et al., 2013). Multi-sequence alignment was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and the result was presented by GeneDoc (Nicholas, 1997). InterPro (https://www.ebi.ac.uk/) was utilized for conserved domain analysis, with findings visualized in Microsoft PowerPoint. To find cis-acting elements localized in the promoter region of Shaker K⁺ channel family genes, 3000 bp of promoter regions upstream of the 5’-UTR of Shaker K⁺ channel family genes were queried. The cis-acting elements were predicted by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Rombauts et al., 1999) and presented with a heatmap by Tbtools (Chen et al., 2020).

This study used the tobacco variety Zhongyan 100 (N. tabacum), grown in a greenhouse under controlled conditions: 16 hours of light and 8 hours of darkness, at temperatures of 23-25°C and 70% relative humidity. Initially, tobacco seeds were sown in soil. After 20 days of germination, seedlings were transplanted onto floating rafts for hydroponic cultivation in a 1/2 Hoagland nutrient solution. Following a 6-day acclimatization period, salt treatment was administered (1/2 Hoagland solution with 100 mM NaCl), while controls received the 1/2 Hoagland solution alone. Nutrient solutions were refreshed every three days.

For the NtSKOR1B RT-qPCR analysis, root, stem, and leaf samples were collected at specific intervals: immediately (0 h), 6 h, 12 h, 30 h, 60 h, and 6 days following a 6-day hydroponic culture period with salt treatment (100 mM NaCl). The samples were immediately flash-frozen in liquid nitrogen and then stored at -80°C.

GUS staining experiments began with the sowing of NtSKOR1B promoter plants (ProNtSKOR1B::GUS). Following 20 days of germination and a 6-day hydroponic culture period, samples of roots, stems, and leaves were collected at designated intervals: immediately (0 h), 12 h, and 30 h after salt treatment exposure. GUS staining was then conducted using β-Galactosidase Reporter Gene Staining Ki (Beijing Leagene Biotech. Co., Ltd., Cat No./ID: DP0013, Beijing, China), with observations made post-staining.

Sampling to measure physiological indicators: Following a 6-day adaptation to hydroculture, tobacco seedlings are subjected to salt stress (1/2 Hoagland nutrient solution with 100 mM NaCl), while the control group receives only 1/2 Hoagland nutrient solution. Young tobacco plants were harvested on the 11th day post-salt stress treatment for the measurement of the following indicators. Plants from various strains and treatments were selected, focusing on the fourth leaf position (the first mature leaf post-salt treatment) to assess leaf length and width, followed by curing the entire plant at 105°C for 30 minutes and drying at 80°C until a constant weight was achieved. Biomass was weighed, n=6. Measurement of ion content: Leaves and roots were separated. Root samples were initially rinsed with ddH2O and subsequently dried with absorbent paper to eliminate external ions. Fresh samples were dried at 105°C until reaching a stable weight, followed by grinding the dried tissue into powder. Approximately 0.01 g of dry sample was placed into a test tube, to which 8 mL of 0.5 M HCl was added, and the mixture was extracted at 20°C and 150 rpm for 30 minutes. Subsequently, the mixture was filtered and adjusted to the extraction volume for testing. A standard K⁺ solution was prepared using 0.5 M HCl, and a flame spectrophotometer (6400A) was employed to quantify the ion content, n=3. Formula for calculating potassium content:

A: Concentration calculated from readings based on standard curve; M: Relative molecular mass of K⁺; V: Reading volume; m: Dry weight of sample.

Total RNA was isolated from tissues of young tobacco plants and purified with the RNeasy Plus Mini Kit (Qiagen, Cat No./ID: 74134) following the manufacturer’s protocol. The RT-qPCR program was described in (Mao et al., 2021). RNA was reverse transcribed into cDNA using Evo M-MLV Mix Kit with gDNA Clean for qPCR (Accurate Biotechnology (Hunan) Co., Ltd, Cat No./ID: AG11728, Changsha, China) and the cDNA was amplified using SYBR Green Pro Taq HS qPCR Premix (Accurate Biotechnology (Hunan) Co., Ltd, Cat No./ID: AG11701, Changsha, China) on the LightCycler^® 96 Instrument (F. Hoffmann-La Roche Ltd, Switzerland). All the primers used for RT-qPCR are listed in Supplementary Table 1 . The amplification reactions were performed in a total volume of 10 μl, containing 5 μl 2×SYBR Green Pro Taq HS qPCR Premix, 0.6 μl forward and reverse primers (10 μM), 1 μl cDNA (10 times diluted), and 3.4 μl ddH2O. The RT-qPCR amplification program was as follows: 95oC for 10 min; 95oC for 10 s, 60oC for 30 s and amplification for 40 cycles. Analysis of the relative gene expression data was conducted using the 2^-ΔC’t method (Mao et al., 2021).

Specific primers, NtSKOR1B-1F and NtSKOR1B-1R, were designed from the LOC104103384 gene sequence for PCR amplification, using N. tabacum L. cDNA to obtain the NtSKOR1B sequence. The knockout site within the NtSKOR1B sequence was identified, leading to the design of knockout primers NtSKOR1B-Crispr-58F and NtSKOR1B-Crispr-58R for constructing the pORE-Cas9/gRNA-NTSKOR1B gene knockout vector. The pORE-Cas9/gRNA-NtSKOR1B construct was introduced into Zhongyan 100 using the Agrobacterium-mediated leaf disk method, generating T0 transgenic mutants. Plants were initially screened for various editing outcomes using primers NTSKOR1B-Crispr-9734F and NtSKOR1B-Crispr-10293R. Nine C1 generation lines were cultivated, and DNA from homozygous mutants was extracted for PCR amplification and sequencing with specific primers NtSKOR1B-Crispr-9734F and NtSKOR1B-Crispr-10293R. This led to the identification of two homozygous knockout lines, characterized by distinct edits: C2 with a T insertion at position 911, and C6 with an 11-base deletion starting at position 989, causing translational termination ( Supplementary Figure 1 ).

Genomic DNA from plants was extracted using the CTAB method, and the NtSKOR1B promoter sequence upstream of the ATG start codon for LOC104103384 was obtained through PCR amplification with primers NtSKOR1Bpro-1F and NtSKOR1Bpro-1R. This yielded a 2439 bp NtSKOR1B promoter sequence, which was then cloned into the plasmid pMD19-T-NtSKOR1Bpro and confirmed by sequencing. Using the plasmid pMD19-T-NtSKOR1Bpro as a template and primers NtSKOR1Bpro-2F and NtSKOR1Bpro-2R for amplification, the binary vector pBI101-NtSKOR1Bpro was obtained, which drives the GUS gene under the NtSKOR1B promoter’s control. The Agrobacterium-mediated leaf disk method was employed for transforming this vector into tobacco plants, generating T0 transgenic lines. The T0 generation lines was confirmed positive ( Supplementary Figure 2 ), using the identification primers NtSKOR1Bpro-3F and NtSKOR1Bpro-1R. Harvest T0 generation seeds from positive plants and sow them on 1/2 MS plates with 50 mg/L kanamycin sulfate. Following resistance screening, strictly self-cross lines displaying a 3:1 resistance ratio, selecting for resistant lines. Staining was performed on T1 generation homozygous plants.

Statistical analysis was done using IBM SPSS Statistics 23 software. Significant differences were examined by one-way ANOVA using the LSD test at p<0.05 and p<0.01. The figures were drawn by GraphPad Prism 6.0.

Genomic screening identified 20 members of the N. tabacum L. Shaker K⁺ channel family using NCBI and SMART tools. Members were systematically numbered according to their similarity to Arabidopsis thaliana Shaker K⁺ channel family counterparts ( Figure 1 , Table 1 ). Ten Shaker K⁺ family members were attributed to the maternal ancestor, Nicotiana sylvestris, and labeled with the suffix “A”, while the remaining ten were derived from the paternal ancestor, Nicotiana tomentosiformis, and marked with “B”. The K⁺ channel proteins varied in length from 598 to 892 amino acids, with molecular weights ranging from 69.5 to 100.5 kDa.

To construct a phylogenetic tree ( Figure 1A ), members were selected from the Shaker K⁺ channel families in Arabidopsis thaliana, Oryza sativa, and N. tabacum. The results showed that, like the other two species, N. tabacum Shaker K⁺ channel family were categorized into five groups.

In 2007, at a time when tobacco genome data were not yet available, Toshio Sano’s team utilized Degenerate PCR and RACE technology to clone four members of the Shaker K⁺ channels from the BY-2 cell line. These channels were designated as NKT1, NKT2, NtKC1, and NTORK1, respectively (Sano et al., 2007). To avoid potential confusion and align with Arabidopsis nomenclature conventions, this study renamed these four genes NKT2B, NKT6B, NKC1B, and NtGORKA. All members of the tobacco Shaker K⁺ channel families possess the signature sequence TXXTXGYG, characteristic of selective K⁺ channels. ( Figure 1B ).

InterPro analysis predicted six conserved domains in N. tabacum. Shaker K⁺ channel family members: K⁺ channel KAT/AKT domain, Ion transport domain, Cyclic nucleotide-binding domain, KHA domain, Ankyrin repeat domain, and voltage-dependent K⁺ channel EAG/ELK/ERG domain ( Figure 2 ). The K⁺ channel KAT/AKT domain, present in all N. tabacum L. Shaker proteins, is the most fundamental conserved domain. The Ion transport domain plays a crucial role in regulating K⁺ transmembrane transport. The Cyclic nucleotide-binding and KHA domains are essential for channel function regulation, whereas the Ankyrin repeat domain primarily mediates protein-protein interactions. The voltage-dependent K⁺ channel EAG/ELK/ERG domain senses voltage changes across the cell membrane.

In Groups I, III, and V, 13 out of 14 members possess all six conserved domains mentioned, whereas Groups II and IV lack the Ankyrin repeat domain. The count of voltage-dependent K⁺ channel EAG/ELK/ERG domains varies among subgroups, with six in Group I, five in Groups II, III, and V, and six in Group IV. Variations in types, lengths, and numbers of conserved domains likely affect the proteins’ response functions.

To elucidate the potential functions and regulatory mechanisms of Shaker K⁺ channels in N. tabacum, we analyzed the cis-acting elements in promoter sequences (3000 bp upstream of the ATG start codon) of 20 Shaker K⁺ channel family using PlantCARE software. Analysis revealed that Shaker K⁺ channel promoters predominantly contain cis-acting elements associated with three major biological processes: abiotic stress response, plant hormone response, and growth and development. Specifically, there are 10 types of elements each for abiotic stress and plant hormones, and 6 for growth and development. ( Figure 3 ) This indicates that the Shaker K⁺ channel family significantly influences tobacco’s response to abiotic stress, hormone regulation, and growth and development processes.

Abiotic stress response elements are the most abundant in the promoter regions of N. tabacum Shaker K⁺ channel genes, comprising 31.58%-71.93% of all identified elements. MYB and MYC elements represent 40% and 19% of stress response elements, respectively. The antioxidant response element (ARE) was found in 19 Shaker members, making up 9% of stress elements. The drought response element (MBS) was present in 17 members (5%), and the W-box, linked to K⁺ uptake and signal transduction, was found in 16 members (6%). Phytohormone response elements constituted 10.53%-38.60% of elements. The ABA response element (ABRE) was identified in 19 Shaker members, comprising 27% of stress elements. Elements related to growth and development accounted for 5.26%-21.05% of the total, with all members containing the light-responsive element (Box 4), comprising 64% of these elements.

In a prior study, we performed transcriptomic analysis on the roots of N. tabacum variety Zhongyan 100 under 100 mM NaCl salt treatment (Mao et al., 2021). Leveraging this dataset, the current study explores the gene expression variations across the tobacco Shaker family members. The findings indicate elevated expression levels of Group I members NKT1A and NKT1B in the roots of tobacco seedlings, in contrast to the lower expression levels of NKT2A and NKT2B. Similarly, Group II members NKT3A, NKT3B, NKT4A, and NKT4B displayed low expression levels. Group III analysis showed low expression levels for NKT5A and NKT5B, with slightly higher levels for NKT6A and NKT6B. Notably, Group IV members NtKC1A and NtKC1B, along with Group V members NtSKOR1A, NtSKOR1B, NtSKOR2A, NtSKOR2B and NtGORKA, exhibited higher expression levels, while NtGORKB was comparatively lower. Post-salt stress treatment comparison revealed that the expression levels of Group V outward rectifying potassium channel members NtSKOR1A and NtSKOR1B were significantly increased due to salt stress. This suggests a potential involvement of these two genes in the ion transport process under salt treatment conditions. Specifically, NtSKOR1A and NtSKOR1B were up-regulated by 1.31 and 3.18 times, respectively, compared to pre-treatment levels. Given the higher expression increase of NtSKOR1B compared to NtSKOR1A, it is posited that NtSKOR1B may play a more significant role in this process ( Figure 4 ).

To further investigate NtSKOR1B expression changes under salt stress, RT-qPCR was conducted. Results indicated a significant upregulation of NtSKOR1B expression in roots with prolonged salt exposure, reaching 9.98-fold after 30 hours compared to the initial level. While NtSKOR1B expression in stems and leaves was lower than in roots, it also exhibited a gradual increase over the duration of salt treatment ( Figure 5A ).

A homozygous Zhongyan 100 transgenic line, with the NtSKOR1B promoter fused to the GUS reporter gene, was then developed. GUS histochemical staining for the NtSKOR1B promoter was consistent with the RT-qPCR findings. GUS staining revealed negligible activity in tobacco seedlings without salt treatment ( Figures 5B–D ). However, GUS activity emerged in the roots, stems, and leaf veins, becoming pronounced after 12 hours and intensifying after 30 hours of salt treatment. This indicates that under standard cultivation conditions, NtSKOR1B expression in tobacco seedlings is minimal. Following salt exposure, expression markedly rises in the root cortex, stems, and leaf veins’ vascular tissues, escalating with prolonged treatment ( Figures 5E–H ).

To elucidate NtSKOR1B’s function under salt stress, two distinct homozygous knockout lines with varied editing methods were exposed to salt treatment (see Materials and Methods for construction and editing details). Under salt stress, NtSKOR1B knockout lines demonstrated higher biomass, increased leaf length, and greater leaf width than wild types, with enhancements of 34.06%-39.24%, 15.87%-21.63%, and 14.89%-17.02%, respectively ( Figures 6A–D ). Knockout of NTSKOR1B led to increased K⁺ levels in both leaf and root tissues of tobacco seedlings over wild type, showing increases of 22.64%-25.47% and 18.06%-21.02%, with significant differences in aerial tissues ( Figures 6E, H ). Furthermore, NTSKOR1B knockout reduced Na⁺ levels in both leaf and root tissues compared to wild types, with reductions of 9.83%-11.48% and 7.52%-7.95%, notably in aerial tissues ( Figures 6F, I ). The NtSKOR1B knockout mutants markedly increased the plant’s K⁺/Na⁺ ratio ( Figures 6G, J ). Under control conditions, no significant differences were observed in these physiological indicators or ion levels ( Figures 6A–J ).

Gene expression varies across species and tissues, reflecting gene regulation complexity and biological adaptive evolution (Hill et al., 2021). Current studies demonstrate that the outward rectifying K⁺ channel SKOR is pivotal in regulating intracellular K⁺ balance through mediating cytoplasmic K⁺ efflux, significantly influencing plant responses to salt, drought, and nutritional stresses. The SKOR analogue, OsK5.2, part of the rice Shaker family, resides within the root vascular bundle. During salt stress, OsK5.2 expression boosts K⁺ efflux from the root vascular bundle to the xylem, elevating plant K⁺ levels and thus enhancing salt tolerance through an increased K⁺/Na⁺ ratio. Furthermore, OsK5.2’s presence in guard cells amplifies K⁺ efflux under salt stress, leading to stomatal closure, diminished water loss, and reduced salt buildup, which in turn decreases Na⁺ transport to leaves(Nguyen et al., 2017). In summary, this potassium channel positively influences plant salt tolerance by ensuring a superior K⁺/Na⁺ balance(Zhou et al., 2022). The NtSKOR1B outward potassium channel, part of the tobacco Shaker family, is located in the root cortex and leaf vascular bundles. Under salt stress, plants activate K⁺ channels in root cortex cells, leading to K⁺ loss. Outward K⁺ channels play a crucial role in this process (Wakeel, 2013; Demidchik, 2014). NtSKOR1B expression speeds up K⁺ efflux, hindering K⁺ level maintenance. K⁺ primarily move from leaves to other plant parts via the phloem. NtSKOR1B is found in leaf vascular bundles, not in guard cells. Under salt stress, leaf vascular bundles accelerate K⁺ loss. Analysis revealed that tobacco with NtSKOR1B knockout exhibited a higher K⁺/Na⁺ ratio and increased salt tolerance under salt stress ( Figure 6 ). Analysis of expression sites ( Figures 5E–H ) shows NtSKOR1B’s abundant expression in the root cortex. Knocking out NtSKOR1B reduces K⁺ efflux under salt stress. NtSKOR1B, also expressed in leaf veins and stem vascular bundles, reduces K⁺ loss in leaves when knocked out. This collectively boosts the plant’s K⁺/Na⁺ ratio and the salt tolerance of tobacco seedlings ( Figure 7 ). This study revealed that NtSKOR1B is involved in the negative regulation of salt tolerance in tobacco seedlings, which lays the foundation for further investigation of the function of this gene in intracellular K⁺ and Na⁺ transport in plant wounding fluid and different leaf positions.

The complexity of gene product interactions and tissue-specific expression patterns, adapted to environmental and physiological demands, are crucial for biological evolution and functional diversity. Shaker K⁺ channels, prevalent in plants, regulate the transmembrane transport of K⁺, playing critical roles in stress response, growth, development, and electrical signal transmission. Arabidopsis AKT1, a classic example of a phosphorylation-regulated Shaker K⁺ channel, is activated by CBL1/9-CIPK23 phosphorylation (Wang et al., 2016). Recent studies have elucidated this regulatory pathway further, revealing how plants modulate the plasma membrane CBL1/9-CIPK9/23-AKT1 in response to external K⁺ levels (Li et al., 2023). Gene expression involves intricate regulatory processes. This study revealed that NTSKOR1B negatively influences the salt tolerance of tobacco seedlings. Bioinformatics analysis identified conserved domains within this gene family related to voltage sensing, K⁺ absorption, and the Ankyrin repeat domain for protein-protein interactions. Specifically, the Ankyrin repeat domain in Arabidopsis thaliana can interact with CIPK family proteins to regulate gene expression and respond to environmental stress (Sedgwick and Smerdon, 1999; Wei and Li, 2009). Furthermore, the promoter region is rich in elements tied to stress response, hormonal signals, and growth, such as MYB for drought, cold, and salt stress responses (Yang et al., 2012; Li et al., 2019; Wang et al., 2022), and the hormone response element ABRE (Gomez-Porras et al., 2007). These findings suggest that NtSKOR1B is regulated by a complex gene network, offering insights for further detailed exploration of its regulatory mechanisms through techniques such as transcriptome analysis and yeast two-hybrid assays.

Under salt stress, excessive Na⁺ competes with K⁺ for plasma membrane uptake sites, inhibiting K⁺ absorption and disrupting plant tissue metabolism (Mulet et al., 2023). Excessive Na⁺ influx leads to cell membrane depolarization, reducing the driving force for K⁺ uptake. Consequently, plants in high salinity environments inevitably suffer from chronic K⁺ deficiency. A stable K⁺/Na⁺ ratio is critical for salt tolerance (Horie et al., 2009; Ordonez et al., 2014). Thus, the regulation and distribution of K⁺ are essential for balancing the ionic imbalance caused by Na⁺. K⁺ activates plasma membrane H⁺-ATPase, crucial for cell functioning (Weng et al., 2020). Plasma membrane H⁺-ATPase creates an electrochemical gradient, powering the Na⁺/H⁺ antiporter to exchange H⁺ for Na⁺, reducing Na⁺’s toxic effects (Xie et al., 2022). This study revealed that NtSKOR1B knockout in N. tabacum L. seedlings under salt stress leads to increased K⁺ and decreased Na⁺ levels, solely at the ionic content level. Further research into Na⁺ transporters, proton pump activities, and expression levels will enhance understanding of NtSKOR1B’s role in regulating the K⁺- Na⁺ relationship under salt stress.

A comprehensive exploration of plant adaptation to salt stress, particularly via analyzing genes that negatively regulate salt tolerance, illuminates the molecular responses of plants to environmental stresses. In response to salt stress, these genes modulate gene expression, preserve ion equilibrium, boost osmoregulator synthesis, fortify antioxidant defenses, and activate distinct signaling pathways. These mechanisms enable plants to regulate ion uptake and distribution, synthesize protective molecules for osmotic balance, and augment antioxidant systems for cellular protection against oxidative stress. Furthermore, the activation of signal transduction pathways triggers a cascade of adaptive and defensive responses in plants. Such research not only enhances our comprehension of plant adaptation mechanisms to environmental stress but also lays the scientific groundwork for developing salt-tolerant crops, pivotal for agricultural productivity and ecological restoration.