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. 2019 Nov 5;182(1):584–596. doi: 10.1104/pp.19.00882

The HKT Transporter HvHKT1;5 Negatively Regulates Salt Tolerance1

Lu Huang 1,2, Liuhui Kuang 1,2, Liyuan Wu 1,✉, Qiufang Shen 1, Yong Han 1, Lixi Jiang 1, Dezhi Wu 1,3,4, Guoping Zhang 1
PMCID: PMC6945855  PMID: 31690708

A high-affinity K+ transporter (HKT) facilitates Na+ loading from roots to shoots in barley and its lower expression can enhance salt tolerance, which shows a potential way to improve salt tolerance of barley as well as other cereal crops.

Abstract

Maintaining low intracellular Na+ concentrations is an essential physiological strategy in salt stress tolerance in most cereal crops. Here, we characterized a member of the high-affinity K+ transporter (HKT) family in barley (Hordeum vulgare), HvHKT1;5, which negatively regulates salt tolerance and has different functions from its homology in other cereal crops. HvHKT1;5 encodes a plasma membrane protein localized to root stele cells, particularly in xylem parenchyma cells adjacent to the xylem vessels. Its expression was highly induced by salt stress. Heterogenous expression of HvHKT1;5 in Xenopus laevis oocytes showed that HvHKT1;5 was permeable to Na+, but not to K+, although its Na+ transport activity was inhibited by external K+. HvHKT1;5 knockdown barley lines showed improved salt tolerance, a dramatic decrease in Na+ translocation from roots to shoots, and increases in K+/Na+ when compared with wild-type plants under salt stress. The negative regulation of HvHKT1;5 in salt tolerance distinguishes it from other HKT1;5 members, indicating that barley has a distinct Na+ transport system. These findings provide a deeper understanding of the functions of HKT family members and the regulation of HvHKT1;5 in improving salt tolerance of barley.

Soil salinization caused by either natural or human activities is a great threat to sustainable agricultural production in the world (Ondrasek et al., 2015). Na+ is the most widespread salt in the environment and a dominant toxic ion in salinity soils. Excess Na+ in plant cells causes ionic toxicity and other physiological damage such as competition with other mineral nutrients (Halfter et al., 2000; Lan et al., 2010; Shabala et al., 2010; Shen et al., 2016; Zhu et al., 2017). To deal with excess Na+, plants have evolved a series of detoxification strategies, including Na+ exclusion and sequestration (Munns and Tester, 2008). Several processes contribute to these mechanisms, including high-affinity K+ transporters (HKTs) and Na+/H+ exchangers, while other transporters such as salt overly sensitive1 also play important roles for salt tolerance in plants (Qiu et al., 2002; Pardo et al., 2006; Munns et al., 2012).

The first member of the HKT gene family was cloned in wheat (Triticum aestivum; Schachtman et al., 1992) and the family has since attracted attention because of its permeability for Na+ (Schachtman and Schroeder, 1994; Rubio et al., 1995, 1999). As a result, many HKT genes have been identified, which has also revealed substantial divergence in function. It is well known that a Ser/Gly residue in the first selectivity pore-forming region (P-loop) is crucial for cation selectivity and subfamily features: the HKT subfamily 1 has a Ser residue (SGGG-type) in the first P-loop region, which is considered to be associated with specific Na+ transport. By contrast, the HKT subfamily 2 has a Gly residue in that site (GGGG-type), which is permeable to both K+ and Na+ (Mäser et al., 2002b; Platten et al., 2006; Rodrígueznavarro and Rubio, 2006; Horie et al., 2009).

At present, many members of the HKT subfamily 1 have been functionally characterized. AtHKT1;1 was shown to mediate large inward Na+ currents in Xenopus laevis oocytes and Na+ hypersensitivity in yeast (Saccharomyces cerevisiae; Uozumi et al., 2000; Kato et al., 2001). It could serve in retrieval of Na+ from xylem, resulting in a decrease in shoot Na+ accumulation and enhancement of salt tolerance in Arabidopsis (Mäser et al., 2002a; Davenport et al., 2007; Møller et al., 2009). In rice (Oryza sativa), OsHKT1;5 encoding a Na+ selective transporter functions in K+/Na+ homeostasis under salt stress (Ren et al., 2005). Kobayashi et al. (2017) used two independent transferred DNA insertion mutants of OsHKT1;5 to reveal its physiological roles in mediating Na+ exclusion in vasculature to protect leaf blades and reproductive tissues under salt stress. In succession, the functions of OsHKT1;1, OsHKT1;3, and OsHKT1;4 have been reported as Na+ transporters in rice (Jabnoune et al., 2009; Wang et al., 2015; Suzuki et al., 2016). Moreover, a major QTL, Nax2, was identified in Einkorn wheat (Triticum monococcum), and TmHKT1;5-A was map-based cloned in the region of Nax2, which reduced Na+ accumulation in leaves (Byrt et al., 2007; James et al., 2011; Munns et al., 2012). Furthermore, it was demonstrated that TaHKT1;5-d from bread wheat (T. aestivum) is a major gene at the Kna1 locus, which plays a role in leaf Na+ exclusion and salt tolerance (Gorham et al., 1990; Byrt et al., 2007, 2014).

Among cereal crops, barley (Hordeum vulgare) is the most salt-tolerant species, and is widely used for salt-tolerance studies (Munns and Tester, 2008). Recently, a genome-wide association study on 2,671 barley genotypes showed that SNPs from HvHKT1;5 were associated with salt tolerance (Hazzouri et al., 2018). However, the exact function of HvHKT1;5 remains to be elucidated. In this study, we cloned and characterized a member of the HKT subfamily 1 transporter in barley, HvHKT1;5. The transport properties of HvHKT1;5 were analyzed in X. laevis oocytes, and subcellular and cellular localization of HvHKT1;5 protein were performed in vitro in onion (Allium cepa) epidermis cells and in vivo in barley roots, respectively. HvHKT1;5 knockdown (RNA interference [RNAi]) transgenic lines were obtained to reveal the gene function. The results show that HvHKT1;5 has a distinct pattern in cellular localization and function in salt tolerance from other reported HKT1;5 transporters, indicating that barley has a distinct Na+ transport system and genetic mechanism for salt tolerance.

RESULTS

Sequence and Phylogenetic Analysis of HvHKT1;5

The genomic sequence of HvHKT1;5 contains 1,722 bp with two introns and three exons, and the full length of its complementary DNA (cDNA) is 1,533 bp, encoding a polypeptide of 510 amino acids (Fig. 1; Supplemental Fig. S1A). Phylogenetic analysis shows that HvHKT1;5 has 39.4% to 85.7% amino acids identity to 28 members of HKT subfamily 1 transporters from 13 plant species (Fig. 1A), with the highest sequence similarity to TaHKT1;5-B2. Based on Ser-76 in the first P-loop (PA), HvHKT1;5 is likely to be a sodium transporter. In addition, three unique amino acid residues (Gln, Ile, and Met) are present in the PA and PB regions of HvHKT1;5 (Fig. 1B), which may be closely associated with its function.

Figure 1.

Figure 1.

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Phylogenetic analysis of HvHKT1;5. A, Phylogenetic tree of HKT subfamily 1 transporters. Accession numbers and species for all sequences are listed in Supplemental Table S2. Scale bar = 0.05 substitutions per site. B, Alignment of HKT1;5 amino acid sequences in rice, barley, and wheat. The conserved Ser/Gly residues in the PA-d region (Mäser et al., 2002b) are indicated by the arrowhead. The amino acid residues specific for HvHKT1;5 are highlighted with boxes.

HvHKT1;5 Is Mainly Expressed in Barley Roots

Reverse transcription quantitative PCR (RT-qPCR) analysis showed that HvHKT1;5 was mainly expressed in root rather than in stem and leaf (Fig. 2A). Spatial expression showed that the expression of HvHKT1;5 was higher in the root hair zone (10–20 mm) than in root tips (0–5 mm) and the root elongation zone (5–10 mm; Fig. 2B). The transcript level of HvHKT1;5 was significantly higher under salt stress than under normal conditions (control). The highest expression level was found when plants were exposed to 300-mm NaCl (Fig. 2C). In a time-course experiment, the expression of HvHKT1;5 increased with exposure time under 200-mm NaCl and reached a peak after level after 3 weeks (Fig. 2D). However, under the control condition, the expression level of HvHKT1;5 in barley roots showed no significant change over time (Fig. 2D). These results indicate that the expression of HvHKT1;5 is tissue-, dose-, and time course-dependent.

Figure 2.

Figure 2.

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Expression pattern of HvHKT1;5. A, Absolute RT-qPCR for HvHKT1;5 in the root, stem, leaf sheath, and leaf blade tissues from 4-week–old seedlings of barley ‘Golden Promise’ grown under normal conditions. B, Absolute RT-qPCR for HvHKT1;5 in the root tips (0–5 mm) and basal root parts (5–10 or 10–20 mm) from 4-week–old seedlings of barley ‘Golden Promise’ grown under normal conditions. C, RT-qPCR for HvHKT1;5 in roots. Three-week–old seedlings of barley ‘Golden Promise’ were prepared by hydroponic cultured and treated by 0, 100, 200, 300, 400, and 500 mm of NaCl for 2 weeks. D, RT-qPCR for HvHKT1;5 in roots. Three-week–old seedlings of barley ‘Golden Promise’ were prepared by hydroponic culture under normal condition and treated by 200 mm of NaCl for 0, 1, 2, 3, and 4 weeks. Data are means ± se (n = 3). Different letters indicate a significant difference (P < 0.05) using Tukey’s test after a one-way ANOVA.

HvHKT1;5 Localizes at the Plasma Membrane of Root Stele Cells

To determine the subcellular localization of HvHKT1;5, a construct containing the coding region of HvHKT1;5 and Superfolder GFP (sGFP) was transiently coexpressed with red fluorescence protein (RFP) or plasma membrane-localized red fluorescence protein (PM-RFP; Nelson et al., 2007) in onion epidermis cells (Fig. 3). When sGFP was expressed together with the RFP marker, fluorescence signals from GFP and RFP were detected across all cells including membranes, nucleus, and other organelles (Fig. 3A). However, the signals from the HvHKT1;5-sGFP fusion were mainly observed at the plasma membrane (Fig. 3B). Furthermore, the signals from the coexpressed HvHKT1;5::sGFP fusion and PM-RFP were only observed at the plasma membrane in onion epidermis cells (Fig. 3C). Relative fluorescence of GFP and RFP signals around the cell periphery also indicated that the signal from HvHKT1;5::sGFP was similar to the signal from the PM-RFP marker (Fig. 3, D–F). After cell plasmolysis, the signal from HvHKT1;5::sGFP was not detected at the cell wall, but was detected at the plasma membrane in onion epidermis cells (Supplemental Fig. S2). Therefore, these results indicate that HvHKT1;5 is a plasma-membrane–localized protein.

Figure 3.

Figure 3.

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Subcellular localization of HvHKT1;5. A to C, Confocal images of onion epidermis cells coexpressing sGFP along with RFP (A); HvHKT1;5-sGFP fusion along with RFP (whole-cell-localized red fluorescence protein; B), or PM-RFP (constructed based on AtPIP2A; C). Microscopic image channels from left to right: GFP-channel, RFP-channel, and merged images. Scale bars = 100 μm. D–F, Relative fluorescence of GFP and RFP signals around the cell periphery. Position indicated by the dotted lines from the left.

The RT-qPCR experiment proved that HvHKT1;5 was mainly expressed in barley roots (Fig. 2A). Therefore, cellular localization of HvHKT1;5 was investigated in barley roots using in situ PCR and immunostaining methods (Figs. 4 and 5). Compared with the negative control without reverse transcription, the HvHKT1;5 transcript showed staining signal predominantly in the root stele sections, particularly xylem parenchyma and pericycle cells adjacent to xylem vessels (Fig. 4, A and E). The signal was also detected at the epidermis, but was much weaker than that at the stele (Fig. 4D). For immunostaining experiments, we used an antibody against GFP to stain the root cells of the wild type and transgenic plants carrying proHvHKT1;5-GFP. Compared with the wild type, the transgenic plants showed staining signal predominantly in root stele cells (Fig. 5, A–I), while signal was not detected at the epidermis (Fig. 5, J–L). Therefore, this indicates that HvHKT1;5 may be mainly involved in ion loading from roots to shoots via xylem.

Figure 4.

Figure 4.

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In situ PCR of HvHKT1;5 in barley roots. A to C, In situ PCR in barley root cross sections (0–2 mm and 5–10 mm from root tip). A, In situ PCR with HvHKT1;5 primers. B, In situ PCR with HvHKT1;5 primers but without the reverse transcription (RT) step (negative control). C, In situ PCR with 18S rRNA primers (positive control). D, High-magnification image of dotted part in (A). E, In situ PCR with HvHKT1;5 primers in barley root tip (0–5 mm) sections lengthwise. All samples were stained with BM-Purple (Roche). Blue color indicates the presence of digoxigenin-labeled cDNA, and brown indicates the absence of the amplified cDNA target. ep, epidermis; c, cortex; en, endodermis; p, pericycle; x, xylem; xp, xylem parenchyma. Scale bars = 100 μm.

Figure 5.

Figure 5.

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Cellular localization of HvHKT1;5. A to C, Immunostaining in barley root cross sections (10 mm from root tip) of the wild type (cv Golden Promise) using the anti-GFP antibody. D to F, Immunostaining in barley root cross sections of the proHvHKT1;5-GFP transgenic plants. G to L, High-magnification image of dotted center parts in (A–C). Seven-d–old seedlings were used. Red fluorescence shows presence of HvHKT1;5 and blue fluorescence is emitted by autofluorescence of cell wall and the counterstain 4′,6-diamidino-2-phenylindole. Cell types are indicated in the enlarged representation with merged signals. en, endodermis; p, pericycle; x, xylem; xp, xylem parenchyma. Scale bars = 100 μm.

HvHKT1;5 Shows Na+ Transport Activity in X. laevis Oocytes

HvHKT1;5 cRNA or water was injected into X. laevis oocytes, which were then assayed by two-electrode voltage clamp (Fig. 6; Supplemental Fig. S3). Water-injected oocytes showed no significant inward or outward currents when clamped in any bath solutions (Supplemental Fig. S3). HvHKT1;5 cRNA-injected oocytes showed large inward currents ∼−16 μA at −140 mV in the presence of 100-mm Na+ in the bath solution, with a positive reversal potential ∼+14 mV (Fig. 6A). Replacing external Na+ with 100 mm of other monovalent cations, including K+, Li+, Rb+, Cs+, and Tris+ resulted in no significant inward currents and very negative reversal potentials (∼−138 mV; Fig. 6A). Elevating external Na+ concentration gradually increased the inward currents of HvHKT1;5 cRNA-injected oocytes. This Na+ concentration-dependent increase of currents was not observed when the external ion was replaced by K+ (Fig. 6B). Interestingly, there was an inhibition of Na+ transport through HvHKT1;5 when the external K+ concentration was 10 mm (Fig. 6, C and D). A significant reduction was found in the magnitude of both inward and outward currents in the presence of 10-mm external K+. Therefore, it may be suggested that HvHKT1;5 is a Na+-selective transporter and is regulated by external K+.

Figure 6.

Figure 6.

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Transport activities of HvHKT1;5 in X. laevis oocytes. A to D, Current-voltage curves of HvHKT1;5-cRNA-injected oocytes in the presence of a series of 100-mm monovalent cations: K+, Li+, Na+, Rb+, Cs+, or Tris+ (A); 1, 10, 30 mm of Na+ or 1, 10, 30 mm of K+ (B); 1, 10, 30 mm of Na+ with or without 10 mm of K+ (C). D, Currents of HvHKT1;5-cRNA-injected oocytes clamped at 40 mm or −140 mm in series Na+ or Na+ and K+ solutions as indicated, plotted from (C). *P < 0.05, **P < 0.01. Student’s t test was used. Data are means ± se (n = 3).

HvHKT1;5 Transgenic Lines Show Different Salt Tolerance from the Wild Type

To reveal the physiological roles of the HvHKT1;5 gene, we generated three independent knockdown (RNAi) barley lines (Fig. 7). The expression levels of HvHKT1;5 in the RNAi lines were dramatically decreased compared with that of the wild type (Supplemental Fig. S4A). In comparison with the wild types (a nontransgenic line [i.e. WT] and a negative-transgenic line isolated from RNAi lines [RNAi-WT]), the RNAi lines showed greater salt tolerance than the wild types (Fig. 7; Supplemental Fig. S4B), on average increasing 76.7% ± 7.9% and 53.1% ± 6.8% (n = 15) of root and shoot dry weights after 3 weeks of 100-mm salt treatment, respectively (Fig. 8, A and B). Exposed to 200-mm salt stress, the RNAi lines also showed greater salt tolerance than the wild types, on average increasing 57.1% ± 10.7% and 53.1% ± 6.7% (n = 9) of root and shoot dry weights, respectively (Fig. 9, A and B; Supplemental Fig. S4B). However, both the wild types and the transgenic lines showed similar growth under the control condition (Fig. 7B; Supplemental Fig. S4C). These results suggest that HvHKT1;5 is involved in salt tolerance in barley.

Figure 7.

Figure 7.

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Effect of knockdown of HvHKT1;5 on plant growth after salt treatment. A and B, Plant growth of the HvHKT1;5 RNAi lines and the wild types (WT) after 3 weeks of 100-mm salt (A) and control conditions (B). Each line was grown in hydroponics. Salt stress was imposed on 2-week–old seedlings for 3 weeks. Scale bar = 10 cm.

Figure 8.

Figure 8.

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Effect of knockdown of HvHKT1;5 on dry weight, Na+, and K+ concentrations after 100 mm of salt treatment. Root (A) and shoot dry (B) weights of the HvHKT1;5 transgenic lines and the wild types (WT) after three weeks of 100-mm salt treatment and the control. K+ contents in the roots (C) and in the shoots of the HvHKT1;5 (D) transgenic lines and the wild types under the control condition. Na+, K+ concentrations and K+/Na+ in the roots (E, G) and shoots (F, H) of the HvHKT1;5 transgenic lines and the wild types under 100-mm salt conditions. Three-week–old seedlings of the transgenic lines and the wild types were prepared by hydroponic culture and then treated by 100-mm NaCl for 3 weeks (n = 5, ± se). Different letters indicate a significant difference (P < 0.05) using Tukey’s test after a one-way ANOVA.

Figure 9.

Figure 9.

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Effect of knockdown of HvHKT1;5 on dry weight, Na, and K+ concentrations after 200-mm salt treatment. Root (A) and shoot (B) dry weights of the HvHKT1;5 transgenic lines and the wild types (WT) after three weeks of 200-mm salt treatment and the control. K+ contents in the roots (C) and in the shoots of the HvHKT1;5 (D) transgenic lines and the wild types under the control condition. Na+, K+ concentrations and K+/Na+ in the roots (E, G) and shoots (F, H) of the HvHKT1;5 transgenic lines and the wild types under 200-mm salt conditions. Three-week–old seedlings of the transgenic lines and the wild types were prepared by hydroponic culture and then treated by 200-mm NaCl for 2 weeks (n = 3, ± se). Different letters indicate a significant difference (P < 0.05) using Tukey’s test after a one-way ANOVA.

HvHKT1;5 Affects Na+ Loading from Roots to Shoots

Under the control condition, tissue K+ concentration showed no significant difference among all lines (Figs. 8, C and D, and 9, C and D). However, under salt stress conditions (100-mm NaCl, treated for 3 weeks; 200-mm NaCl, treated for 2 weeks), tissue Na+ and K+ concentrations showed significant differences between the transgenic lines and the wild types (Figs. 8 and 9). In contrast, tissue Ca2+ and Mg2+ concentrations showed no significant difference (Supplemental Fig. S5). In the roots, the RNAi lines had higher Na+ and K+ concentrations than the wild types after 100-mm salt treatment (Fig. 8E). In the shoots, the RNAi lines clearly showed lower Na+ concentrations than the wild types, but no significant difference in K+ concentrations (Fig. 8F). Exposed to 200-mm salt stress, the RNAi lines had similar root Na+ and K+ concentrations with the wild types, while they showed dramatically lower shoot Na+ and K+ concentrations than the wild types (Fig. 9, E and F). Correspondingly, the RNAi lines had higher K+/Na+ in both roots and shoots than the wild types (Figs. 8, G and H, and 9, G and H). Moreover, for Na+ uptake by roots (total Na+ content in the whole plant/root dry weight), the RNAi lines showed no significant difference with the wild types (Supplemental Fig. S6, A and B). It can be concluded that a lower expression level of HvHKT1;5 gene could reduce Na+ translocation from roots to shoots, and indirectly resulted in tissue K+ concentration changes under salt stress.

The physiological role of HvHKT1;5 in Na+ translocation from roots to shoots was further confirmed by xylem sap assay. After 2 d of 100-mm salt treatment, the K+ and Na+ concentrations in the xylem sap showed no significant difference among all lines (Fig. 10A). However, after 4-d treatment, the Na+ concentration in the xylem sap of the RNAi lines was significantly lower than that in the wild types (Fig. 10B). For K+ concentration in the xylem sap, there was a slight difference among these lines (Fig. 10B). Under the control condition, K+ concentration in the xylem sap was similar among all lines (Supplemental Fig. S6C). These results indicate that HvHKT1;5 is indeed involved in Na+ translocation from roots to shoots, and negatively regulates salt tolerance in barley.

Figure 10.

Figure 10.

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Na+ and K+ concentrations in xylem sap of the HvHKT1;5 transgenic lines and the wild types (WT). A and B, Na+ and K+ concentrations of xylem sap in the HvHKT1;5 transgenic lines and the wild types after salt treatments for 2 d (A) and for 4 d (B). Four-week–old seedlings of the transgenic lines and the wild types were prepared by hydroponic culture and then treated by 100-mm NaCl (n = 5, ± se). Different letters indicate a significant difference (P < 0.05) using Tukey’s test after a one-way ANOVA.

DISCUSSION

Previous studies functionally identified HKT1;5 transporters involved in salt tolerance, including OsHKT1;5, TmHKT1;5-A, and TaHKT1;5-d (Munns et al., 2012; Byrt et al., 2014; Kobayashi et al., 2017). Here, we characterized a homology of HKT1;5, HvHKT1;5, in barley. Functional analysis revealed that HvHKT1;5 showed a distinct pattern in cellular localization and functional roles in salt tolerance. Previously reported HKT1;5 transporters were involved in Na+ unloading from the xylem in roots and contribute to salt tolerance in plants (Munns et al., 2012; Byrt et al., 2014; Kobayashi et al., 2017). Conversely, HvHKT1;5 was involved in Na+ loading from roots to shoots via the xylem, negatively regulating salt tolerance in barley. In detail, HvHKT1;5 was mainly expressed in roots, which was similar to its homologous gene in wheat (Munns et al., 2012). The expression of HvHKT1;5 was induced by salt stress, similar to OsHKT1;5 in rice (Kobayashi et al., 2017). Recently, Hazzouri et al. (2018) reported a link between HvHKT1;5 and salt tolerance in barley using a genome-wide association study. However, the exact function of HvHKT1;5 in salt tolerance remains to be elucidated.

In this study, HvHKT1;5 was mainly localized to the plasma membrane of stele cells, particularly xylem parenchyma cells adjacent to the xylem vessels (Figs. 3–5). Moreover, HvHKT1;5 showed the ability to transport Na+, but not K+ in X. laevis oocytes (Fig. 6). These results indicate that HvHKT1;5 is likely a plasma membrane transporter responsible for Na+ transport. To investigate the function of HvHKT1;5 in barley salt tolerance, RNAi transgenic lines were produced. To date, many physiological parameters are commonly used to identify salt tolerance, including dry weights of shoot and root (Qiu et al., 2011; Wu et al., 2011), Na+ and K+ contents (Chen et al., 2005; Tajbakhsh et al., 2006), and K+/Na+ (Chen et al., 2007b, 2007a; Kronzucker et al., 2008; Shabala et al., 2010). Under salt stress, knocking down HvHKT1;5 resulted in reduced Na+ translocation from roots to shoots, causing a decrease in the Na+ concentration in xylem saps and shoots and an increase in K+/Na+ in plants, leading to increased salt tolerance when compared with the wild-type plants (Figs. 7–10). These findings indicate that HvHKT1;5 is involved in Na+ loading from roots to shoots via xylem. Its negative regulation in salt tolerance of barley distinguishes HvHKT1;5 from other HKT1;5 transporters in rice and wheat.

Notably, tissue K+ concentrations showed similar changes as Na+ among all lines under salt stress (Figs. 8E and 9F). Interestingly, heterologous expression of HvHKT1;5 in X. laevis oocytes proved that HvHKT1;5 was permeable to Na+, but not to K+, and the Na+ transport activity of HvHKT1;5 was inhibited by external K+ (Fig. 6). We further compared the tissue K+ concentrations among all lines under the normal (control) condition, and no difference was found (Figs. 8, C and D and 9, C and D). Thus, we supposed that knockdown of HvHKT1;5 in barley does not have a direct effect on K+ uptake or translocation under normal condition. Although K+ concentrations showed similar patterns as Na+ concentrations did among all lines (Figs. 8E and 9F), the RNAi lines still had higher K+/Na+ values compared with the wild types (Figs. 8, G and H, and 9, G and H), indicating that the knockdown of HvHKT1;5 in barley leads to lower shoot Na+ concentration directly and lower shoot K+ concentration indirectly. As a result, K+/Na+ increased.

In rice, OsHKT1;5 (SKC1) is associated with shoot K+ concentration (Ren et al., 2005). The near-isogenic line containing the SKC1 allele from the salt-tolerant Nona Bokra genotype had lower Na+ concentrations in shoots and in xylem saps than the susceptible variety Koshihikari under salt condition (Ren et al., 2005). Function loss of OsHKT1;5 resulted in higher Na+ accumulation in rice shoots, especially in leaf blades (Kobayashi et al., 2017). These results indicate that OsHKT1;5 (SKC1) participates in Na+ unloading from the xylem in roots. Moreover, OsHKT1;5 was also involved in Na+ exclusion in the phloem in shoots (Kobayashi et al., 2017). Similarly, Na+ exclusion from leaf blades was regulated by OsHKT1;4 at the vegetative growth stage (Suzuki et al., 2016). In wheat, the TmHKT1;5-A gene was reported to have a function of Na+ unloading from the xylem in roots, similar to TaHKT1;5-D (Munns et al., 2012; Byrt et al., 2014). Thus, HKT1;5 can be considered as a Na+ transporter responsible for Na+ exclusion from the xylem and beneficial for enhancement of salt tolerance.

Barley is more salt tolerant than other cereal crops (Munns and Tester, 2008). Only two HKT transporters have been functionally characterized in barley, namely HvHKT2;1 and HvHKT1;1 (Mian et al., 2011; Han et al., 2018). HvHKT2;1 localizes in the cortex cells of barley roots. Overexpression of HvHKT2;1 led to higher Na+ uptake, higher Na+ concentration in the xylem sap, and enhanced translocation of Na+ to leaves when plants were exposed to 50- or 100-mm NaCl. Interestingly, these responses correlated with enhanced salt tolerance by reinforcing the salt-including behavior of barley plants under low or moderate salinity conditions (Mian et al., 2011). In wheat, reducing the expression of TaHKT2;1 resulted in a decrease in Na+ uptake and translocation, thereby enhancing salt tolerance (Laurie et al., 2002). Another HKT transporter gene, HvHKT1;1, is mainly expressed in xylem parenchyma cells and epidermis cells. However, constitutive expression of HvHKT1;1 did not increase Na+ influx into plant roots, and may take part in Na+ redistribution to root epidermal cells for efflux. Knockdown of HvHKT1;1 in barley led to higher Na+ accumulation in both roots and leaves, while overexpression of HvHKT1;1 in salt-sensitive Arabidopsis hkt1-4 and salt overly sensitive1 to salt overly sensitive12 mutants resulted in significantly lower Na+ accumulation (Han et al., 2018). In this study, the HvHKT1;5 gene showed different functions in barley compared with its homologous genes in rice and wheat. We speculate that the different localization of HvHKT1;5 around xylem mainly leads to the opposite direction of Na+ transport and a different physiological function in barley.

Plant HKT proteins contain four conserved P-loop domains, and the Ser residue (SGGG-type) in the first P-loop region primarily determines Na+ permeability for HKT subfamily 1 transporters (Mäser et al., 2002b; Horie et al., 2009; Hauser and Horie, 2010). Previous research found that substitution of Asn by Asp in the second P-loop region of HKT1-type transporters altered cation selectivity and uptake dynamics (Ali et al., 2016). Phylogenetic analysis showed that HvHKT1;5 was similar to HKT1;5s in rice and wheat. Interestingly, HvHKT1;5 has similar ion affinity as other HKT1;5 transporters, but has a different physiological function under salt stress. We propose that the difference among these HKT1;5s may be related to amino acid residues in the PA and PB regions of HvHKT1;5 (Q/V, I/V, M/V; Fig. 1B). TmHKT1;5-A and TaHKT1;5-d were mainly expressed in xylem parenchyma and pericycle cells adjacent to xylem vessels (Munns et al., 2012; Byrt et al., 2014), while in rice, the expression of OsHKT1;5 was also detected in the phloem of diffuse vascular bundles in basal nodes (Kobayashi et al., 2017). Compared with HKT1;5 transporter genes in rice and wheat, the expression of HvHKT1;5 was detected in root stele cells, particularly in xylem parenchyma cells adjacent to the xylem vessels (Figs. 4 and 5). The special cellular localization of HvHKT1;5 could be one of the reasons why the function of HvHKT1;5 in Na+ translocation from roots to shoots in barley is different from other HKT1;5 transporters in rice and wheat.

In conclusion, HvHKT1;5 acts as a membrane protein with Na+ transport ability. In barley plants, HvHKT1;5 is mainly expressed in root stele cells. Knockdown of HvHKT1;5 leads to lower Na+ translocation from roots to shoots, resulting in enhanced salt tolerance. Unlike previously reported HKT1;5 transporters in graminaceous crops, HvHKT1;5 negatively regulates salt tolerance in barley. This study showed a potential use of HvHKT1;5 in improving salt tolerance of barley as well as other cereal crops.

MATERIALS AND METHODS

Cloning and Sequencing of the HvHKT1;5 Coding Region

To clone the full-length sequence of the HvHKT1;5 coding sequence (CDS) region, total RNA was extracted from root tissues of barley (Hordeum vulgare) ‘Golden Promise’ using the MiniBEST Plant RNA Extraction Kit (TaKaRa) according to the manufacturer’s manual. cDNA was synthesized using the PrimeScript II First Strand cDNA Synthesis Kit (TaKaRa). Full-length cDNA was amplified by PCR with primers based on a reference sequence of gene ID DQ912169 by blasting the mRNA sequence of OsHKT1;5 (Kobayashi et al., 2017) against the barley genome database (http://webblast.ipk-gatersleben.de/barley/). Primer information is provided in Supplemental Table S1. The purified amplified product was then introduced into the pGEM vector by pGEM-T (Easy) Vector Systems (Promega), and then used for sequencing by a sequence analyzer (ABI 310; Perkin-Elmer Biosystems) according to the manufacturer’s manual.

Phylogenetic Analysis

After the full-length CDS sequence of HKT1;5 was obtained, the amino acid sequence of HvHKT1;5 was translated using the program DNASTAR (http://www.dnastar.com/). Amino acid sequence alignment of HKT1;5 homologs from rice (Oryza sativa), barley, and wheat (Triticum aestivum and Triticum monococcum) was performed using the software ClustalW (http://clustalw.ddbj.nig.ac.jp/). The phylogenetic tree was constructed by the software MEGA 7 (http://www.megasoftware.net/), using a minimum-evolution method (Poisson model) with 1,000 bootstrap replicates. All accession numbers and species for all amino acid sequences are listed in Supplemental Table S2.

Expression Patterns of HvHKT1;5

For spatial expression of HvHKT1;5, 4-week–old seedlings of cultivar Golden Promise were separated into roots, stems, leaf sheaths, and leaf blades. To further investigate the spatial expression of HvHKT1;5 in subdivided roots, the roots were separated into different segments (0–5, 5–10, 10–20 mm from the root tips) with a razor. Additionally, 3-week–old seedlings were exposed to 200-mm NaCl for 0, 1, 2, 3, and 4 weeks to determine the expression time course of HvHKT1;5. Moreover, to determine the effects of salt concentration on HvHKT1;5 expression, 3-week–old seedlings were treated with different concentrations of NaCl (100, 200, 300, 400, and 500 mm) for 1 week. After salt treatments, samples were harvested and used for total RNA extraction. The cDNA was synthesized as described in "Cloning and Sequencing of the HvHKT1;5 Coding Region". RT-qPCR was performed using SYBR premix EX Taq (TaKaRa) in a volume of 20 μL, consisting of 1-μL cDNA template, 0.5-μL primers, and 10-μL iTaq Universal SYBR Green Supermix. The RT-qPCR reaction was conducted using a CFX96 Real-Time PCR Detection System (Bio-Rad). HvActin was used as an internal reference gene. Primers are provided in Supplemental Table S1. The relative expression level was calculated by a 2−ΔΔCT method using the software CFX Manager (Bio-Rad) with the lowest expression being defined as “1.” For absolute quantification, a series of dilutions (from 1× 10−1 to 10−6 ng) of the plasmids of HvHKT1;5 coding region into pGEM vector as mentioned in "Cloning and Sequencing of the HvHKT1;5 Coding Region" were prepared, and then assayed by RT-qPCR to generate a standard curve. CT values of samples in the spatial expression were converted into absolute copy numbers and quantified using the standard curves (Supplemental Fig. S1).

Subcellular Localization of HvHKT1;5

To determine the subcellular localization of HvHKT1;5, the HvHKT1;5 CDS sequence containing KpnI and XbaI restriction sites (without the stop codon) was amplified by PCR and confirmed by sequencing. The primers are listed in Supplemental Table S1. The amplified cDNA fragment was then subcloned in-frame in front of the GFP-coding region in a pCAMBIA 1300 vector, housing an sGFP driven by the CaMV 35S promoter. Two RFP markers were used, including RFP (whole-cell–localized red fluorescence protein; Matz et al., 1999) and PM-RFP (based on AtPIP2A), a marker of plasma membrane (Nelson et al., 2007). Gold particles with a diameter of 1 μm coated with HvHKT1;5-sGFP or sGFP alone with RFP were introduced into onion (Allium cepa) epidermal cells using particle bombardment (PDS1000/He particle delivery system; Bio-Rad) with 1,100-psi rupture disks under a vacuum of 27 inches of Hg. After incubation in dark condition at room temperature for 16 h, the fluorescence of onion epidermal cells was imaged using a LSM 780 Exciter confocal laser scanning microscope (Zeiss). Cell plasmolysis was treated with 30% (w/v) Suc solution for 20 min. Distributions and fluorescence tracking were quantified using both the softwares LSM 510 AIM (v3.2; Zeiss) and ImageJ (http://www.rsbweb.nih.gov/ij).

Cellular Localization of HvHKT1;5

To examine the cellular localization of HvHKT1;5, in situ PCR and immunostaining experiments were conducted using barley roots. For in situ PCR, HvHKT1;5 in situ mRNA transcripts were amplified according to Athman et al. (2014). Root samples from 7-d–old seedlings of cv Golden Promise were immersed in fixative containing 63% (v/v) ethanol, 5% (v/v) acetic acid, and 2% (v/v) formaldehyde for 12 h. After that, the samples were embedded into 5% (w/v) agarose, and then sectioned to 50 μm. The PCR was performed with in situ PCR primers listed in Supplemental Table S1. The PCR amplification program started at 98°C for 30 s, followed by 30 cycles of 98°C for 10 s, 58°C for 25 s, and 72°C for 5 s, with a final extension at 72°C for 5 min. Samples were stained using BM-Purple AP substrate (Roche) for 2 h. After staining, the sections were washed and mounted in 40% (v/v) glycerol, and then observed on a model no. DM2500M microscope (Leica).

For immunostaining experiments, we introduced the transformation vector carrying ProHvHKT1;5-GFP fusion into barley cv Golden Promise. The promoter was amplified from cv Golden Promise genomic DNA with HindIII and BamHI restriction sites. The amplified fragment was cloned into pCAMBIA1300-GFP vector carrying the GFP gene and the terminator of the nopaline synthase gene, producing the proHvHKT1;5-GFP construct. Immunostaining was performed using the roots of the wild-type barley and the transgenic lines carrying proHvHKT1;5-GFP by an antibody against GFP as described by Yamaji and Ma (2007). Barley seeds were surface-sterilized, rinsed, and germinated on paper towels in the dark at 20°C for 7 d. Before sampling, the paper towels were transferred into a beaker and saturated in the solution containing 2.0 of mm K+ and 0.5 of mm Ca2+ with 150 mm of Na+ for 16 h. Ten root segments with lengths of 1.0 cm from the apex were excised, rinsed, and then fixed in formalin-acetic acid-alcohol solution (Beyotime) for immunostaining according to Han et al. (2018). The GFP signal was observed using a confocal laser scanning microscope (TCS SP8; Leica).

Oocyte Voltage Clamp

To study the ion affinity and transport ability, HvHKT1;5 was heterologously expressed in Xenopus laevis oocytes. Gene cloning, cRNA synthesis, oocytes isolation, injection, and incubation were carried out according to previous studies (Grefen et al., 2010; Byrt et al., 2014; Pornsiriwong et al., 2017). For monovalent cation selectivity analysis, oocytes were bathed in an HMg solution (6 mm of MgCl2, 1.8 mm of CaCl2, and 10 mm of MES, at pH 6.5) with different concentrations of cation-chloride salts (or cation-Glu salts). The osmolality of all bath solutions was adjusted to 240–260 mOsmol kg−1 using a vapor pressure osmometer (Wescor) by adding d-mannitol. Voltage steps were applied from +40 to −140 in −20 mV decrements, with a holding potential of –20 mV. All experiments were performed at room temperature with three biological replicates. Water-injected oocytes were used as negative control. For voltage-clamp analysis, voltage-pulse protocols, data acquisition, and data analysis were performed using Henry’s Electrophysiological Suite Version 3.5.1 (University of Glasgow) and the SigmaPlot 12.5 software (Systat Software, IBM).

Barley Transformation and Identification of Transgenic Lines

To generate the hairpin HvHKT1;5 RNAi construct, we cloned a 196-bp fragment (9–204 bp from ATG) of HvHKT1;5 cDNA as inverted repeats into the pANDA vector (Miki and Shimamoto, 2004) driven by the maize (Zea mays) ubiquitin 1 promoter using the Gateway technology (Invitrogen; http://www.invitrogen.com). The primers used for the RNAi constructs are listed in Supplemental Table S1. The recombinant vector (HvHKT1;5::pANDA) was transformed into Agrobacterium tumefaciens (strain AGL1). Immature embryos of barley ‘Golden Promise’ were used for Agrobacterium-mediated transformation according to a previous protocol (Harwood, 2014). The transgenic lines containing HvHKT1;5::pANDA were named as RNAi lines.

We obtained more than 10 independent transgenic lines for the RNAi transformation, which were confirmed by PCR using the primers listed in Supplemental Table S1. Three independent transgenic lines (T2 generation) of the RNAi lines were used for further analysis. RT-qPCR was performed to determine the expression levels of HvHKT1;5 in the roots and shoots of the RNAi lines and the wild types using the primers listed in Supplemental Table S1.

Plant Materials and Growth Conditions

The seeds of the wild-type cv Golden Promise homozygous negative RNAi line (RNAi-WT) and RNAi lines were sterilized with 2% (w/v) H2O2 for 30 min and rinsed three times with distilled water, then soaked at room temperature for 4 h. The seeds were planted into moist sands in germination boxes and then placed in a growth chamber at 25°C/20°C (d/n) under dark conditions for 3 d. After the seeds germinated, light was provided at 250 μmol photons m−2 s−1 of photosynthetically active radiation. Ten-d–old seedlings were transplanted in 6-L black plastic pots containing one-fifth Hoagland’s solution (pH 6.0) and aerated with pumps. Each pot contained five individual plants of wild-type, RNAi-wild-type, and three independent RNAi lines. The solution contained 1 mm of KNO3, 1 mm of Ca(NO3)2, 0.4 mm of MgSO4, 0.2 mm of NH4H2PO4, and micronutrients comprising 20 μm of Fe-EDTA, 3 μm of H3BO3, 1.0 μm of (NH4)6Mo7O24, 0.5 μm of MnCl2, 0.4 μm of ZnSO4, and 0.2 μm of CuSO4. The solution was renewed every 3 d. Seedlings were grown in a controlled growth room at 22°C of 14-h d/18°C of 10-h n with 250-μmol photons m−2 s−1 (photosynthetically active radiation).

For salt treatment, seedlings were grown in hydroponics for 2 weeks and then treated with salt. Salt was added into the hydroponic solutions at a rate of 50- or 100-mm NaCl increment per day to reach a final concentration of 100 or 200 mm. After 3 weeks of 100-mm NaCl or 2 weeks of 200-mm salt treatment, roots and shoots of each seedling were separated and harvested. Five biological replicates for 100-mm treatment and three biological replicates for 200-mm treatment were set for each line. Root and shoot samples were then dried at 70°C for 2 d for ion concentration determination.

Ion Concentration Determination

Dried root and shoot samples were digested in concentrated nitric acid at 140°C. The concentration of Na, K, Ca, and Mg in the digested solution was determined by an inductively coupled plasma-optical emission spectrometer (iCAP 6000 series; Thermo Fisher Scientific) as described by Wu et al. (2013). Ion uptake was calculated by (total ion content in the whole plant/root dry weight) at the end of salt treatment according to Wu et al. (2016).

Xylem Sap Analysis

For determination of Na+ and K+ concentrations in the xylem sap, 4-week–old seedlings of the RNAi lines and wild types were grown under 100-mm NaCl and the control conditions. After 2 d and 4 d of salt treatment, barley plants were cut 20 mm above the root-shoot junction in a pressure chamber (EL540-300; Wagtech; http://www.wagtech.co.uk). Xylem sap was collected for 30 min and three replicates were set for each line. Finally, the concentration of Na+ and K+ in the xylem sap was measured by inductively coupled plasma optical emission spectrometry as previously mentioned.

Statistical Analysis

Significance analysis was performed by Student’s t test or Tukey’s test using the software SPSS (v16; IBM SPSS Statistics). The difference at P < 0.05 was considered as significant.

Accession Numbers

All accession numbers and species for all amino acid sequences are listed in Supplemental Table S2.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We specifically thank Michael R. Blatt (University of Glasgow) and Zhong-Hua Chen (Western Sydney University) for helpful discussion and article revising, and Dr. Jiming Xu (Zhejiang University, China) and Jixing Xia (Guangxi University, China) for the technical support.

Footnotes

1

This study was supported by the National Natural Science Foundation of China (31620103912 and 31771685), the China Agriculture Research System (CARS-05), and the Jiangsu Collaborative Innovation Centre for Modern Crop Production.

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