Genome-wide identification of kiwifruit K⁺ channel Shaker family members and their response to low-K⁺ stress

Abstract

Background

‘Hongyang’ kiwifruit (Actinidia chinensis cv ‘Hongyang’) is a high-quality variety of A. chinensis with the advantages of high yield, early ripening, and high stress tolerance. Studies have confirmed that the Shaker K⁺ genes family is involved in plant uptake and distribution of potassium (K⁺).

Results

Twenty-eight Shaker genes were identified and analyzed from the ‘Hongyang’ kiwifruit (A. chinensis cv ‘Hongyang’) genome. Subcellular localization results showed that more than one-third of the AcShaker genes were on the cell membrane. Phylogenetic analysis indicated that the AcShaker genes were divided into six subfamilies (I-VI). Conservative model, gene structure, and structural domain analyses showed that AcShaker genes of the same subfamily have similar sequence features and structure. The promoter cis-elements of the AcShaker genes were classified into hormone-associated cis-elements and environmentally stress-associated cis-elements. The results of chromosomal localization and duplicated gene analysis demonstrated that AcShaker genes were distributed on 18 chromosomes, and segmental duplication was the prime mode of gene duplication in the AcShaker family. GO enrichment analysis manifested that the ion-conducting pathway of the AcShaker family plays a crucial role in regulating plant growth and development and adversity stress. Compared with the transcriptome data of the control group, all AcShaker genes were expressed under low-K⁺stress, and the expression differences of three genes (AcShaker15, AcShaker17, and AcShaker22) were highly significant. The qRT-PCR results showed a high correlation with the transcriptome data, which indicated that these three differentially expressed genes could regulate low-K⁺ stress and reduce K⁺ damage in kiwifruit plants, thus improving the resistance to low-K⁺ stress. Comparison between the salt stress and control transcriptomic data revealed that the expression of AcShaker11 and AcShaker18 genes was significantly different and lower under salt stress, indicating that both genes could be involved in salt stress resistance in kiwifruit.

Conclusion

The results showed that 28 AcShaker genes were identified and all expressed under low K⁺ stress, among which AcShaker22 was differentially and significantly upregulated. The AcShaker22 gene can be used as a candidate gene to cultivate new varieties of kiwifruit resistant to low K⁺ and provide a reference for exploring more properties and functions of the AcShaker genes.

Introduction

Kiwifruit is a plant of the genus Actinidia in the family Actinidiaceae [1, 2]. It has a high vitamin content and is known as the ‘king of fruits’ [3, 4]. Kiwifruit is one of several fruits that has been successfully adopted by the international market in recent years; it is the most widely grown and economically valuable cultivar in the world, and it is a high-value export crop for countries such as New Zealand, Italy, and Chile [5]. China is the prime source of wild kiwifruit and the largest producer of kiwifruit [6]. ‘Hongyang’ kiwifruit (A. chinensis cv ‘Hongyang’) is the first new red-fleshed kiwifruit variety selected and bred by the Cangxi County Agricultural Bureau of Guangyuan City, Sichuan Province, and the Sichuan Institute of Natural Resources, and it is a high-quality variety of A. chinensis [7]. With early ripening, high yield, and suitability for a wide range of plantings, ‘Hongyang’ kiwifruit is rich in nutrients and has high economic value and development prospects.

Potassium (K) is an essential macroelement for plant growth and is second only to nitrogen (N) in its internal content [8]. K⁺ has a direct regulatory effect on stomatal opening and closing and cellular water uptake [9]. In 1943, Boyer et al. [10] first reported the ability of K⁺ to catalyze the activation of pyruvate kinase. More than 60 enzymes, including synthases, transferases, and oxidases, are known to require K⁺ as an activator and thus participate in the synthesis and metabolism of plant proteins. Previous studies showed that K⁺ is also involved in the H+-ATPase enzyme activation, which allows K⁺ to pass through the plasma membrane from the extracellular solution into the cell, suggesting that K⁺ can facilitate the transmembrane transport of intracellular substances [11]. Kaiser et al. [12] and Pier and Berkowitz [13] reported that the color effect of K⁺ on photosynthesis is also manifested in the rate at which CO₂ is fixed by photosynthesis; when the external K⁺ concentration increases, the rate at which CO₂ is fixed by photosynthesis increases; conversely, the opposite is true. Fontana et al. [14] reported that K deficiency significantly affects the growth and development of cotton plants, as evidenced by a reduction in plant height and total leaf-root area and further reduced fresh and dry biomass of the whole plant. The results of tomato soilless culture experiments suggested that K can reduce the negative effects of Cd by improving photosynthesis and promoting chlorophyll synthesis, and optimizing nutrient composition and concentration may be a better strategy for reducing the impact of Cd on plant growth and physiology [15]. In addition, K⁺ plays the most vital role in improving plant protein stability [16]. As an opposite-phase charged ion, K⁺ is usually involved in the long-range transport of NO₃⁻ in the xylem and the storage of NO₃⁻ in vesicles; in plants, after the reduction of NO₃⁻, comparable organic acids are synthesized from K⁺ to maintain intracellular charge balance and pH stability [17, 18]. K⁺ can promote the release of photosynthates from chloroplasts or directly affect the activity of ATPase in sieve tubes, and it can also regulate the osmotic potential of photosynthates from source to reservoir by increasing the pH in the sieve tubes of the phloem [19].

Despite the abundance of K⁺ in the soil, K⁺ uptake and utilization by plant roots are low, resulting in lower crop yields [20, 21]. Potash resources are scarce in China, and most potassium is imported. The shortage of potassium in the soil has become a prime factor limiting agricultural development in China and the world [22].

In K⁺-deficient plants, sucrose loading in the phloem is inhibited [23, 24], and sucrose transport to the roots is reduced, resulting in a significant decrease in the root sucrose concentration [25]. Under K⁺ deficiency stress, the rate of outward transport of photosynthesis products is significantly reduced, forcing a significant increase in the content of photosynthesis products in the source leaves [25, 26]. Therefore, the growth and development of aboveground plant parts and the root system are inhibited [27]. Severely K⁺-deficient plants wilt, and the cell wall lignification process is blocked. As a result, the cells are more susceptible to fungal infestation, ultimately leading to lower yield and quality [21].

Shaker channels are considered the most vital proteins for maintaining K⁺ in plants, and they are involved in K⁺ nutrient uptake, regulation of intracellular K⁺ homeostasis, and transport [28]. The first K⁺ channel gene, AKT, was cloned in A. thaliana [29] and plays a vital role in K⁺ uptake and tolerance to low-K⁺ stress and adapts to high salt environments by maintaining intracellular K⁺/Na⁺. Similarly, Ródenas et al. [30] noted that the AtSKOR potassium channel mediates potassium transport to the xylem and is significantly upregulated under salt stress, thereby increasing salt tolerance. Nine shaker gene family members have been identified in A. thaliana and are categorized into five subfamilies based on their role in K⁺ channel proteins [31]. Currently, with the development of genomics and genetics, the study of the shaker K⁺ channel protein-encoding gene family has been completed with genome-wide identification and analysis in a variety of plants, including cabbage [32], cereal [33], Rosa canina [34], and sweet potato [35]. The shaker gene family plays a vital role in K⁺ uptake and stress resistance [32]. Chen et al. showed that the GmAKT1 gene increased the tolerance of soybeans to abiotic stresses [36]. Domestic researchers have found that the IbAKT gene may play a crucial role in K⁺ deficiency tolerance regulation in sweet potatoes [35]. More Shaker family genes have been identified in plants, the understanding of Shaker family genes has gradually deepened, and the biological functions of these genes have been elucidated. However, there are no relevant studies on the Shaker gene family in kiwifruit.

This study aimed to comprehensively analyze the identification, physicochemical property analysis, phylogenetic analysis, gene structure, structural domains and conserved motifs, cis-acting regulatory elements, chromosomal localization, gene duplication, covariate analysis, and GO enrichment analysis of Shaker family members across the whole genome of kiwifruit and to explore the expression pattern of kiwifruit in response to low-K⁺ and salt stress. This study lays the foundation for understanding the evolution and biological functions of the Shaker transcription factor family in kiwifruit. Moreover, this study provides a primary basis for further research on breeding for kiwifruit resistance to low-K⁺ stress.

Materials and methods

Plant materials

‘Hongyang’ kiwifruit from the Aziying orchard, Panlong District, Yunnan Province, were selected as explants, and kiwifruit seedlings were obtained by histoculture techniques. A. chinensis ‘Hongyang’ was cultivated from seedlings collected from an orchard in Henan Province by the Institute of Natural Resources of Sichuan Province and Guangxi County Agricultural Bureau of Sichuan Province. It has excellent traits, such as a large fruit, thin skin, fast growth, early maturity, and red flesh [7].

Access to data

The ‘Hongyang’ kiwifruit genetic data were downloaded from the kiwifruit gene database KGD: Kiwifruit Genome Database (http://kiwifruitgenome.org/) (Actinidia chinensis (Hong Yang V3)). Sequence information on the conserved structural domains of the Shaker gene family (ID: PF00027, ID: PF00520, ID: PF11834) was downloaded from the Pfam website (http://pfam.xfam.org/), and hidden Markov model files were constructed using the HMMER3 soft package [33]. The protein sequences and genomic data of nine Arabidopsis Shaker K⁺ channel protein families were downloaded from the Arabidopsis genome website TAIR (https://www.Arabidopsis.org/) [34].

Identification and physicochemical characterization of kiwifruit shaker gene family members

Based on the data downloaded from the above steps, the BLAST program in TBtools software was used to perform a BLAST comparison of the kiwifruit genomic protein data using the protein sequences of the Arabidopsis Shaker gene family as a reference. The protein sequences in the kiwifruit genomic protein data that had high homology (E-value < 1 × 10^− 5) sequences were screened out [37]. Second, the hmmsearch program was used to search the kiwifruit genome-wide protein structural domains (ID: PF00027, ID: PF00520, ID: PF11834), and protein sequences with an E-value of < 1 × 10^− 5 were filtered out [38]. Finally, the protein sequence number columns obtained in the above two steps were combined, and duplicates were selected. The selected protein sequences were extracted from the kiwifruit gene protein database using the TBtools tool to obtain the corresponding sequences, which were saved as *.fast files [37]. The online protein structural domain analysis tools SMART (http://smart.embl-heidelberg.de/) and CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) were used to determine whether the candidate sequences contained Shaker structural domains [39], and the missing or incomplete structural domains were eliminated to obtain the kiwifruit Shaker gene family members. The identified Shaker genes were renamed according to their chromosomal locations and named AcShaker + sequence number. The obtained protein sequences were submitted to the ExPASy ProtParam (https://web.ExPASy.org/protparam/) online tool for the detection of physicochemical properties such as protein sequence length, relative molecular weight, theoretical isoelectric point, and hydrophilicity. Finally, subcellular localization analysis was performed using WoLF PSORT (https://wolfpsort.hgc.jp/) [40]. TBtools was utilized to calculate the gene length and coding region length 2000 bp upstream of the encoded gene [37].

Phylogenetic analysis of the kiwifruit Shaker gene family

To understand the evolutionary relationship between kiwifruit and Arabidopsis Shaker gene families. The sequences of the Shaker K⁺ proteins obtained from kiwifruit and A. thaliana were combined, and an amino acid sequence phylogenetic tree of 28 kiwifruit Shaker proteins (AcShaker) and nine A. thaliana Shaker proteins [38] was constructed using the neighbor-joining (NJ) method with a bootstrap value of 1000. The bootstrap value was 1000. The phylogenetic tree was built using the iTOL (https://itol.embl.de/) online tool [41]. Finally, 28 Shaker proteins of kiwifruit were grouped according to the grouping of Arabidopsis Shaker K⁺ channel proteins [42]. The amino acid sequences of AcShaker22 and AtAKT1 were subjected to multiple sequence comparisons using MAFFT (version 7.5) comparison software, and the results were visualized using Geneious (version 9.1.4) comparison [43, 44]. The tertiary structures of the AcShaker22 and AtAKT1 proteins were constructed using SWISS-MODEL (https://swissmodel.ExPASy.org/).

Analysis of gene structure, structural domains, and conserved motifs

Based on the genome annotation file (GFF3) of kiwifruit, TBtools software was used to analyze the gene structure of each gene [36]. The structural domains of the kiwifruit Shaker K⁺ channel proteins were then predicted using the CD-search online tool (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) from the NCBI database with default parameters [45]. Motif analysis of AcShaker-encoded proteins was performed using the online tool MEME (https://meme-suite.org/meme/) [46] (the maximum number of conserved motifs retrieved was ten, and the other parameters were the defaults) to analyze the differences between members of the Shaker gene family. Finally, the above results were visualized by TBtools software.

Analysis of cis-acting elements

To analyze the cis-acting elements in the promoter region of the AcShaker genes, the base sequences 2000 bp upstream of each gene were extracted separately from the genome annotation file (GFF3) using TBtools software, and then the promoter cis-acting element analyses were carried out using the online analysis software PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/HTML/) [47].

Chromosome localization and covariance analysis

Chromosome position information was obtained from the genome annotation file (GFF3), and chromosome position mapping was performed by TBtools software. The chromosome length information, position information, and density information of the AcShaker gene were extracted by TBtools, and the intraspecific covariance of AcShaker family members was analyzed using MCSanX in TBtools software [37].

GO functional enrichment analysis of the Shaker genes

Based on the kiwifruit whole-genome protein sequence files, the protein sequences of the AcShaker protein-encoding gene family were extracted, and the proteins of the 28 AcShaker genes were functionally annotated through the EggNOG-MAPPER database (http://eggnog-mapper.embl.de/) [48]. The Shaker gene families of kiwifruit were then subjected to GO functional enrichment analysis using TBtools and plotted and visualized by the online tool ChiPlot (https://www.chiplot.online/bar_plot_width_category) [49].

Expression pattern analysis of Kiwifruit Shaker genes in response to low-K⁺ and salt stress

Determination of low-K⁺ and salt concentrations

In the MS media, the K⁺ in the media was reduced by decreasing the concentration of KNO₃ and increasing the appropriate concentration of NH₄NO₃ to compensate for the loss of NO₃⁻ from KNO₃. With the modified ‘Hongyang’ kiwifruit MS medium by Wu et al., ‘Hongyang’ kiwifruit histocultured seedlings grew best in the NH₄NO₃ concentration range of 250 mg/L − 650 mg/L, and the MS medium with NH₄NO₃ concentration in MS medium was 450 mg/L [50]. A concentration of KNO₃ in the 300 mg/L-700 mg/L was the best for growth [51]. In this experiment, a one-way experimental design was adopted, and the concentrations of KNO₃ were 300 mg/L, 250 mg/L, 200 mg/L, 150 mg/L, and 100 mg/L, with corresponding NH₄NO₃ concentrations of 450 mg/L, 500 mg/L, 550 mg/L, 600 mg/L and 650 mg/L, respectively. Regular MS medium was used as the control to screen out the best K⁺-deficient formula. The optimum K⁺-deficient formulation was screened. Tissue culture-generated seedlings with roughly equal root lengths were selected and cultured in media supplemented with different concentrations of K⁺. Each bottle was inoculated with one plant, and three replicates were made for each combination. Each replicate had ten bottles of seedlings, which were randomly placed in the incubation room at 26 ± 1 °C after inoculation. After inoculation, the plants were randomly placed in an incubation room at 26 ± 1 °C. Phenotypic changes were observed, and growth was measured every 5 d.

Phenotypic observations of K⁺-deficient kiwifruit leaves and roots

Sixty ordinary plants with almost uniform growth were randomly selected, and 30 were placed in K⁺-deficient or regular media. The number of roots per plant was controlled to three, and the excess root hairs were removed before the plants were planted in the media. The plant roots were placed close to the surface of the culture medium. After 30 d of incubation, the seedlings were removed. Then, the number of roots, number of root hairs, and number of red leaves were observed and statistically analyzed.

Low-K⁺ and salt stress treatments and transcriptome sequencing

A total of 18 ordinary plants with almost uniform growth were randomly selected, and nine were added to K⁺-deficient or regular media. After 10 d of incubation [49], the seedlings were removed. Then, the roots of the plants were removed in liquid nitrogen for transcriptome sequencing. Nine plants in K⁺-deficient culture media were randomly grouped into T1, T2, and T3, and nine plants in regular culture media were randomly grouped into CK1, CK2, and CK3. Each group contained three sample plants, and the roots of each sample plant were taken as the experimental materials in turn. The samples were removed, stored on dry ice, and then sent to BQ Biotech for transcriptome sequencing and analysis.

Twelve kiwifruit histocultures with 4–5 leaves were selected and treated with salt (NaCl). Before the experiments, many pretests were carried out to determine the optimal NaCl concentration (3 g/L). The salt treatment times were set at 24 h, 48 h, and 72 h for the T1, T2, and T3 groups, respectively, and a salt treatment time of 0 h was used for the CK group. Three biological replicates were performed for each group. The samples were sequentially removed and stored in liquid nitrogen for transcriptome sequencing and analysis by Wuhan Baiyihui Energy Biotechnology Co., Ltd. TBtools software (version 2.012) was used to construct heatmaps.

Expression pattern analysis of the Shaker gene family in ‘Hongyang’ kiwifruit plants under low-K⁺ and salt stresses

The whole genome of A. chinensis was selected as the reference genome for transcriptome sequencing data, and Bowtie2 software was used for sequence comparison. The comparison results of Bowtie2 were statistically analyzed to obtain the number of reads compared to each transcript for each sample using RSEM v1.3.0 software [52], and the average expression level (fragments per kilobase per million bases, FPKM) conversion was calculated for each transcribed region. Paired-end reads from the same fragment were counted as fragments, which provided the expression level EXPECTED count data for genes and transcripts, where genes with FPKM values greater than 0 were considered expressed genes. The domain p value was determined by controlling the FDR (false discovery rate) [53]. Then, using the read count information, differentially expressed genes (DEGs) were analyzed using the R package edge R. The genes with thresholds of FDR < 0.05, log₂FC > 1, or log₂FC<-1 were screened as the final DEGs.

qRT-PCR analysis

To further validate the accuracy of the transcriptome data, we selected ten genes (including AcShaker15, 17, and 22) whose expression significantly differed under low-K⁺ stress for qRT-PCR validation. Kiwifruit leaves cultured in regular media for 10 d composed the control group (CK1, CK2, and CK3), and kiwifruit leaves cultured in low-K⁺ media for 10 d composed the treatment group (T1, T2, and T3). Total RNA was extracted using a Vazyme kit (Nanjing, China) from Novozymes Biotechnology Co., Ltd. To reverse-transcribe total RNA into cDNA for qRT-PCR analysis, HiScript^®ll QRT SuperMix was used for qPCR (+ gDNA wiper) (R223-01) for the First Strand Synthesis Kit. The qRT-PCR primers (Table S1) were then designed using Primer 6.0. The WDR1 gene was used as an internal reference gene for standard RT-qPCR using the ChamQ Universal SYBR qPCR Master Mix (Q711-02) kit with three replicates per gene. Finally, expression analysis was performed using the 2^−ΔΔCt method.

Results

Identification and physicochemical characterization of kiwifruit Shaker gene family members

BLAST and hmmsearch searches were performed on kiwifruit genomic protein data using protein sequences of the Arabidopsis Shaker K⁺ channel protein-encoding gene family as a control. Next, the kiwifruit Shaker gene family was predicted by CDD and Pfam to remove missing or absent structural domains, and a total of 28 Shaker genes were ultimately identified. These genes were named AcShaker1-AcShaker28 according to their chromosomal positions. Further analysis of the AcShaker protein sequences revealed that the protein lengths of the Shaker genes varied considerably, ranging from 338 (AcShaker12)-900 (AcShaker30) amino acids; the molecular weights ranged from 14.21 (AcShaker23)-100.69 (AcShaker30) kDa; the pI ranged from 4.36 (AcShaker23)-9.87 (AcShaker6); and the instability coefficients ranged from 25.66 (AcShaker23)-56.83 (AcShaker11). A total of nine genes, AcShaker5, AcShaker11, AcShaker15, AcShaker17, AcShaker18, AcShaker20, AcShaker22, AcShaker27, and AcShaker28, had instability coefficients less than 40, which indicated that they were stable proteins, and the other AcShaker genes were all unstable proteins. Second, K⁺ can improve the stability of proteins [16]; the lipolysis index ranged from 77.82 (AcShaker6) to 101.15 (AcShaker4) (Table 1). The subcellular localization prediction of the Plant-mPLoc tool showed that nine AcShakers were located in the cell membrane. Plant K⁺ uptake requires the involvement of a membrane [20], which is favorable for improving the efficiency of K⁺ uptake in plants. Twelve AcShakers were located in the nucleus, four AcShakers were located on chloroplasts, and the remaining three AcShakers were located on the plasma membrane, Golgi apparatus, and extracellular matrix (Fig. 1, Table S2).

Table 1 Analysis of physicochemical properties of AcShaker TFs

Fig. 1

Physicochemical property analysis diagram of AcShaker genes. The numbers in the heatmap indicate numerical values. Different genes are indicated by different colors. The gene name is indicated by the outermost circle, followed by the number of amino acids (aa), molecular weight (kDa), aliphatic index, theoretical pi, and instability index

Phylogeny of the kiwifruit Shaker gene family

To understand the evolutionary relationship between the kiwifruit Shaker gene family and other Shaker gene families, 28 kiwifruit Shaker protein sequences were aligned with 9 Arabidopsis Shaker protein sequences. The phylogenetic tree was constructed using the ML method of MERGA and IQtree, and the results showed a consistent tree topology (Fig. 2). Based on the clustering results, the kiwifruit Shaker gene family can be divided into six subfamilies, namely, subfamilies I, II, III, IV, V, and VI. Among them, subfamily VI contained only 13 Shaker genes in kiwifruit, indicating that the kiwifruit AcShaker13, AcShaker14, AcShaker6, AcShaker24, AcShaker9, AcShaker23, AcShaker8, AcShaker1, AcShaker25, AcShaker20, AcShaker7, AcShaker20, and AcShaker4 genes are distantly related to the Shaker genes of Arabidopsis. Except for subclade VI, subclade I had the greatest number of AcShaker genes, containing nine gene family members, including six in kiwifruit and three in Arabidopsis. Subclade IV contained two genes, AcShaker22 and AtKC1, showing that the kiwifruit AcShaker22 gene was most closely related to Arabidopsis AtKC1. In addition, two, two, and four AcShaker family member genes were identified in subclades II, III, and V, respectively.

Fig. 2

Phylogenetic tree of the Arabidopsis and kiwifruit Shaker gene families. The phylogenetic tree was constructed using the maximum likelihood method with 1000 replicate bootstrap values, with different groupings indicated by different colors

Multiple sequence comparison and three-dimensional structure of proteins encoded by the AcShaker22 and AtKC1 genes

Multiple alignments of the protein sequences encoded by the AcShaker22 and AtKC1 genes showed (Fig. 3A) that the two genes were highly conserved and had high protein sequence similarity, suggesting similar functions. Proteins with structurally alike sequences have conserved three-dimensional structures, and conserved structural domains in different proteins have conserved functions. The tertiary protein structures of AcShaker22 and AtKC1 were constructed using SWISS-MODEL software. The results showed (Fig. 3B) that the protein structures of the two genes were highly similar, and the ratios of α-helices, β-sheets, elongated strands, and free coiling were very close to each other. This finding suggested that the structural domains of the two proteins are very similar and have conserved functions.

Fig. 3

Sequence comparison and tertiary structure maps of the AcShaker22 and AtKC1 proteins. (A) Multiple comparisons of protein sequences encoded by the AcShaker22 and AtKC1 genes; black and gray shading indicate identical and similar amino acid residues, respectively. (B) Three-dimensional structure maps of the AcShaker22 and AtKC1 proteins

Gene structure, structural domains, and conserved motifs of the kiwifruit Shaker gene family

To investigate the sequence characteristics of the AcShaker protein, the motif composition of this protein was analyzed in this study using the MEME online tool (Fig. 4B). The results showed that motifs 1, 2, 4, 5, and 10 were highly conserved. However, the same motifs had different positions in different protein sequences, which might be related to the structure and function of this protein. Motif 8 appeared twice in the protein sequences of AcShaker2, AcShaker11, AcShaker15, AcShaker17, AcShaker18, AcShaker21, AcShaker26, AcShaker27, and AcShaker28 (Fig. 4A, B), suggesting that subfamily I, II and III AcShaker genes may be involved in specific functions. All genes in subfamily II contain eight motifs, and it can be hypothesized that the motif distribution of members of this subfamily is highly conserved. However, even for members of the same subfamily, there were some differences in motif distribution, such as motif 8 appearing twice in the protein sequences of AcShaker2 and AcShaker26 in subfamily V (Fig. 4A, B). This may be because differences exist between transcription factor family genes under specific conditions.

Differences in the distribution of conserved motifs within AcShaker gene families may also be influenced by gene structure and structural domains. The distribution of gene structure is crucial for studying evolutionary traits within gene families, and 28 AcShaker genes were subjected to sequence comparison and analyzed for gene structure based on genome annotation files. The results showed (Fig. 4A, D) that the majority (27) of the 28 kiwifruit Shaker gene family members were within 25 kb in length, and the remaining gene (AcShaker2) was approximately 55 kb. The number of introns and exons in the kiwifruit Shaker gene varied, with the number of introns and exons ranging from 1 to 13 and 2–16, respectively. AcShaker4 contains only one intron, but AcShaker19 has 13 introns. In addition, this study revealed that genes with closer relationships have more similar gene structures. For example, in subclade I, three genes, AcShaker17, AcShaker21, and AcShaker28, have the same gene structure, although they are different in length.

The conserved structural domains were also analyzed in this study. The results showed that all 27 AcShaker proteins, except AcShaker20, had the structural domain of the PLN03192 superfamily, and subclades IV and AcShaker12, AcShaker17, and AcShaker19 contained special KHA structural domains (dimerization structural domains of the K⁺ channel). AcShaker12 and AcShaker19 both contain two special structural domains. AcShaker20 has a special structural domain in the PTZ0032 superfamily. AcShaker1, AcShaker6, AcShaker8, AcShaker10, AcShaker13, AcShaker14, AcShaker23, AcShaker24, and AcShaker25 in subfamily VI all have special CAP_ED structural domains (Fig. 4A, C). These findings further verify that regardless of the distribution of conserved motifs, gene structures, or structural domains, there is a certain correlation with phylogenetic relationships, and the closer the evolutionary relationship is, the more similar the sequence features of AcShaker gene family members are, and the more identical their structures are.

Fig. 4

Phylogenetic relationships, gene structures, structural domains, and conserved motif distributions of AcShaker K⁺ channel protein family genes. (A) Phylogenetic tree analysis of 28 Shaker family genes constructed by MERGA. (B) Distribution of conserved motifs in AcShaker. Motifs are indicated by colored boxes, and black lines indicate the relative length of the proteins. (C) Conserved structural domains of AcShaker genes. (D) The exon-intron structure of the AcShaker gene

Prediction of cis-acting elements of the AcShaker gene

To further study the response mechanism of the kiwifruit Shaker gene and the response mechanism, 28 promoter cis-acting elements in the 2000 bp sequence upstream of the kiwifruit Shaker gene were preprocessed using the online software PlantCARE. The prediction results (Fig. 5, Table S3) revealed a total of 330 promoter cis-acting elements, which could be classified into six main categories: phytohormone response elements (47.58%), environmental stress response elements (22.41%), MYB binding sites (3.64%), protein binding sites (2.12%), light response elements (14.41%) and plant-specific regulatory elements (9.70%). Among the phytohormone response elements, abscisic acid response elements (53) and jasmonic acid response elements (40) accounted for the greatest proportion; among the environmental stress response elements, low-temperature response elements (20) were the most numerous, followed by drought response elements (12). The greater number of drought stress response elements may be related to the fact that K⁺ can guide stomatal opening and closing and cellular water uptake [54]. In summary, in addition to light response elements, 85.5% of promoter cis-acting elements can regulate gene expression and material metabolism in plants. Promoter cis-acting element analysis suggested that kiwifruit Shaker gene family expression may improve kiwifruit adaptation under various adverse conditions, including low-K⁺ stress.

Fig. 5

Distribution of cis-acting elements in the 2000 bp sequence upstream of the 28 AcShaker K⁺ channel protein family genes, with different cis-acting elements indicated by different colors

Chromosome mapping and replication of the Shaker gene in kiwifruit

The results of chromosomal localization (Fig. 6) indicated that the 28 Shaker genes of kiwifruit were unevenly distributed on 18 chromosomes. The greatest number of genes were located on chromosomes 1 and 2 (3); an equal number of genes were located on chromosomes 7, 11, 15, 16, 20, 22, and 28 (1); and two genes were located on chromosomes 2, 6, 13, 17, 19, and 26. There was no significant correlation between the number of Shaker genes on chromosomes and chromosomes.

Gene tandem duplication and fragment duplication are crucial means of gene family expansion in plants; after multiple duplications, the number of gene family members increases, and plants can better cope with complex environmental changes, including low K⁺ levels. There are more AcShaker genes than Arabidopsis genes [32]. To understand the duplication of AcShaker family genes in kiwifruit, we performed a gene duplication analysis of 28 AcShaker genes (Fig. 4). The results revealed no tandemly duplicated genes, but 13 segmentally duplicated gene pairs were identified. These results suggest that segmental duplications may play a key role in gene duplication events in the AcShaker family, consistent with findings in cabbage [32].

Fig. 6

Covariance analysis of kiwifruit AcShaker genes. The gray lines indicate all duplicated genes; the blue-green lines represent segmentally duplicated Shaker gene pairs. The heatmaps and line plots are gene densities, with line densities increasing sequentially from blue to white to red, and the yellow rectangles are chromosomes, with chromosome names shown between each chromosome and gene density

GO functional enrichment of the Shaker genes

To further elucidate the biological functions of AcShaker family genes, in this study, the proteins of 28 AcShaker genes were annotated for gene functions using the EggNOG-MAPPER database and plotted and visualized with the online tool ChiPlot (Fig. 7). The 28 AcShaker genes were annotated and assigned to three broad categories, namely, biological process, cellular component, and molecular function. In particular, in terms of biological processes, transmembrane transport, metal cation transport, monoprotic cation transport, inorganic ion transmembrane transport, biomass regulation, and bioregulatory pathways were enriched with more AcShaker genes. The modes of K⁺ transport were transmembrane transport, metal cation transport, and inorganic ion transmembrane transport [55], which suggests that the genes of the AcShaker family of genes may enable plants to adapt to the environment by regulating ion transport, including K⁺ transport, to adapt to adverse environments. All AcShaker genes were enriched in membrane and membrane-intrinsic components. Among molecular functions, the number of enriched AcShaker genes was greater for monogenic cation transmembrane-transporter protein activity, voltage-gated monogenic cation through rice activity, and inorganic molecular entity transmembrane transporter activity, indicating that molecular functions play a vital role in the regulation carried out by AcShaker family genes. The number of genes enriched in each prime category was significantly greater than 28, suggesting that the same gene may play different roles in different functional entries.

Fig. 7

GO annotation results for AcShaker genes. Biological process, cellular component, molecular function, and gene count are shown in green, orange, blue-green, and purple, respectively. The x-axis indicates the value and number of gene functions, and the y-axis represents the annotation function of the gene

Response of the Shaker gene in Kiwifruit to low-K⁺ stress and salt stress

Determination of low-K⁺ concentrations and phenotypic observations of leaves and roots

The uniformly grown histocultured seedlings were connected to media with different K⁺ concentrations, and their growth was observed every 5 d. First, the weight of the histocultured seedlings in each period was recorded, the growth was calculated and recorded, and the average value was taken every 5 d (Fig. S1, Table S4). Second, the growth results of each period were analyzed by ANOVA using SPSS (Table S5), and the differences in growth characteristics of the group-cultured seedlings were analyzed for each weight (Table S6). Finally, the growth of K⁺-deficient kiwifruit was analyzed by discriminant analysis (Fig. S2, Tables S7, S8). The results showed that treatment group 5 had the greatest effect on K⁺ deficiency in ‘Hongyang’ kiwifruit.

After controlling the length of the roots, the kiwifruit plants were cultured under low-K⁺ conditions and in MS media for 30 d. The results indicated (Fig. 8) that under low-K⁺ stress, the kiwifruit leaves turned red, the number of root hairs increased, and heel growth accelerated. For roots and root hairs, the K⁺-deficient group had many more roots than the control group, and the average root length was approximately twice as long as that of the control group. The data on red leaves, roots, root hairs, and mean root length of CK and T plants (Table S9) were analyzed by ANOVA using SPSS, and the differences were highly significant (P < 0.001).

Fig. 8

Phenotypic observation plots of kiwifruit in K⁺-deficient and control media. A, comparison of kiwifruit roots and leaves cultured in K⁺-deficient and regular media for 10 d. B, mean values of red leaves, roots, root hairs, and root length of kiwifruit plants cultured for 10 d in K⁺-deficient medium (T) or normal medium (CK). **: P < 0.01

Expression pattern of the Shaker K⁺ protein in kiwifruit under low-K⁺ stress

In this study, we analyzed the transcriptome data of ‘Hongyang’ kiwifruit plants after low-K + stress to understand the expression pattern of AcShaker genes in ‘Hongyang’ kiwifruit under low-K⁺ stress. The results showed (Fig. 9) that all 28 AcShaker genes were expressed, with 12 genes showing upregulation and 16 genes showing downregulation. The differential expression of three genes (AcShaker15, AcShaker17, and AcShaker22) was significantly varied from that of the control, with p-values less than 0.001. Among the three DEGs, the AcShaker15 and AcShaker17 genes were downregulated, and the AcShaker22 gene was upregulated. There were more downregulated genes with significant differential expression than upregulated genes, which suggests that AcShaker genes may negatively regulate the damage caused by low-K⁺ stress to ‘Hongyang’, thus improving the ability of plants to resist low-K⁺ stress.

Fig. 9

Heatmap of the expression profiles of 28 AcShaker genes under low-K⁺ stress. The various colored boxes indicate different log₂ (FPKM) values; the expression gradually increased from blue to yellow to red. CK is the control group, and T is the treatment group; **: P < 0.01

qRT-PCR analysis

To verify the accuracy of the transcriptome data, ten genes whose expression significantly differed were selected for qRT-PCR validation. Tables S10 and S11 show that the correlation between the qRT-PCR and RNA-Seq data was high, which confirmed the accuracy of the transcriptome data and verified the reliability of the transcriptome data. The qRT-PCR and RNA-Seq results of three AcShake genes (AcShake15, AcShake17, and AcShake22) showed (Fig. 10) that the correlation between the qRT-PCR and RNA-Seq expression of the three AcShake genes was high, indicating that the AcShake15, AcShake17, and AcShake22 genes play crucial regulatory roles in the low-K⁺ stress response.

Fig. 10

Plot of qRT-PCR versus RNA-Seq expression of three differentially expressed AcShaker genes (AcShaker15, AcShaker17, and AcShaker22) under low-K⁺ stress. Blue: RNA-Seq data; orange: qRT-PCR data; gray: standard deviation of the qRT-PCR data. r: correlation coefficient of qRT-PCR and RNA-Seq expression. CK1, CK2, and CK3 were used as the controls, and T1, T2, and T3 were used as treatments

Expression pattern of the Shaker gene in kiwifruit under salt stress

To understand the expression pattern of AcShaker genes in kiwifruit under salt stress, transcriptome data from kiwifruit plants after salt stress were analyzed in this study. The results showed (Fig. 11) that there were 28 AcShaker-expressed genes in both groups of kiwifruit under salt stress. Seventeen genes were downregulated, and 11 were upregulated in the T1 treatment group. Compared with those in the control group, six genes were significantly differentially expressed, among which the AcShaker9 and AcShaker27 genes were upregulated, and the AcShaker11, AcShaker18, AcShaker21, and AcShaker24 genes were downregulated. In the T2 treatment group, 15 genes were downregulated, and 13 were upregulated. Both genes (AcShaker11 and AcShaker18) showed significant differential expression compared to the control group. In the T3 treatment group, 11 genes were downregulated, and 17 were upregulated. Eight genes were significantly differentially expressed compared to those in the control group, among which the AcShaker9, AcShaker12, and AcShaker19 genes were upregulated, and the AcShaker11, AcShaker18, AcShaker21, AcShaker23, and AcShaker24 genes were downregulated. The AcShaker11 and AcShaker18 genes differed significantly among the three groups of salt treatments, and all were downregulated. The number of downregulated genes with significant differential expression was greater than that of upregulated genes, suggesting that AcShaker genes may enhance plant resistance to salt stress by negatively regulating the damage caused by salt stress in kiwifruit.

Fig. 11

Heatmap of the expression profiles of 28 AcShaker genes under salt stress. A, B, and C show the gene expression heatmaps of the T1, T2, and T3 groups and the CK comparison. The various colored boxes indicate different log2 (FPKM) values; the expression gradually increased from blue to yellow to red. CK is the control group, and T is the treatment group

Discussion

Shaker K⁺ channels are typical K⁺ channels involved in K⁺ uptake and stress resistance in higher plants [36]. Shaker K⁺ channel proteins in plants are highly similar in structure to Shaker K⁺ proteins in Drosophila [56]. In recent years, Shaker K⁺ channels have been categorized into three main types, namely, inwardly rectifying, outwardly rectifying, and weakly rectifying K⁺ channels, based on voltage dependence and the direction of K⁺ transmembrane transport [57]. In this study, 28 Shaker genes were identified in ‘Hongyang’ kiwifruit. Phylogenetic analysis revealed that 13 genes, including AcShaker6, AcShaker13, and AcShaker14, did not cluster with AtShaker genes, suggesting unique functions. The Shaker genes of kiwifruit can be divided into six subfamilies (Fig. 2). Among them, the AtAKT1 gene belongs to subfamily I, and it has been shown that AtAKT1 plays a vital role in K⁺ uptake, tolerance to low-K⁺ stress and tolerance to high salt stress [29]. It is hypothesized that the genes in clade I have similar functions. This classification was more apparent in the comprehensive analysis of conserved motifs, gene structures, and structural domains. According to the conserved motif analysis, similar genes were clustered in almost the same subfamily, and there were multiple motifs in specific proteins, which implies that they may have exceptional functions [36].

Structural analyses of the genes revealed that the number of introns in the kiwifruit Shaker gene varied considerably, with the number of introns ranging from 1 to 13 (Fig. 4). This gene was similar to the Shaker genes in cabbage [32], pear [34], sweet potato [35], soybean [36], and woolly cotton [28]. Many variations in the number of introns in the Shaker genes of kiwifruit may be due to chromosomal variation resulting from paleopolyploid events [58], which may have led to the diversity of gene functions. The clustering of the phylogenetic tree was highly similar to the structural features of the conserved motifs, gene structures, and structural domains, further suggesting that genes with similar evolutionary processes may also have similar functions, thus contributing to screening for Shaker genes with similar functions. For example, the overexpression of OsAKT1 in rice increased the K⁺ content in tissues and improved drought tolerance [59]. Overexpression of GmAKT1 in A. thaliana increased root length and potassium concentration under drought and salt stress, suggesting that GmAKT1 plays a role in the response of soybean plants to these stresses [36]. Overexpression of HvAKT1 in barley increased the K⁺ content in tissues and improved plant tolerance to drought [60]. The roots of sweet potato plants overexpressing IbAKT1 showed greater K⁺ influx than adventitious roots and lower K⁺ efflux than adventitious roots under K⁺ deficiency stress, indicating that IbAKT1 may play a vital role in the regulation of K⁺ deficiency tolerance in sweet potato plants [35]. It can be hypothesized that AcShaker genes play an important role in abiotic stress regulation, especially under abiotic stresses, including K⁺ deficiency stress [61, 62]. This finding was further confirmed by expression profiling in this study.

Promoter homeopathic action elements play vital roles in regulating transcription and gene expression [63,64,65]. In this study, a large number of homeostatic elements related to kiwifruit resistance, such as ABA response elements, MeJA response elements, SA response elements, and hypoxia-induced response elements, were identified in the 2000 bp sequence upstream of the kiwifruit Shaker gene (Fig. 5). Numerous previous studies have shown that both ABA [66, 67] and MeJA [68,69,70] can stimulate the expression of plant defense genes and induce chemical defenses and some physiological and functional stress responses. Anaerobic induction [71] enhances plant tolerance to hypoxia, confers the ability to adapt to survival under hypoxic conditions, and effectively improves plant stress tolerance. In conclusion, from the results of homologous promoter role element prediction, the kiwifruit vibrational gene family plays a vital role in kiwifruit resistance to various stresses. Moreover, the GO enrichment analysis in this study revealed that transmembrane transport, inorganic ion transmembrane transport, and bioregulatory pathways were enriched with a greater number of AcShaker genes (Fig. 7), suggesting that genes in the AcShaker gene family can adapt plants to adverse environments by regulating ion transport [32, 34, 57].

Many studies have shown that gene duplication is the prime mode of gene amplification and one of the main molecular mechanisms by which genes evolve to produce genes with new functions [72, 73]. We analyzed the gene duplication events occurring in the Shaker gene family of kiwifruit (Fig. 5). We detected 13 pairs of gene duplications in 28 Shaker genes, all of which were segmental duplications without tandem duplications, indicating that gene duplication events are also vital means of expanding the Shaker gene family in kiwifruit. In addition, the greatest number of Shaker gene duplications occurred on chromosomes 17 and 26, along with a relatively high number of genes chromosomally localized to chromosomes 17 and 26, suggesting that kiwifruit chromosomes 17 and 26 may contain relatively vital kiwifruit gene resources. The efficiency of K⁺ uptake may increase after multiple replication events. The replication results suggest that there should be a polyploidization event in kiwifruit, thus confirming the findings of Shi et al. [58].

In this study, ‘Hongyang’ kiwifruit showed the most severe deficiency in seedlings in MS media with an NH₄NO₃ concentration of 600 mg/L and a KNO₃ concentration of 100 mg/L (Fig. S1 and S2). Although the seedlings could grow, the growth quality was almost negative. The edges of the leaves of the seedlings of this combination were red or brown at the later stage, which was in agreement with the results of the study conducted by Lin et al. [74], and kiwifruit was at a K⁺-deficient level. It is reasonable to use this combination for subsequent transcriptome analysis and consequent analysis of the expression profiles of Shaker gene family members.

Observations of leaf and root phenotypes after 30 d of K⁺ deficiency revealed a generalized reddening of leaf margins, a significant increase in the number of roots and root hairs, and a longer root length in K⁺-deficient plants than in controls (Fig. 8). These are typical K⁺ deficiency phenomena in plants. In rice, when subjected to low-K⁺ stress, leaves become darker in color, and the appearance of old leaves resembles burnt symptoms [75]. In kiwifruit, the symptoms of K⁺ deficiency in the field were greenish leaves and slight wilting of the leaf margins of old leaves, which were consistent with the results of this experiment [76]. According to the results of this study, the number of roots and root hairs and the length of the roots of ‘Hongyang’ kiwifruit increased significantly after K⁺ deficiency compared to those in the control group; therefore, ‘Hongyang’ kiwifruit is a plant with high K⁺ uptake efficiency.

To further elucidate the biological functions of Shaker genes and the principles of their regulation, we analyzed the expression patterns of the Shaker gene family in conjunction with the transcriptome of kiwifruit under low-K⁺ stress. The results showed that all 28 Shaker genes could be expressed under low-K⁺ stress with high transcript levels (Fig. 9), among which three genes were differentially expressed at significantly different levels. Among the three DEGs, the AcShaker15 and AcShaker17 genes were phenotypically downregulated, and the AcShaker22 gene was upregulated. The three AcShake DEGs were subjected to qRT-PCR verification, and the correlation between the results of qRT-PCR and transcriptome sequencing was high (Fig. 10), which confirmed the accuracy of the transcriptome data and verified the reliability of the transcriptome data with the DEGs. The AcShaker22 gene belongs to subfamily IV (KC1) (Fig. 2), a subunit of the K-channel protein family Shaker, and it is primarily expressed in the roots. In A. thaliana, the AtKC1 gene is localized in root hairs as a channel to regulate K⁺ uptake by roots [77]. The kiwifruit AcShaker22 gene can regulate the root uptake of K⁺. In 2002, Birgit investigated the role of the AtKC1 gene in A. thaliana roots and demonstrated that the AtKC1/AKT1 tetramer, formed by AtKC1 and AKT1, was able to regulate the uptake of K in root hairs [78]. The AcShaker15 and AcShaker17 genes belonging to subgroup III (AKT2) and subgroup I (AKT1) were downregulated, whereas AKT1 and AKT2 exhibited similar K⁺ penetration in leaf sclerotial cells [35].

In this study, the phylogenetic tree grouped the AtKC1 gene of A. thaliana into the same (IV) subclade as the AcShaker22 gene of kiwifruit and the AtAKT1 gene and Shaker17 into the same (I) subclade (Fig. 2), suggesting that they may have similar gene functions. Sequence comparison and protein three-site structure prediction revealed (Fig. 3) that AcShaker22 and AtKC1 have similar protein sequences and similar protein structures, and it is hypothesized that AcShaker22 and AtKC1 have similar functions. Posttranslational regulation is a vital regulatory mechanism for K⁺ channel activity in plant cells [78,79,80]. Transcriptional regulation of K⁺ transporters is another important mechanism for plants to cope with environmental stresses, and K⁺ deprivation may induce the transcription of some K⁺ transporters [81, 82]. Wang et al. demonstrated that the AKT1 gene in Agrobacterium dalianensis was negatively regulated by low-K⁺ stress [77]. The AtKC1 gene, a regulator of AKT1, negatively regulates the activity of AKT1 under low-K⁺ stress.

Moreover, AtKT1 belongs to a subunit of the A. thaliana Shaker family of inwardly rectifying channels [34]. Under low-K⁺ stress (Fig. 9), the AcShaeker22 and AcShaker17 genes were significantly expressed in kiwifruit; the AcShaeker22 gene was upregulated, and the AcShaeker17 gene was downregulated. These findings suggest that the kiwifruit AcShaeker22 and AcShaeke17 genes and the A. thaliana AtKC1 gene may be regulated similarly to AtAKT1 (Fig. 12). When kiwifruit perceives low-K⁺ stress, the transcript level of AcShaker may increase. It is hypothesized that the AcShaker22 gene product may interact with other Shaker K⁺ channel subunits, such as the AcShaker17 gene product. Thus, it may regulate K⁺ uptake and transport in kiwifruit and contribute to adaptation to environmental changes. However, further studies and functional analyses (e.g., heterologous expression of AcShaker22 in yeast or Arabidopsis) are needed to better understand the role of the AcShaker22 gene in response to changes in K⁺ concentration.

Fig. 12

Schematic model of the mechanism by which AcShaker22 regulates K⁺ channels. x indicates the inwardly rectifying channel subunit (AcShaker17) of the kiwifruit Shaker family. This is a schematic model of the AcShaker22 gene mechanism based on a model of the AtAKT1 gene. When the external K⁺ concentration is low, the intracellular K⁺ concentration in the plant also becomes low, which may induce transcription of the AcShaker22, thereby increasing the rate of K⁺ uptake and enhancing the plant’s tolerance to low K⁺

It has been suggested that potassium and sodium ion channels are associated. In this study, nine AcShaker genes were significantly differentially expressed under salt stress (Fig. 11). Four of these genes were upregulated (AcShaker9, 12, 19, and 27), and five were downregulated (AcShaker11, 18, 21, 23, and 24). The isotropic bidirectional transport of potassium and sodium ions contributes to the maintenance of intracellular Na⁺/K⁺ homeostasis, which may play a crucial role in the mechanism of salt tolerance in sugar-leaf plants [83]. The AcShaker12 and AcShaker19 genes are in the same subclade V as SKOR in A. thaliana, suggesting that AcShaker12 and AcShaker19 are closely related to AtSKOR. The salt-stressed outwardly rectifying K⁺ channel protein SKOR may be the only Shaker K⁺ channel that plays a role in K⁺ secretion from xylem sap. Continued accumulation of Na⁺ in xylem cells causes depolarization of the plasma membrane of thin-walled xylem cells, which activates its activity and the transport of K⁺ from the xylem to the above-ground parts [84], and it was inferred that the AcShaker12 and AcShaker19 genes may have the same function. In conclusion, based on the transcriptional data, kiwifruit Shaker genes may play a crucial role in the response of kiwifruit to abiotic stresses such as low-K⁺ stress and salt stress.

Conclusion

Twenty-eight AcShaker genes were identified from the kiwifruit genome and classified into six subfamilies by phylogenetic analysis. All 28 AcShaker genes were found to be expressed in the transcriptome data under both salt stress and low-K⁺ stress. The three salt stress transcriptome datasets showed that the expression levels of AcShaker11 and AcShaker18 varied significantly relative to control conditions. Both genes appeared to be downregulated, suggesting that the AcShaker gene may negatively regulate plant injury caused by salt stress. The transcriptome data of plants subjected to low-K⁺ stress showed significant differential expression of three AcShaker genes compared with the control. The qRT-PCR results were strongly correlated with the transcriptome data, indicating that the transcriptome data are reliable. Among these genes, AcShaker22 expression was differentially and significantly upregulated under low-K⁺ stress and could be a candidate gene for breeding new low-K⁺-resistant kiwifruit varieties. Our results on AcShaker genes could be utilized in developing transgenic kiwifruit plants that are tolerant to abiotic stress and more efficient at K⁺ uptake.