Genome-wide characterization and expression analysis of the HAK gene family in response to abiotic stresses in Medicago
BMC Genomics volume 23, Article number: 791 (2022)
The high-affinity K+ transporter (HAK) family plays a vital role in K+ uptake and transport as well as in salt and drought stress responses. In the present study, we identified 22 HAK genes in each Medicago truncatula and Medicago sativa genome. Phylogenetic analysis suggested that these HAK proteins could be divided into four clades, and the members of the same subgroup share similar gene structure and conserved motifs. Many cis-acting elements related with defense and stress were found in their promoter region. In addition, gene expression profiles analyzed with genechip and transcriptome data showed that these HAK genes exhibited distinct expression pattern in different tissues, and in response to salt and drought treatments. Furthermore, co-expression analysis showed that 6 homologous HAK hub gene pairs involved in direct network interactions. RT-qPCR verified that the expression level of six HAK gene pairs was induced by NaCl and mannitol treatment to different extents. In particular, MtHK2/7/12 from M. truncatula and MsHAK2/6/7 from M. sativa were highly induced. The expression level of MsHAK1/2/11 determined by RT-qPCR showed significantly positive correlation with transcriptome data. In conclusion, our study shows that HAK genes play a key role in response to various abiotic stresses in Medicago, and the highly inducible candidate HAK genes could be used for further functional studies and molecular breeding in Medicago.
Potassium (K+) is one of the most essential mineral nutrients for plant growth and development, and it is also the most abundant monovalent cation in plants, accounting for approximately 2–10% of plant dry weight . The role of K+ is not only essential for plants to maintain normal physiological and biochemical processes such as stomatal movement, photosynthesis, but also involved in responses to biotic and abiotic stresses [2,3,4]. The storage of K+ in vacuoles plays an important role in maintaining the concentration of K+ in cytoplasm. However, the optimal concentration of K+ must be maintained for the cells to function normally. In the cytoplasm, K+ concentration is related to the tolerance of plants to different stresses, such as drought  and salinity . In plants, roots are the first place to sense the lack of K+ and has the ability to sense the change of external K+ concentration . K+ is firstly absorbed by the root, then transferred to the aerial part and distributed to different organs within the cell . As a fixed organism, plants have evolved an efficient K+ transport system to maintain the optimal growth state under lower K+ levels .
According to their structure and function, K+ transport proteins in plants can be divided into five families: (1) Shaker-like K+ channels; (2) tandem-pore K+ (TPK) channels; (3) HAK/KUP/KT K+ transporters; (4) HKT transporters; and (5) cation-proton antiporters (CPAs) . Among them, the HAK/KUP/KT family is the largest families and KUP was first found in bacteria and HAK in fungi [11, 12]. In plants, based on gene homology, the HAK family members were initially identified in barley (HAK1) , and KUP1/KT1 and KUP2/KT2 in Arabidopsis [14, 15]. However, HAKs are absent in animal. Therefore, name of HAK/KUP/KT was widely used for the whole transporter family in plants [8, 10]. Subsequently, HAK/KUP/KT genes were found in other plants, such as maize , Ipomoea , tea , and Gossypium hirsutum .
HAK transporters play diverse roles in K+ uptake and transport, salt and drought stress responses, and morphological development of roots and shoots . So far, the physiological functions of several plant K+ transporters have been elucidated. In Arabidopsis, AtHAK5 is involved in high-affinity K+ uptake and it is capable of absorbing K+ in solutions below 10 µM [8, 9]. AtHAK5 is required for Arabidopsis growth and K+ acquisition from low K+ solutions under saline conditions . In rice, the expression of OsHAK5 was up-regulated under K+ deficiency conditions, and over-expression of OsHAK5 increased salt stress tolerance by K+/Na+ ratio . Under normal K+ supply conditions, the expression of OsHAK1 is up-regulated under salt stress. However, when K+ is deficient, salt stress reduced net K+ uptake rate of OsHAK1 mutants, indicating that OsHAK1 plays a key role in enhancing salt tolerance . When OsHAK1 was over-expressed in rice, it enhanced drought tolerance with lower level of lipid peroxidation and higher activities of antioxidant enzymes, and it positively regulated the expression levels of genes involved in K+ homeostasis and stress responses . While OsHAK21 was knockout, the ratio of K+ uptake and the salt tolerance decreased . These findings implied HAK/KUP/KT genes had potential functions in promoting drought and salt tolerance in plants. In addition, it was also reported that the HAK gene expression level is also related to the roots, and the expression level of AtHAK5 was induced in root under K+-limitation conditions . It was also found that AtKUP5 was only expressed in root hairs and could be used as a K+ flux sensor .
Although genome-wide identification of HAK gene family has been accomplished in rapeseed  and cassava , but not in Medicago. The release of the Medicago sativa genome data and the newly improved Medicago truncatula annotated reference genome assembly provided the possibility for genome-wide identification of the HAK gene family of these two representative species of the Medicago genus. The molecular basis and stress resistance mechanisms of K+ transport and homeostasis in M. sativa are largely unknown. In the present study, we identified 22 HAK genes from M. truncatula and 22 from M. sativa in their genomes, and analyzed their phylogenetic relationships, conserved motifs and domains, gene structure, cis-acting elements, syntenic relationships, tissue expression patterns and expression profiling in responses to salt stress and osmotic stress. The data provided in this study are reliable to screen key candidate genes from the HAK family in Medicago for further functional investigation at molecular level, and for molecular breeding of M. sativa with stress tolerance.
Genome survey to identify HAK genes in M. truncatula and M. sativa
Based on comparative genomics, a total of 44 HAK candidate genes were identified from M. truncatula and M. sativa genome. Characteristics of HAK genes, including TIGR locus, homologous gene, protein length, number of intron, transmembrane domains (TM), isoelectric point (pI), molecular weight (MW), and putative subcellular localization, were shown in Tables 1 and 2. MtHAK and MsHAK genes encode proteins ranging from 395 to 871, 385 to 855 amino acids in length, respectively. The genomic sequences of MtHAK and MsHAK contained 3–9, 4–12 exons, respectively. All MtHAK members contain 20 TM structural domains except MtHAK8 and MtHAK16 that have 13 and 19 TM domains respectively. Members of MsHAK family contained 8–20 TM domains. Subcellular location analyses showed that HAK proteins from M. truncatula and M. sativa were all predicated to be located in plasma membrane.
Multiple sequence alignment, phylogenetic analysis and classification of HAK genes in Medicago
In order to better understand the characteristics of HAK protein sequence, the most conservative region covering potassium ion transporter HAK were analyzed using MEGA-X, and displayed via jalview (Additional Fig. S2). The conserved amino acid sequences of six KUP/HAK/KT domains are GDLGTSPLY, ANDDNGEGG, GDGVLTPAIS, GSEAMFADLGHF, AYGIAVV, and FRCIVIYGYKD, respectively. All HAK members contain at least two KUP/HAK/KT domains, which were similar to those HAKs members from Gossypium raimondii  and Cajanus cajan .
To analyze the phylogenetic relationship and evolution of the HAK family in Medicago and different plants, we used 113 HAK genes from several plants to construct a phylogenetic tree by using neighbor-joining (NJ) method, including A. thaliana, M. truncatula, M. sativa, Oryza sativa, and Glycine max (Fig. 1). These HAK proteins could be divided into four clades as in a previous report , and each of them could be further subdivided into sub-cluster A and B. Cluster I consisted of six MtHAKs, seven MsHAKs and only one AtHAK (AtHAK5), Cluster II contained the most HAKs members (47) with 15 members from Medicago. Cluster III has two subclusters (IIIA and IIIB) with the same number (Fig. 1), and three AtKUP members within each subcluster. Cluster IV has the least HAK with only 10 members. Notably, subcluster IVB has only two HAK members from rice (OsHAK26 and OsHAK6). This indicated that the amplification rate in distinct groups varies, which might reflect the specific function during the process of evolution.
Analysis of gene structure and conserved motifs of HAK genes in Medicago
To comprehensively study the function of HAK genes, we performed analysis on gene structure and conserved motifs (Fig. 2). Over long evolutionary time intervals, exon and intron positions are generally conserved in orthologous genes, whereas intron/exon structure of HAK family varied, but sufficiently conserved in paralogous genes [31, 32]. The HAK gene structure analyzed in Medicago revealed the intron/exon organization and conservation among them. Cluster I contains 5–10 exons and none of them contain UTRs except MtHAK6 and MtHAK20 (Fig. 2A, B). In cluster II, great diversity was observed in exon length. Cluster III and cluster IV contain 4–10 and 7–8 exons, respectively, and the gene structure and the number of the cluster IV is highly consistent (Fig. 2A, B).
We identified 20 conserved motifs with sizes ranging from 15 to 50 residues in width, which were annotated as K+ potassium transporter motif (Additional Fig. S3). Generally, the motifs were almost evenly distributed, and a similar number of motifs were present in HAK proteins from each of the four clusters (Fig. 2 A, C). Motifs 1–10, 12 and 16–18 were conserved in all four HAK clusters, with a few exceptions where a particular motif was missing in 3–5 genes (Fig. 2C). Together, the common K+ potassium transporter motifs and similar gene structure in the same cluster supported the phylogenetic classification of the HAK family and implied functional similarities among these HAK genes.
Analyses of chromosomal distribution and synteny of HAK genes in Medicago
The genomic distribution of the HAK genes in Medicago was determined by mapping the ORFs of all identified genes onto their corresponding chromosome (Fig. 3). It was shown that the distribution of HAK genes were uneven in both M. truncatula and M. sativa, and they were distributed on chromosomes 2–7, but none on chromosome 1. In M. truncatula and M. sativa, 1, 2, 1 HAK members were found on chromosomes 3, 6, and 7, respectively, and these members were in approximately the same chromosomal position in both species (Fig. 3). Differently, most MtHAK members were found on chromosome 8 with eight members, followed by chromosome 5 with five members (Fig. 3A). Most MsHAK members were found on chromosome 4 with six members, followed by chromosome 8 with five members (Fig. 3B).
Tandem, segmental and whole-genome duplication are the main impetus for gene family expansion . Two pairs of segmental duplication were found in M. truncatula (MtHAK2/MtHAK5) and M. sativa (MsHAK2/MsHAK6), respectively (Fig. 3A, B and Table S2). In addition, only two pair of tandem repeat events (MsHAK4/MsHAK5 and MsHAK8/MsHAK9) were found in M. sativa (Fig. 3B and Table S2), while this event was absent in M. truncatula (Fig. 3A).
Furthermore, comparative syntenic maps of M. sativa, G. max and A. thaliana associated with M. truncatula, and G. max and A. thaliana associated with M. sativa were constructed to illustrate the evolution relationship of HAK gene family (Fig. 3C). Notably, 10, 28 and 6 orthologous pairs were found between M. truncatula and M. sativa, M. truncatula and G. max, M. truncatula and A. thaliana, respectively (Fig. 3C and Table S2). Three genes in M. truncatula (MtHAK1, 4 and 7) showed a collinear relationship with those in M. sativa, G. max and A. thaliana (Fig. 3C and Table S2). Meanwhile, 22 and 4 orthologous pairs were found between M. sativa and G. max, M. sativa and A. thaliana, respectively (Fig. 3C and Table S2), and four genes in M. sativa (MsHAK1, 3, 11 and 7) showed a collinear relationship with those in A. thaliana and G. max (Fig. 3C and Table S2). These genes may play irreplaceable role in evolution of the HAK family.
To better understand the evolutionary selection pressure during the formation of HAK gene family, the Ka/Ks values of HAK gene pairs were analyzed for Mt-Mt, Ms-Ms, Mt-Ms, Mt-Gm, Mt-At, Ms-Gm and Ms-At (Fig. 3D and Table S2). The Ka/Ks values of these orthologous gene pairs were all less than 1, indicating that HAK genes may have undergone strong purification selection pressure during evolution.
Analysis of cis-acting elements in the promoter sequences of HAK genes in Medicago
Cis-acting elements serve as potential regulators of abiotic stress. The online tool PlantCARE was utilized to identify several cis-acting elements in HAK genes. The promoter sequence of 2,000 bp for the 22 MtHAK and 22 MsHAK genes were analyzed. The cis-acting elements identified were functionally categorized into 11 categories, including: auxin responsive (AuxRE-core), gibberellin-responsive (GARE-motif, P-box, TATC-box), MeJA-responsive (TGACG-motif, CGTCA-motif), abscisic acid-responsive (ABRE), defense and stress responsiveness (TC-rich repeats, W-box), MYB binding site involved in drought-inducibility (MBS), ethylene-responsive (ERE), salicylic acid responsiveness (TCA-element), wound responses (WUN motif), low temperature-responsive (LTR), and anaerobic induction (ARE) (Fig. 4 and Table S3).
The promoters of HAK genes contained various cis-acting elements with different numbers. In particular, most HAK genes contain ARE elements and they may play a crucial role in anaerobic induction response in roots of Medicago. Methyl jasmonate (MeJA), as a wounding-related phytohormone, it is able to stimulate the expression of defense-related genes . Interestingly, HAK genes have relatively more MeJA-responsive elements than the other types (Fig. 4B), in particular MsHAK15 with 8 MeJA-responsive elements, indicating that HAK with more MeJA-responsive elements maybe play a specific role in wounding stress resistance induced by MeJA. It is generally known that three cis-acting elements, ABRE, MBS and W-box, are related with responsiveness to drought-induced signaling and regulation of downstream gene expression . Our results showed that many HAK genes contained more ABRE were grouped in cluster I, II, IV (Fig. 4B, C), indicating that HAK gene family plays a role in drought resistance in Medicago. Notably, salicylic acid responsiveness elements were presented with high numbers in cluster IV, indicating that these five genes of this cluster play a key role in resistance to salicylic acid-related stress (Fig. 4A, B).
Expression patterns of HAK genes in different tissues and under stress treatments
We investigated the expression patterns of HAKs in various tissues of M. truncatula with genechip dataset from the MtGEA web server, including roots, stems, leaves, flowers, pods and seeds (Fig. 5A, Table S4). Overall, half (8) of the 16 MtHAK genes with available probe sets were expressed at relatively low level in these tissues, and the other half were expressed at relatively high level (Fig. 5A). Among the eight genes with low expression level, MtHAK15 was highly expressed in seeds (Fig. 5A). Among the other eight genes with high expression level, MtHAK17 were expressed at relatively low level in seeds and flowers, and MtHAK7 in pods (Fig. 5A). As for M. sativa, gene expression levels in six tissues were analyzed based on transcriptome data, including roots, elonged-stems, pre-elonged-stems, leaves, flowers and nodules (Fig. 5B, Table S4). According to their expression level, these 19 MsHAK genes could be divided into three categories, genes with high expression level in each tissue (MsHAK2,15/16,3,10), genes with low expression level (MsHAK20, 17/22, 13, 14, 11), and genes with moderate expression level (MsHAK6/7, 4/5/8/9, 1, 12) (Fig. 5B). Notably, among genes with high expression level, MsHAK2 was expressed with relatively low level in nodules. Among the genes with low expression level, MsHAK20 was expressed at relatively high level in roots and nodules, and MsHAK11 in nodules. Among genes with moderate expression level, MsHAK6/7 were expressed with relatively low in nodules (Fig. 5B).
Expression profiles of MtHAK genes under stress were initially analyzed based on the data retrieved from the MtGEA web server, including samples from roots and shoots under drought treatment, and roots under in vitro culture salinity and under hydroponic salinity conditions (Fig. 5C, E, Table S4). The expression level of MtHAK3, MtHAK2, MtHAK7 and MtHAK12 were highly induced in both in vitro culture and hydroponic salinity conditions (Fig. 5C). In addition, the expression level of several genes were significantly increased under drought treatment, but decreased after re-watering, including MtHAK12, MtHAK7 and MtHAK5 in roots and MtHAK12, MtHAK3 and MtHAK2 in shoots (Fig. 5E).
As for M. sativa, the expression level of MsHAK genes were analyzed under NaCl and mannitol treatment with transcriptome data, and it was found that most genes were induced at different level (Fig. 5D, F, Table S4). Under both treatments, MsHAK1, MsHAK3, and MsHAK10 maintained a relatively high level than all the other genes (Fig. 5D, F). The expression level of MsHAK1, MsHAK2 and MsHAK3 were also significantly increased under both treatments (Fig. 5D, F).
Co-expression network analysis in M. truncatula under stress treatment
To further investigate the function of the MtHAK gene family under NaCl stress in M. truncatula, a weighted gene co-expression network analysis (WGCNA) was performed with 10,658 genes that were differentially expressed, and a total of 13 modules were generated (Fig. 6A). It is worth noting that three up-regulated modules were screened and shown in green, red, and brown, and one down-regulated module was found and shown in blue (Fig. 6A). MtHAK7 and MtHAK2 were recognized in the green modules, MtHAK5 in red modules, MtHAK13 in brown modules, and MtHAK12 in blue modules (Fig. 6E). Green module contained 766 genes and 22,362 interactions, with 92 and 123 genes are closely related with MtHAK7 and MtHAK2, respectively. Red module contains 1076 genes and 32,555 interactions, 12 genes are closely related with MtHAK5. Brown module contained 1337 genes and 41,798 interactions, and 58 genes are closely related with MtHAK13. The blue module contains 1702 genes and 219,316 interactions, with 83 genes are closely related with MtHAK12 (Fig. 6C, E). We found that in the co-expression network with 5 MtHAK genes, the main GO enrichment pathways included macromolecule biosynthetic process, response to endogenous stimulus, cellular component assembly and nitrogen compound metabolic process (Fig. 6G). The above results suggest that these five MtHAK genes may respond to salt stress by participating in these metabolic pathways.
In order to explore the response mechanism of M. truncatula to drought stress, the co-expression network analysis was conducted with 15,175 differentially expressed genes, resulting in a total of 12 modules (Fig. 6B). Among them, two up-regulated modules were screened and shown in green and turquoise, and they included 838 and 2,976 genes, and 47,581 and 307,210 interactions, respectively (Fig. 6D). MtHAK5 and MtHAK12 were identified in the green module, of which 342 and 83 genes were closely associated with MtHAK5 and MtHAK12, and MtHAK7 and MtHAK9 were identified in the turquoise module, with 16 and 324 genes were directly related with MtHAK7 and MtHAK9, respectively (Fig. 6F). The GO enrichment analysis of the co-expression network of the four identified HAKs revealed that they were mainly enriched in several pathways such as response to abiotic stimulus, response to stress and response to stimulus, indicating that these HAK genes are likely involved in these pathways (Fig. 6H). Taken together, among 363 and 770 genes that were screened under salt stress or drought stress, 9 genes were co-expressed in both treatments, including 3 HAK genes (MtHAK5, MtHAK7, and MtHAK12) (Fig. 6I, Table S5).
Co-expression network analysis in M. sativa under stress treatment
In order to study the response mechanisms of M. sativa under salt and drought stress, co-expression network analysis was performed on 14,144 and 12,677 differentially expressed genes in M. sativa, respectively (Fig. 7). Under salt stress, a total of 27 expression modules were generated (Fig. 7A), and HAK genes were identified in 5 modules (Fig. 7C). A total of 2,533 genes and 334,585 interactions in the blue module, and a total of 572 genes and 20,049 interactions in the red module, 441 genes and 13,866 interactions in green module, 253 genes and 5,056 interactions in tan module, and 264 genes and 5,807 interactions in greenyellow module (Fig. 7C, E). MsHAK1/3/14 were identified in the blue module, with 278/319/98 genes were closely related to MsHAK1/3/14, MsHAK6/12/2/11 were identified in red/green/tan/greenyellow, with 31/28/30/48 genes closely related with them, respectively (Fig. 7E). The GO enrichment analysis showed that M. sativa mainly responded to salt stress through catabolic process, response to external stimulus, response to abiotic stimulus, regulation of developmental process and regulation of reductive process (Fig. 7G).
A total of 23 modules were generated under drought stress in M. sativa (Fig. 7B). Finally, four modules of blue, salmon, midnightblue and yellow were identified to contain HAK genes. The blue module included 2,160 genes and 173,756 interactions. MsHAK1/10/12 were found in this module, which were closely related to 860/282/76 genes, respectively. MsHAK2 was identified in the salmon module, which was closely related with 7 genes; MsHAK6 was identified in the midnightblue module, which was closely related with 28 genes. MsHAK11 was identified in the yellow module and it was closely related with 37 genes (Fig. 7F). GO enrichment analysis mainly enriched metabolic processes such as metabolic process, developmental process, response to abiotic stimulus and response to stress (Fig. 7H), indicating M. sativa may respond to drought through these pathways. Of the 780 and 1,040 genes screened under salt or drought stress in M. sativa, 261 genes were induced by these two stresses simultaneously, in which 5 HAK genes (MsHAK1, MsHAK2, MsHAK6, MsHAK11, MsHAK12) were co-expressed in both treatments (Fig. 7I, Table S5).
Validation of the expression profile of stress-responsive HAK genes by RT-qPCR
By co-expression analyses, we found that three genes HAK genes (MtHAK5, MtHAK7, and MtHAK12) in M. truncatula were induced by both salt and drought stress, and their homologous genes in M. sativa were MsHAK2, MsHAK7, and MsHAK11 (Fig. 6I). Meanwhile, five genes were screened in the co-expression network from M. sativa (MsHAK1, MsHAK2, MsHAK6, MsHAK11, and MsHAK12), and their homologous genes in M. truncatula are MtHAK1, MtHAK2, MtHAK5, MtHAK7, and MtHAK9 (Fig. 7I). In these 16 HAK genes, 4 of them are duplicate genes (MtHAK5, MtHAK7, MsHAK2 and MsHAK11), and 6 genes from M. truncatula (MtHAK1, MsHAK2, MtHAK5, MtHAK7, MtHAK9, and MtHAK12), and 6 genes from M. sativa (MsHAK1, MsHAK2, MsHAK6, MsHAK7, MsHAK11, and MsHAK12) were selected for further analyses.
RT-qPCR were performed to verify the transcript abundance of selective HAK genes in seedlings of M. truncatula and M. sativa under salt and drought treatments at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h (Figs. 8 and 9). In M. truncatula, all genes were highly induced under both stresses except MtHAK9 that was not induced under NaCl treatment. The expression levels of MtHAK5, MtHAK7, and MtHAK12 increased gradually from 2 to 24 h for the two treatments. In particular, the expression level of MtHAK7 was induced by more than 10 folds at 6 h for both treatments (Fig. 8). MtHAK2 responded to salt stress rapidly by more than 10-fold increase at 1 h and maintained a relatively high level from 3 to 24 h (Fig. 8).
RT-qPCR data showed that all MsHAK genes were highly induced by salt and drought treatment, except MsHAK12 that was only slightly induced by both treatment at 24 h, which is the same as its homologous MtHAK9 (Fig. 9). MsHAK1 and MsHAK2 showed identical trends at different time points under the two stresses. Notably, expression level of MsHAK6 was the highest 1 h under drought treatment, while its expression level was significantly higher and increased at 6, 12, 24 h induced by NaCl stress, which was the same for MsHAK7.
Since the treatment time points for RT-qPCR were the same as for the transcriptome data, these two datasets thus were also compared for correlation analysis (Fig. 9). All RT-qPCR for six MsHAK genes were positively correlated with transcriptome data, and MsHAK2 had the highest correlation coefficient of 0.710 than for the other five genes.
The plant HAK/KUP/KT family showed a major function in root K+ acquisition and they also play a vital role in environmental stress adaptation and regulation of cell size . K+ also play crucial roles in enzyme activation, anion neutralization, osmoregulation, and ultimate improvement of plant yield . In the current study, we identified a total of 22 HAK genes in M. truncatula and 22 HAK genes in M. sativa. Multiple sequence alignment confirmed that almost all M. truncatula and M. sativa HAK members contained HAK/KUP/KT domains (Additional Fig. S2) and there is no clear preference for the N-terminus or C-terminus. They shared highly conserved amino acid length, which was consistent with those of the model plants such as Arabidopsis , rice  and maize . AtHAKs and OsHAKs were divided into four clusters on the basis of phylogenetic analysis, with cluster I, II, II and IV, respectively. HAK genes of Medicago can also be classified into four clusters according to the classification criteria for Arabidopsis and rice, with cluster I (A, B), II (A, B), III (A, B), and IV (A, B) (Fig. 1), which implied the taxonomy and evolution of HAK gene family members were quite conservative in Medicago. Notably, cluster IA and IV do not contain members from Arabidopsis, and OsHAK13 was positioned in cluster IIB previously , while it was positioned in cluster III B in this study. This inconsistency might be due to different methods or parameters were used for protein alignment and phylogenetic construction. Different cluster showed varied functions, the HAK genes in groups I was proven to be mainly involved in high-affinity K+ uptake and translocation. Some genes of Cluster IB (Fig. 2), such as AtHAK5, was associated with high-affinity K+ uptake and mostly expressed in root parts , which can help roots to absorb K+ under K+ deficiency stress environment. Remarkably, more genes in groups II, III, and IV have been reported to be associated with various plant developmental processes, for example, AtKUP5 (IIIb) . These genes served different functions in different evolutionary processes, providing theoretical reference for the understanding of functions of HAK gene in Medicago.
In plants, members of the HAK gene family display a low conservation in their exon/introns structures. The number of exons in Mt/MsHAK genes ranged from 4 to 10 (Fig. 2B), which is very similar to that of rice (2 to 10)  and maize (3 to 10) . Mt/MsHAK family members with two or three homologies contained different exon numbers. For instance, MsHAK7 and MtHAK5 had 8 exons, whereas MsHAK6 had three exons. This suggests that exon acquisition or loss might have occurred in this gene family during evolution, leading to various structures of homoeologous genes.
Duplication and divergence play critical role in the expansion and evolution of gene families [38, 39]. In previous studies, segmental duplications (SD) and tandem duplications (TD) events were reported as the major contributors to the large HAK gene expansion in many plant genomes [16, 40]. However, in this study, we found that not only intraspecies duplication events (Mt-SD, 10%; Ms-TD, 20%) (Fig. 3A, B) but also interspecies duplication (Mt-Gm, 28; Ms-Gm, 22) were the major contributors to the rapid gene expansion of the HAK family in Medicago (Fig. 3C). This same scenario was also demonstrated in Medicago (Mt-Ms, 10) HAK family (Fig. 3C), three genes in M. truncatula (MtHAK1, 4 and 7) showed a collinear relationship with those in M. sativa, G. max and A. thaliana (Fig. 3C and Table S2); Four genes in M. sativa (MsHAK1, 3, 11 and 7) showed a collinear relationship with those in A. thaliana and G. max (Fig. 3C and Table S2). Finally, two pairs of homologous genes from Medicago, MtHAK1/MsHAK1, and MtHAK7/MsHAK11, are collinear with A. thaliana and G. max. Thus, the expansion of the HAK genes could be an indication that HAK genes play key roles in multiple biological processes. This study showed that around 46% (20/44) of HAK genes from M. truncatula and M. sativa were involved in duplicated genomic blocks, and the Ka/Ks values of all orthologous gene pairs are less than 1 (Fig. 3D, Table S2). This suggests that a strong purifying selection on the Medicago orthologous HAKs to remove deleterious mutations at protein level .
Cis-acting elements played pivotal function in the regulation of gene expression by controlling the efficiency of the promoters. Studies on cis-acting elements could provide key foundation for further functional characterization of the HAK gene family . Previous studies have demonstrated that HAK plays an important role in salt and drought resistance [21, 22]. In our studies, TGACG-motif (MeJA-responsive), ABRE (abscisic acid-responsive), ERE (ethylene-responsive), TC-rich repeats, W-box (defense and stress responsiveness) and ARE (anaerobic induction) elements were found widely distributed in HAK genes (Fig. 4). MtHAK9, MsHAK6 and MsHAK12 contain 4 MeJA-responsive element, MtHAK1, MtHAK2 and MsHAK7 had 8, 4 and 3 abscisic acid-responsive element, respectively; MsHAK7 contained 4 drought-inducibility elements (Fig. 4). And these results suggested that HAK genes of Medicago are likely involved in salt and drought stress.
Drought and salt are the most prevalent and severe abiotic stress factors for plant growth . It is urgent to improve the salinity and drought tolerance of M. sativa to increase yield. Genechip data and transcriptome data under NaCl and drought treatments, along with expression profiles in various tissues suggested that the expressions of six homologous gene pairs (except for MtHAK1) were highly induced or drastically changed in Medicago (Fig. 5). In addition, the expression pattern of all six genes under NaCl and drought treatments were verified by RT-qPCR analyses in M. truncatula and M. sativa (Figs. 6 and 7A), and the results suggested that the vast majority genes are involved in NaCl and/or drought induction. Correspondingly, five genes pair (MtHAK1/MsHAK1, MtHAK2/MsHAK2, MtHAK5/MsHAK7, MtHAK7/MsHAK11 and MtHAK12/MsHAK2) were significantly up-regulated under NaCl and mannitol treatments, and their expression pattern were the same in M. truncatula and M. sativa. Meanwhile, MtHAK1/MsHAK1, and MtHAK7/MsHAK11 were identified by WGCNA analysis (Figs. 6I and 7I). These evidences indicated that these genes are likely key genes in response to NaCl and drought stress in Medicago.
Previous studies have confirmed that maintaining efficient K+ uptake is prerequisite for K+/Na+ homeostasis and salt tolerance when plants are exposed to salt stress . In this study, the expression of five HAK genes pairs (except MtHAK9/MsHAK12) was up-regulated under salt stress (Figs. 6 and 7A), contributing to high K+/Na+ ratio and salt tolerance. The similar results were also observed for OsHAKs in rice . AtKUP7 is responsible for K+ uptake at high K+ concentrations, and may be also involved in K+ transport into xylem sap, affecting K+ translocation from roots to shoots . The homologous gene pair MtHAK1/MsHAK1 are more closely related with AtKUP7, and they exhibited high expression under salt and drought stress, indicating that these two genes are possibly involved in the transport of K+ under stress conditions. HAKs could enhance drought tolerance through absorbing and accumulating K+ to increase cytosolic ion concentration for plants [44, 45]. In the present study, the expression level of several Mt/MsHAK genes were up-regulated when plants were exposed to drought stress (Figs. 6 and 7 A), being consistent with the previous reports on TaHAKs in wheat  and MeKUPs in cassava . In addition, AtKUP2 mutation affects cell expansion and leads to developmental defects in shoots , and its close homologous gene MtHAK12 was highly up-regulated under both treatments, suggesting that MtHAK12 may also be involved in early stem development and stress responses.
A total of 22 and 22 HAK genes were identified in M. truncatula and M. sativa, respectively. Phylogenetic analysis suggested that these HAK proteins could be divided into four groups, and the members of the same subgroup share similar gene structure characteristics and conserved motifs. Many cis-acting elements related with defense and stress, and anaerobic induction was found in their promoter region. In addition, gene expression profiles showed that these HAK genes exhibited distinct expression pattern in different tissues, and in response to salt and drought treatments. Furthermore, co-expression analysis showed that 6 homologous HAK hub gene pairs involved in direct network interactions. RT-qPCR verified that the expression level of six HAK gene pairs were induced by NaCl and mannitol treatment to different extent. In particular, MtHK2/7/12 from M. truncatula and MsHAK2/6/7 from M. sativa were dramatically induced. The expression level of MsHAK1/2/11 determined by RT-qPCR showed significantly positive correlation with transcriptome data.
Materials and methods
Identification of HAK family members in Medicago
The genomic data of M. truncatula and M. sativa were downloaded from the website of https://figshare.com/articles/dataset/Medicago_sativa_genome_(accessed on 1 May 2022) and_annotation_files/12,623,960 and http://www.medicagogenome.org/ (accessed on 1 May 2022), respectively. We used the Hidden Markov Model (HMM) profile of the K+ transporter domain (PF02705) to query the candidate HAKs from Medicago genomic database using HMMER3.0 with a cutoff of 0.01 . Then, we examined the candidate HAK protein sequences, which include the K+ transporter domain, using the Pfam database (http://pfam.xfam.org) (accessed on 5 May 2022) and SMART program (http://smart.embl-heidelberg.de/) (accessed on 5 May 2022). Further BLASTP search with an E-value cutoff of e− 10 was performed to sort HAK family members using AtHAK , OsHAK  and ZmHAK  as queries, which were retrieved from TAIR (http://www.arabidopsis.org) (accessed on 10 May 2022) and RICEDATA (http://www.ricedata.cn/gene) (accessed on 10 May 2022), respectively. In order to ensure the correctness of the selected genes, output putative HAK protein sequences were submitted to InterProScan (https://www.ebi.ac.uk/interpro/search/sequence-search) (accessed on 12 May 2022), CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) (accessed on 12 May 2022), Pfam (https://pfam.xfam.org/) (accessed on 15 May 2022), and SMART (http://smart.embl-heidelberg.de/) (accessed on 15 May 2022). Finally, all predicted protein sequences were curated manually by softberry (http://linux1.softberry.com/) (Additional Fig. S1). In total, 22 MtHAK and 22 MsHAK genes were identified, and assigned based on their locations on chromosome (Table 1). Correspondingly, ExPASy (https://web.expasy.org/compute_pi/) (accessed on 17 May 2022) was used to determine the isoelectric point (pI) and molecular weight (MW) of HAK proteins. Subcellular localization of HAK proteins were predicted by using the Softberry Home Page (http://linux1.softberry.com/berry.phtm) (accessed on 17 May 2022).
Analyses on sequence, conserved motif and structural characterization
Conserved motifs were identified by selecting motifs from the MEME program (http://meme-suite.org/tools/meme) (accessed on 24 May 2022) with the motif number of HAK was set as 20, and the width range of 10 to 200 amino acids (aa). Subsequently, sequence alignment was carried out by using jalview (http://www.jalview.org/Web_Installers/install.htm). The visualization of exon-intron positions and conserved motifs were executed through the TBtools software.
Analyses of phylogenetic relationship
The HAK protein sequences of A. thaliana, O. sativa and G. max were downloaded from Phytozome (https://phytozome-next.jgi.doe.gov/) (accessed on 20 May 2022). We constructed phylogenetic trees using MEGA7.0 according to the maximum likelihood method and performed bootstrap testing with 1000 replicates . Meanwhile, subfamily clustering on phylogenetic tree was determined based on that of Arabidopsis. Subsequently, EvolView (https://evolgenius.info/evolview-v2/) (accessed on 25 May 2022) was used to view the phylogenetic tree.
Analysis of chromosome locations and collinearity
The loci of HAK genes were obtained from the genome annotation data. TBtools software was applied to map the chromosome locations for each genes. Next, these sequences were analyzed to identify collinearity blocks against the whole genome using MCSCAN (http://chibba.agtec.uga.edu/duplication/mcscan/) (accessed on 26 May 2022) . Moreover, intraspecific synteny relationship (M. truncatula and M. sativa) and interspecific synteny relationship (M. truncatula and M. sativa, M. truncatula and G. max, M. truncatula and A. thaliana, M. sativa and G. max, M. sativa and A. thaliana) were analyzed, and they were mapped to the chromosomes of M. truncatula and M. sativa using TBtools, respectively . Lastly, the synonymous (Ks) and nonsynonymous (Ka) substitution rates were estimated using TBtools .
Analyses of cis-acting elements and location of HAK genes in Medicago
Cis-acting elements were searched from upstream regions (2000 bp) of all HAK genes. The cis-acting elements in the promoter region were analyzed at the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 27 May 2022) . The visualize models of cis-acting elements in the promoters were carried out with TBtools.
Analysis of expression level of HAK genes
Genechip data from roots and shoots and those under drought and salt stress conditions for MtHAK genes were downloaded from the M. truncatula Gene Expression Atlas (https://Mtgea.noble.org/v3/) (accessed on 28 May 2022). Two-week-old M. truncatula seedlings (cv. Jemalong line A17) were treated with 200 mM salt solution at 0 h, 6 h, 24 and 48 h, with biological triplicates. The roots under hydroponics condition were treated with 200 mM salt solution at 0 h, 1 h, 2 h, 5 h, 10 and 24 h. When the fourth shoot emerging out, roots and shoots were harvested separately at 2 d, 3 d, 4 d, 7 d, 10 d and 14 d after water withholding, 2-day well-watered plants were treated as control, respectively, and all experiments were performed with biological triplicates. The expression level of MtHAK genes in root, stem, leaf, flower, pod, seed were also analyzed. Amazing HeatMap software was used to generate heatmap . The original transcriptome data from M. sativa under NaCl and mannitol treatments at 0 h, 1 h, 3 h, 6 h, 12 h, 24 h (SRR7160314-15, 22–23, 25–49, 51–52, 56–57) were downloaded from NCBI database . Twelve-day-old alfalfa (Zhongmu No. 1) seedlings under hydroponics condition were separated into three groups: (1) control (1/2 MS nutrient solution), (2) salt (250 mM NaCl), (3) drought (400 mM mannitol). The treatment time during were 1 h, 3 h, 6 h, 12 h, and 24 h with biological triplicates.
Then the data was converted to fastq files using SRA-Toolkit v2.9 . Raw reads were trimmed using the Trimmomatic-0.39 . Gene expression level was determined by mapping cleaned reads to the corresponding M. sativa reference genomes using the StringTie v2.1.3 package .
Co-expression network construction and GO enrichment analysis of Mt/MsHAK gene family
The WGCNA package were used for co-expression network analysis, four co-expression networks were constructed, including M. truncatula/M. sativa response to NaCl/drought stresses, respectively. To identify differentially expressed genes, the probe sets of M. truncatula (RPKM less than 10) and the genes of M. sativa (FPKM less than 1) that show very low expression levels were removed. Then, genes with different expression levels were determined by comparing different stress treatments and control using DEseq by multiple-factor design (fold change greater than 2 and Padj < 0.05). After these filtering steps, 10,658/15,175 differential expressed genes of M. truncatula response to NaCl/drought stresses, and 14,144/12,677 differentially expressed genes of M. sativa response to NaCl/drought stresses remained. The co-expression gene network for those selected genes were constructed, a β (soft thresholding power) value of 9 was selected based on the scale-free topology criterion. The Pearson algorithm was used to calculate the correlation coefficient, and the results were stored as a signed co-expression matrix. The resulting adjacency matrix was converted to a topological overlap (TO) matrix via TOM similarity algorithm, and the genes were hierarchically clustered based on TO similarity. The parameters for TOMType were: MaxBlocksize was 60,000, minModuleSize was 30, deepSplit was 2, and mergeCutHeight was 0.25. Finally, network visualization for each module was performed with genes directly/indirectly related to Mt/MsHAK genes using the Cytoscape version 3.7.2, and the edges were filtered with weights below 0.1 to ensure reliability and readability of the results. Gene Ontology pathway enrichment analysis for the involved modules was performed using TBtools. The GO terms with a corrected p-value ≤ 0.05 were considered to be significantly enriched.
Plant materials and treatments
M. truncatula (cv. Jemalong A17) and M. sativa (cv. Zhongmu No.1) plants used in this study were generated and stored at the Institute of Animal Sciences of Chinese Academy of Agricultural Sciences. Roots, stems, leaves, flowers of mature M. truncatula and M. sativa plants, were collected separately for RNA extraction and RT-qPCR analysis. To investigate the expression pattern of HAK genes in response to NaCl and mannitol stress, seeds were germinated and transferred into the MS solid medium (MS basal salts supplemented with 30 g/L sucrose, 0.8% (w/v) agar), then kept in a growth chamber at 25 °C under a photoperiod of 16/8 light/dark regime. When the third leaf was fully expanded, seedlings were transferred to fresh MS solid medium supplied with 300 mM NaCl and 15% mannitol, respectively, and the whole plants were collected at 0 h, 1 h, 3 h, 6 h, 12 and 24 h for each treatment. The sample were frozen in liquid nitrogen and stored at -80 °C for subsequent analysis.
RNA extraction, analysis of the gene expression by RT-qPCR and correlation analyses
The detailed procedures of total RNA extraction, first strand cDNA synthesis and RT-qPCR were described according to the manufacturer’s instructions (TIANGEN, Beijing). Each reaction was performed in biological triplicates with actin gene as references and the data from RT-qPCR was analyzed using 2−△△CT method. The results were analyzed by means ± standard deviation (student’s t test n = 3, * p < 0.05, ** p < 0.01). The primer sequences used in this study were listed in Table S1. GraphPad Prism 6.0 was used to draw scatter plots of gene expression, and linear regression analysis and Pearson correlation analysis were performed between the expression levels analyzed by transcriptome and by RT-qPCR.
Availability of data and materials
All the data presented in this manuscript are available in the main text or in the supplementary material.
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This project was supported by the National Nature Science Foundation of China (31860675 and U1906201), the China Agriculture Research System of MOF and MARA (CARS-34).
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Fig. S1. Conserved protein motif analysis of HAK family members before and after correction manually by softberry. ‘ori’ represents the original predicted gene sequence, and ‘new’ represents the new predicted gene sequence. Fig. S2. Multiple sequence alignment of MtHAKs and MsHAKs. The alignment were performed by MEGA and visualized by Jalview. Residues with more than 50% similarity were shaded. Conserved regions (KUP/HAK/KT) were indicated at the top. Fig. 3. The sequence information of 20 conserved motifs of HAK gene in Medicago, including the sequence logo and amino acids, as well as amino acids numbers of each motif.
Supplementary Table 1. List of primers used in this research. Supplementary Table 2. The synteny gene pairs of Medicagotruncatula, Medicago sativa, Glycine max and Arabidopsisthaliana, as well as the KaKs of comparative synteny gene pairs. SupplementaryTable 3. List of all identified cis-acting elements in all HAKgenes found in Medicago. Supplementary Table4-1. Detailed information on the available tissue expression levels of MtHAKgenes retrieved from microarray data for M. truncatula. Supplementary Table4-2. Detailed information on the available tissue expression levels of MsHAKgenes retrieved from transcriptome data for M. sativa. Supplementary Table4-3. Detailed information on the available expression levels of MsHAKgenes response to salinity stress treatments retrieved from microarray data forM. truncatula. Supplementary Table4. Detailed information on the available expression levels of MsHAKgenes response to drought stress treatments retrieved from microarray data for M.truncatula. Supplementary Table4-5. Detailed information on the available expression levels of MsHAKgenes response to salinity stress treatment retrieved from transcriptome datafor M. sativa. Supplementary Table4-6. Detailed information on the available expression levels of MsHAK genesresponse to drought stress treatment retrieved from transcriptome data for M.sativa. Supplementary Table5-1. The co-expression network of MtHAK genes in DEGs under salt stress. Supplementary Table5-2. The co-expression network of MtHAK genes in DEGs under drought stress. Supplementary Table5-3. The co-expression network of MsHAK genes in DEGs under salt stress. Supplementary Table5-4. The co-expression network of MsHAK genes in DEGs under drought stress.
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Li, Q., Du, W., Tian, X. et al. Genome-wide characterization and expression analysis of the HAK gene family in response to abiotic stresses in Medicago. BMC Genomics 23, 791 (2022). https://doi.org/10.1186/s12864-022-09009-2