Genome-wide identification, characterization and expression analysis of HAK genes and decoding their role in responding to potassium deficiency and abiotic stress in Medicago truncatula

Abstract

Background: The HAK family is the largest potassium (K⁺) transporter family, vital in K⁺ uptake, plant growth, and both plant biotic and abiotic stress responses. Although HAK family members have been characterized and functionally investigated in many species, these genes are still not studied in detail in Medicago truncatula, a good model system for studying legume genetics.

Methods: In this study, we screened the M. truncatula HAK family members (MtHAKs). Furthermore, we also conducted the identification, phylogenetic analysis, and prediction of conserved motifs of MtHAKs. Moreover, we studied the expression levels of MtHAKs under K⁺ deficiency, drought, and salt stresses using quantitative real-time PCR (qRT-PCR).

Results: We identified 20 MtHAK family members and classified them into three clusters based on phylogenetic relationships. Conserved motif analyses showed that all MtHAK proteins besides MtHAK10 contained the highly conserved K⁺ transport domain (GVVYGDLGTSPLY). qRT-PCR analysis showed that several MtHAK genes in roots were induced by abiotic stress. In particular, MtHAK15, MtHAK17, and MtHAK18 were strongly up-regulated in the M. truncatula roots under K⁺ deficiency, drought, and salt stress conditions, thereby implying that these genes are good candidates for high-affinity K⁺ uptake and therefore have essential roles in drought and salt tolerance.

Discussions: Our results not only provided the first genetic description and evolutionary relationships of the K⁺ transporter family in M. truncatula, but also the potential information responding to K⁺ deficiency and abiotic stresses, thereby laying the foundation for molecular breeding of stress-resistant legume crops in the future.

Keywords: Expression pattern; Genome-wide analysis; HAKs; Medicago truncatula.

Figure 1. Phylogenetic analysis of HAK proteins in M. truncatula (red circle), A. thaliana (green triangle), and O. sativa (blue square).

The tree was constructed using MEGA7.0 software by the neighbor-joining method. The numbers next to the branch represent the 1,000 bootstrap replicates expressed in percentage.

Figure 2. Phylogenetic tree, gene structure, and conserved motifs of the HAKs in M. truncatula.

(A) Phylogenetic tree of the MtHAK proteins. (B) Exon-intron structure distribution. (C) Conserved protein motifs.

Figure 3. The synteny analysis of MtHAKs displayed between the M. truncatula and Arabidopsis genomes.

The M. truncatula and Arabidopsis chromosomes are represented by yellow and green boxes, respectively. Blue lines indicate the collinear relationship of MtHAKs between M. truncatula and Arabidopsis, while green lines indicate the MtHAK gene pairs.

Figure 4. Analysis of the cis-acting regulatory elements in the promoter region of the MtHAK genes.

Depending on the functional annotation, the elements were classified into three main categories: phytohormone-responsive, abiotic stress-responsive, and plant growth and development-related. The frequency of these elements in the promoter region was represented by the numbers and the depth of the red color.

Figure 5. Expression patterns of the MtHAK genes in different developmental tissues.

The microarray data were normalized based on the mean value of each gene in all the analyzed plant organs. The heat map was portrayed by the relative expressions after log₂ transformed.

Figure 6. Relative expression of the MtHAK genes in response to K⁺ deficiency treatment.

Two-week-old seedlings were placed in K⁺ deficient conditions for 0, 1, 6, 12, 24, and 48 h. Mean values and standard errors were calculated from three biological replicates. An asterisk (*) indicates the significant difference between K⁺ deficiency and control at p < 0.05.

Figure 7. Relative expression of the MtHAK genes in response to salt stress.

Two-week-old seedlings were treated with 300 mM NaCl for 0, 1, 6, 12, 24, and 48 h. Mean values and standard errors were calculated from three biological replicates. An asterisk (*) indicates the significant difference between the salt-stressed and control at p < 0.05.

Figure 8. Relative expression of the MtHAK genes in response to drought stress.

Two-week-old seedlings were treated with 18% PEG6000 for 0, 1, 6, 12, 24, and 48 h. Mean values and standard errors were calculated from three biological replicates. An asterisk (*) indicates the significant difference between the drought-stressed and control at p < 0.05.