Identification of HAK genes in sugarcane
Based on comparative genomics, 29 members of SbHAK genes were identified from sorghum (sorghum bicolor, sugarcane’s nearest relative). Using the protein sequences of sorghum HAK genes as reference, 30 distinct S. spontaneum HAK genes (Table 1), excluding alleles, were identified from the genome of tetraploidy S. spontaneum AP85-441 [28]. Each of these genes contain one to four alleles with an average of 3 (Additional file 1). The 30 SsHAK genes are distributed on seven S. spontaneum chromosome: chromosome 1 contains six genes; chromosome 2 contains seven genes; chromosome 3 contains four genes; chromosome 4 contains two genes; chromosome 5 contains five genes; chromosome 6 and 8 each contains three genes; No SsHAK genes were identified only on chromosome 7 (Additional file 1).
All the 30 predicted SsHAK proteins have a typical “K_trans” domain (PF02705), which is specific to HAK/KUP/KT potassium transporter family members. For consistency, these SsHAK genes were named based on the previously reported O. sativa HAK nomenclature and phylogenetic relationships [17]. If two SsHAK genes were equally close to a single OsHAK gene, then the same name was used followed by the letters “a” and “b” (Table 1). Two paralogous SsHAK genes (SsHAK19a and SsHAK19b) were identified corresponding to the same sorghum gene, Sobic.006G062100, which may be caused by gene loss in sorghum or gene duplication in sugarcane. Amino acid number of the identified 30 SsHAKs ranged from 487 to 967, with an average of 758. The predicted isoelectric points (pI) of the SsHAKs varied from 5.88 to 9.26, the average pI was 8.15. The molecular weight ranged from 55.84 kDa to 106.49 kDa, with an average of 84.47 kDa (Table 1). Prediction of transmembrane domains in SsHAK proteins indicated that most of them contained 11 or 12 transmembrane helices, which was similar to the situation in sorghum. Subcellular location of the SsHAK proteins predicted by WoLF PSORT showed that the SsHAK proteins were mainly on plasma membrane, which was best suited for their roles as transporters to maintain K+ homeostasis in sugarcane. In addition, the SsHAK proteins also showed localization on some organelles, including endoplasmic reticulum, vacuole, cytoplasm, golgi body and chloroplast. Protein sequence alignment of SsHAKs with their orthologs in sorghum showed that S. spontaneum and sorghum bicolor shared identities ranging from 81% to 98% with an average of 92.5% (Table 1). Four hundred thirty-five pair-wise protein sequence comparisons among these SsHAKs showed that SsHAK19a and SsHAK19b shared the highest identity (96%), other gene pairs had protein sequence similarities ranging from 28% to 82% with an average of 46%, indicating the SsHAKs are an ancient gene family with high sequence divergence (Additional file 2).
To investigate the possible evolutionary functional constraints after the split of sorghum and sugarcane, nonsynonymous to synonymous substitution ratio (Ka/Ks) between SsHAK and its orthologous gene in sorghum was calculated. The results showed that Ka/Ks ratios were less than 0.5, except for SsHAK13, suggesting that purifying selection was the main force to drive the evolution of HAK genes (Fig. 1).
Phylogenic analysis of HAK genes in S. spontaneum and some representative angiosperms
To analyze the evolution of HAK gene family in S. spontaneum and different plants, a total of 278 HAK genes from 14 representative angiosperms and a HAK member from Chlamydomonas reinhardtii as the outgroup were used to construct phylogenetic tree using the Neighbor-Joining method (Fig. 2, Additional file 3). The 278 HAK genes included 6 in Amborella trichopoda, 8 in Solanum lycopersicum, 13 in Vitis vinifera, 8 in Carica papaya, 13 in Arabidopsis thaliana, 12 in Ananas comosus, 25 in Brachypodium distachyon, 27 in Oryza sativa, 28 in Setaria italica, 28 in Setaria viridis, 27 in Zea mays, 29 in Sorghum bicolor, 30 in Saccharum spontaneum and 24 in Saccharum hybrid R570 [29]. Amino acid sequence of the 279 HAK/KUP/KT transporters from 15 representative plant species was uploaded as supplementary data (Additional file 4).
Table 1
Overview and comparison of HAK genes in Saccharum spontaneum and Sorghum bicolor
Sorghum bicolor
|
|
Saccharum spontaneum
|
Similarityf
|
Gene
|
AAa
|
pIb
|
Mwc (kDa)
|
TMSd
|
P.L.e
|
|
Gene
|
AAa
|
pIb
|
Mwc
(kDa)
|
TMSd
|
P.L.e
|
Sobic.006G061300
|
788
|
8.75
|
87.13
|
12
|
PM
|
|
SsHAK1
|
780
|
8.83
|
86.84
|
12
|
PM
|
94.42%
|
Sobic.003G418100
|
783
|
8.91
|
87.53
|
12
|
PM
|
|
SsHAK2
|
788
|
8.85
|
88.18
|
12
|
PM
|
94.61%
|
Sobic.003G164400
|
811
|
8.4
|
89.60
|
10
|
PM/ER
|
|
SsHAK3
|
785
|
8.69
|
86.79
|
11
|
PM
|
97.34%
|
Sobic.007G153001
|
706
|
8.37
|
78.02
|
9
|
PM/ER
|
|
SsHAK4
|
702
|
8.90
|
78.08
|
9
|
PM
|
92.92%
|
Sobic.003G413600
|
775
|
8.78
|
86.36
|
11
|
PM
|
|
SsHAK5a
|
705
|
8.39
|
78.76
|
11
|
PM
|
85.64%
|
Sobic.003G413700
|
775
|
8.54
|
86.42
|
11
|
PM
|
|
SsHAK5b
|
750
|
7.58
|
83.86
|
10
|
PM
|
93.35%
|
Sobic.002G411500
|
788
|
8.8
|
87.72
|
13
|
PM
|
|
SsHAK7
|
818
|
8.81
|
91.32
|
13
|
PM/Vacu
|
90.95%
|
Sobic.001G379900
|
805
|
7.36
|
89.80
|
12
|
PM/Cyto
|
|
SsHAK8
|
770
|
8.36
|
85.88
|
11
|
PM/ER
|
93.18%
|
Sobic.002G417500
|
792
|
6.96
|
87.53
|
12
|
PM/Cyto
|
|
SsHAK9
|
743
|
8.39
|
82.35
|
11
|
PM/ER
|
91.34%
|
Sobic.010G197500
|
820
|
8.37
|
91.15
|
10
|
PM/ER
|
|
SsHAK10
|
755
|
8.94
|
83.57
|
10
|
PM/Vacu
|
90.52%
|
Sobic.006G213500
|
805
|
8.33
|
89.66
|
13
|
PM/ER
|
|
SsHAK11
|
719
|
7.24
|
80.33
|
12
|
PM/ER
|
92.06%
|
Sobic.007G075100
|
790
|
8.21
|
88.50
|
14
|
PM
|
|
SsHAK12
|
509
|
8.54
|
57.87
|
8
|
PM
|
87.93%
|
Sobic.010G224400
|
779
|
8.97
|
85.92
|
12
|
PM/Cyto
|
|
SsHAK13
|
757
|
8.62
|
83.38
|
12
|
PM/ER
|
95.76%
|
Sobic.002G313900
|
843
|
5.71
|
93.38
|
12
|
PM/ER
|
|
SsHAK14
|
811
|
5.88
|
90.03
|
11
|
PM
|
91.12%
|
Sobic.006G210700
|
743
|
8.85
|
82.93
|
12
|
PM/ER
|
|
SsHAK15
|
852
|
6.00
|
95.04
|
12
|
PM/ER
|
90.12%
|
Sobic.001G184000
|
817
|
8.91
|
92.60
|
12
|
PM
|
|
SsHAK16a
|
487
|
9.26
|
55.84
|
8
|
PM/Cyto
|
81.06%
|
Sobic.001G184100
|
810
|
8.61
|
91.65
|
11
|
PM/ER
|
|
SsHAK16b
|
802
|
8.69
|
91.07
|
12
|
PM/ER
|
96.03%
|
Sobic.002G220600
|
708
|
8.77
|
78.15
|
12
|
PM
|
|
SsHAK17
|
701
|
9.06
|
78.01
|
12
|
PM
|
93.57%
|
Sobic.002G130800
|
787
|
8.69
|
88.61
|
14
|
PM/ER
|
|
SsHAK18
|
788
|
8.35
|
88.56
|
14
|
PM/ER
|
96.45%
|
Sobic.006G062100
|
746
|
7.29
|
83.31
|
12
|
PM/Golgi
|
|
SsHAK19a
|
767
|
7.00
|
85.62
|
10
|
PM/Golgi
|
94.78%
|
Sobic.006G062100
|
746
|
7.29
|
83.31
|
12
|
PM/Golgi
|
|
SsHAK19b
|
730
|
6.65
|
81.30
|
9
|
PM/Vacu
|
93.33%
|
Sobic.004G160000
|
735
|
8.46
|
80.43
|
12
|
PM/ER
|
|
SsHAK20a
|
730
|
8.81
|
80.09
|
12
|
PM/ER
|
97.01%
|
Sobic.006G061700
|
788
|
8.66
|
88.27
|
11
|
PM/Cyto
|
|
SsHAK20b
|
794
|
8.60
|
89.03
|
11
|
PM/Golgi
|
83.01%
|
Sobic.001G183700
|
828
|
8.51
|
92.29
|
11
|
PM/Cyto
|
|
SsHAK21
|
818
|
8.22
|
91.50
|
11
|
PM/ER
|
95.17%
|
Sobic.002G001800
|
931
|
8.61
|
102.07
|
12
|
PM/Chlo
|
|
SsHAK22
|
967
|
9.08
|
106.49
|
11
|
PM/Vacu
|
88.52%
|
Sobic.002G188600
|
852
|
6.78
|
93.82
|
12
|
PM/ER
|
|
SsHAK23
|
846
|
6.55
|
93.13
|
12
|
PM
|
98.00%
|
Sobic.010G112800
|
773
|
8.39
|
85.44
|
12
|
PM/Chlo
|
|
SsHAK24
|
698
|
7.62
|
77.44
|
10
|
PM/Chlo
|
96.94%
|
Sobic.004G250700
|
774
|
7.34
|
86.29
|
13
|
PM/ER
|
|
SsHAK25
|
800
|
7.13
|
89.27
|
14
|
PM/ER
|
94.62%
|
Sobic.007G209900
|
774
|
9.08
|
82.47
|
10
|
PM/Chlo
|
|
SsHAK26
|
744
|
8.98
|
82.93
|
10
|
PM/Chlo
|
89.63%
|
Sobic.001G184300
|
814
|
8.32
|
91.82
|
11
|
PM/ER
|
|
SsHAK27
|
812
|
8.44
|
91.41
|
11
|
PM/ER
|
97.67%
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
PM= plasma membrane, ER= endoplasmic reticulum, Vacu= vacuole, Cyto= cytoplasm, Golgi= golgi body, Chlo= chloroplast
a Amino acids number of HAK protein sequence
b Isoelectric point (pI) predicted by ExPASy (https://web.expasy.org/compute_pi/)
c Molecular weight (Mw) predicted by ExPASy (https://web.expasy.org/compute_pi/)
d Number of transmembrane domains possessed by HAK predicted by TMHHM Server v.2.0 (http://www.cbs.dtu.dk/ services/TMHMM/)
e Subcellular location of the HAK proteins predicted by WoLF PSORT (https://www.genscript.com/wolf-psort.html)
f Protein sequence similarity between sorghum and sugarcane calculated by BLASTP
These HAK genes could be divided into four clades (I, II, III, IV) based on previously reported OsHAKs [17]. In A. trichopoda, the earliest diverging angiosperm, there were only 6 HAK genes, while in dicots and monocots, the number of HAKs ranged from 8 to 30 (Fig. 2, Fig. 3), indicating that the ancient whole-genome duplication (WGD) contributed to the expansion of the HAK gene family in both dicots and monocots. Clade II and clade III included HAK genes from all 14 angiosperm genomes, indicating that the progenitors of these genes may have already existed prior to the split of angiosperm (Fig. 2, Fig. 3). Clade I and clade IV mainly contained HAK genes from monocotyledons. Eighty-three HAK genes were identified in clade I, in which only one HAK gene was from A. comosus (Aco006685, homologous with SsHAK5) and Arabidopsis (AtHAK5) respectively, and the other 81 HAK genes were from all eight examined Poaceae species (Fig. 2, Fig. 3). Twenty-nine HAKs were grouped into clade IV, and only 2 out of them were from dicotyledon, these results indicated that the HAKs was unevenly distributed.
Based on the pairwise synonymous substitution rates (Ks) in Sorghum bicolor and S. spontaneum (Additional file 5), the divergence time among four clades of HAK family was estimated. The median values of pairwise Ks varied from 1.644 to 2.851, corresponding to a divergence time ranging from 134.8 to 233.7 Mya, suggesting that HAKs in the four clades were ancient and divergent. Moreover, the divergence time between two pairs of duplicated SsHAKs (SsHAK5a/5b and SsHAK16a/16b ) ranged from 18.94 to 58.14 Mya (Additional file 6). These results suggested that the SsHAK family is an ancient gene family with recent gene duplication events.
Exon/intron organization of HAK family in S. spontaneum and other angiosperms
To investigate the structural characteristics and evolution of the HAK gene family, the exon/intron of the HAKs was mapped to the phylogenic tree, and the gene feature and pattern was analyzed (Fig. 2). The exon number in the HAK family of the 15 examined plant species ranged from 2 to 16 with an average of 8.4, and 217 out of 279 (77.8%) HAK genes possessed 8 to 10 exons (Additional file 7 and 8). This result suggested that the last common ancestor (LCA) of angiosperm HAK genes had 8 to 10 exons.
The exon number of SsHAKs varied from 2 to 12, and half of the SsHAKs possessed 8 or 9 exons. The pattern of SsHAKs gene structure was similar to that of HAK genes from sorghum and maize in the same clade, which suggesting that the HAK gene structure in the Panicoideae was relatively conserved. In clade I, exon number of HAK genes varied from 2 to 12, which was also varied the most among these 4 clades. Noteworthy, HAK genes in the subfamily where SsHAK22 located had only 2 to 4 exons, however, the protein size remained consistent, which were likely due to the loss of intron. Clade II had the largest number of HAK genes, with 60 out of 98 HAKs possessed 9 exons, while 5 out of 9 SsHAKs harbored 8 exons. SsHAK3/8/10 had one less exon than their orthologous genes in sorghum; the first exon in SsHAK13 and seventh exon in SsHAK24 were smaller than the corresponding exons in sorghum, both cases caused shorter amino acid sequence in S. spontaneum (Table 1, Fig. 2). In clade III, exon number was relatively conserved, with 61 out of 68 HAK genes possessing 8 to 10 exons, while the gene size varied greatly, which was mainly due to the different size of introns. In clade IV, exon number ranged from 2 to 8 with an average of 7, which was smaller than in other clades. Noteworthy, HAK genes in the subfamily where SsHAK4 located had only 2 to 5 exons, which was likely caused by intron loss during the process of evolution. The results indicated that HAKs underwent gene structure reconstruction under different evolutionary dynamics in S. spontaneum and other angiosperms in this study.
Expression analysis of HAK genes in Saccharum species
To study the expression profiles and potential functions of HAKs in Saccharum, we compared the gene expression patterns according to 4 sets of RNA-seq data: 1) Different developmental stages and tissues; 2) Leaf gradient; 3) Circadian rhythm; 4) Treatment under low potassium stress. FPKM values of HAK1, HAK7 and HAK20b in YT55 at 0 h, 6 h, 12 h, 24 h, 48 h and 72h under K+-starvation were verified by RT-qPCR. The relative expression level was positively correlated with FPKM value (R² = 0.8419, Additional file 9), suggesting the reliability of gene expression based on RNA-seq analysis.
Expression pattern of HAKs in different tissues at different stages
To study gene functional divergence among the Saccharum species, transcriptome profiles of HAKs between two Saccharum species, S. officinarum and S. spontaneum were analyzed based on RNA-seq at three developmental stage (seedling, premature and mature stage) in five different tissues including 2 leaf (leaf and leaf roll) and 3 stalks (immature, maturing and mature) (Fig. 4). Among the 30 HAK genes analyzed, 18 genes (HAK3/4/5a/5b/12/13/14/15/16a/16b/17/19a/19b/20a/20b/21/22/26) showed very low or undetectable expression level in all examined tissues of the two Saccharum species. HAK1 and HAK2 had different expression pattern in the two Saccharum species, HAK1 had higher expression levels in S. spontaneum than in S. officinarum and the expression level in leaf were higher than that in stems at three different stages, while HAK2 had higher expression levels in S. officinarum than in S. spontaneum, and the expression level in stems were higher than that in leaf. HAK8 mainly expressed in the upper stems, while the expression level in middle and lower stems were very low. HAK9 and HAK10 were observed to have higher expression level in stem than in leaf. HAK18 was expressed in all examined tissues, with higher expression level especially in leaf at seedling stage and mature stem. Noteworthy, HAK27 was highly expressed in leaf at all examined three stage, but the expression level in stem was very low or undetectable.
Expression pattern of HAKs across leaf segment gradient
To further explore functional divergence of HAK genes for photosynthesis in the source tissues, we studied the expression pattern of HAKs in continuously developing leaf segment gradient from S. officinarum and S. spontaneum (Fig. 5). Saccharum leaf was divided into four zone: basal zone (sink tissue), transitional zone (undergoing sink-source transition), maturing zone and mature zone (fully differentiated zone with active photosynthesis) following the method described in maize [30]. Consistent with the expression pattern at different developmental stages, 18 HAK genes (HAK3/4/5a/5b/12/13/14/15/16a/16b/17/19a/19b/20a/20b/21/22/26) showed very low or undetected expression level in all examined leaf segments, suggesting their limited roles in sugar transport (Fig. 5). HAK1 and HAK2 showed higher expression level in basal zone than in other 3 zones. The expression level of HAK7 increased gradually from the base to the tip of the leaf of S. spontaneum, while in S. officinarum, HAK7 displayed higher expression level in the maturing zone than in other 3 zones. The expression level of HAK8 decreased gradually from the base to the tip of leaf both in S. officinarum and S. spontaneum. HAK9 showed different expression pattern in S. spontaneum and S. officinarum. In S. spontaneum, the expression level of HAK9 increased gradually from base zone to maturing zone, then decreased in mature zone; while in S. spontaneum, the expression level of HAK9 decreased from transition zone to maturing zone then increased in the mature zone, and the expression level was much higher in S. officinarum, suggesting the gene functional divergence after the split of this two Saccharum spcies. HAK10 showed higher expression level in the transition zone in S. spontaneum, and higher expression level in the mature zone in S. officinarum. HAK18 displayed higher expression level in maturing zone both in S. spontaneum and S. officinarum, while HAK23 showed higher expression level in basal zone both in the two Saccharum species. HAK25 displayed higher expression level in maturing zone in S. officinarum, but showed higher expression level in basal zone in S. spontaneum.
Expression pattern of HAKs during the circadian rhythms
Acting as an enzyme activator, potassium ions participate in a series of photosynthesis process [31]. To analyze the expression pattern of HAKs during the diurnal cycles, we investigated the transcriptome profiles of the mature leaves in the two Saccharum species at 2 h intervals over 24 h period and at 4 h intervals over an additional 24 h. Consistent with the transcriptome profiles at different developmental stages and in leaf segment gradient, 18 genes (HAK3/4/5a/5b/12/13/14/15/16a/16b/17
/19a/19b/20a/20b/21/22/26) displayed very low or undetectable expression level in the two Saccharum species, further supporting their limited roles in growth and development (Fig. 6). Besides, HAK8 and HAK24 also showed low expression level over two 24 h period. HAK1, HAK2, HAK7, HAK18 and HAK27 showed higher expression level in S. officinarum than in S. spontaneum, while HAK9 and HAK10 displayed higher expression level in S. spontaneum than in S. officinarum. HAK1 and HAK2 were observed to have no diurnal expression pattern in the two saccharum species. HAK7 displayed higher expression level at night than in the daytime and showed the lowest expression level at noon in S. officinarum, but showed no diurnal expression pattern in S. spontaneum; While HAK10 displayed higher expression level at night than in the daytime in S. spontaneum, but showed no diurnal expression pattern in S. officinarum. HAK9 displayed higher expression level at night than in the daytime in both saccharum species. HAK18 and HAK27 displayed higher expression in the morning in the two Saccharum species. These findings suggested the functional divergence of the HAK genes in diurnal rhythms.
Expression pattern of HAKs under K+-deficient stress
To investigate the functional divergence of HAK genes in response to low potassium stress in sugarcane, we studied the expression profiles of HAKs in root from the Saccharum hybrid variety YT55 at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h under low K+ stress (Fig. 7). Among the 30 HAK genes analyzed, 14 genes (HAK3/4/5a/5b/11/13/16a
/16b/19a/19b/20a/22/26/27) showed very low or undetectable expression level before and after exposure to low K+ stress. Noteworthy, HAK1 showed strong induction in root under low K+ condition and reached the highest level at 24 h, then decreased subsequently at 48 h and 72 h. HAK21 was strongly induced after exposure to low K+ stress within 12 hours, but was down-regulated to a low expression level subsequently. HAK20b was down-regulated within 12 hours, then showed up-regulated and to the highest level at 72 h. HAK7, HAK10, HAK18 and HAK24 were down-regulated after exposure to low K+ stress. Some other HAKs, such as HAK12/14/15/25 showed constitutive expression.
Functional complementation validation of SsHAK1 and SsHAK21 in yeast mutant strain R5421
SsHAK1 and SsHAK21 were selected for complementary validation in yeast as they were both induced in response to low K+ stress. The transformed yeast strain carrying only an empty vector pYES2.0 was used as control. There was no observable growth differences between transformed yeast with pYES2.0 and pYES2.0-SsHAK1, pYES2.0-SsHAK21 in SC/-ura medium containing 100 mM KCl (Fig. 8). However, when KCl concentration decreased to 10 mM, the growth of transformed yeast with SsHAK1 and SsHAK21 were better than that of transformed yeast with empty vector. And when KCl concentration decreased to 1mM, the growth of transformed yeast with empty vector was significantly inhibited, while the growth of transformed yeast with SsHAK1 or SsHAK21 were almost unaffected (Fig. 8). This results suggested that both SsHAK1 and SsHAK21 could recover the K+ absorption function in the yeast mutant strain R5421, indicating that they had potassium transporter activity.