Characterization and Expression of KT/HAK/KUP Transporter Family Genes in Willow under Potassium Deficiency, Drought, and Salt Stresses

The K+ transporter/high-affinity K+/K+ uptake (KT/HAK/KUP) transporters dominate K+ uptake, transport, and allocation that play a pivotal role in mineral homeostasis and plant adaptation to adverse abiotic stresses. However, molecular mechanisms towards K+ nutrition in forest trees are extremely rare, especially in willow. In this study, we identified 22 KT/HAK/KUP transporter genes in purple osier willow (designated as SpuHAK1 to SpuHAK22) and examined their expression under K+ deficiency, drought, and salt stress conditions. Both transcriptomic and quantitative real-time PCR (qRT-PCR) analyses demonstrated that SpuHAKs were predominantly expressed in stems, and the expression levels of SpuHAK1, SpuHAK2, SpuHAK3, SpuHAK7, and SpuHAK8 were higher at the whole plant level, whereas SpuHAK9, SpuHAK11, SpuHAK20, and SpuHAK22 were hardly detected in tested tissues. In addition, both K+ deficiency and salt stress decreased the tissue K+ content, while drought increased the tissue K+ content in purple osier plant. Moreover, SpuHAK genes were differentially responsive to K+ deficiency, drought, and salt stresses in roots. K+ deficiency and salt stress mainly enhanced the expression level of responsive SpuHAK genes. Fifteen putative cis-acting regulatory elements, including the stress response, hormone response, circadian regulation, and nutrition and development, were identified in the promoter region of SpuHAK genes. Our findings provide a foundation for further functional characterization of KT/HAK/KUP transporters in forest trees and may be useful for breeding willow rootstocks that utilize potassium more efficiently.

As one of the most popular diploid willow plants, purple osier willow (Salix purpurea) plays an important role in soil and water conservation, shelter forest, and biomass energy, and its genome has been successfully sequenced [40][41][42]. The molecular basis and mechanisms towards K + nutrition and homeostasis in strawberry are essentially unknown. In this study, we identified 22 KT/HAK/KUP family transporter genes (SpuHAKs) in diploid purple osier willow and analyzed their expression profiles under both normal and K + deficiency conditions, which provided gene resources for revealing the mechanism of K + uptake, transport, and distribution in woody trees and provided a theoretical basis for the control of K + fertilizer application in woody trees and efficient genetic manipulation and breeding of willow plants.

Plant Material and Growth
Condition. The 1-year old female purple osier plants (a gift from Nanjing Forest University in Nanjing, China) were obtained by cutting asexual cloning and used throughout this study. Plants were grown in a growth chamber with 12 h light at 25°C followed by 12 h dark at 20°C (with 60% relative humidity). Leaf, stem, root, full blooming flower, young fruit (with a diameter of 0.5 cm in green colour), and mature fruit (with a full size in red color) tissues of Yellow Wonder 5AF8 were collected from the same plant on April 15th, 2018 and frozen immediately in liquid nitrogen for further RNA extraction and quantitative real-time PCR analyses.
Plants were grown in the control conditions (half-strength MS basal medium [43], supplemented with 1 mM K + , 2% sucrose, 1% agar, and 0.5 g L -1 of MES, pH 5.7) in a growth chamber for 2 weeks, and then transferred to the1/2MS solution in plastic containers. The nutrient solution was changed every other day. For K + deficiency treatment, K + was omitted from the 1/2MS medium by adding equal molar Na + to keep the concentration of N stabilization. In drought treatments, plants were exposed to 1/2MS supplemented with 15% (w/v) PEG6000. In salt treatments, plants were suffered to 1/2MS supplemented with 150 mmol·L -1 NaCl. Plants were exposed to K + deficiency treatment for 72 h, and then suffered to qRT-PCR and K + content determination. K + content was measured as described by Song and Su [33]; plant samples were dried and digested using the HNO 3 -HCLO 4 method and subjected to ICP-AES (IRIS Advantage, Thermo Electron, Waltham, MA, USA).

Identification of SpuHAK Genes in Purple Osier Willow.
Genome information of purple osier willow was screened from The Plant Genomics Resource from Phytozome 12 (https:// phytozome.jgi.doe.gov/pz/portal.html). To obtain all the KT/HAK/KUP family genes in purple osier willow, BLAST searches against the genome database were carried out with the full-length of 13 Arabidopsis KT/HAK/KUP protein sequences as references. The amino acid sequences of proteins codified by candidate purple osier willow KT/HAK/KUP genes were verified using the InterProScan 4.8 (http://www.ebi.ac.uk/ Tools/pfa/iprscan/) and Pfam (http://pfam.xfam.org), to confirm the existence of K + transporter (PF02705) domains. Candidate genes without K + transporter domains were removed. Intron numbers were gathered on Phytozome Genomics Resources. Physicochemical properties of KT/HAK/KUP transporters were calculated using the ProtParam tool (http://web.expasy.org/protparam/), including amino acid length, theoretical isoelectric point (PI), molecular weight, instability index, aliphatic index, and grand average of hydropathicity (GRAVY). Subcellular localization prediction was performed on the PSORT Server (https://www.genscript .com/psort.html). Putative cis-acting regulatory elements were predicted on the PlantCARE (http://bioinformatics.psb .ugent.be/webtools/plantcare/html/) online server.
2.6. Statistical Analysis. All data were statistically analyzed using independent samples t test in SPSS 13.0 software (SPSS Chicago, Illinois, USA). Asterisks indicate statistical differences between plants under control and stress treatment ( * P < 0:05, * * P < 0:01, independent-samples t test). Data were compared between plants under control and stress treatment. Details are described in figure legends. Graphs were produced using Origin 8.0 software.

Identification of SpuHAK Genes in Purple Osier Willow.
By BLAST searching of the Phytozome Genomics Resources (purple osier willow), 22 putative strawberry SpuHAK genes were identified, which were entitled as SpuHAK1 to Spu-HAK22. Protein domain verification analyses showed that all of them contain the K + transporter transmembrane domain (PF02705). Except SpuHAK14 and 16 which are still unclear, all the other SpuHAK genes were distributed on 9 distinct chromosomes, in which 6 genes on the 3rd chromosome and 5 genes on the 1st chromosome. All SpuHAK genes possess 5 to 9 introns that varied distinctly in length. Detailed information about these SpuHAK genes, including gene ID, gene location, CDS (coding sequence) length, peptide length, and intron number, is provided in Table 1.
The properties of SpuHAK proteins were also analyzed. The molecular weight of these predicted proteins range from 64.65 to 95.39 kDa correspondingly ( Table 2). The amino acid sequences of s SpuHAK proteins share an overall identity of 51.09% (data not shown). Instability index assays implicated that 15 of the 22 SpuHAK proteins were stable proteins, whereas the remaining 7 members were unstable proteins ( Table 2). According to the value of theoretical PI, 17 of the 22 SpuHAK proteins were alkalescent, and the remaining 5 members were acidic (Table 2). Moreover, the GRAVY index indicated that all of the SpuHAK proteins in purple osier willow are hydrophobic proteins with positive values, and aliphatic index analyses illustrated that all SpuHAK proteins had high values above 100, which supports the predication that SpuHAKs are hydrophilic proteins (Table 2).

Phylogenetic and Protein Motif Analysis of SpuHAK
Proteins. To confirm the evolutionary relationships of Spu-HAK proteins, a Maximum Likelihood (ML) phylogenetic tree was generated based on the alignment of the KT/HAK/KUP amino acid sequences in purple osier willow, Arabidopsis, rice, strawberry, peach, pear, and poplar. All plant KT/HAK/KUP transporters were classified into 4 major groups (I-IV, Figure 1). The SpuHAK proteins were randomly distributed in Groups I-IV, each with 8, 4, 7, and 3 members, respectively ( Figure 1 and Table 2). Purple osier willow and poplar belong to the same family of Salicaceae; all of the 22 SpuHAK members were closely clustered with the corresponding poplar orthologs in the phylogenetic tree, one to one or two to one, respectively ( Figure 1). Moreover, all Roseaceae orthologs from strawberry, peach, and pear have the closest genetic relationship ( Figure 1).

The Cis-Acting Regulatory Elements in the Promoter
Regions of SpuHAK Genes in Purple Osier Willow. Promoter regions of the SpuHAK family genes were obtained from the Phytozome Genome Database via retrieving 2 kb range genomic DNA sequences upstream of the translation start sites of the SpuHAK family genes. Prediction showed that at least 13 kinds of cis-acting regulatory elements were observed in the promoter regions of SpuHAK genes (Table 4), which were involved in abiotic stress response (light, drought inducibility, low temperature, anaerobic induction and defense, and stress), hormone response (salicylic acid, gibberellin, methyl jasmonate, abscisic acid, and auxin), circadian regulation, and nutrition and development (meristem expression and endosperm expression). In particular, two abiotic stress response regulatory elements (light response and anaerobic induction) were detected in all SpuHAK family genes, while the other cis-acting regulatory elements were found in distinct SpuHAK genes with different numbers (Table 4).

Transcriptomic Expression Profiles of SpuHAK in Purple
Osier Willow. To gain insights into the possible functions of SpuHAK genes during the willow growth and development, the transcriptomic data during purple osier willow 3 BioMed Research International   5 BioMed Research International development were obtained from Phytozome online database. In general, the percentages of SpuHAK gene expression in different tissues and organs are 32% in stem, 26% in the apical bud, and 9% in catkin, followed by 7% in predormant bud, 6% in dormant bud, 6% in root, 5% in female receptive, 5% in stem nod, and 4% in full leaf ( Figure 2).
In particular, SpuHAK genes were expressed differently in distinct tissues and organs ( Figure 3). The expression levels of SpuHAK1, SpuHAK2, SpuHAK3, SpuHAK7, and SpuHAK8 were higher at the whole plant level, whereas SpuHAK9, SpuHAK11, SpuHAK20, and SpuHAK22 were extremely low in tested tissues (Figure 3).

qRT-PCR Determination of SpuHAK Genes in Purple
Osier. We further performed qRT-PCR to determine the expression profiles of SpuHAK genes in different tissues of 1-year old female purple osier. Results showed that SpuHAK genes were unevenly expressed in the tested organs, including leaves, stems, and roots ( Figure 4). The expression levels of SpuHAK1, SpuHAK2, SpuHAK3, SpuHAK5, SpuHAK6, SpuHAK7, and SpuHAK8 was higher than that of the other genes, at the whole plant level, whereas SpuHAK9, SpuHAK11, SpuHAK15, SpuHAK20, and SpuHAK22 were extremely low in tested tissues (Figure 4), which was exactly consistent with that of the transcriptomic expression profiles (Figure 3). In details, 14 of the 22 SpuHAK genes (i.e., SpuHAK1, SpuHAK2, SpuHAK3, SpuHAK5, SpuHAK6, SpuHAK7, SpuHAK10, SpuHAK12, SpuHAK13, SpuKU14, SpuHAK16, SpuHAK17, SpuHAK18, and SpuHAK19) were mainly expressed in stems, more than in the other tissues, and SpuHAK4 and SpuHAK21 were mainly expressed in roots, whereas SpuHAK8 was evenly expressed throughout the whole plant ( Figure 4).

SpuHAK Gene Expression under K + Deficiency, Drought, and Salt
Conditions. The transcript level changes after stress treatment is an important factor for a transporter functioning as a high-affinity K + uptake in the root epidermis [46]. To investigate the role of SpuHAK in uptaking and maintaining K + homeostasis in willow, especially under adverse conditions, we analyzed the expression profiles of SpuHAK genes in roots of purple osier under K + deficiency, drought, and salt stresses. Results showed that the expression of SpuHAKs was differentially affected by K + deficiency, drought, and salt stresses in roots ( Figure 5). In particular, 13 out of 22 SpuHAKs were responsive to K + deficiency, in which 10 genes (SpuHAK3, SpuHAK4, SpuHAK9, SpuHAK12, SpuKU13, SpuHAK14, SpuHAK16, SpuHAK17, SpuHAK19, and SpuHAK21) were significantly enhanced, and the remaining 3 genes (SpuHAK1, SpuHAK6, and SpuHAK18) were dramatically reduced ( Figure 5). Expression of 6 genes were affected by drought stress, in which SpuHAK5, SpuHAK7, and SpuHAK21 were increased and SpuHAK3, SpuHAK4, and SpuHAK12 were decreased ( Figure 5). Expression of 8 genes was altered by salt stress, which were all significantly induced except for SpuHAK1 that was reduced ( Figure 5).

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Notably, SpuHAK8 was evenly expressed in tested tissues of purple osier at a moderate level and had no response to any treatment. Expression of SpuHAK15 and SpuHAK20 were extremely low in purple osier and also had no effect to any treatment ( Figure 5).
In addition, the tissue K + accumulation was higher in shoots than in roots, and the highest was observed in stems ( Figure 6). Both K + deficiency and salt stresses decreased the tissue K + concentration, while drought stress strengthened the tissue K + concentration, in all tested tissues ( Figure 6).

Discussion
KT/HAK/KUP family transporters play a major role in catalyzing K + acquisition and uptake and maintaining plant cation homeostasis, which further contributes to plant growth and development [1,5,13,26]. Genome-wide analysis of the KT/HAK/KUP gene family has been reported in various plants [1,12]. However, the molecular mechanism of K + absorption in willow has not been mentioned yet.
Purple osier grows fast and has strong adaptability that plays important roles in water and soil conservation, shelter forest, and bioenergy [47][48][49][50]. In this present study, we identified and characterized 22 KT/HAK/KUP transporters in purple osier willow. In terms of the woody plants, the number of KT/HAK/KUP transporters genes in purple osier willow is more than that in peach (16) [26], but less than that in poplar (31) [10]. These findings may reflect the fact that purple osier willow has a larger genome of 392 Mbp [40,41], more than that of peach (230 Mbp) [51], much shorter than that of poplar (520 Mbp) [52]. Although a similar number of genes is similar to that in pear (21) [37,38], the genome of purple osier willow was much shorter than that of pear (512 Mbp) [53]. Moreover, the phylogenetic tree of KT/HAK/KUP transporters in purple osier willow strictly followed the same distribution into four groups, which is similar to that in rice [30], poplar [10], and pear [37,38]. In particular, all of KT/HAK/KUP transporters in purple osier were closely clustered with corresponding orthologs of poplar (Figure 1), implying that KT/HAK/KUP family transporters may be highly conserved among Salicaceae plants.
Eukaryotic genes contain exons and introns, which is one of the characteristics of the former to distinguish prokaryotes. Introns play an important role in alternative splicing, and a gene can produce many different proteins. Introns being the essential entities of eukaryotic gene families during the evolution of multiple gene families. In this present study, the intron No. was quite different among SpuHAK family genes, and SpuHAK5 and SpuHAK10 possess the most (9) introns, while SpuHAK13 possesses the least (5) introns that varied distinctly in length. These findings may reflect the distinct roles in catalyzing K + acquisition and uptake and maintaining plant cation homeostasis, which still lacks molecular evidences. In addition, the KT/HAK/KUP family transporters have extensive and delicate localizations in plant cells that may share diverse functions or have common and recent evolutionary origins [30,32,37]. In purple osier willow, SpuHAK transporters were predicted to mainly localize in the plasma membrane and organelle membranes, including mitochondrion and vacuole. Genomic transcriptomic data indicated that SpuHAK genes are differently expressed in distinct tissues or organs of purple osier (Figures 2 and 3). Further, qRT-PCR analysis demonstrated that SpuHAK genes were unevenly expressed in tested organs, including leaves, stems, and roots ( Figure 4). All these findings indicate that the KT/HAK/KUP family members have extensive and delicate localization and expression patterns in purple, and each gene may have a unique role during distinct biological or developmental processes. Furthermore, it is worth mentioning that a majority of SpuHAK genes were highly expressed in stems, more than that in leaves and roots, which was in accordance with the expression profiles of transcriptomic data, implying that these genes are prone to be functional K + transporters, especially during maintaining K + accumulation and homeostasis in willow stems. These findings again support the proposition that K + favorably accumulates in stems that will be further allocated to different parts of the aboveground [26,33,38].
In plants, the root system provides sufficient surface area, where K + nutrients need to be transported through the root surface via K + transporters [24,33]. We found that K + deficiency significantly reduced tissue K + accumulation, which was similar to the previous reports [15,21,22,26,33,38]. When suffered of adverse abiotic stresses, plants have to urgently adjust their nutritional status or enhance the internal metabolic systems to maintain basic growth [22,23,33,38]. Undoubtedly, the ubiquitous presence of KT/HAK/KUP transporters in plants implies that they play critical roles in acquiring nutrients and improving plant tolerance to adverse environmental conditions, including K + deficiency, drought, and salt [15][16][17][18][19][20][21]33]. Cis-acting regulatory elements, especially of abiotic stress response elements, can control promoter efficiency by combining with key elements in the promoter region and then regulate target gene   BioMed Research International expression [22,30,32]. In this work, at least 5 kinds of abiotic stress-responsive elements, including drought inducibility and defense and stress, were found in SpuHAK genes (Table 4). Further qRT-PCR analyses showed that SpuHAK genes respond differently to K + deficiency, drought, and salt stresses, implying that they may contribute to stress resistance favorably in willow plants. In details, 13 out of 22 SpuHAK genes were responsive to K + deficiency, and 10 genes (in which 5 genes of SpuHAK3, SpuHAK4, SpuHAK9, SpuHAK14, and SpuHAK16 belong to Group I) were significantly upregulated, indicating that these genes are prone to be active in K + uptake or transport in willow roots to maintain "optimal utilization" of K + under limited K + conditions. Again, these findings support the proposition that Group I members exhibit a more specialized K + transporter function during K + deficiency condition [12,31,38,54]. Drought treatment strengthened the K + content especially in aboveground parts, indicating that the water status makes a great contribution to the internal K + concentration. Simultaneously, drought stress stimulated the expressions of 3 genes (SpuHAK5, SpuHAK7, and SpuHAK21), in which drought inducibility regulatory elements were detected, but reduced the expressions of 3 SpuHAK genes, implying that these SpuHAK genes may be involved in adaptation to drought stress in quite a complicate way. When plant faced salinity stress, the radius of Na + and K + ions is close and a great amount of Na + may compete with K + that could be preferentially absorbed. Salt treatment greatly decreased the K + content throughout the whole plant and upregulated 7 out of 8 salt-responsive SpuHAK genes ( Figure 5), suggesting that these SpuHAK transporters may play crucial roles in mobilizing the maximum root uptake and accumulation of external K + to maintain "maximal uptake" of external K + for vital activities under salt stress. Moderate K + transport has to be accumulated and maintained in plants to cope with adverse abiotic stresses by regulating the expression of specific genes [37,55]. Nonetheless, SpuHAK transporters that respond to such abiotic stresses are likely to function in K + transport, which is indispensible for purple osier willow adaptation to undesired stresses.   Relative expression Leaf Stem Root Figure 4: Quantitative real time-PCR analysis of SpuHAKs in purple osier willow. 1-year old female purple osier plants were obtained by cutting asexual cloning. Plants were grown in 1/2MS liquid medium in a growth chamber with 12 h light at 25°C followed by 12 h dark at 20°C (with 60% relative humidity). Leaf, stem, and root tissues of Yellow Wonder 5AF8 were collected from the same plant on April 15th, 2018, and frozen immediately in liquid nitrogen for RNA extraction and quantitative real-time PCR.

Conclusions
22 KT/HAK/KUP transporter genes were identified and characterized which were predominantly expressed in stems, in purple osier willow. Both K + deficiency and salt stress decreased the tissue K + content, while drought increased the tissue K + content in the purple osier plant. SpuHAK genes were unevenly expressed in the tested organs, and K + deficiency and salt stress mainly enhanced the expression of responsive SpuHAK genes. Physiological and molecular function determination of SpuHAK genes will be further studied.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
We declare that we do not have any commercial or associative interest that represents a conflict of interest with the work submitted. SpuHAK22 K + deficiency Figure 5: Expression changes of SpuHAKs under K + deficiency, drought, and salt stress treatments. 1-year-old purple osier plants were subjected to K + deficiency, drought (15% (w/v) PEG6000), and salt (150 mmol·L -1 NaCl) treatments for 72 h before examination. Each SpuHAK gene was analyzed in leaves, stems, and roots. The relative expression level of genes was presented after normalization to the internal control from three independent biological repeats. 1-year-old purple osier plants were subjected to K + deficiency, drought (15% (w/v) PEG6000), and salt (150 mmol·L -1 NaCl) treatments for 72 h, and then digested using the HNO 3 -HCLO 4 method and determined by ICP-AES. Asterisks indicate statistical differences between plants under control and stress treatment ( * * P < 0:01, independent-samples t test).

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