Identification of the MaHAKs in banana
A total of 26 HAK genes were originally obtained from the banana A genome using the HMM search. Two erroneously predicted genes (Ma02_04550, Ma07_18090) were manually removed after confirming the presence of the K+ transporter domain. Finally, 24 genes were identified as banana HAK genes and were renamed according to their corresponding homologs from rice and maize based on phylogenetic relationship analysis. Fourteen family members and the letters a, b, c and d were added when more than one gene clustered together with the specific rice gene, for instance, MaHAK10a, MaHAK10b, MaHAK10c and MaHAK10d (Table 1). The composition of HAK members in the banana genome was very different from that in rice, maize or Arabidopsis. No MaHAK genes homologous to OsHAK3, OsHAK4, OsHAK6, OsHAK15, OsHAK16, OsHAK19, OsHAK20, OsHAK21, OsHAK22, OsHAK24, OsHAK25, OsHAK26 or OsHAK27 were identified. However, seven MaHAKs had two or three or even four homologs, including MaHAK2, MaHAK7, MaHAK8, MaHAK10, MaHAK11, MaHAK13 and MaHAK14. The length of MaHAK genes ranged from 565 to 839 amino acids (aa). Each MaHAK protein contained at least 8 transmembrane segments. The molecular weight of the MaHAK proteins ranged from 63.54 to 93.82 kDa.
Table 1
The HAK/HUP/KT genes in Musa acuminata L.
Gene Name
|
Gene ID
|
Amino Acid Length
|
TMs
|
MW(kDa)
|
MaHAK1
|
Ma05_g07130
|
633
|
11
|
70.02
|
MaHAK2a
|
Ma11_g18830
|
785
|
13
|
87.66
|
MaHAK2b
|
Ma08_g09830
|
638
|
8
|
71.27
|
MaHAK5
|
Ma10_g17110
|
752
|
12
|
83.28
|
MaHAK7a
|
Ma10_g01080
|
782
|
13
|
88.14
|
MaHAK7b
|
Ma05_g07770
|
781
|
13
|
87.76
|
MaHAK8a
|
Ma11_g23250
|
769
|
13
|
86.12
|
MaHAK8b
|
Ma08_g01620
|
779
|
11
|
86.39
|
MaHAK9
|
Ma07_g23610
|
771
|
11
|
85.62
|
MaHAK10a
|
Ma09_g23160
|
774
|
11
|
85.90
|
MaHAK10b
|
Ma01_g00350
|
781
|
10
|
86.43
|
MaHAK10c
|
Ma07_g21520
|
771
|
11
|
86.31
|
MaHAK10d
|
Ma03_g06090
|
782
|
13
|
87.28
|
MaHAK11a
|
Ma05_g00190
|
789
|
14
|
88.16
|
MaHAK11b
|
Ma08_g33820
|
794
|
14
|
88.38
|
MaHAK11c
|
Ma11_g16690
|
780
|
14
|
87.64
|
MaHAK12
|
Ma02_g04540
|
565
|
11
|
63.54
|
MaHAK13a
|
Ma08_g06810
|
736
|
12
|
81.47
|
MaHAK13b
|
Ma03_g24780
|
740
|
11
|
81.88
|
MaHAK14a
|
Ma08_g19550
|
836
|
10
|
93.82
|
MaHAK14b
|
Ma09_g11150
|
839
|
10
|
93.66
|
MaHAK17
|
Ma07_g12190
|
704
|
9
|
78.10
|
MaHAK18
|
Ma04_g39690
|
774
|
13
|
86.34
|
MaHAK23
|
Ma07_g21540
|
829
|
13
|
92.70
|
Phylogenetic analysis of MaHAKs in banana
The evolutionary relationship of HAKs in banana, rice, maize and Arabidopsis was analyzed by constructing a phylogenetic tree (Figure 1). Based on previous studies of HAK proteins in Arabidopsis and rice (Maser et al. 2001; Gupta et al. 2008), the MaHAK proteins were classified into four groups (Ⅰ-Ⅳ). Only MaHAK5 was placed in Group Ⅰ; MaHAK1, 2, 7, 8, 9, 10, 13 were placed in Group Ⅱ; MaHAK11, 12, 14, 18 and 23 were placed in Group Ⅲ; and only MaHAK17 was placed in Group Ⅳ. Group Ⅱ and Ⅲ were the most abundant in banana, comprising 54% and 40% proteins of all MaHAKs, respectively.
Gene structure and conserved motif examination of MaHAKs in banana
To further gain more insight into the evolution of the HAK family in banana, the exon-intron organizations of all identified MaHAK genes were examined. As shown in Figure 2, the coding sequences of all MaHAK genes were disrupted by introns, with numbers ranging from 5 to 10. Homologous genes usually have similar structures; for example, the three MaHAK11 genes contained 9 exons and 8 introns.
To assess the conservation and diversification of MaHAK proteins, protein motifs were further analyzed using the online program MEME. Motifs 1, 2, 3, 4, 6, 9 and 10 were conserved in all MaHAK proteins. It was also obvious that the motif composition was similar among homologous genes. Interestingly, the lengths of introns between the two homologous genes MaHAK7a and MaHAK7b were quite different, while the motif distributions were highly similar.
Genomic distribution and evolutionary analysis of MaHAKs in banana
MaHAK genes were unevenly distributed in 10 of 11 banana chromosomes (chr), except chr06 (Figure 3). Overall, the MaHAK gene was likely to be located in the distal parts of the chromosomes. Chr08 contained the largest number of MaHAK genes (MaHAK2b, MaHAK8b, MaHAK11b, MaHAK13a, MaHAK14a). To further investigate the duplication event occurring in the MaHAK genes, a synteny analysis was conducted. Six pairs of syntenic genes, MaHAK2a (chr11)-MaHAK2b (chr08), MaHAK8b (chr08)-MaHAK9 (chr07), MaHAK10c (chr07)-MaHAK10d (chr03), MaHAK11a (chr05)-MaHAK11c (chr11), MaHAK13a (chr08)-MaHAK13b (chr03) and MaHAK14a (chr08)-MaHAK14b (chr09), were found in MaHAKs (Figure 4). These results indicated that gene duplication events play an important driving force for MaHAK evolution.
Expression profile of MaHAKs under different potassium treatments
To study the expression profiles of MaHAK genes in different tissues under different potassium stresses, transcriptome analyses of the roots, pseudostems and leaves in the Baxi cultivar under normal (+K+) and potassium- deficient (-K+) conditions were performed. As shown in Figure 5a, most MaHAK genes exhibited moderate expression in different tissues under the different treatments, and the expression of MaHAK13a was very low or undetectable. MaHAK1, 2b and 10d showed low expression levels in roots, and MaHAK17 was expressed at low levels in both roots and pseudostems under both K+ treatment conditions. MaHAK14b showed very low expression in roots under normal K+ conditions but was upregulated under K+-deficient conditions. In addition, MaHAK14b was normally expressed at low levels in leaves in the K+ treatment conditions but was highly expressed in the pseudostem. MaHAK7b was indeed upregulated under K+-deficient treatment in roots and leaves. To analyze the expression pattern of MaHAK genes when symptoms appeared (60 d after treatment) under potassium stress, FPKM (kilobase/million mapped reads) data of MaHAKs were extracted (Xu et al. 2019). Six MaHAK genes were differentially expressed in normal-K+ roots (NKRs) versus low-K+ roots (LKRs) (Figure 5b). MaHAK10c and MaHAK10d were strongly upregulated and downregulated after low-K+ treatment, respectively. These results implied the different roles of these MaHAK genes in different tissues under different K+ conditions.
Functional characterization of MaHAK1 in potassium uptake
According to the increased expression of MaHAK14b induced by K+ deficiency in banana roots, MaHAK14b might be a high-affinity K+ uptake transporter from banana. First, the subcellular location of MaHAK14b was examined by generating a construct expressing a fusion protein of MaHAK14b-GFP (green fluorescent protein). The recombinant construct and control vector were introduced into the leaves of Nicotiana benthamiana. As shown in Figure 6a, the results indicate that MaHAK14b was solely located in the plasma membrane, while the green fluorescence of the GFP control was located in the nucleus, cytoplasm and plasma membrane.
Several reported HAK proteins could complement the growth defect of potassium-deficient yeast strain R5421, e.g., OsHAK1, OsHAK5 and ZmHAK5 (Yang et al. 2014; Chen et al. 2015; Qin et al. 2019). To examine the potassium uptake activity of MaHAK14b, R5421 yeast were transformed with MaHAK14b and the vector as a control. The growth of transformants was compared on AP medium containing various K+ concentrations. Yeast R5421 expressing MaHAK14b grew in 1 mM KCl, while no growth was observed in R5421 transformed with empty vector (Figure 6b). This result indicates that MaHAK14b is a high-affinity K+ transporter with K+ uptake activity.