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Article

Genome-Wide Identification of the Shaker Potassium Channel Family in Chinese Cabbage and Functional Studies of BrKAT1 in Yeast

by
Jin-Yan Zhou
1,
Ze-Chen Gu
1 and
Dong-Li Hao
2,*
1
Department of Agronomy and Horticulture, Jiangsu Vocational College of Agriculture and Forest, Jurong 212400, China
2
The National Forestry and Grassland Administration Engineering Research Center for Germplasm Innovation and Utilization of Warm-Season Turfgrasses, Jiangsu Key Laboratory for the Research and Utilization of Plant Resources, Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun Yat-Sen), Nanjing 210014, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(9), 1954; https://doi.org/10.3390/agronomy14091954 (registering DOI)
Submission received: 2 August 2024 / Revised: 26 August 2024 / Accepted: 28 August 2024 / Published: 29 August 2024
(This article belongs to the Topic Plant Responses to Environmental Stress)
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Abstract

:
Shaker potassium channels play a crucial role in potassium (K+) nutrition and stress resistance in plants. However, systematic research on Shaker K+ channels in Chinese cabbage [Brassica rapa var. chinensis (L.) Kitamura] remains scarce. This study identified 13 Shaker K+ channel members within the cabbage genome, which are unevenly distributed across eight chromosomes. Notably, the number of Shaker K+ channel members in Chinese cabbage exceeds that found in the model plants Arabidopsis (9) and rice (10). This discrepancy is attributed to a higher number of homologous proteins in Groups II and V of Chinese cabbage, with gene segmental duplication in these two subgroups being a significant factor contributing to the expansion of the Shaker K+ channel gene family. Interspecies collinearity analysis revealed that the whole genome and the Shaker K+ channel family of Chinese cabbage show greater similarity to those of Arabidopsis than to those of rice, indicating that Shaker K+ channels from the Brassicaceae family have a closer relationship than that from the Poaceae family. Given that gene expansion occurs in Group II, we investigated whether a functional difference exists between BrKAT1.1 and BrKAT1.2 using yeast assays and promoter analysis. The expression of two BrKAT1 genes in the potassium uptake-deficient yeast mutant R5421 can restore growth under low potassium conditions, indicating their role in potassium absorption. Truncation of the N-terminal 63 amino acids of BrKAT1.2 resulted in the loss of potassium absorption capability, suggesting that the N-terminus is essential for maintaining the potassium absorption function of BrKAT1.2. Furthermore, the expression of the two BrKAT1 genes in the salt-sensitive yeast G19 enhances yeast tolerance to salt stress. These results demonstrate that BrKAT1.1 and BrKAT1.2 exhibit similar abilities in potassium uptake and salt tolerance. The difference between BrKAT1.1 and BrKAT1.2 lay in their promoter regulatory elements, suggesting that differences in transcriptional regulation contributed to the functional differentiation of BrKAT1.1 and BrKAT1.2. These findings provide a foundation for understanding the evolution and functional mechanisms of the Shaker K+ channel family in Chinese cabbage and for improving potassium nutrition and salt tolerance in this species through the manipulation of BrKAT1.

1. Introduction

Potassium (K+) is an essential nutrient for plant growth, playing critical roles in cell elongation, stomatal movement, signal transduction, protein synthesis, ion balance, and the maintenance of transmembrane potential gradients [1]. Shaker K+ channel-mediated K+ uptake is vital for potassium nutrition in plants [2]. This process primarily involves the uptake of K+ from the soil solution, K+-dependent changes in guard cell volume that regulate stomatal opening and closure, K+ release from xylem parenchyma cells into the transpiration stream, and K+ fluxes during pollen tube growth [3]. Shaker K+ channels are classified into five subtypes. Groups I and II are categorized as inwardly rectifying potassium ion channels, Group III consists of weakly rectifying K+ channels, Group IV includes inward Shaker K+ channel regulatory subunits, and Group V comprises outwardly rectifying K+ channels. In the model plant Arabidopsis, Group I members include AtAKT1, AtAKT5, and AtAKT6. AtAKT1 is predominantly expressed in the root epidermis and cortex cells [4], mediating potassium absorption in the roots. The knockout of AtAKT1 significantly reduces the potassium absorption capacity of roots, resulting in leaf chlorosis under low potassium conditions [5]. AtAKT6 is primarily expressed in pollen and pollen tubes, and its knockout reduces potassium absorption in pollen tubes, thereby inhibiting their growth [6]. AtAKT5 is mainly expressed in flowers and may contribute to meeting the potassium nutritional demands of these tissues [7]. The two members of Group II, AtKAT1 and AtKAT2, are located in leaf guard cells. Disruption of AtKAT1/2 activity leads to a significant loss of potassium influx into guard cells, indicating their essential roles in maintaining potassium nutrition in this tissue [8]. Group III contains only one member, AtAKT2/3, which is crucial for K+ transport in the phloem and sucrose transport to the roots [9]. AtKAT3, a member of Group IV, is expressed in both roots and leaves; however, it does not independently facilitate potassium absorption [10]. Instead, it forms heteropolymers with AtAKT1 [11,12], AtKAT1, AtKAT2, and AtAKT2/3 [13] to regulate ion transport activity and voltage dependence, thereby preventing potassium loss. Group V includes AtGORK and AtSKOR. AtSKOR is primarily expressed in the stellar cortex and pericycle cells of roots, playing a critical role in releasing K+ into the xylem and transporting it upward [14]. AtGORK is expressed in both leaf guard cells and root hairs; its expression in guard cells is essential for potassium efflux [15,16], while its expression in root hairs likely contributes to regulating osmotic potential and membrane potential [17].
Shaker K+ channels, as the primary initiators of potassium absorption in plants, play crucial roles in stress responses, particularly to salt stress and drought. In Zygophyllum xanthoxylum, plants with ZxAKT1 knockdown exposed to salt exhibited a decrease in K+ concentration, leading to K+/Na+ imbalance [18]. Complementary experiments analyzing atakt1 mutant lines demonstrated that SmAKT1 mediates K+ uptake in response to salt stress [19]. Transgenic Arabidopsis and soybean plants overexpressing GmAKT1 show enhanced growth, increased K+ concentrations, reduced Na+ concentrations, and a higher K+/Na+ ratio under salt stress [20]. In rice, overexpression of OsAKT1 was found to positively influence potassium nutrition and drought tolerance [21]. Additionally, in barley, HvAKT1 improved drought tolerance by enhancing K+ uptake in roots, thereby regulating root ion homeostasis [22].
Given the crucial role of Shaker K+ channels in potassium nutrition and stress resistance, an increasing number of Shaker K+ channel members have been identified with the aid of published plant genomic information [1,23,24,25,26,27]. Notably, there are differences in the number of family members and evolutionary relationships among these Shaker K+ channels, as well as functional variations among homologous proteins [9,28]. This underscores the urgency of studying Shaker K+ channels in specific species. Although Chinese cabbage is a widely consumed vegetable, systematic research on the Shaker K+ channel members in this species is lacking. This study aimed to identify the Shaker K+ channel members and their evolutionary relationships in cabbage using various bioinformatics techniques, including whole-genome identification, gene structure analysis, chromosome position analysis, intraspecific collinearity, interspecific collinearity analysis, and promoter analysis. Given that gene expansion occurs in Group II, we investigated whether a functional difference exists between BrKAT1.1 and BrKAT1.2 using yeast assays. Two BrKAT1 homologs were transferred into the potassium absorption-deficient yeast R5421 to evaluate their growth performance under different potassium supply intensities, thereby determining their potassium absorption function. Additionally, the N-terminus of BrKAT1 was truncated to investigate its effect on BrKAT1 function. Finally, the two BrKAT1 genes were transformed into the salt-sensitive yeast G19, and their roles in salt tolerance were assessed through growth performance under various salt stress conditions. This study is expected to contribute to a better understanding of the evolution and functional mechanisms of the Shaker K+ channel in cabbage.

2. Materials and Methods

2.1. Genome-Wide Identification of Shaker K+ Channel Family Members

In accordance with our previous methods [29], we performed a search for the protein sequence of Brara_Chiifu, Arabidopsis, and rice in the Pfam database using the Pfam_stcan program (version 14.0, https://github.com/SMRUCC/GCModeller/tree/master/src/interops/scripts/PfamScan, accessed on 20 October 2023). Protein sequences containing the conserved domains of the Shaker K+ channel (PF00027.28: cNMP_binding + PF00520.30: Ion_trans) were screened. Thirteen, nine, and ten Shaker K+ channel family members were identified from Brara_Chiifu, Arabidopsis, and rice. The Chinese cabbage genome was downloaded from http://39.100.233.196:82/download_genome/Brassica_Genome_data/Brara_Chiifu_V3.5/, the Arabidopsis genome was downloaded from https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001433935.1/, and the rice genome was downloaded from https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_001433935.1/.

2.2. Phylogenetic Tree Analysis, Gene Structure, and Chromosome Position

Phylogenetic tree analysis: The protein sequences of the Shaker K+ channel members identified in all three species were combined and input into the MUSCLE (version 5.0, https://drive5.com/muscle/) program for global alignment. The phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap replicates. The comparison results were then input into the MEGA program (version 11, https://www.megasoftware.net/) to construct a maximum likelihood (ML) tree, which was subsequently visualized using the iTOL program (version 5.0, https://itol.embl.de/) [29].
Gene structure: Motif information for the Shaker K+ channel was extracted from the Pfam annotation results, and a motif distribution map was generated using the GSDS program (version 2.0, http://gsds.gao-lab.org/, accessed on 10 December 2023) [29].
Chromosome position: Information on Shaker K+ channels was extracted from the GFF file of the centipedegrass genomic database. The positions of these genes on the chromosomes were visualized using the MG2C program (version 2.1, http://mg2c.iask.in/mg2c_v2.1/) [30].

2.3. Intraspecific Collinearity

First, the MCScanX program (version 2.0, https://github.com/wyp1125/MCScanx) was used to identify collinear blocks in the Brara_Chiifu genome. Subsequently, Shaker K+ channel members within these collinear blocks were extracted, and finally, the Circos program (version 0.69, http://circos.ca/) was employed to visualize them [25].

2.4. Interspecific Collinearity

Collinear blocks among the Brara_Chiifu genome, Arabidopsis genome, and rice genome were identified using the JCVI program (version 0.9.13, https://github.com/tanghaibao/jcvi). Subsequently, the Shaker K+ channel members distributed within these collinear blocks were extracted. The final visualization was generated using the JCVI (version 0.9.13, https://github.com/tanghaibao/jcvi) drawing subroutine [31].

2.5. Promoter Analysis

For each Shaker K+ channel member, a 2000 bp sequence upstream of the ATG translational initiation site was extracted and identified as the promoter region. Cis-acting regulatory elements were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and subsequently visualized with TBtools.

2.6. Plant Culture, Gene Cloning, Vector Construction, and Yeast Transformation

Seeds of Chinese cabbage [Brassica rapa var. chinensis (L.) Kitamura] were directly sown in the soil. Uniform seedlings with a spacing of 25 cm × 15 cm were collected when they reached the three-leaf stage. After one month of growth, the plants were harvested. The base fertilizer used was 225 kg/hm2 of compound fertilizer (N-P2O5-K2O: 15-15-15).
The mature leaves of Chinese cabbage were harvested, cut, and ground into a fine powder. RNA was extracted using an RNA extraction kit (#RC201, Vazyme, Nanjing, China) and subsequently reverse transcribed into cDNA using a reverse transcription kit (#R211, Vazyme, Nanjing, China). The BrKAT1.1, BrKAT1.2, and N-terminal truncated BrKAT1.2 were then amplified via PCR. The PCR procedure was as follows: predenaturation at 95 °C for 5 min, denaturation at 95 °C for 15 s, annealing at 65 °C for 15 s, extension at 72 °C for 2 min, followed by 30 cycles of reaction, and a final extension at 72 °C for 5 min [32]. The BrKAT1.1, BrKAT1.2, and N-terminal truncated BrKAT1.2 were cloned and inserted into the pYES2 vector, and the sequence was verified by sequencing (General Biology Company, Chuzhou, China). The primers used are listed in Table 1 and were synthesized (purification method: PAGE) by the General Biology Company.
Yeast transformation was performed according to the instructions provided in the yeast transformation kit (#SK2400, Coolaber, Beijing, China). The yeast transformants screened on SD-URA media were verified by sequencing (General Biology, Chuzhou, China). The SsKAT1.1 from Stenotaphrum secundatum used as a comparison for KAT1.1 from other plant species is kept by our laboratory. More information about this gene is currently being submitted to another journal.

2.7. Yeast Assays

The potassium ion absorption-deficient yeast R5421 was purchased from Coolaber (Beijing, China). The sequenced transformants were cultured in SD-URA liquid media supplemented with 50 mM KCl at 30 °C for 2 days. When the optical density at 600 nm (OD600) reached 0.6–0.8, the cells were collected, and the OD600 was adjusted to 1.0 with sterile water. The suspension was subsequently diluted by factors of 10, 100, and 1000. Five microliters of the suspension were spotted on AP-URA solid media supplemented with different concentrations of KCl (100, 2, 0.2, and 0.02 mM). The resulting yeast was cultured at 30 °C, and photographs were taken 2 days after spotting [27].
The salt-sensitive yeast G19 was purchased from Baosai (Beijing, China). The sequenced transformants were cultured in SG-URA liquid media at 30 °C for 2 days. When the OD600 reached 0.6–0.8, the cells were collected, and the OD600 was adjusted to 1.0 with sterile water. The OD600 of the suspension was subsequently diluted to 0.1, 0.01, and 0.001 with sterile water. Five microliters of the suspension were spotted on AP-URA solid media supplemented with different concentrations of NaCl (0, 100, and 200 mM) and a fixed concentration of KCl (2 mM). The resulting yeast was cultured at 30 °C, and photographs were taken 2 days after spotting [33].
The experiments contained three replicates and were repeated three times.

3. Results

3.1. Phylogenetic Tree Analysis

A search of the Chinese cabbage protein database revealed a total of 13 Shaker K+ channel members, which is significantly more than the 9 found in Arabidopsis and 10 in rice (Figure 1). The phylogenetic tree analysis indicated that these Shaker K+ channels can be divided into five groups. Among the 13 Shaker K+ channel members in Chinese cabbage, 3 are clustered in Group I (BrAKT1, BrAKT5, and BrAKT6), 4 in Group II (BrKAT1.1, BrKAT1.2, BrKAT2.1, and BrKAT2.2), 1 in Group III (BrAKT2/3), 2 in Group IV (BrKAT3.1 and BrKAT3.2), and 3 in Group V (BrSKOR1.1, BrSKOR1.2, and BrGORK). Among the nine Shaker K+ channel members in Arabidopsis, three are clustered in Group I (AtAKT1, AtAKT5, and AtAKT6), two in Group II (AtKAT1 and AtKAT2), one in Group III (AtAKT2/3), one in Group IV (AtKAT3), and two in Group V (AtSKOR1.1 and AtGORK). Among the 10 Shaker K+ channel members in rice, 2 are clustered in Group I (OsAKT1 and OsAKT3), 3 in Group II (OsKAT1, OsKAT2, and OsKAT3), 1 in Group III (OsAKT2), 2 in Group IV (OsKAT4 and OsKAT6), and 2 in Group V (OsKOR1 and OsKOR2). The higher number of Shaker K+ channel members in cabbage compared to Arabidopsis and rice can be attributed to the expansion of homologous protein numbers in Groups II and V. The number of amino acids in the Shaker K+ channels of Chinese cabbage varies between 615 and 877 amino acids. The molecular weight (MW) ranges from 74.51 kDa to 98.16 kDa, and the isoelectric point (pI) ranges from 6.05 to 9.18 (Table 2).

3.2. Protein Domains of the Chinese Cabbage Shaker K+ Channel

Genetic structure analysis revealed that members of the cabbage Shaker K+ channel within the same group exhibit similar protein domains (Figure 2). Specifically, Group I members possess ion-trans, cNMP-binding, Ank-2, and KHA protein domains, while Group II proteins contain ion-trans, cNMP-binding, and KHA domains. Group III proteins possess ion-trans, cNMP-binding, Ank-2, and KHA domains. Group IV proteins consist of ion-trans, cNMP-binding, and KHA domains, whereas Group V proteins include ion-trans, cNMP-binding, and Ank-2 domains. Notably, Shaker K+ channel members within the same group share similar protein domain layouts; in contrast, members from different groups, although possessing similar domains, exhibit distinct protein domain arrangements compared to those of the same group.

3.3. Chromosome Position and Intraspecific Collinearity of Shaker K+ Channels in Chinese Cabbage

The 13 Shaker K+ channel members of Chinese cabbage are unevenly distributed across 8 chromosomes (Figure 3). Among these, chromosome A01 (BrKAT3.2, BrKAT2.1, BrAKT5, and BrAKT2/3) contains four Shaker K+ channel members. Chromosomes A03 (BrSKOR1.2 and BrKAT3.1) and A09 (BrKAT1.1 and BrAKT6) each contain two Shaker K+ channel members, while chromosomes A04 (BrAKT1), A05 (BrSKOR1.1), A06 (BrKAT1.2), A07 (BrGORK), and A08 (BrKAT2.2) each have one member. Notably, Shaker K+ channel members are absent from chromosomes A02 and A10.
The results of the intraspecific collinearity analysis revealed nine collinear gene pairs in the Shaker K+ channel of Chinese cabbage (Figure 4). These gene pairs include BrKAT1.1-BrKAT2.2, BrKAT2.2-BrKAT1.2, BrKAT1.1-BrKAT1.2, BrKAT2-BrKAT2.2, BrKAT1.2-BrKAT2.1, BrSKOR1.1-BrSKOR1.2, BrAKT6-BrKAT3.1, BrKAT3.1-BrKAT3.2, and BrAKT6-BrKAT3.2. Among these, seven pairs occur within the same subclass, with five pairs in Group II (BrKAT1.1-BrKAT1.2, BrKAT2.1-BrKAT2.2, BrKAT1.1-BrKAT2.2, BrKAT1.2-BrKAT2.2, and BrKAT1.2-BrKAT2.1), one pair in Group IV (BrKAT3.1-BrKAT3.2), and one pair in Group V (BrSKOR1.1-BrSKOR1.2). The other two collinear gene pairs (BrAKT6-BrKAT3.1 and BrAKT6-BrKAT3.2) exist in different subtypes, both occurring between Group I and Group IV. These collinear gene pairs represent segmental duplications rather than tandem duplications.
The Ka/Ks values of nine duplicated Shaker K+ channel gene pairs were calculated for selective analysis. The results indicate that the Ka/Ks ratios varied from 0.10 to 0.28 (Table 3).

3.4. Interspecific Collinearity

The collinearity analysis of the genomes of Chinese cabbage, Arabidopsis, and rice suggested that there are more collinear gene pairs between the Chinese cabbage genome and the Arabidopsis genome, whereas there are particularly few collinear gene pairs between the Chinese cabbage genome and the rice genome (Figure 5). This finding indicates that Chinese cabbage is more closely related to Arabidopsis in terms of evolution. Shaker K+ channel collinear gene pairs exist only in Chinese cabbage and Arabidopsis but not in Chinese cabbage and rice, suggesting a differentiation of Shaker K+ channels between the Brassicaceae and Poaceae families. A total of 15 Shaker K+ channel collinear gene pairs were identified in Chinese cabbage and Arabidopsis. These gene pairs are BrAKT5-AtAKT6, BrAKT6-AtAKT6, BrSKOR1.2-AtSKOR, BrSKOR1.1-AtSKOR, BrKAT2.1-AtKAT2, BrAKT2/3-AtAKT2/3, BrAKT5-AtAKT5, BrKAT3.2-AtKAT3, BrKAT3.1-AtKAT3, BrKAT1.2-AtKAT2, BrKAT2.2-AtKAT2, BrAKT6-AtAKT5, BrKAT2.1-AtKAT1, BrKAT1.2-AtKAT1, and BrKAT1.1-AtKAT1. These collinear gene pairs are distributed across five groups. Among them, there are six pairs in Group II (BrKAT2.1-AtKAT2, BrKAT1.2-AtKAT2, BrKAT2.2-AtKAT2, BrKAT2.1-AtKAT1, BrKAT1.2-AtKAT1, and BrKAT1.1-AtKAT1), two pairs in Group V (BrSKOR1.2-AtSKOR and BrSKOR1.1-AtSKOR), four pairs in Group I (BrAKT5-AtAKT6, BrAKT6-AtAKT6, BrAKT5-AtAKT5, and BrAKT6-AtAKT5), two pairs in Group IV (BrKAT3.2-AtKAT3 and BrKAT3.1-AtKAT3), and one pair in Group III (BrAKT2/3-AtAKT2/3).

3.5. Promoter Analysis

An analysis of the promoter in the upstream 2 kb region of the Shaker K+ channel gene revealed the presence of multiple regulatory elements associated with Shaker K+ channels (Figure 6). The same group of genes possess distinct promoter regulatory elements. Specifically, these differences are outlined as follows.
A total of 15 regulatory elements were identified in BrAKT1, including the auxin-responsive element (1), cis-acting element involved in low-temperature responsiveness (2), cis-acting element involved in the abscisic acid responsiveness (3), cis-acting regulatory element involved in light responsiveness (4), cis-acting regulatory element involved in the MeJA responsiveness (2), MYB binding site involved in drought-inducibility (1), part of a conserved DNA module involved in light responsiveness (1), and protein binding site (1).
A total of 23 regulatory elements were identified in BrAKT5, including the cis-acting element involved in defense and stress responsiveness (1), cis-acting element involved in light responsiveness (1), cis-acting element involved in low-temperature responsiveness (1), cis-acting element involved in the abscisic acid responsiveness (2), cis-acting regulatory element essential for the anaerobic induction (1), cis-acting regulatory element involved in light responsiveness (3), cis-acting regulatory element involved in the MeJA responsiveness (2), cis-regulatory element involved in endosperm expression (1), gibberellin-responsive element (1), light responsive element (2), MYBHv1 binding site (2), part of a conserved DNA module involved in light responsiveness (2), and part of a light responsive element (4).
A total of 21 regulatory elements were identified in BrAKT6, including the binding site of AT-rich DNA binding protein (ATBP-1) (1), cis-acting element involved in defense and stress responsiveness (1), cis-acting element involved in the abscisic acid responsiveness (2), cis-acting regulatory element essential for the anaerobic induction (1), cis-acting regulatory element involved in auxin responsiveness (1), cis-acting regulatory element involved in light responsiveness (2), cis-acting regulatory element involved in the MeJA responsiveness (2), cis-acting regulatory element related to meristem expression (1), gibberellin-responsive element (1), light responsive element (1), MYB binding site involved in flavonoid biosynthetic genes regulation (1), and part of a conserved DNA module involved in light responsiveness (7).
A total of 17 regulatory elements were identified in BrKAT1.1, including the binding site of AT-rich DNA binding protein (ATBP-1) (1), cis-acting element involved in defense and stress responsiveness (2), cis-acting element involved in the abscisic acid responsiveness (1), cis-acting regulatory element essential for the anaerobic induction (4), cis-acting regulatory element involved in auxin responsiveness (3), cis-acting regulatory element involved in light responsiveness (1), light responsive element (1), part of a conserved DNA module involved in light responsiveness (1), part of a light responsive element (2), and part of a module for light response (1).
A total of 21 regulatory elements were identified in BrKAT1.2, including the cis-acting element involved in low-temperature responsiveness (2), cis-acting element involved in salicylic acid responsiveness (1), cis-acting element involved in the abscisic acid responsiveness (1), cis-acting regulatory element essential for the anaerobic induction (1), cis-acting regulatory element involved in auxin responsiveness (1), cis-acting regulatory element involved in light responsiveness (1), cis-acting regulatory element involved in the MeJA responsiveness (2), gibberellin-responsive element (1), light responsive element (1), MYB binding site involved in drought-inducibility (1), part of a light responsive element (7), and part of a module for light response (2).
A total of 24 regulatory elements were identified in BrKAT2.1, including the auxin-responsive element (1), cis-acting element involved in defense and stress responsiveness (2), cis-acting element involved in low-temperature responsiveness (1), cis-acting element involved in salicylic acid responsiveness (1), cis-acting element involved in the abscisic acid responsiveness (1), cis-acting regulatory element essential for the anaerobic induction (2), cis-acting regulatory element involved in auxin responsiveness (1), cis-acting regulatory element involved in light responsiveness (2), cis-acting regulatory element related to meristem expression (2), cis-regulatory element involved in endosperm expression (1), element for maximal elicitor-mediated activation (1), light responsive element (1), part of a conserved DNA module involved in light responsiveness (2), part of a light responsive element (4), protein binding site (1), and wound-responsive element (1).
A total of 28 regulatory elements were identified in BrKAT2.2, including the binding site of AT-rich DNA binding protein (ATBP-1) (1), cis-acting element involved in defense and stress responsiveness (1), cis-acting element involved in low-temperature responsiveness (1), cis-acting element involved in salicylic acid responsiveness (1), cis-acting regulatory element essential for the anaerobic induction (3), cis-acting regulatory element involved in auxin responsiveness (1), cis-acting regulatory element involved in circadian control (1), cis-acting regulatory element involved in light responsiveness (1), cis-acting regulatory element involved in the MeJA responsiveness (2), gibberellin-responsive element (1), light responsive element (3), MYB binding site involved in light responsiveness (1), MYBHv1 binding site (1), part of a conserved DNA module involved in light responsiveness (5), part of a light responsive element (4), and part of a module for light response (1).
A total of 29 regulatory elements were identified in BrAKT2/3, including the auxin-responsive element (2), cis-acting element involved in defense and stress responsiveness (1), cis-acting element involved in low-temperature responsiveness (1), cis-acting element involved in salicylic acid responsiveness (1), cis-acting element involved in the abscisic acid responsiveness (2), cis-acting regulatory element (1), cis-acting regulatory element essential for the anaerobic induction (3), cis-acting regulatory element involved in circadian control (2), cis-acting regulatory element involved in light responsiveness (1), cis-acting regulatory element involved in the MeJA responsiveness (2), enhancer-like element involved in anoxic-specific inducibility (1), gibberellin-responsive element (1), light responsive element (1), MYB binding site involved in drought-inducibility (3), MYBHv1 binding site (1), part of a conserved DNA module involved in light responsiveness (5), and part of a light responsive element (1).
A total of 29 regulatory elements were identified in BrKAT3.1, including the binding site of AT-rich DNA binding protein (ATBP-1) (1), cis-acting element involved in defense and stress responsiveness (1), cis-acting element involved in low-temperature responsiveness (3), cis-acting element involved in the abscisic acid responsiveness (3), cis-acting regulatory element essential for the anaerobic induction (3), cis-acting regulatory element involved in light responsiveness (3), cis-acting regulatory element involved in the MeJA responsiveness (6), cis-acting regulatory element involved in zein metabolism regulation (1), cis-acting regulatory element related to meristem expression (1), gibberellin-responsive element (1), MYB binding site involved in light responsiveness (1), part of a conserved DNA module involved in light responsiveness (3), part of a module for light response (1), and part of a module for light response (1).
A total of 32 regulatory elements were identified in BrKAT3.2, including the cis-acting element involved in defense and stress responsiveness (1), cis-acting element involved in low-temperature responsiveness (1), cis-acting element involved in the abscisic acid responsiveness (4), cis-acting regulatory element essential for the anaerobic induction (3), cis-acting regulatory element involved in light responsiveness (4), cis-acting regulatory element involved in the MeJA responsiveness (2), light responsive element (3), part of a conserved DNA module involved in light responsiveness (3), and part of a light responsive element (11).
A total of 31 regulatory elements were identified in BrSKOR1.1, including the cis-acting element involved in the abscisic acid responsiveness (5), cis-acting regulatory element essential for the anaerobic induction (2), cis-acting regulatory element involved in light responsiveness (6), cis-acting regulatory element involved in the MeJA responsiveness (8), cis-acting regulatory element involved in zein metabolism regulation (3), light responsive element (1), MYB binding site involved in light responsiveness (2), and part of a light responsive element (4).
A total of 39 regulatory elements were identified in BrSKOR1.2, including the cis-acting element involved in defense and stress responsiveness (1), cis-acting element involved in salicylic acid responsiveness (1), cis-acting element involved in the abscisic acid responsiveness (5), cis-acting regulatory element essential for the anaerobic induction (1), cis-acting regulatory element involved in auxin responsiveness (1), cis-acting regulatory element involved in light responsiveness (8), cis-acting regulatory element involved in the MeJA responsiveness (6), light responsive element (4), MYB binding site involved in drought-inducibility (1), MYBHv1 binding site (2), part of a conserved DNA module involved in light responsiveness (3), and part of a light responsive element (6).
A total of 23 regulatory elements were identified in BrGORK, including the auxin-responsive element (1), binding site of AT-rich DNA binding protein (ATBP-1) (1), cis-acting element involved in salicylic acid responsiveness (2), cis-acting element involved in the abscisic acid responsiveness (4), cis-acting regulatory element essential for the anaerobic induction (3), cis-acting regulatory element involved in light responsiveness (4), cis-acting regulatory element involved in the MeJA responsiveness (2), light responsive element (1), MYB binding site involved in light responsiveness (2), and part of a light responsive element (3).

3.6. Both BrKAT1 Proteins Have Potassium Absorption Functions, and the N-Terminus Is Necessary for Maintaining Potassium Channel Function

Two wild-type BrKAT1 and an N-terminal truncated BrKAT1.2 mutant were transferred into the potassium uptake-deficient yeast R5421, and their growth performance under different potassium supply intensities was studied to determine whether they have potassium absorption activity (Figure 7). The results showed that yeast transformed with various plasmids could grow normally under sufficient potassium supply (100 mM), indicating that the transfer of different plasmids does not affect yeast growth. Under restricted potassium supply (≤2 mM), yeast with the empty vector could not survive, which is consistent with previous reports [27]. Both BrKAT1 genes can restore yeast growth under low potassium supply, indicating that they both have potassium absorption functions. The growth performance of yeast transformed with the N-terminal truncated BrKAT1.2 was similar to that of yeast transformed with the empty vector; neither could survive under restricted potassium supply, indicating that truncation of the N-terminus of BrKAT1.2 resulted in the loss of potassium absorption activity.

3.7. BrKAT1 Enhances Cell Salt Tolerance

Two wild-type BrKAT1 genes and one SsKAT1.1 (from Stenotaphrum secundatum) gene were transferred into the salt-sensitive yeast G19, and their effects on salt tolerance were studied by observing growth performance at different salt concentrations (Figure 8). The results showed that yeast transformed with different plasmids could grow normally under salt-free conditions, indicating that the transfer of different plasmids did not affect yeast growth. After high concentrations of NaCl were added to the culture medium, the growth of yeast transformed with the empty vector was significantly inhibited, and the degree of growth inhibition increased with increasing NaCl concentration. This finding is consistent with previously reported results [33]. Compared to growth performance under non-salt stress, BrKAT1-expressing yeast did not show significant growth inhibition under high NaCl conditions. In comparison to the empty vector-expressing yeast, the BrKAT1-expressing yeast exhibited greater salt tolerance. These results indicate that BrKAT1 can enhance cell salt tolerance. However, the SsKAT1.1-expressing yeast showed no significant growth difference compared to the empty vector-expressing yeast, indicating that under the experimental salt concentrations, SsKAT1.1 does not affect the salt tolerance of yeast cells.

4. Discussion

4.1. Gene Expansion Exists in the Shaker K+ Channel of Chinese Cabbage, and the Segmental Duplication of the Genes in Group II and Group V as the Main Reason for the Above Phenomenon

There are 13 members of the Shaker K+ channel in Chinese cabbage, a number significantly greater than that found in Arabidopsis (9), rice (10), foxtail millet (10) [27], peach (7) [26], sweet potato (11) [34], Vigna radiata (8) [24], pear (8) [23], apple (10) [23], and strawberry (6) [23], indicating that gene expansion has occurred in the Shaker K+ channel of Chinese cabbage (Figure 1). Phylogenetic tree analyses showed that the expansion of the Shaker K+ channel gene in Chinese cabbage primarily occurred in two subclasses: Group II and Group V (Figure 1). This gene expansion is attributed to gene segment duplication events in the two groups (Figure 4). The KAT1 and KAT2 genes in Group II produced two paralogous genes, KAT1.1, KAT1.2, KAT2.1, and KAT2.2, while the SKOR gene in Group V produced two paralogues, SKOR1.1 and SKOR1.2. In conclusion, these studies reveal that, unlike in other species, there is significant gene expansion in the Shaker K+ channel of Chinese cabbage, primarily due to the segmental duplication of genes in Groups II and V. This segmental duplication may have been caused by whole-genome duplication (WGD) events that occurred during the evolution of Chinese cabbage [35,36]. These findings are consistent with reports that segmental duplication is an important driving force behind the expansion of plant ARF (auxin-responsive factor) gene families [37]. Together, these results demonstrate that segmental duplication plays a crucial role in driving the expansion of plant gene families.
The results of interspecific collinearity suggest that both the entire genome and the Shaker K+ channel family of Chinese cabbage have a close evolutionary relationship with those of Arabidopsis but a distant evolutionary relationship with those of rice (Figure 5). This may be because cabbage and Arabidopsis belong to the Brassicaceae family, whereas rice belongs to the Poaceae family. The fact that Brassica species not only shared a whole-genome duplication event approximately 13–17 million years ago with their close relative Arabidopsis but also underwent a Brassica-specific whole-genome triplication event approximately 5–9 million years ago [38,39] may explain the expansion of the Shaker K+ channel gene in Chinese cabbage and the lack of expansion of the Arabidopsis gene.

4.2. The Two Homologs of Group II, BrKAT1.1 and BrKAT1.2, Have Similar Potassium Uptake and Salt Tolerance Ability but Possess Distinct Promoter Elements

Gene duplication and subsequent functional differentiation of new genes promote the expansion and new functionalization of important gene families, enabling plants to better adapt to their environments [40]. Expression-level variation and protein-level functional variation are two major routes to gene functional differentiation [41]. It is found that the regulatory elements located on the promoter regions are different between BrKAT1.1 and BrKAT1.2 (Figure 7), suggesting that differences in transcriptional regulation contributed to the functional differentiation of BrKAT1.1 and BrKAT1.2. The protein-level functional investigation demonstrated that both BrKAT1 genes can mediate similar potassium absorption and enhance cell salt tolerance (Figure 6, Figure 7 and Figure 8). Previous studies have shown that genes homologous to BrKAT1, such as AtKAT1 and OsKAT1, can mediate potassium absorption and that their expression in yeast or rice can increase salt tolerance [42]. These results indicate that BrKAT1 has basic functions similar to those of its homologous genes, specifically mediating potassium absorption and playing a role in salt tolerance. However, the SsKAT1.1 from Stenotaphrum secundatum did not affect the salt tolerance of yeast cells, indicating the distinct roles of homologous KAT1 in salt tolerance. The Ka/Ks value between the BrKAT1.1 and BrKAT1.2 gene pairs, being less than 1, suggests that segmental duplication is associated with purifying selection on amino acid substitutions [37]. This results in limited functional differentiation. In the future, both of these genes can be utilized as candidate genes to improve plant potassium utilization efficiency and salt tolerance. Although there is no difference in the basic functions of the two paralogues, their unique protein functions may differ due to variations in amino acid sequences. For example, a substitution of a key amino acid results in OsAKT2 functioning as an inward-rectifying potassium ion channel, whereas AtAKT2/3 behaves as a weakly rectifying potassium ion channel [9]. AtAKT1 requires CBL1-CIPK23 activation to perform potassium absorption functions, whereas its homologous gene, OsAKT1, does not require CBL1-CIPK23 for potassium absorption [28]. Therefore, further research is needed to determine whether these two BrKAT1 paralogous genes have different functional regulatory mechanisms.

4.3. The N-Terminus Is Necessary for Maintaining the Function of BrKAT1

Structurally, plant Shaker K+ channels typically exhibit a relatively short intracytoplasmic N-terminal domain (approximately 60 amino acids), followed by a hydrophobic core composed of six transmembrane segments (S1 to S6, with the pore domain inserted between S5 and S6), and a long intracytoplasmic region represents more than half of the sequence [43]. The fifth and sixth transmembrane segments, along with the membrane P domain linking these segments, form the walls of the channel pore. The P domain contains a highly conserved stretch of residues (TV/TGYG), which constitutes the narrowest region of the pore and acts as a selectivity filter [44]. The fourth transmembrane segment, which harbors positively charged amino acids (R or K), is responsible for regulating the voltage gating of the Shaker channel. Movements of this segment within the membrane, in response to changes in transmembrane electrical potential, result in conformational changes that favor the opening or closing of the pore. In plant Shaker K+ channels, a large cytosolic region downstream of the hydrophobic core contains several domains, including a putative cyclic nucleotide-binding site and, in the majority of Shaker channels, an ankyrin domain, which serves as a site of interaction with regulatory proteins (e.g., kinases or phosphatases) (Figure 2) [2,45]. The N-terminus of the Shaker K+ channel AtKAT1 regulates its voltage-dependent gating by altering the membrane electric field, but it does not determine its K+ uptake capacity [46]. This study found that completely truncating the N-terminus of BrKAT1 resulted in loss of function, indicating the crucial role of the N-terminus in maintaining potassium ion channel activity (Figure 7). The main reason for this phenomenon is that we used complete truncation of the N-terminus, while the previous experiment employed partial truncation (△20–34). Prior reports have shown that removing half of the N-terminus (△2–34) can cause AtKAT1 to lose its potassium absorption capacity [47]. In this study, the entire N-terminus (△1–63) of BrKAT1 was truncated, resulting in the loss of potassium absorption capacity. Combined with the observation that a single amino acid variation (C8A) or partial truncation (△1–15) in the N-terminus can shift AtAKT1 from an inactive state to an active state [48], we speculate that the N-terminus functions by modulating the transition between the inactive and active states of BrKAT1.

5. Conclusions

There are 13 members of the Shaker K+ channel in Chinese cabbage, a number that exceeds that found in other species. The expansion of the Shaker K+ channel gene family in Chinese cabbage is primarily attributed to segmental duplication events involving genes in Groups II and V. Further analysis reveals that the two homologs in Group II, BrKAT1.1 and BrKAT1.2, exhibit similar abilities in potassium uptake and salt tolerance, but with distinct promoter response elements. These results suggest that differences in transcriptional regulation contributed to the functional differentiation of BrKAT1.1 and BrKAT1.2. Structurally, the N-terminus is essential for maintaining the function of BrKAT1. These findings provide a foundation for understanding the evolution and functional mechanisms of the Shaker K+ channel family in Chinese cabbage, as well as for improving potassium nutrition and salt tolerance through the manipulation of BrKAT1.

Author Contributions

D.-L.H. designed the work. J.-Y.Z. carried out the experiments. D.-L.H. and J.-Y.Z. wrote the paper. Z.-C.G. revised the paper. All authors contributed to the article. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Jiangsu Provincial Double-Innovation Doctor Program (Grant No. JSSCBS20221643) and the Program for the Young Innovative Talents of Jiangsu Vocational College of Agriculture and Forest (Grant No. 2021kj26).

Data Availability Statement

The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no competing interests.

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Figure 1. Phylogenetic tree of the relationships among the Shaker K+ channel proteins of Chinese cabbage, Arabidopsis, and rice. Shaker K+ channels from Chinese cabbage are indicated by red dots. Shaker K+ channels from Arabidopsis are indicated by blue squares. Shaker K+ channels from rice are represented by green stars.
Figure 1. Phylogenetic tree of the relationships among the Shaker K+ channel proteins of Chinese cabbage, Arabidopsis, and rice. Shaker K+ channels from Chinese cabbage are indicated by red dots. Shaker K+ channels from Arabidopsis are indicated by blue squares. Shaker K+ channels from rice are represented by green stars.
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Figure 2. Phylogenetic tree and protein domains among the Chinese cabbage Shaker K+ channel family. The left panel shows a phylogenetic tree, and the right panel shows the protein domain results. Both are constructed on the basis of the Shaker K+ channel amino acid sequence.
Figure 2. Phylogenetic tree and protein domains among the Chinese cabbage Shaker K+ channel family. The left panel shows a phylogenetic tree, and the right panel shows the protein domain results. Both are constructed on the basis of the Shaker K+ channel amino acid sequence.
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Figure 3. Position of Shaker K+ channel members on the chromosomes. The name and gene ID of the Shaker K+ channel are indicated.
Figure 3. Position of Shaker K+ channel members on the chromosomes. The name and gene ID of the Shaker K+ channel are indicated.
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Figure 4. Intraspecies collinearity analysis. The gray line represents the collinear gene pairs, whereas the red line represents the collinear Shaker K+ channel gene pairs in the genome of Chinese cabbage. The gene name (blue) and gene ID number are marked at the corresponding positions.
Figure 4. Intraspecies collinearity analysis. The gray line represents the collinear gene pairs, whereas the red line represents the collinear Shaker K+ channel gene pairs in the genome of Chinese cabbage. The gene name (blue) and gene ID number are marked at the corresponding positions.
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Figure 5. Collinearity analyses of Shaker K+ channel genes among Chinese cabbage, rice, and Arabidopsis. The gray lines among the three plants represent collinear blocks in wide regions of the genome, whereas the red lines represent the orthologous relationships of the Shaker K+ channel genes.
Figure 5. Collinearity analyses of Shaker K+ channel genes among Chinese cabbage, rice, and Arabidopsis. The gray lines among the three plants represent collinear blocks in wide regions of the genome, whereas the red lines represent the orthologous relationships of the Shaker K+ channel genes.
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Figure 6. Promoter analysis of Shaker K+ channels. The various regulatory elements were represented using distinct colors.
Figure 6. Promoter analysis of Shaker K+ channels. The various regulatory elements were represented using distinct colors.
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Figure 7. Yeast functional complementation. The growth performance of a yeast mutant (deficient in K+ uptake) harboring BrKAT1.1, BrKAT1.2, or one BrKAT1 gene with a truncated N-terminus is presented. The K+ concentrations used were 0.02, 0.2, 2, and 100 mM. The dilution factor of the cell suspension is indicated.
Figure 7. Yeast functional complementation. The growth performance of a yeast mutant (deficient in K+ uptake) harboring BrKAT1.1, BrKAT1.2, or one BrKAT1 gene with a truncated N-terminus is presented. The K+ concentrations used were 0.02, 0.2, 2, and 100 mM. The dilution factor of the cell suspension is indicated.
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Figure 8. The expression of BrKAT1 increases salt tolerance in yeast. The growth performance of a BrKAT1.1-, BrKAT1.2-, and SsKAT1.1-expressing yeast mutant (G19, a salt-sensitive yeast strain) on AP media supplemented with different concentrations of Na+ is presented. The dilution factor of the cell suspension is indicated.
Figure 8. The expression of BrKAT1 increases salt tolerance in yeast. The growth performance of a BrKAT1.1-, BrKAT1.2-, and SsKAT1.1-expressing yeast mutant (G19, a salt-sensitive yeast strain) on AP media supplemented with different concentrations of Na+ is presented. The dilution factor of the cell suspension is indicated.
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Table 1. Primers used for gene cloning.
Table 1. Primers used for gene cloning.
NamePrimers (5′ to 3′)
BrKAT1.1-FactatagggaatattaagcttATGTCCATCTCTTGCACCAGAAACT
BrKAT1.1-RtgatggatatctgcagaattcTCAGCTTGATGAGAAAAACAAATGA
BrKAT1.2Ncut-FactatagggaatattaagcttATGTGGCTTGTCCTTCTAGTTATTTACT
BrKAT1.2Ncut-RtgatggatatctgcagaattcTCAATGTCTACCTTCAAACTCAATTTG
BrKAT1.2-FactatagggaatattaagcttATGCCAATCTCTTGTACCAGAAACT
BrKAT1.2-RtgatggatatctgcagaattcTCAATGTCTACCTTCAAACTCAATTTG
Table 2. The basic protein information of Shaker K+ channels in Chinese cabbage.
Table 2. The basic protein information of Shaker K+ channels in Chinese cabbage.
Protein NameGene_IDLengthMW (kDa)pI
BrKAT3.2BraA01g005450.3.5C65174.517.24
BrAKT5BraA01g005630.3.5C87698.166.94
BrKAT2.1BraA01g009830.3.5C78790.799.18
BrAKT2/3BraA01g012890.3.5C86297.926.73
BrSKOR1.2BraA03g031580.3.5C72382.546.84
BrKAT3.1BraA03g058380.3.5C65675.198.19
BrAKT1BraA04g019690.3.5C86097.447.9
BrSKOR1.1BraA05g042590.3.5C82994.056.65
BrKAT1.2BraA06g043430.3.5C68879.056.4
BrGORKBraA07g005880.3.5C81794.116.05
BrKAT2.2BraA08g013430.3.5C67076.948.67
BrKAT1.1BraA09g022550.3.5C65775.697.11
BrAKT6BraA09g054710.3.5C877986.64
Table 3. The Ka/Ks values of 9 duplicated Shaker K+ channel gene pairs.
Table 3. The Ka/Ks values of 9 duplicated Shaker K+ channel gene pairs.
Gene PairsKsKaKa/Ks
BrKAT1.1-BrKAT2.20.171.110.15
BrKAT2.2-BrKAT1.20.211.160.18
BrKAT1.1-BrKAT1.20.050.300.18
BrKAT2.1-BrKAT2.20.090.470.20
BrKAT1.2-BrKAT2.10.161.480.11
BrSKOR1.1-BrSKOR1.20.040.350.10
BrAKT6-BrKAT3.10.744.650.16
BrKAT3.1-BrKAT3.20.060.220.28
BrAKT6-BrKAT3.20.564.550.12
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Zhou, J.-Y.; Gu, Z.-C.; Hao, D.-L. Genome-Wide Identification of the Shaker Potassium Channel Family in Chinese Cabbage and Functional Studies of BrKAT1 in Yeast. Agronomy 2024, 14, 1954. https://doi.org/10.3390/agronomy14091954

AMA Style

Zhou J-Y, Gu Z-C, Hao D-L. Genome-Wide Identification of the Shaker Potassium Channel Family in Chinese Cabbage and Functional Studies of BrKAT1 in Yeast. Agronomy. 2024; 14(9):1954. https://doi.org/10.3390/agronomy14091954

Chicago/Turabian Style

Zhou, Jin-Yan, Ze-Chen Gu, and Dong-Li Hao. 2024. "Genome-Wide Identification of the Shaker Potassium Channel Family in Chinese Cabbage and Functional Studies of BrKAT1 in Yeast" Agronomy 14, no. 9: 1954. https://doi.org/10.3390/agronomy14091954

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