Identification of the High-Affinity Potassium Transporter Gene Family (HKT) in Brassica U-Triangle Species and Its Potential Roles in Abiotic Stress in Brassica napus L.

Members of the high-affinity potassium transporter (HKT) protein family regulate the uptake and homeostasis of sodium and potassium ions, but little research describes their roles in response to abiotic stresses in rapeseed (Brassica napus L.). In this study, we identified and characterized a total of 36 HKT genes from the species comprising the triangle of U model (U-triangle species): B. rapa, B. nigra, B. oleracea, B. juncea, B. napus, and B. carinata. We analyzed the phylogenetic relationships, gene structures, motif compositions, and chromosomal distributions of the HKT family members of rapeseed. Based on their phylogenetic relationships and assemblage of functional domains, we classified the HKT members into four subgroups, HKT1;1 to HKT1;4. Analysis of the nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios of HKT gene pairs suggested that these genes have experienced strong purifying selective pressure after duplication, with their evolutionary relationships supporting the U-triangle theory. Furthermore, the expression profiles of BnaHKT genes varies among potassium, phytohormone and heavy-metal treatment. Their repression provides resistance to heavy-metal stress, possibly by limiting uptake. Our results systematically reveal the characteristics of HKT family proteins and their encoding genes in six Brassica species and lay a foundation for further exploration of the role of HKT family genes in heavy-metal tolerance.


Introduction
Plant growth, development, and productivity are negatively affected by multiple abiotic stresses, including heavy metals and salinity, due to the unfavorable conditions they impose.In response, plants have developed compensatory systems allowing them to adjust to abiotic stress conditions through a complex cascade of specific physiological, molecular, and cellular adaptations [1].The potassium cation (K + ) is an essential plant nutrient and plays crucial roles in response to a variety of abiotic stresses such as salt stress, drought stress, oxidative stress, and so on [2,3].Potassium transporters form several protein families: the KT (K + transporter)/HAK (high-affinity K + )/KUP (K + uptake) family, the Trk (K + transporter)/HKT family, the KEA (K + efflux antiporter) family, and the CHX (cation/hydrogen exchanger) family [4,5].Of these, the Trk/HKT family occurs only in Plants 2023, 12, 3768 3 of 20

Identification of HKT Family Genes in the Brassica U-Triangle Species
We identified a total of 38 putative HKT proteins in the six Brassica U-triangle species: B. rapa (Bra), B. nigra (Bni), B. oleracea (Bol), B. juncea (Bju), B. napus (Bna) and B. carinata (Bca).However, we eliminated BniB08g052450.2N and Bol007717 due to their incomplete structures.We thus retained 36 putative HKT proteins, consisting of five BraHKTs, four BniHKTs, three BolHKTs, eight BjuHKTs, eight BnaHKTs and eight BcaHKTs, for analysis (Table 1).We renamed these HKT members as HKT1;1 to HKT1;4: the first number behind HKT represented class I or II, and the second number behind HKT corresponds to the classified subfamilies in this study, with the two numbers separated by a semicolon (;).The characteristics of the encoded HKT proteins are listed in Table 1 (gene ID, protein size, molecular weight (MW), isoelectric point (pI), and predicted subcellular location).The lengths of the proteins ranged from 350 to 585 amino acid residues, and their MWs varied from 39.60 to 65.84 kDa.The pIs of all of the HKTs were greater than 7, ranging from 8.84 to 9.76.Subcellular localization predictions revealed that nearly all of the family members may localize to cell membranes, with BcaHKT1;2a being the only one predicted to localize to chloroplasts.

Phylogenetic Analysis of HKT Proteins in Arabidopsis and the Brassica U-Triangle Species
The HKT family members of the U-triangle species all belong to subfamily I (with the S-G-G-G sequence) [7].To reveal the evolutionary relationships among the HKT family members, we reconstructed a phylogenetic tree using protein sequences from Arabidopsis AtHKT1;1 and the 36 proteins from the Brassica U-triangle species.Based on their relative positions in the tree and their functional domains, we classified all proteins into one of four subgroups: HKT1;1 to HKT1;4 (Figure 1).HKT1;3 and HKT1;4 members were located in the same larger phylogenetic branch.The HKT1;1 subgroup contained sequences from all seven species, while the HKT1;2 and HKT1;3 subgroup included members of all six U-triangle species, but not Arabidopsis.HKT1;4 members were present in B. nigra, B. juncea, B. carinata, B. napus and B. rapa.The number of members in the four subgroups is approximately equal, with 10 in HKT1;1 (including AtHKT1;1), nine in HKT1;2, 10 in HKT1;3 and eight in HKT1;4.The phylogenetic tree had clear and reliable clusters, and adjacent sequences within the same clade had close evolutionary relationships, consistent with the U-triangle pattern between the allotetraploids (B.napus, B. juncea, and B. carinata) and their diploid progenitors (B.rapa, B. oleracea, and B. nigra).
Proteins in the HKT family have a characteristic structure containing four loops forming the pore [7,11].The members of the HKT family can be classified into subfamily I and subfamily II based on the residues in the first pore loop that form the selectivity filter-a Ser residue (S-G-G-G) for subfamily I and a Gly residue (G-G-G-G) for subfamily II [7].As dicotyledons, U-triangle species only have subfamily I HKTs [7,11].The presence of a Ser residue supported this idea (Figure 2A).There are four conserved selectivity filter-pore regions (P-loops: P1 to P4, Figure 2).Among the four pore domains, P4 was less conserved than the other three.BraHKT1;3a, BjuHKT1;3a, BniHKT1;4 and BraHKT1;4 lacked P4, while BnaHKT1;4b and BolHKT1;3 lacked some fragments in P4 but still contained the Gly residue (Figure 2).These characteristic sequences may have been lost during evolution.Even so, the pore regions for the Brassica species are highly conserved, confirming that the species share high similarity.

Conserved Protein Motifs of HKT and Variations in Gene Structure
To further investigate relationships among the HKT family members in each subgroup, we identified their conserved motifs and corresponding gene structures (Figure 3).We detected five conserved motifs among the 37 HKT proteins between Arabidopsis and the U-triangle species.All HKT members contained motifs 1 to 4. The first conserved residue (Ser) was in Motif 3, and the second conserved residue (Gly) in Motif 2. The third conserved residue (Gly) was in Motif 1, and the fourth conserved residue (Gly) was between Motif 1 and Motif 5 (Figure 3).For renaming, a species-specific prefix is included, and a lowercase letter suffix is used to represent the gene number within each clade.The dark red numbers beside the main nodes are bootstrap scores.

Multiple Sequence Alignment of HKT Proteins in the U-Triangle Species
To visualize the conserved domains in the HKT protein sequences, we performed a multiple sequence alignment using AtHKT1;1 from Arabidopsis, and 36 HKT protein sequences from the six Brassica U-triangle species.Using NCBI and Interpro conserved domain analysis, we determined that all identified HKT family members contain TrkH [37]  and 2a38euk (https://www.ncbi.nlm.nih.gov/Structure/cdd/TIGR00934(accessed on 26 November 2022)) [38] conserved domains, although the number and position of the domains varied.DeepTMHMM detected eight distinct transmembrane helices in all HKT proteins, except for BcaHKT1;2a, BraHKT1;3a, BraHKT1;4, BjuHKT1;3a, BnaHKT1;4b, BniHKT1;4, BolHKT1;3, which have seven, six, six, six, seven, six, and seven helices, respectively (Figure 2C, I-VIII).Proteins in the HKT family have a characteristic structure containing four loops forming the pore [7,11].The members of the HKT family can be classified into subfamily I and subfamily II based on the residues in the first pore loop that form the selectivity filter-a Ser residue (S-G-G-G) for subfamily I and a Gly residue (G-G-G-G) for subfamily II [7].As dicotyledons, U-triangle species only have subfamily I HKTs [7,11].The presence of a Ser residue supported this idea (Figure 2A).There are four conserved selectivity filter-pore regions (P-loops: P1 to P4, Figure 2).Among the four pore domains, P4 was less conserved than the other three.BraHKT1;3a, BjuHKT1;3a, BniHKT1;4 and BraHKT1;4 lacked P4, while BnaHKT1;4b and BolHKT1;3 lacked some fragments in P4 but still contained the Gly residue (Figure 2).These characteristic sequences may have been lost during The exon and intron structure of HKT genes was more complex.BjuHKT1;2b and BnaHKT1;2b contained the most exons (six), while BraHKT1;3a, BjuHKT1;3a and BniHKT1;4 had the fewest exons (one).The number of exons varied among members of the HKT family with diversity gene structures (Figure 3).However, the numbers of exons and exon-intron patterns were similar within each HKT family subgroup, supporting the notion that our classification method was reliable.

Chromosomal Distribution of HKT Genes in the U-Triangle Species
In total, the 36 HKT genes mapped onto 24 chromosomes of U-triangle species, with chromosomes in the A (13 genes), B (12 genes), and C (11 genes) subgenomes, which are unevenly distributed (Figure 4).Moreover, HKT1;3 and HKT1;4 are closely associated; some even belong to the tandem duplication event (Figure 4).We also found that HKTs in the same subgenome were generally located in parallel physical positions, suggesting that extensive collinearity existed among them in U-triangle species.In addition, some HKT family genes were just detected in B. carinata, suggesting that duplicated genes have been lost during evolution.chromosomes in the A (13 genes), B (12 genes), and C (11 genes) subgenomes, which are unevenly distributed (Figure 4).Moreover, HKT1;3 and HKT1;4 are closely associated; some even belong to the tandem duplication event (Figure 4).We also found that HKTs in the same subgenome were generally located in parallel physical positions, suggesting that extensive collinearity existed among them in U-triangle species.In addition, some HKT family genes were just detected in B. carinata, suggesting that duplicated genes have been lost during evolution.

Collinearity Analysis of HKT Genes in U-Triangle Species
Arabidopsis, the allotetraploids (B.napus, B. juncea, and B. carinata) and their diploid progenitors (B.rapa, B. oleracea, and B. nigra) were divided into three groups according to the subgenome relationships, respectively, from which we identified orthologous gene pairs (Figure 5).We calculated the numbers of nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios for the HKT gene pairs to reveal the evolutionary constraints acting on the HKT gene pairs.Among the 563 gene pairs compared, only BcaHKT1;1b and BolHKT1;1, BnaHKT1;1b and BolHKT1;1 had Ka/Ks ratios > 2, while the Ka/Ks ratios of other gene pairs were all below 1, with most of the values ranging between 0.1 and 0.3.This observation suggested that the HKT family genes in the seven species have experienced strong purifying selective pressure following duplication.We calculated the numbers of nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios for the HKT gene pairs to reveal the evolutionary constraints acting on the HKT gene pairs.Among the 563 gene pairs compared, only BcaHKT1;1b and BolHKT1;1, BnaHKT1;1b and BolHKT1;1 had Ka/Ks ratios > 2, while the Ka/Ks ratios of other gene pairs were all below 1, with most of the values ranging between 0.1 and 0.3.This observation suggested that the HKT family genes in the seven species have experienced strong purifying selective pressure following duplication.

Expression Profiles of HKT Family Genes in B. napus under Potassium and Phytohormone Treatment
Based on the published RNA-Seq data (http://www.bnagadb.cn/),we examined the expression of the HKT genes in rapeseed was affected by abiotic stresses, including the different potassium concentrations and different phytohormones (IAA, indole-3-acetic acid or heteroauxin; ACC, 1-aminocyclopropanecarboxylic acid; ABA, abscisic acid; GA3, gibberellin; 6-BA, N-6-benzyladenine), respectively.Overall, BnaHKT1;1a and BnaHKT1;1b were most significantly induced upon low potassium conditions in roots (Figure 6).BnaHKT1;2a, BnaHKT1;3a and BnaHKT1;3b displayed the highest expression level in leaves among both control (CK) groups and low potassium (LK)-treated groups, and BnaHKT1;3a and BnaHKT1;3b were significantly upregulated when subjected to a low potassium concentration.The HKT genes were upregulated, presumably to counteract potassium deficiency by raising the uptake of this element, suggesting that the different BnaHKTs have distinct roles in different tissues.
BnaHKT1;1b were most significantly induced upon low potassium conditions in roots (Figure 6).BnaHKT1;2a, BnaHKT1;3a and BnaHKT1;3b displayed the highest expression level in leaves among both control (CK) groups and low potassium (LK)-treated groups, and BnaHKT1;3a and BnaHKT1;3b were significantly upregulated when subjected to a low potassium concentration.The HKT genes were upregulated, presumably to counteract potassium deficiency by raising the uptake of this element, suggesting that the different BnaHKTs have distinct roles in different tissues.To well understand the phytohormone-induced expression profiles of the BnaHKTs, we investigated their expression levels in seeding roots of ZS11 under different phytohormone treatments.Notably, BnaHKT1;2b, BnaHKT1;2a, BnaHKT1;3a and BnaHKT1;3b were sensitive to phytohormones, and showed the similar trend (Figure 7).Generally, for IAA and ACC treatment, we observed a significant downregulation between 1 and 3 h and an upregulation between 3 and 12 h, and another downregulation during 12-24 h (Figure 7).However, expression under ABA, GA3 and 6-BA treatments followed a unimodal pattern, with a decrease in expression after reaching a peak during 3-24 h.Based on the variation of transcript levels, we suggest that BnaHKT1;3a and BnaHKT1;3b should play crucial roles in response to phytohormones, but the members of BnaHKT1;1a, BnaHKT1;1b, BnaHKT1;4a and BnaHKT1;4b showed less sensitivity to phytohormones treatments.In To well understand the phytohormone-induced expression profiles of the BnaHKTs, we investigated their expression levels in seeding roots of ZS11 under different phytohormone treatments.Notably, BnaHKT1;2b, BnaHKT1;2a, BnaHKT1;3a and BnaHKT1;3b were sensitive to phytohormones, and showed the similar trend (Figure 7).Generally, for IAA and ACC treatment, we observed a significant downregulation between 1 and 3 h and an upregulation between 3 and 12 h, and another downregulation during 12-24 h (Figure 7).However, expression under ABA, GA3 and 6-BA treatments followed a unimodal pattern, with a decrease in expression after reaching a peak during 3-24 h.Based on the variation of transcript levels, we suggest that BnaHKT1;3a and BnaHKT1;3b should play crucial roles in response to phytohormones, but the members of BnaHKT1;1a, BnaHKT1;1b, BnaHKT1;4a and BnaHKT1;4b showed less sensitivity to phytohormones treatments.In conclusion, BnaHKTs had a variety of responses to treatments with several phytohormones.

Expression Patterns of HKT Genes in Rapeseed under Heavy-Metal Stress
To reveal the possible functions of BnaHKT genes in response to heavy-metal stress in B. napus seedling, we detected the expression profiles of eight BnaHKTs under heavy-metal stress by RT-qPCR analysis in roots, hypocotyls, and cotyledons, respectively.As shown in Figure 8, these members showed the variation expression patterns.Under As 3+ treatment, all BnaHKTs were repressed in roots and BnaHKT1;3a, BnaHKT1;3b and BnaHKT1;4b were inhibited in different tissues, while BnaHKT1;1a, BnaHKT1;1b, BnaHKT1;2a, BnaHKT1;2b and BnaHKT1;4a showed the highest expression levels in hypocotyls (Figure 8).Under Cd 2+ treatment, all BnaHKTs were obviously downregulated, including BnaHKT1;1a, and BnaHKT1;1b in roots, hypocotyls, and cotyledons; BnaHKT1;2a and BnaHKT1;4a in roots and cotyledons.Our findings indicate that these BnaHKTs showed the different expression levels in response to heavy-metal stress, suggesting that they may have potential functions in protection from heavy-metal stress.
conclusion, BnaHKTs had a variety of responses to treatments with several phytohormones.

Expression Patterns of HKT Genes in Rapeseed under Heavy-Metal Stress
To reveal the possible functions of BnaHKT genes in response to heavy-metal stress in B. napus seedling, we detected the expression profiles of eight BnaHKTs under heavymetal stress by RT-qPCR analysis in roots, hypocotyls, and cotyledons, respectively.As shown in Figure 8, these members showed the variation expression patterns.Under As 3+ treatment, all BnaHKTs were repressed in roots and BnaHKT1;3a, BnaHKT1;3b and BnaHKT1;4b were inhibited in different tissues, while BnaHKT1;1a, BnaHKT1;1b, BnaHKT1;2a, BnaHKT1;2b and BnaHKT1;4a showed the highest expression levels in hypocotyls (Figure 8).Under Cd 2+ treatment, all BnaHKTs were obviously downregulated, including BnaHKT1;1a, and BnaHKT1;1b in roots, hypocotyls, and cotyledons; BnaHKT1;2a and BnaHKT1;4a in roots and cotyledons.Our findings indicate that these BnaHKTs showed the different expression levels in response to heavy-metal stress, suggesting that they may have potential functions in protection from heavy-metal stress.

Cis-Element Analysis of BnaHKT Promoters
We carried out cis-element analyses in the 2000 bp regions upstream of the BnaHKT gene transcription start sites, and identified several fundamental cis-acting elements related to hormone, plant development and abiotic responses.In this study, each BnaHKT promoter contained the light-responsive elements (LRE) [39] and cis-acting regulatory element involved in the MeJA (methyl jasmonate response element) responsiveness [40].Moreover, in BnaHKT promoters, our predictions revealed six types of phytohormone-responsive cis-elements (including ABA, auxin and GA-responsive elements), three types of stressresistance cis-elements (defense, drought-inducibility, and low-temperature responsiveness) and six types of cis-elements involved in plant growth and development (Figure 9).These data indicate that BnaHKT genes play important roles in stress resistance, plant biology and hormone signaling pathways.
responsive cis-elements (including ABA, auxin and GA-responsive elements), three types of stress-resistance cis-elements (defense, drought-inducibility, and low-temperature responsiveness) and six types of cis-elements involved in plant growth and development (Figure 9).These data indicate that BnaHKT genes play important roles in stress resistance, plant biology and hormone signaling pathways.

Discussion
The HKT family is a plant-specific protein family that is believed to control a variety of developmental and stress responses in plants [6,7].Previous studies showed that the HKT family genes regulate K + concentration and homeostasis in plants [7,36].To date, the HKT family has been identified in different species [8][9][10][11][12][14][15][16][17][18][19][20].In this study, we identified 36 HKT family members in the six Brassica U-triangle species.Although divergences were found among the number of HKT genes in Brassica species, the number of HKT genes in B. juncea (AABB, 8), B. napus (AACC, 8), and B. carinata (BBCC, 8) was roughly equal to the sum of those in their corresponding diploid ancestors B. rapa (AA, 5) plus B. nigra (BB, 4), B. rapa (AA, 5) plus B. oleracea (CC, 3), and B. nigra (BB, 4) plus B. oleracea (CC, 3), respectively.In addition, based on our analysis of Ka/Ks values, most of the HKT genes experienced strong purifying selective pressure during evolution, and they retained a high degree of collinearity along their respective chromosomes after diverting from the same ancestor.
Previous studies have shown that HKT family proteins can be categorized as class I and class II according to the presence of different functional residues [7].Moreover, HKT members in dicots contain only subgroup I (S-G-G-G) [7].In agreement, all HKT genes in the six Brassica species were all assigned to subgroup I (S-G-G-G) (Figure 2.).In this study, Plants 2023, 12, 3768 14 of 20 we also categorized the HKT family members into four subgroups (HKT1;1 to HKT1;4) based on their evolutionary relationships and reconstructed a phylogenetic tree.Located in the same larger phylogenetic branch, members in HKT1;3 and HKT1;4 are more closely related to each other than to members of other subgroups.The HKT family members showed a higher similarity in motif pattern, gene structure, parallel physical positions within the same subgroups, and variation between the different branches and members.In addition, we detected Motifs 1, 2, 3, and 4 in all HKT family members, suggesting that these four motifs represent the functional domains that play ion transport roles in the HKT family, a result consistent with previous studies [41].By contrast, Motif 5 was lacking in some of the family members but might still retained some properties as HKT proteins.These results suggest that, in each subgroup, HKT family genes might have experienced the same evolutionary pressures and have similar functions.
In modern agricultural practices, abiotic stresses are a major constraint to crop production [42].HKT family members have many functions, including Na + and K + uptake and Na + -K + homeostasis [7,13].Our results confirmed that the expression of HKT genes was induced under low K + concentration, corroborating the basic function of this family under high-salinity conditions.In this study, BnaHKT1;1a and BnaHKT1;1b were induced by low-K + (Figure 6), suggesting that they may play positive roles in regulating potassium content and maintaining homeostasis [13,43].However, because ion transport involves many internal mechanisms, we also examined some other possible functions, such as phytohormone treatments and heavy-metal stress.Among these, ABA, auxin, ethylene, and gibberellin play important roles in signaling and response to various stresses such as salinity by influencing physiological mechanisms, activating related proteins, changing root structure, and interacting with each other to regulate plant growth and development [13,[43][44][45][46][47][48][49].Research has also described the functions of HKT genes in response to phytohormones in vivo [13,44].The raise of ethylene levels can increase sodium accumulation in the shoot and salt sensitivity in rice by inducing OsHKT2;1 [43].However, the detailed expression patterns of HKT genes have been lacking in B. napus.In the present study, phytohormones obviously activate the expression of BnaHKT1;2b, BnaHKT1;2a, BnaHKT1;3a and BnaHKT1;3b (Figure 7).
Promoter cis-elements play vital roles in environmental adaptation by plants, and analysis of these elements provides a promising method for investigating the possible functions of their associated genes [50].All of the BnaHKT genes have phytohormone, plant development and abiotic response cis-elements (Figure 9).Research shows inclusion of ABI4-binding cis-element reduces SlSOS2 expression, decreasing salt resistance in the cultivated tomato [51].Moreover, ABRE and G-box elements are favorable binding sites of bZIP TFs that regulate stress responses [52].AtbZIP24 and ABI4 can also negatively regulate AtHKT1;1, affecting salt stress tolerance [53].Therefore, cis-elements play crucial roles in stress tolerance.Based on our findings, BnaHKT play a role in response to low K + stress and are induced by hormones in response to the stress response, growth, and development.
Heavy-metal pollution has become one of the most important factors restricting crop yield and quality [42].Considerable evidence supports the idea that Brassica species have a high tolerance to heavy metals [54], and Brassica species are considered heavy-metal accumulators [35].In this study, we predicted the potential roles of BnaHKTs under heavymetal stress.Notably, the expression level of BnaHKTs could be sensitive to heavy metals in B. napus, but they display the functional redundancy in different conditions (Figure 8).Overall, our study systematically studied the characteristics of HKT family genes in Utriangle species and documented the expression profiles of BnaHKT genes under low K + treatment, phytohormone treatments and heavy-metal stress.These results lay a foundation for further elucidating the function of BnaHKTs, and can contribute to improve capacity of the B. napus in response to abiotic stresses.

Structural Analysis of Conserved Motifs and Genes of the HKT Family Members
The online program Multiple Expectation Maximization for Motif Elucidation (MEME) (https://meme-suite.org/meme/doc/meme.html(accessed on 1 February 2022)) was used to identify and analyze the conserved motif of the HKT family members [68].The specific parameters were as follows: zero or one occurrence per sequence, maximum number of motifs 5; and optimum width of each motif between 6 wide and 50 wide (inclusive) [69].The motif structure and gene structure are shown together with the evolutionary tree using the Gene Structure View in TBtools software (https://github.com/CJ-Chen/TBtools(accessed on 1 February 2022)) [57].

Chromosomal Localization and Colinearity Analysis of HKT Genes
The chromosomal location information of HKT family genes was extracted using the genome sequence annotation of Brassica species (BnIR, https://yanglab.hzau.edu.cn/BnIR(accessed on 16 November 2022)) [55]; the HKT family genes were mapped to their corresponding chromosomes by using MapChart V2.32 [70].Moreover, tandem repeats were identified based on the physical location of HKT tandem repeats on a single chromosome.To better understand the colinearity relationship, TBtools-One Step MCScanX and TBtools-Amazing Super Circos were used to analyze the homologous genetic relationships among HKT genes [57].Then, the nonsynonymous substitution rates (Ka), synonymous substitution rates (Ks) and Ka/Ks ratios were calculated to estimate selection pressures during evolution.

Expression Profile Analysis of HKT Genes
The rapeseed cultivar Zhongshuang 11 (ZS11) was used as the plant material for potassium and hormone treatments (IAA, GA3, 6-BA, ABA, and ACC).The plant materials and treatments were as described previously [71], and RNA-Seq data were obtained from BnaGADB v1.0 (http://www.bnagadb.cn/(accessed on 2 January 2022)) to investigate the phytohormone-responsive expression patterns of BnaHKTs.For heavy-metal treatments, 15 mg/L As and 30 mg/L Cd were used as a most optimal concentration due to the previous study [33].The healthy seeds were selected from P163 and P087 and treated with distilled water (the control) and 15 mg/L As or 30 mg/L Cd.The relative expression levels of all BnaHKT genes were calculated using TopHat and Cufflinks, and normalized into FPKM values (fragments per kilobase of transcript per million mapped reads) [72,73].Finally, to better display the data, the data were converted into Log 10 (FPKM value + 1) followed by TBtools [57] to generate the heatmaps to show BnaHKT expression.The RNA-seq datasets, calculation methods and so on were as described elsewhere [72].

RT-qPCR Analysis of BnaHKT Genes
Total RNA extraction and RT-qPCR analysis are the same as described in our previous research [33].The qualified RNA was reverse transcribed into complementary DNA (cDNA) with an RNA PCR Kit (AMV) Ver.3.0 (Takara, Dalian, China).The subsequent qPCR analysis was performed using SYBR Premix Ex Taq II (Takara, Dalian, China) on a Bio-Rad CFX96 Real Time System (Bio-Rad Laboratories, Hercules, CA, USA) as previously described [33].Finally, the results were normalized to the reference gene BnaActin7 (EV116054) [74] via the 2 −∆∆Ct method [75].Error bars represent standard errors from three independent biological replicates in this study.The primers are listed in Table S1.

Conclusions
In this study, we identified 36 HKT family genes in the six Brassica U-triangle species, and classified them into four subgroups according to their evolutionary relationships.Most of the HKT family genes experienced strong purifying selective pressure after duplication.On the basis of BnaHKTs transcript levels, functional diversity is displayed: BnaHKT1;1a and BnaHKT1;1b induced by low K + , BnaHKT1;2a, BnaHKT1;2b, BnaHKT1;3a and BnaHKT1;3b sensitive to phytohormones.Furthermore, most BnaHKTs were strongly suppressed by heavy-metal stress.Our findings provide novel clues for future studies on the potential roles of BnaHKTs in response to heavy-metal stress.In summary, these results provided valuable information to further explore the functional BnaHKTs, and improve the tolerance to heavy-metal stress and other abiotic stresses in Brassica species.

Figure 1 .
Figure 1.Phylogenetic tree of the HKT protein family from Arabidopsis and the Brassica U-triangle species.The HKT family was divided into four subgroups (HKT1;1-4), which are shown in red, blue, purple and green for HKT1;1-4, respectively.Arabidopsis, red star; B. napus, yellow star; B. rapa, white star; B. juncea, black star; B. oleracea, purple star; B. carinata, blue star; B. nigra, green star.For renaming, a species-specific prefix is included, and a lowercase letter suffix is used to represent the gene number within each clade.The dark red numbers beside the main nodes are bootstrap scores.

Figure 1 .
Figure 1.Phylogenetic tree of the HKT protein family from Arabidopsis and the Brassica U-triangle species.The HKT family was divided into four subgroups (HKT1;1-4), which are shown in red, blue, purple and green for HKT1;1-4, respectively.Arabidopsis, red star; B. napus, yellow star; B. rapa, white star; B. juncea, black star; B. oleracea, purple star; B. carinata, blue star; B. nigra, green star.For renaming, a species-specific prefix is included, and a lowercase letter suffix is used to represent the gene number within each clade.The dark red numbers beside the main nodes are bootstrap scores.

Figure 2 .
Figure 2. Sequence analysis of HKT family members in Arabidopsis and the Brassica U-triangle species.(A) Sequence alignment of the conserved pore domain is shown in the figure, and P1-P4 indicate the four pore domains.The red triangles indicate the locations of sequences that are less conserved.The yellow shading represents the crucial residues that were used for classification.The subgroups are labeled in the same colors as in Figure 1.(B) WebLogos analysis of the conservation at the S1 and G2 amino acid residue pore-forming regions.(C) The transmembrane helices are labeled I to VIII with the conserved residues (S-G-G-G).

Figure 2 .
Figure 2. Sequence analysis of HKT family members in Arabidopsis and the Brassica U-triangle species.(A) Sequence alignment of the conserved pore domain is shown in the figure, and P1-P4 indicate the four pore domains.The red triangles indicate the locations of sequences that are less conserved.The yellow shading represents the crucial residues that were used for classification.The subgroups are labeled in the same colors as in Figure 1.(B) WebLogos analysis of the conservation at the S1 and G2 amino acid residue pore-forming regions.(C) The transmembrane helices are labeled I to VIII with the conserved residues (S-G-G-G).

Figure 3 .
Figure 3. Phylogenetic tree, motif distributions, and gene structure analysis of HKTs between Arabidopsis and the U-triangle species.The conserved motif map and gene structure map are shown alongside the phylogenetic tree.(A) Phylogenetic tree of the HKT protein family from Arabidopsis and the Brassica U-triangle species (same with Figure 1).(B) Conserved motifs of the HKT proteins.Five motifs were identified using the MEME program, and are indicated as differently colored boxes.The black lines indicate relative protein lengths.(C) Gene structure of HKT family genes.Coding sequences (CDS), green boxes; introns, gray lines; untranslated regions (UTRs), orange boxes.(D) The sequences of the 5 Motifs for Figure 3B.Each subgroup on the left side is labeled in the same color as in Figure 1.

Figure 3 .
Figure 3. Phylogenetic tree, motif distributions, and gene structure analysis of HKTs between Arabidopsis and the U-triangle species.The conserved motif map and gene structure map are shown alongside the phylogenetic tree.(A) Phylogenetic tree of the HKT protein family from Arabidopsis and the Brassica U-triangle species (same with Figure 1).(B) Conserved motifs of the HKT proteins.Five motifs were identified using the MEME program, and are indicated as differently colored boxes.The black lines indicate relative protein lengths.(C) Gene structure of HKT family genes.Coding sequences (CDS), green boxes; introns, gray lines; untranslated regions (UTRs), orange boxes.(D) The sequences of the 5 Motifs for Figure 3B.Each subgroup on the left side is labeled in the same color as in Figure 1.

Figure 4 .
Figure 4. Chromosome localization of HKT genes across U-triangle species.(A) HKT genes located on the A subgenome in B. juncea, B. napus, and B. rapa.(B) HKT genes located on the B subgenome

Figure 4 .
Figure 4. Chromosome localization of HKT genes across U-triangle species.(A) HKT genes located on the A subgenome in B. juncea, B. napus, and B. rapa.(B) HKT genes located on the B subgenome in B. juncea, B. carinata, and B. nigra.(C) HKT genes located on the C subgenome in B. napus, B. carinata, and B. oleracea.The distributions of the HKT genes are in accordance with the evolutionary relationships in U-triangle species.The labels above the corresponding chromosomes indicate the name of the source organisms and their subgenomes.The scales to the left indicate the sizes of the various Brassica chromosomes in Mb.Genes in the same subgroup are shaded using the same colors as in Figure 1.

Figure 5 .
Figure 5. Collinearity analysis of HKT family genes between Arabidopsis and U-triangle species.(A) Collinearity analysis of HKT family genes among Arabidopsis, B. nigra, B. oleracea, and B. carinata.(B) Collinearity analysis of HKT family genes among Arabidopsis, B. nigra, B. rapa, and B. juncea.(C) Collinearity analysis of HKT family genes among Arabidopsis, B. oleracea, B. rapa, and B. napus.AtChr4 is the only chromosome with one HKT gene in Arabidopsis.The chromosomes are shown in different colors from the U-triangle species.The different colored lines represent the syntenic regions.The scales represent the length of the chromosomes.

Figure 5 .
Figure 5. Collinearity analysis of HKT family genes between Arabidopsis and U-triangle species.(A) Collinearity analysis of HKT family genes among Arabidopsis, B. nigra, B. oleracea, and B. carinata.(B) Collinearity analysis of HKT family genes among Arabidopsis, B. nigra, B. rapa, and B. juncea.(C) Collinearity analysis of HKT family genes among Arabidopsis, B. oleracea, B. rapa, and B. napus.AtChr4 is the only chromosome with one HKT gene in Arabidopsis.The chromosomes are shown in different colors from the U-triangle species.The different colored lines represent the syntenic regions.The scales represent the length of the chromosomes.

Figure 6 .
Figure 6.Expression profiles of BnaHKTs under potassium treatment.The expression profiles of each BnaHKT were calculated as Log10 (FPKM value + 1).L, leaves; R, roots; d: days; LK, low-potassium treatment; CK (control) indicates normal nutrient solution with no potassium deficiency.FPKM, fragments per kilobase of exon model per million.

Figure 6 .
Figure 6.Expression profiles of BnaHKTs under potassium treatment.The expression profiles of each BnaHKT were calculated as Log 10 (FPKM value + 1).L, leaves; R, roots; d: days; LK, low-potassium treatment; CK (control) indicates normal nutrient solution with no potassium deficiency.FPKM, fragments per kilobase of exon model per million.

Figure 8 .
Figure 8. Expression patterns of BnaHKTs under As 3+ or Cd 2+ treatment.Data obtained by RT-qPCR were normalized to the expression level of BnaActin7, and error bars represent the standard deviation (SD) of three biological replicates Statistically significant differences were analyzed by Student t-test.* p < 0.05; ** p < 0.01; *** p < 0.001.P087 and P163 are the two accessions used for heavy-metal treatment.

Figure 9 .
Figure 9. Predicted cis-elements in BnaHKT promoters.The analysis was based on the 2000-bp regions 5 to BnaHKT transcription start sites.The cis-acting elements identified are marked with differently colored boxes, and the functional categories are noted at the right.

Table 1 .
Characteristics of HKTs from Arabidopsis and the Brassica U-triangle species.