Genome-wide systematic characterization of HAK/KUP/KT gene family and their expression profiles during plant growth and in response to low K+ stress in Saccharum

Background: Plant genomes contain large number of HAK/KUP/KT transporters, and they play important roles in potassium uptake and translocation, osmotic potential regulation, salt tolerance, root morphogenesis and plant development. Potassium deficiency in soil of sugarcane main planting area is serious. However, the HAK/KUP/KT gene family remains to be characterized in sugarcane (Saccharum). Results: In this study, 30 HAK/KUP/KT genes were identified from Saccharum spontaneum. Phylogenetics, duplication events, gene structure and expression pattern were analyzed. Phylogenetic analysis of HAK/KUP/KT genes from 15 representative plants showed that this gene family were divided into four groups (clade I-IV). Both ancient whole-genome duplication (WGD) and recent gene duplication contributed to the expansion of HAK/KUP/KT gene family. Nonsynonymous to synonymous substitution ratio (Ka/Ks) analysis showed that purifying selection was the main force to drive the evolution of HAK/KUP/KT genes. The divergence time of HAK/KUP/KT gene family was estimated to range from 134.8 to 233.7 Mya based on Ks analysis, suggesting that it is an ancient gene family in plants. Gene structure analysis showed that HAK/KUP/KT genes was accompanied by intron gain/loss in the process of evolution. RNA-seq data analysis demonstrated that HAK/KUP/KT genes from clade II and III mainly displayed constitutive expression in various tissues, while most genes from clade I and IV had no or very low expression in the tested tissues at different developmental stages. SsHAK1 and SsHAK21 displayed upregulated expression in response to low K+ stress. Yeast functional complementation analysis suggested that SsHAK1 and SsHAK21 could rescue K+ uptake ability in the yeast mutant. Conclusions: This study provided insight into the gene evolutionary history of HAK/KUP/KT genes. HAK7/9/18 were mainly expressed in the high photosynthetic zone and mature zone of stem. Moreover HAK7/9/18/25 were regulated by sunlight. SsHAK1 and SsHAK21 played important mediating potassium acquisition under limited K+ supply. Our results provide valuable information and key candidate genes for further study on the function of HAK/KUP/KT in monoclonal subsequent yeast Competent cells of transformed. Yeast strains with empty vector and target genes isolated, then used for gradient dilution and inoculated in the SC/-ura medium 100 mM, 5 and 0 mM KCl, observing results after 3-5 days culture at 30

plant species was uploaded as supplementary data (Additional file 4). 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). Twentynine 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. (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 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].

Expression pattern of HAKs during the circadian rhythms
Acting as an enzyme activator, potassium ions participate in a series of photosynthesis process [31]. 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).

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.

Discussion
The HAK/KUP/KT family of potassium transporters have been widely reported to be associated with K + transport across membranes in plants. Plant genome contains large number of HAK/KUP/KT transporters whose function involve the K + absorption and transport, salt tolerance, osmotic potential regulation and in controlling root morphology and shoot phenotyping [7]. However, genome-wide analysis of the HAK/KUP/KT gene family have not been conducted in Saccharum due to its complex genetic background. Recently released S. spontaneum genome allowed us to indentified 30 HAK genes from S.
spontaneum. Besides, 248 HAK genes from other 13 representative plant species and an outgroup were used to construct phylogenetic tree and study the evolution of HAK genes in Saccharum. Furthermore, expression analysis based on RNA-seq revealed spatiotemporal expression and functional divergence in HAK family, which provides valuable information and robust candidate genes for future functional analysis.

Evolution of HAK gene family in Saccharum and representative angiosperms
WGD or polyploidization, gene loss and diploidization are considered to be important evolutionary forces in plants [32,33]. Angiosperms, pan-core eudicots and monocots were originated from the ε, γ and σ WGD event, which have been revealed by rigorous phylogenomic approach [33]. Recent study showed that pineapple had one fewer ancient ρ WGD event than other gramineous plants [34]. in angiosperms ( about 130 Mya) and after ε GWD event (about 220 Mya) [33].
The HAKs number in four clade varied greatly (from 29 to 98, Fig. 3 Arabidopsis, which may be due to the gene functional redundancy of the HAK family.
HAK18 was retained in all monocotyledonous and dicotyledonous plants, showing its functional constraint for the HAK family and the expression profiles analysis of HAK18 also confirmed this .
In clade II and clade III, SsHAK2 and SsHAK7 were retained from the ε GWD event, and in dicotyledons these two orthologous genes were lost. SsHAK3 and SsHAK13 originated after A. trichopoda had evolved separately from other angiosperms. SsHAK8, SsHAK9 and SsHAK10 were assumed to be retained from the ε GWD event; SsHAK11, SsHAK12, SsHAK15, SsHAK24 and SsHAK25 were retained from σ GWD event since only monocotyledons contained these genes. SsHAK14 and SsHAK23 were assumed to be retained from the ε GWD event, but HAK14 was probably lost in dicotyledons. Clade IV contained the least number of HAKs, SsHAK4 and SsHAK17 were originated before the split of monocotyledons and dicotyledons and after the split of A. trichopoda from angiosperms.
The LCA of SsHAK26 originated after the split of the Gramineae and pineapple.
HAK gene family in plant showed a less conserved exon/intron structure. The exon number in Saccharrum ranged from 2 to 12 (Fig. 1, Additional file 7), and the variation range in Saccharrum was larger than that in rice [17], maize [19] and wheat [36]. Three types of mechanism: exon/intron gain/loss, exonization/pseudo-exonization and insertion/deletion mainly lead to the exon-intron structure difference in paralogous or orthologous genes [37]. Although the gene structure of SsHAKs changed greatly, the protein size was relatively conserved, suggesting that exon-intron structure difference in SsHAKs was mainly caused by intron gain/loss. Clade I and clade IV belong to the older HAK family in Saccharrum, so the HAKs in these two clades were speculated to have more intron gain/loss events based on the 'introns-early' theory during the lengthy evolutionary process [38,39]. The results in this study also support this view since the variation of exon number in clade I and clade IV was much greater than that in clade II and clade III.

Gene expression and functional divergence of HAKs in Saccharrum
Transcriptional regulation of K + transporters is a common mechanism for plant species responding to low K + stress [8], and expression pattern analysis can provide insight into the potential functions of HAK gene family. In this study, we found that most HAK genes in clade I and clade IV showed low or undetectable expression levels across all examined samples. While most HAK genes in clade II and clade III were strongly expressed in all tested tissues. These results were consistent with the results of previous studies on HAK genes in rice [17], Arabidopsis [25] and wheat [36]. Five OsHAK genes (OsHAK2/10/15/23/25) from clade II and III were expressed in all examined tissues of three different genotype [17]. In Arabidopsis, 12 out of 13 HAK/KUP/KT genes were from clade II and III, most of them were expressed in roots, leaves, siliques and flowers [25]. Similarly, most TaHAKs in wheat belonging to clade II and III were constitutively expressed in all tissues [36].
Low K + stress tends to induce the upregulated expression of K + transporter genes [40].
previous studies demonstrated that the expression of OsHAK1 in rice [20], TaHAK1 in wheat [36] and PbrHAK1 in pear [41] were induced by K + starvation. In this study, the expression level of SsHAK1 increased rapidly under low K + stress, and this result is in good agreement with previous studies. Noteworthy, SsHAK21 was upregulated greatly after a short period of K + starvation treatment and then rapidly downregulated (transient activation), indicating that SsHAK21 was involved in the low K + stress response in sugarcane. And similar results were found in rice that OsHAK21 functions in the maintenance of ion homeostasis and tolerance to salt stress [42]. SsHAK1, SsHAK17 and SsHAK21 displayed upregulated expression, suggesting that they may play important role in maintaining normal growth and mediating potassium acquisition under K + deficiency. In addition, nearly half the SsHAK genes were not expressed or had very low levels of expression in all tested tissues, at all stages or even under low K + stress, this may be caused by the gene functional redundancy due to WGD events in sugarcane.
Root system acquire K + from soil solution then K + is transported among compartment within cells and from root to shoot. A schematic model was proposed based on the expression profiles of the 30 SsHAK genes to illustrate the spatial and temporal gene expression in plant tissues and root hair cell of sugarcane (Fig. 8). HAK7/9/18 were mainly expressed in the tissues of maturing and mature stem and leave, indicating their important roles in K + transport in these tissues. HAK7/9/18/25 also showed circadian rhythm expression pattern, suggesting these genes were regulated by sunlight. Low K + stress induced up-regulated transcriptional expression of HAK genes. In Arabidopsis, transcription factor such as DDF2, JLO, ARF2, RAP2.11, TFII_A, bHLH121 directly bind the promoter of AtHAK5 to induce its expression and increase tolerance to low K + and salt stress [26]. In this study, the expression of HAK1 and HAK21 were greatly up-regulated, which may also be positively regulated by the transcription factors (TFs) such as DDF2 and JLO and further experiment like yeast one-hybrid can be used to screen the TFs. AtHAK5 and its homologs from pepper and tomato can be activated by the CIPK23 (CBL-interacting protein kinase 23)/CBL1 (calcineurin B-like protein) complex [27]. In rice, OsHAK1/19/20 can be phosphorylated by a receptor like protein kinase, RUPO (ruptured pollen tube) [43].
In this study, CBL-CIPK complex and receptor-like kinase RUPO may also act as a regulator of high-affinity potassium transporters, such as HAK1 via phosphorylationdependent interaction.

Conclusions
In this study, 30 HAK (high affinity K + transporter) genes were identified through comparative genomics from sugarcane. Evolution analysis revealed that both ancient and SsHAK21 mediated K + transport under low K + stress. Altogether, these results provides valuable information and robust candidate genes for future functional analysis for genetic improvement of potassium utilization efficiency in sugarcane. March, 2017 in Fuzhou were 6:25 a.m. and 6:05 p.m. respectively. The tissues collection followed the approach as previously described [34]. respectively, and stored in liquid nitrogen for total RNA isolation.

Homology search analysis
According to previous reports, the protein sequences of 13, 27 and 27 HAK/KUP/ KT gene family identified in Arabidopsis thaliana, Oryza sativa and Zea mays [17][18][19] were obtained from Phytozome V12.1 (https://phytozome.jgi.doe.gov/pz/portal.html). With these protein sequences as queries, putative members of HAK/KUP/ KT gene family were cDNA from RNA of YT55 after 12h of low potassium stress treatment was used as template   The expression pattern of HAK/KUP/KT genes based on FPKM across leaf gradients in S. officinarum and S. spontaneum.

Figure 6
The expression pattern of HAK/KUP/KT genes based on FPKM during the diurnal cycles in S. officinarum and S. spontaneum.