The HKT Transporter HvHKT1;5 Negatively Regulates Salt ...The HKT Transporter HvHKT1;5 Negatively Regulates Salt Tolerance1 Lu Huang,2 Liuhui Kuang,2 Liyuan Wu, Qiufang Shen, Yong

The HKT Transporter HvHKT1;5 Negatively RegulatesSalt Tolerance1

Lu Huang,2 Liuhui Kuang,2 Liyuan Wu, Qiufang Shen, Yong Han, Lixi Jiang, Dezhi Wu,3,4 andGuoping Zhang

Department of Agronomy, Key Laboratory of Crop Germplasm Resource of Zhejiang Province,Zhejiang University, Hangzhou, 310058 China

ORCID IDs: 0000-0002-8579-0763 (L.J.); 0000-0002-7900-0542 (D.W.); 0000-0002-9042-2607 (G.Z.).

Maintaining low intracellular Na1 concentrations is an essential physiological strategy in salt stress tolerance in most cerealcrops. Here, we characterized a member of the high-affinity K1 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 adjacentto the xylem vessels. Its expression was highly induced by salt stress. Heterogenous expression of HvHKT1;5 in Xenopus laevisoocytes showed that HvHKT1;5 was permeable to Na1, but not to K1, although its Na1 transport activity was inhibited byexternal K1. HvHKT1;5 knockdown barley lines showed improved salt tolerance, a dramatic decrease in Na1 translocation fromroots to shoots, and increases in K1/Na1 when compared with wild-type plants under salt stress. The negative regulation ofHvHKT1;5 in salt tolerance distinguishes it from other HKT1;5 members, indicating that barley has a distinct Na1 transportsystem. These findings provide a deeper understanding of the functions of HKT family members and the regulation ofHvHKT1;5 in improving salt tolerance of barley.

Soil salinization caused by either natural or humanactivities is a great threat to sustainable agriculturalproduction in the world (Ondrasek et al., 2015). Na1 isthe most widespread salt in the environment and adominant toxic ion in salinity soils. Excess Na1 in plantcells causes ionic toxicity and other physiologicaldamage such as competition with other mineral nutri-ents (Halfter et al., 2000; Lan et al., 2010; Shabala et al.,2010; Shen et al., 2016; Zhu et al., 2017). To deal withexcess Na1, plants have evolved a series of detoxifica-tion strategies, including Na1 exclusion and seques-tration (Munns and Tester, 2008). Several processescontribute to these mechanisms, including high-affinityK1 transporters (HKTs) and Na1/H1 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 clonedin wheat (Triticum aestivum; Schachtman et al., 1992)and the family has since attracted attention because ofits permeability for Na1 (Schachtman and Schroeder,1994; Rubio et al., 1995, 1999). As a result, many HKTgenes have been identified, which has also revealedsubstantial divergence in function. It is well known thata Ser/Gly residue in the first selectivity pore-formingregion (P-loop) is crucial for cation selectivity andsubfamily features: the HKT subfamily 1 has a Serresidue (SGGG-type) in the first P-loop region, which isconsidered to be associatedwith specific Na1 transport.By contrast, the HKT subfamily 2 has a Gly residue inthat site (GGGG-type), which is permeable to both K1and Na1 (Mäser et al., 2002b; Platten et al., 2006;Rodrígueznavarro and Rubio, 2006; Horie et al., 2009).

At present, many members of the HKT subfamily1 have been functionally characterized. AtHKT1;1 wasshown tomediate large inward Na1 currents in Xenopuslaevis oocytes and Na1 hypersensitivity in yeast(Saccharomyces cerevisiae; Uozumi et al., 2000; Kato et al.,2001). It could serve in retrieval of Na1 from xylem,resulting in a decrease in shoot Na1 accumulation andenhancement of salt tolerance in Arabidopsis (Mäseret al., 2002a; Davenport et al., 2007; Møller et al.,2009). In rice (Oryza sativa), OsHKT1;5 encoding aNa1 selective transporter functions in K1/Na1 homeo-stasis under salt stress (Ren et al., 2005). Kobayashi et al.(2017) used two independent transferred DNA insertionmutants of OsHKT1;5 to reveal its physiological roles in

1This study was supported by the National Natural ScienceFoundation of China (31620103912 and 31771685), the China Agricul-ture Research System (CARS-05), and the Jiangsu Collaborative In-novation Centre for Modern Crop Production.

2These authors contributed equally to this article.3Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Dezhi Wu ([email protected]).

L.H., D.W., and G.Z. designed the experiments and wrote the ar-ticle; L.H., L.K., L.W., Q.S., and Y.H. performed the experiments; L.J.gave comments on the article; all authors discussed the results andcommented on the article.

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584 Plant Physiology�, January 2020, Vol. 182, pp. 584–596, www.plantphysiol.org � 2020 American Society of Plant Biologists. All Rights Reserved.

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https://orcid.org/0000-0002-8579-0763https://orcid.org/0000-0002-8579-0763https://orcid.org/0000-0002-7900-0542https://orcid.org/0000-0002-7900-0542https://orcid.org/0000-0002-9042-2607https://orcid.org/0000-0002-9042-2607https://orcid.org/0000-0002-8579-0763https://orcid.org/0000-0002-7900-0542https://orcid.org/0000-0002-9042-2607http://crossmark.crossref.org/dialog/?doi=10.1104/pp.19.00882&domain=pdf&date_stamp=2019-12-27https://doi.org/10.13039/501100001809https://doi.org/10.13039/501100001809mailto:[email protected]://www.plantphysiol.orgmailto:[email protected]://www.plantphysiol.org/cgi/doi/10.1104/pp.19.00882

mediating Na1 exclusion in vasculature to protect leafblades and reproductive tissues under salt stress. Insuccession, the functions of OsHKT1;1, OsHKT1;3, andOsHKT1;4 have been reported as Na1 transporters inrice (Jabnoune et al., 2009; Wang et al., 2015; Suzukiet al., 2016). Moreover, a major QTL, Nax2, wasidentified in Einkorn wheat (Triticum monococcum),and TmHKT1;5-A was map-based cloned in the re-gion of Nax2, which reduced Na1 accumulation inleaves (Byrt et al., 2007; James et al., 2011; Munnset al., 2012). Furthermore, it was demonstrated thatTaHKT1;5-D from bread wheat (T. aestivum) is a majorgene at the Kna1 locus, which plays a role in leaf Na1exclusion and salt tolerance (Gorham et al., 1990; Byrtet 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 gen-otypes showed that SNPs from HvHKT1;5 were as-sociated with salt tolerance (Hazzouri et al., 2018).However, the exact function ofHvHKT1;5 remains to beelucidated. In this study, we cloned and characterized amember of the HKT subfamily 1 transporter in barley,HvHKT1;5. The transport properties of HvHKT1;5were analyzed in X. laevis oocytes, and subcellular andcellular localization of HvHKT1;5 protein were per-formed in vitro in onion (Allium cepa) epidermis cellsand in vivo in barley roots, respectively. HvHKT1;5knockdown (RNA interference [RNAi]) transgenic lineswere obtained to reveal the gene function. The resultsshow that HvHKT1;5 has a distinct pattern in cellularlocalization and function in salt tolerance from otherreported HKT1;5 transporters, indicating that barleyhas a distinct Na1 transport system and geneticmechanism for salt tolerance.

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

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HvHKT1;5 Negatively Regulates Salt Tolerance

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RESULTS

Sequence and Phylogenetic Analysis of HvHKT1;5

The genomic sequence of HvHKT1;5 contains1,722 bp with two introns and three exons, and the fulllength of its complementary DNA (cDNA) is 1,533 bp,encoding a polypeptide of 510 amino acids (Fig. 1;Supplemental Fig. S1A). Phylogenetic analysis showsthat HvHKT1;5 has 39.4% to 85.7% amino acids identityto 28 members of HKT subfamily 1 transporters from13 plant species (Fig. 1A), with the highest sequencesimilarity to TaHKT1;5-B2. Based on Ser-76 in thefirst P-loop (PA), HvHKT1;5 is likely to be a sodiumtransporter. In addition, three unique amino acid resi-dues (Gln, Ile, and Met) are present in the PA and PBregions of HvHKT1;5 (Fig. 1B), which may be closelyassociated with its function.

HvHKT1;5 Is Mainly Expressed in Barley Roots

Reverse transcription quantitative PCR (RT-qPCR)analysis showed that HvHKT1;5 was mainly expressedin root rather than in stem and leaf (Fig. 2A). Spatialexpression showed that the expression of HvHKT1;5was higher in the root hair zone (10–20mm) than in roottips (0–5 mm) and the root elongation zone (5–10 mm;Fig. 2B). The transcript level of HvHKT1;5 was signifi-cantly higher under salt stress than under normal con-ditions (control). The highest expression level wasfound when plants were exposed to 300-mM NaCl

(Fig. 2C). In a time-course experiment, the expression ofHvHKT1;5 increased with exposure time under 200-mMNaCl and reached a peak after level after 3 weeks(Fig. 2D). However, under the control condition, theexpression level ofHvHKT1;5 in barley roots showed nosignificant change over time (Fig. 2D). These resultsindicate that the expression of HvHKT1;5 is tissue-,dose-, and time course-dependent.

HvHKT1;5 Localizes at the Plasma Membrane of RootStele Cells

To determine the subcellular localization ofHvHKT1;5, a construct containing the coding region ofHvHKT1;5 and Superfolder GFP (sGFP) was transientlycoexpressed with red fluorescence protein (RFP) orplasma membrane-localized red fluorescence protein(PM-RFP; Nelson et al., 2007) in onion epidermis cells(Fig. 3). When sGFP was expressed together with theRFP marker, fluorescence signals from GFP and RFPwere detected across all cells including membranes,nucleus, and other organelles (Fig. 3A). However,the signals from the HvHKT1;5-sGFP fusion weremainly observed at the plasma membrane (Fig. 3B).Furthermore, the signals from the coexpressedHvHKT1;5::sGFP fusion and PM-RFP were only ob-served at the plasma membrane in onion epidermiscells (Fig. 3C). Relative fluorescence of GFP and RFPsignals around the cell periphery also indicated that thesignal from HvHKT1;5::sGFP was similar to the signal

Figure 2. Expression pattern ofHvHKT1;5. A, Absolute RT-qPCR forHvHKT1;5 in the root, stem, leafsheath, and leaf blade tissues from4-week–old seedlings of barley ‘GoldenPromise’ grown under normal conditions.B, AbsoluteRT-qPCR forHvHKT1;5 in theroot tips (0–5 mm) and basal root parts(5–10 or 10–20 mm) from 4-week–oldseedlings of barley ‘Golden Promise’grown under normal conditions. C,RT-qPCR forHvHKT1;5 in roots. Three-week–old seedlings of barley ‘GoldenPromise’ were prepared by hydroponiccultured and treated by 0, 100, 200,300, 400, and 500 mM of NaCl for2 weeks. D, RT-qPCR for HvHKT1;5 inroots. Three-week–old seedlings ofbarley ‘Golden Promise’ were preparedby hydroponic culture under normalcondition and treated by 200 mM ofNaCl for 0, 1, 2, 3, and 4 weeks. Dataare means 6 SE (n 5 3). Different let-ters indicate a significant difference(P, 0.05) using Tukey’s test after a one-way ANOVA.

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from the PM-RFP marker (Fig. 3, D–F). After cell plas-molysis, the signal from HvHKT1;5::sGFP was notdetected at the cell wall, but was detected at the plasmamembrane in onion epidermis cells (Supplemental Fig.S2). Therefore, these results indicate that HvHKT1;5 is aplasma-membrane–localized protein.The RT-qPCR experiment proved that HvHKT1;5

was mainly expressed in barley roots (Fig. 2A). There-fore, cellular localization of HvHKT1;5 was investigatedin barley roots using in situ PCR and immunostainingmethods (Figs. 4 and 5). Compared with the negativecontrol without reverse transcription, the HvHKT1;5transcript showed staining signal predominantly inthe root stele sections, particularly xylem parenchymaand pericycle cells adjacent to xylem vessels (Fig. 4, Aand 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 anti-body against GFP to stain the root cells of the wildtype and transgenic plants carrying proHvHKT1;5-GFP.Compared with the wild type, the transgenic plantsshowed staining signal predominantly in root stelecells (Fig. 5, A–I), while signal was not detected atthe epidermis (Fig. 5, J–L). Therefore, this indicatesthat HvHKT1;5 may be mainly involved in ion load-ing from roots to shoots via xylem.

HvHKT1;5 Shows Na1 Transport Activity inX. laevis Oocytes

HvHKT1;5 cRNA or water was injected into X. laevisoocytes, which were then assayed by two-electrodevoltage clamp (Fig. 6; Supplemental Fig. S3). Water-injected oocytes showed no significant inward oroutward currents when clamped in any bath solu-tions (Supplemental Fig. S3). HvHKT1;5 cRNA-injected oocytes showed large inward currents;216 mA at2140 mV in the presence of 100-mM Na1in the bath solution, with a positive reversal potential;114 mV (Fig. 6A). Replacing external Na1 with100 mM of other monovalent cations, including K1,Li1, Rb1, Cs1, and Tris1 resulted in no significantinward currents and very negative reversal poten-tials (;2138 mV; Fig. 6A). Elevating external Na1concentration gradually increased the inward cur-rents of HvHKT1;5 cRNA-injected oocytes. This Na1concentration-dependent increase of currents was notobserved when the external ion was replaced by K1(Fig. 6B). Interestingly, there was an inhibition of Na1transport through HvHKT1;5 when the external K1concentration was 10 mM (Fig. 6, C and D). A signif-icant reduction was found in the magnitude of bothinward and outward currents in the presence of10-mM external K1. Therefore, it may be suggested

Figure 3. Subcellular localization of HvHKT1;5. A to C, Confocal images of onion epidermis cells coexpressing sGFP along withRFP (A);HvHKT1;5-sGFP fusion alongwithRFP (whole-cell-localized red fluorescence protein; B), or PM-RFP (constructed basedonAtPIP2A; C). Microscopic image channels from left to right: GFP-channel, RFP-channel, andmerged images. Scale bars5 100mm. D–F, Relative fluorescence of GFP and RFP signals around the cell periphery. Position indicated by the dotted linesfrom the left.



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that HvHKT1;5 is a Na1-selective transporter and isregulated by external K1.

HvHKT1;5 Transgenic Lines Show Different Salt Tolerancefrom the Wild Type

To reveal the physiological roles of the HvHKT1;5gene, we generated three independent knockdown(RNAi) barley lines (Fig. 7). The expression levels ofHvHKT1;5 in the RNAi lines were dramatically de-creased compared with that of the wild type(Supplemental Fig. S4A). In comparison with the wildtypes (a nontransgenic line [i.e. WT] and a negative-transgenic line isolated from RNAi lines [RNAi-WT]),the RNAi lines showed greater salt tolerance thanthe wild types (Fig. 7; Supplemental Fig. S4B), onaverage increasing 76.7% 6 7.9% and 53.1% 6 6.8%(n 5 15) of root and shoot dry weights after 3 weeksof 100-mM salt treatment, respectively (Fig. 8, A andB). Exposed to 200-mM salt stress, the RNAi linesalso showed greater salt tolerance than the wildtypes, on average increasing 57.1% 6 10.7% and53.1% 6 6.7% (n 5 9) of root and shoot dry weights,respectively (Fig. 9, A and B; Supplemental Fig.S4B). However, both the wild types and the trans-genic lines showed similar growth under the controlcondition (Fig. 7B; Supplemental Fig. S4C). These

results suggest that HvHKT1;5 is involved in salttolerance in barley.

HvHKT1;5 Affects Na1 Loading from Roots to Shoots

Under the control condition, tissue K1 concentrationshowed no significant difference among all lines (Figs.8, C and D, and 9, C and D). However, under salt stressconditions (100-mM NaCl, treated for 3 weeks; 200-mMNaCl, treated for 2 weeks), tissue Na1 and K1 con-centrations showed significant differences between thetransgenic lines and the wild types (Figs. 8 and 9). Incontrast, tissue Ca21 and Mg21 concentrations showedno significant difference (Supplemental Fig. S5). In theroots, the RNAi lines had higher Na1 and K1 concen-trations than the wild types after 100-mM salt treatment(Fig. 8E). In the shoots, the RNAi lines clearly showedlower Na1 concentrations than the wild types, but nosignificant difference in K1 concentrations (Fig. 8F).Exposed to 200-mM salt stress, the RNAi lines hadsimilar root Na1 and K1 concentrations with the wildtypes, while they showed dramatically lower shoot Na1andK1 concentrations than thewild types (Fig. 9, E andF).Correspondingly, the RNAi lines had higher K1/Na1 inboth roots and shoots than thewild types (Figs. 8,GandH,and 9, G andH). Moreover, for Na1 uptake by roots (totalNa1 content in the whole plant/root dry weight), the

Figure 4. In situ PCR of HvHKT1;5 inbarley roots. A to C, In situ PCR inbarley root cross sections (0–2 mm and5–10 mm from root tip). A, In situ PCRwith HvHKT1;5 primers. B, In situ PCRwith HvHKT1;5 primers but withoutthe reverse transcription (RT) step(negative control). C, In situ PCR with18S rRNA primers (positive control). D,High-magnification image of dottedpart in (A). E, In situ PCR withHvHKT1;5 primers in barley root tip(0–5 mm) sections lengthwise. Allsamples were stained with BM-Purple(Roche). Blue color indicates the pres-ence of digoxigenin-labeled cDNA,and brown indicates the absence of theamplified cDNA target. ep, epidermis;c, cortex; en, endodermis; p, pericycle;x, xylem; xp, xylem parenchyma. Scalebars 5 100 mm.


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RNAi lines showed no significant difference with thewild types (Supplemental Fig. S6, A and B). It can beconcluded that a lower expression level of HvHKT1;5gene could reduce Na1 translocation from roots toshoots, and indirectly resulted in tissue K1 concentra-tion changes under salt stress.The physiological role of HvHKT1;5 in Na1 trans-

location from roots to shoots was further confirmedby xylem sap assay. After 2 d of 100-mM salt treat-ment, the K1 and Na1 concentrations in the xylemsap showed no significant difference among all lines(Fig. 10A). However, after 4-d treatment, the Na1concentration in the xylem sap of the RNAi lines wassignificantly lower than that in the wild types(Fig. 10B). For K1 concentration in the xylem sap,there was a slight difference among these lines(Fig. 10B). Under the control condition, K1 concen-tration in the xylem sap was similar among all lines(Supplemental Fig. S6C). These results indicate thatHvHKT1;5 is indeed involved in Na1 translocationfrom roots to shoots, and negatively regulates salttolerance in barley.

DISCUSSION

Previous studies functionally identified HKT1;5 trans-porters involved in salt tolerance, including OsHKT1;5,TmHKT1;5-A, and TaHKT1;5-D (Munns et al., 2012; Byrtet al., 2014; Kobayashi et al., 2017). Here, we charac-terized a homology of HKT1;5, HvHKT1;5, in barley.Functional analysis revealed that HvHKT1;5 showed adistinct pattern in cellular localization and functionalroles in salt tolerance. Previously reported HKT1;5transporters were involved in Na1 unloading from thexylem 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 Na1loading from roots to shoots via the xylem, negativelyregulating salt tolerance in barley. In detail,HvHKT1;5was mainly expressed in roots, which was similar toits homologous gene in wheat (Munns et al., 2012).The expression of HvHKT1;5 was induced by saltstress, similar to OsHKT1;5 in rice (Kobayashi et al.,2017). Recently, Hazzouri et al. (2018) reported alink between HvHKT1;5 and salt tolerance in barley

Figure 5. Cellular localization of HvHKT1;5. A toC, Immunostaining in barley root cross sections(10 mm from root tip) of the wild type (cv GoldenPromise) using the anti-GFP antibody. D to F,Immunostaining in barley root cross sections ofthe proHvHKT1;5-GFP transgenic plants. G to L,High-magnification image of dotted center partsin (A–C). Seven-d–old seedlings were used. Redfluorescence shows presence of HvHKT1;5 andblue fluorescence is emitted by autofluorescenceof cell wall and the counterstain 49,6-diamidino-2-phenylindole. Cell types are indicated in theenlarged representation with merged signals. en,endodermis; p, pericycle; x, xylem; xp, xylemparenchyma. Scale bars 5 100 mm.



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using a genome-wide association study. However, theexact function of HvHKT1;5 in salt tolerance remains tobe elucidated.

In this study, HvHKT1;5 was mainly localized to theplasma membrane of stele cells, particularly xylemparenchyma cells adjacent to the xylem vessels (Figs.3–5). Moreover, HvHKT1;5 showed the ability totransport Na1, but not K1 in X. laevis oocytes (Fig. 6).These results indicate that HvHKT1;5 is likely a plasmamembrane transporter responsible for Na1 transport.To investigate the function of HvHKT1;5 in barleysalt tolerance, RNAi transgenic lines were produced. Todate, many physiological parameters are commonlyused to identify salt tolerance, including dry weightsof shoot and root (Qiu et al., 2011; Wu et al., 2011),Na1 and K1 contents (Chen et al., 2005; Tajbakhshet al., 2006), and K1/Na1 ((Chen et al., 2007b, 2007a;

Kronzucker et al., 2008; Shabala et al., 2010)). Under saltstress, knocking down HvHKT1;5 resulted in reducedNa1 translocation from roots to shoots, causing a de-crease in the Na1 concentration in xylem saps andshoots and an increase in K1/Na1 in plants, leading toincreased salt tolerance when compared with the wild-type plants (Figs. 7–10). These findings indicate thatHvHKT1;5 is involved in Na1 loading from roots toshoots via xylem. Its negative regulation in salt toler-ance of barley distinguishes HvHKT1;5 from otherHKT1;5 transporters in rice and wheat.

Notably, tissue K1 concentrations showed similarchanges as Na1 among all lines under salt stress (Figs.8E and 9F). Interestingly, heterologous expression ofHvHKT1;5 in X. laevis oocytes proved that HvHKT1;5was permeable to Na1, but not to K1, and the Na1transport activity ofHvHKT1;5was inhibited by external

Figure 6. Transport activities of HvHKT1;5 in X. laevis oocytes. A to D, Current-voltage curves of HvHKT1;5-cRNA-injectedoocytes in the presence of a series of 100-mMmonovalent cations: K1, Li1, Na1, Rb1, Cs1, or Tris1 (A); 1, 10, 30 mM of Na1 or 1,10, 30 mM of K1 (B); 1, 10, 30 mM of Na1 with or without 10 mM of K1 (C). D, Currents of HvHKT1;5-cRNA-injected oocytesclamped at 40 mM or 2140 mM in series Na1 or Na1 and K1 solutions as indicated, plotted from (C). *P , 0.05, **P , 0.01.Student’s t test was used. Data are means 6 SE (n 5 3).


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K1 (Fig. 6). We further compared the tissue K1 con-centrations among all lines under the normal (control)condition, and no difference was found (Figs. 8, C andD and 9, C and D). Thus, we supposed that knockdownof HvHKT1;5 in barley does not have a direct effect onK1 uptake or translocation under normal condition.Although K1 concentrations showed similar patternsas Na1 concentrations did among all lines (Figs. 8E and9F), the RNAi lines still had higher K1/Na1 valuescompared with the wild types (Figs. 8, G and H, and 9,G and H), indicating that the knockdown of HvHKT1;5in barley leads to lower shoot Na1 concentration di-rectly and lower shoot K1 concentration indirectly. As aresult, K1/Na1 increased.In rice,OsHKT1;5 (SKC1) is associated with shoot K1

concentration (Ren et al., 2005). The near-isogenic linecontaining the SKC1 allele from the salt-tolerant NonaBokra genotype had lower Na1 concentrations inshoots and in xylem saps than the susceptible varietyKoshihikari under salt condition (Ren et al., 2005).

Function loss of OsHKT1;5 resulted in higher Na1 ac-cumulation in rice shoots, especially in leaf blades(Kobayashi et al., 2017). These results indicate thatOsHKT1;5 (SKC1) participates in Na1 unloading fromthe xylem in roots. Moreover, OsHKT1;5 was also in-volved in Na1 exclusion in the phloem in shoots(Kobayashi et al., 2017). Similarly, Na1 exclusion fromleaf blades was regulated by OsHKT1;4 at the vegeta-tive growth stage (Suzuki et al., 2016). In wheat, theTmHKT1;5-A gene was reported to have a function ofNa1 unloading from the xylem in roots, similar toTaHKT1;5-D (Munns et al., 2012; Byrt et al., 2014). Thus,HKT1;5 can be considered as a Na1 transporter re-sponsible for Na1 exclusion from the xylem and bene-ficial for enhancement of salt tolerance.Barley is more salt tolerant than other cereal crops

(Munns and Tester, 2008). Only two HKT transportershave been functionally characterized in barley, namelyHvHKT2;1 andHvHKT1;1 (Mian et al., 2011; Han et al.,2018). HvHKT2;1 localizes in the cortex cells of barleyroots. Overexpression of HvHKT2;1 led to higher Na1uptake, higher Na1 concentration in the xylem sap, andenhanced translocation of Na1 to leaves when plantswere exposed to 50- or 100-mM NaCl. Interestingly,these responses correlated with enhanced salt toleranceby reinforcing the salt-including behavior of barleyplants under low or moderate salinity conditions (Mianet al., 2011). In wheat, reducing the expression ofTaHKT2;1 resulted in a decrease in Na1 uptake andtranslocation, thereby enhancing salt tolerance (Laurieet al., 2002). Another HKT transporter gene,HvHKT1;1,is mainly expressed in xylem parenchyma cells andepidermis cells. However, constitutive expression ofHvHKT1;1 did not increase Na1 influx into plant roots,and may take part in Na1 redistribution to root epi-dermal cells for efflux. Knockdown of HvHKT1;1 inbarley led to higher Na1 accumulation in both rootsand leaves, while overexpression of HvHKT1;1 in salt-sensitive Arabidopsis hkt1-4 and salt overly sensitive1 tosalt overly sensitive12 mutants resulted in significantlylower Na1 accumulation (Han et al., 2018). In thisstudy, the HvHKT1;5 gene showed different functionsin barley compared with its homologous genes in riceand wheat. We speculate that the different localizationof HvHKT1;5 around xylem mainly leads to the oppo-site direction of Na1 transport and a different physio-logical function in barley.Plant HKT proteins contain four conserved P-loop

domains, and the Ser residue (SGGG-type) in the firstP-loop region primarily determines Na1 permeabilityfor HKT subfamily 1 transporters (Mäser et al., 2002b;Horie et al., 2009; Hauser and Horie, 2010). Previousresearch found that substitution of Asn by Asp in thesecond P-loop region of HKT1-type transporters alteredcation selectivity and uptake dynamics (Ali et al., 2016).Phylogenetic analysis showed that HvHKT1;5 wassimilar to HKT1;5s in rice and wheat. Interestingly,HvHKT1;5 has similar ion affinity as other HKT1;5transporters, but has a different physiological functionunder salt stress. We propose that the difference among

Figure 7. Effect of knockdown of HvHKT1;5 on plant growth after salttreatment. A and B, Plant growth of the HvHKT1;5 RNAi lines and thewild types (WT) after 3 weeks of 100-mM salt (A) and control conditions(B). Each line was grown in hydroponics. Salt stress was imposed on2-week–old seedlings for 3 weeks. Scale bar 5 10 cm.



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these HKT1;5s may be related to amino acid residuesin the PA and PB regions of HvHKT1;5 (Q/V, I/V,M/V; Fig. 1B). TmHKT1;5-A and TaHKT1;5-D weremainly expressed in xylem parenchyma and pericyclecells adjacent to xylem vessels (Munns et al., 2012; Byrtet al., 2014), while in rice, the expression of OsHKT1;5was also detected in the phloem of diffuse vascularbundles in basal nodes (Kobayashi et al., 2017).Compared with HKT1;5 transporter genes in rice andwheat, the expression of HvHKT1;5 was detected inroot stele cells, particularly in xylem parenchymacells adjacent to the xylem vessels (Figs. 4 and 5). Thespecial cellular localization of HvHKT1;5 could beone of the reasons why the function of HvHKT1;5in Na1 translocation from roots to shoots in barleyis different from other HKT1;5 transporters in riceand wheat.

In conclusion, HvHKT1;5 acts as amembrane proteinwith Na1 transport ability. In barley plants, HvHKT1;5is mainly expressed in root stele cells. Knockdownof HvHKT1;5 leads to lower Na1 translocation fromroots to shoots, resulting in enhanced salt tolerance.

Unlike previously reported HKT1;5 transporters ingraminaceous crops, HvHKT1;5 negatively regulatessalt tolerance in barley. This study showed a potentialuse of HvHKT1;5 in improving salt tolerance of barleyas 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 (Hor-deum vulgare) ‘Golden Promise’ using the MiniBEST Plant RNA ExtractionKit (TaKaRa) according to the manufacturer’s manual. cDNA was syn-thesized using the PrimeScript II First Strand cDNA Synthesis Kit(TaKaRa). Full-length cDNAwas amplified by PCR with primers based ona reference sequence of gene ID DQ912169 by blasting the mRNA se-quence of OsHKT1;5 (Kobayashi et al., 2017) against the barley genomedatabase (http://webblast.ipk-gatersleben.de/barley/). Primer infor-mation is provided in Supplemental Table S1. The purified amplifiedproduct was then introduced into the pGEM vector by pGEM-T (Easy)Vector Systems (Promega), and then used for sequencing by a sequenceanalyzer (ABI 310; Perkin-Elmer Biosystems) according to the manufac-turer’s manual.

Figure 8. Effect of knockdown ofHvHKT1;5 on dry weight, Na1, and K1

concentrations after 100 mM of salttreatment. Root (A) and shoot dry (B)weights of the HvHKT1;5 transgeniclines and the wild types (WT) after threeweeks of 100-mM salt treatment and thecontrol. K1 contents in the roots (C) andin the shoots of the HvHKT1;5 (D)transgenic lines and the wild types underthe control condition. Na1, K1 concen-trations and K1/Na1 in the roots (E, G)and shoots (F, H) of the HvHKT1;5transgenic lines and the wild types under100-mM salt conditions. Three-week–oldseedlings of the transgenic lines and thewild types were prepared by hydroponicculture and then treated by 100-mMNaClfor 3 weeks (n5 5,6 SE). Different lettersindicate a significant difference (P ,0.05) using Tukey’s test after a one-wayANOVA.


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Phylogenetic Analysis

After the full-length CDS sequence of HKT1;5was obtained, the amino acidsequence of HvHKT1;5 was translated using the program DNASTAR (http://www.dnastar.com/). Amino acid sequence alignment of HKT1;5 homologsfrom rice (Oryza sativa), barley, and wheat (Triticum aestivum and Triticummonococcum) was performed using the software ClustalW (http://clustalw.ddbj.nig.ac.jp/). The phylogenetic tree was constructed by the software MEGA7 (http://www.megasoftware.net/), using a minimum-evolution method

(Poisson model) with 1,000 bootstrap replicates. All accession numbers andspecies for all amino acid sequences are listed in Supplemental Table S2.

Expression Patterns of HvHKT1;5

For spatial expression ofHvHKT1;5, 4-week–old seedlings of cultivarGoldenPromise were separated into roots, stems, leaf sheaths, and leaf blades. Tofurther investigate the spatial expression of HvHKT1;5 in subdivided roots, the

Figure 9. Effect of knockdown ofHvHKT1;5 on dry weight, Na, and K1

concentrations after 200-mM salt treat-ment. Root (A) and shoot (B) dryweights of the HvHKT1;5 transgeniclines and the wild types (WT) after threeweeks of 200-mM salt treatment and thecontrol. K1 contents in the roots (C) andin the shoots of the HvHKT1;5 (D)transgenic lines and the wild types un-der the control condition. Na1, K1

concentrations and K1/Na1 in the roots(E, G) and shoots (F, H) of theHvHKT1;5 transgenic lines and thewild types under 200-mM salt condi-tions. Three-week–old seedlings of thetransgenic lines and thewild typeswereprepared by hydroponic culture andthen treated by 200-mM NaCl for2 weeks (n 5 3, 6 SE). Different lettersindicate a significant difference (P ,0.05) using Tukey’s test after a one-wayANOVA.

Figure 10. Na1 and K1 concentrationsin xylem sap of the HvHKT1;5 trans-genic lines and the wild types (WT). Aand B, Na1 and K1 concentrations ofxylem sap in the HvHKT1;5 transgeniclines and the wild types after salttreatments for 2 d (A) and for 4 d (B).Four-week–old seedlings of the trans-genic lines and the wild types wereprepared by hydroponic culture andthen treated by 100-mM NaCl (n5 5,6SE). Different letters indicate a signifi-cant difference (P, 0.05) using Tukey’stest after a one-way ANOVA.



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roots were separated into different segments (0–5, 5–10, 10–20mm from the roottips) with a razor. Additionally, 3-week–old seedlings were exposed to 200-mMNaCl for 0, 1, 2, 3, and 4 weeks to determine the expression time course ofHvHKT1;5. Moreover, to determine the effects of salt concentration onHvHKT1;5 expression, 3-week–old seedlings were treated with different con-centrations of NaCl (100, 200, 300, 400, and 500 mM) for 1 week. After salttreatments, samples were harvested and used for total RNA extraction. ThecDNA was synthesized as described in "Cloning and Sequencing of theHvHKT1;5 Coding Region". RT-qPCR was performed using SYBR premix EXTaq (TaKaRa) in a volume of 20 mL, consisting of 1-mL cDNA template, 0.5-mLprimers, and 10-mL iTaq Universal SYBR Green Supermix. The RT-qPCR re-action was conducted using a CFX96 Real-Time PCR Detection System (Bio-Rad). HvActin was used as an internal reference gene. Primers are provided inSupplemental Table S1. The relative expression level was calculated by a22DDCT method using the software CFX Manager (Bio-Rad) with the lowestexpression being defined as “1.” For absolute quantification, a series of dilu-tions (from 13 1021 to 1026 ng) of the plasmids of HvHKT1;5 coding region intopGEM vector as mentioned in "Cloning and Sequencing of the HvHKT1;5Coding Region" were prepared, and then assayed by RT-qPCR to generate astandard curve. CT values of samples in the spatial expression were convertedinto 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 CDSsequence containing KpnI and XbaI restriction sites (without the stop codon)was amplified by PCR and confirmed by sequencing. The primers are listed inSupplemental Table S1. The amplified cDNA fragment was then subcloned in-frame in front of the GFP-coding region in a pCAMBIA 1300 vector, housing ansGFP driven by the CaMV 35S promoter. Two RFP markers were used, in-cluding 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 mm coated with HvHKT1;5-sGFP orsGFP alone with RFP were introduced into onion (Allium cepa) epidermal cellsusing particle bombardment (PDS1000/He particle delivery system; Bio-Rad)with 1,100-psi rupture disks under a vacuum of 27 inches of Hg. After incu-bation in dark condition at room temperature for 16 h, the fluorescence of onionepidermal cells was imaged using a LSM 780 Exciter confocal laser scanningmicroscope (Zeiss). Cell plasmolysis was treated with 30% (w/v) Suc solutionfor 20 min. Distributions and fluorescence tracking were quantified using boththe 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 immu-nostaining 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 im-mersed 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 mm. The PCR was performed within situ PCR primers listed in Supplemental Table S1. The PCR amplificationprogram started at 98°C for 30 s, followed by 30 cycles of 98°C for 10 s, 58°C for25 s, and 72°C for 5 s, with a final extension at 72°C for 5 min. Samples werestained using BM-Purple AP substrate (Roche) for 2 h. After staining, the sec-tions were washed and mounted in 40% (v/v) glycerol, and then observed on amodel no. DM2500M microscope (Leica).

For immunostaining experiments, we introduced the transformation vectorcarrying ProHvHKT1;5-GFP fusion into barley cv Golden Promise. The pro-moter was amplified from cv Golden Promise genomic DNA with HindIII andBamHI restriction sites. The amplified fragment was cloned intopCAMBIA1300-GFP vector carrying the GFP gene and the terminator of thenopaline synthase gene, producing the proHvHKT1;5-GFP construct. Immu-nostaining was performed using the roots of the wild-type barley and thetransgenic lines carrying proHvHKT1;5-GFP by an antibody against GFP asdescribed byYamaji andMa (2007). Barley seedswere surface-sterilized, rinsed,and germinated on paper towels in the dark at 20°C for 7 d. Before sampling, thepaper towels were transferred into a beaker and saturated in the solutioncontaining 2.0 of mM K1 and 0.5 of mM Ca21 with 150 mM of Na1 for 16 h. Tenroot segments with lengths of 1.0 cm from the apex were excised, rinsed, and

then fixed in formalin-acetic acid-alcohol solution (Beyotime) for immunos-taining according to Han et al. (2018). The GFP signal was observed using aconfocal laser scanning microscope (TCS SP8; Leica).

Oocyte Voltage Clamp

To study the ion affinity and transport ability,HvHKT1;5was heterologouslyexpressed in Xenopus laevis oocytes. Gene cloning, cRNA synthesis, oocytesisolation, injection, and incubation were carried out according to previousstudies (Grefen et al., 2010; Byrt et al., 2014; Pornsiriwong et al., 2017). Formonovalent cation selectivity analysis, oocytes were bathed in anHMg solution(6 mM of MgCl2, 1.8 mM of CaCl2, and 10 mM of MES, at pH 6.5) with differentconcentrations of cation-chloride salts (or cation-Glu salts). The osmolality of allbath solutions was adjusted to 240–260 mOsmol kg21 using a vapor pressureosmometer (Wescor) by adding D-mannitol. Voltage steps were applied from140 to 2140 in 220 mV decrements, with a holding potential of –20 mV. Allexperiments were performed at room temperature with three biological repli-cates. Water-injected oocytes were used as negative control. For voltage-clampanalysis, voltage-pulse protocols, data acquisition, and data analysis wereperformed using Henry’s Electrophysiological Suite Version 3.5.1 (Universityof Glasgow) and the SigmaPlot 12.5 software (Systat Software, IBM).

Barley Transformation and Identification ofTransgenic Lines

To generate the hairpin HvHKT1;5 RNAi construct, we cloned a 196-bpfragment (9–204 bp from ATG) of HvHKT1;5 cDNA as inverted repeats intothe 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 inSupplemental Table S1. The recombinant vector (HvHKT1;5::pANDA) wastransformed into Agrobacterium tumefaciens (strain AGL1). Immature embryosof barley ‘Golden Promise’ were used for Agrobacterium-mediated transfor-mation according to a previous protocol (Harwood, 2014). The transgenic linescontaining HvHKT1;5::pANDA were named as RNAi lines.

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

Plant Materials and Growth Conditions

The seeds of the wild-type cv Golden Promise homozygous negative RNAiline (RNAi-WT) and RNAi lines were sterilized with 2% (w/v) H2O2 for 30 minand rinsed three times with distilled water, then soaked at room temperaturefor 4 h. The seeds were planted into moist sands in germination boxes and thenplaced 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 mmol photons m22 s21 ofphotosynthetically active radiation. Ten-d–old seedlings were transplanted in6-L black plastic pots containing one-fifth Hoagland’s solution (pH 6.0) andaerated with pumps. Each pot contained five individual plants of wild-type,RNAi-wild-type, and three independent RNAi lines. The solution contained1 mM of KNO3, 1 mM of Ca(NO3)2, 0.4 mM of MgSO4, 0.2 mM of NH4H2PO4, andmicronutrients comprising 20 mM of Fe-EDTA, 3 mM of H3BO3, 1.0 mM of(NH4)6Mo7O24, 0.5 mM of MnCl2, 0.4 mM of ZnSO4, and 0.2 mM of CuSO4. Thesolution was renewed every 3 d. Seedlings were grown in a controlled growthroom at 22°C of 14-h d/18°C of 10-h n with 250-mmol photons m22 s21 (pho-tosynthetically active radiation).

For salt treatment, seedlingsweregrown inhydroponics for 2weeks and thentreatedwith salt. Salt was added into the hydroponic solutions at a rate of 50- or100-mMNaCl 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 andshoots of each seedling were separated and harvested. Five biological replicatesfor 100-mM treatment and three biological replicates for 200-mM treatment wereset for each line. Root and shoot samples were then dried at 70°C for 2 d for ionconcentration determination.


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Ion Concentration Determination

Dried root and shoot samples were digested in concentrated nitric acid at140°C. The concentration of Na, K, Ca, and Mg in the digested solution wasdetermined by an inductively coupled plasma-optical emission spectrometer(iCAP 6000 series; Thermo Fisher Scientific) as described byWu et al. (2013). Ionuptake 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 Na1 and K1 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, barleyplants were cut 20 mm above the root-shoot junction in a pressure chamber(EL540-300; Wagtech; http://www.wagtech.co.uk). Xylem sap was collectedfor 30 min and three replicates were set for each line. Finally, the concentrationof Na1 and K1 in the xylem sap was measured by inductively coupled plasmaoptical emission spectrometry as previously mentioned.

Statistical Analysis

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

Accession Numbers

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

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Full-length cDNA sequence and standard curvefor absolute quantification of HvHKT1;5.

Supplemental Figure S2. Subcellular localization of HvHKT1;5.

Supplemental Figure S3. Transport activities of water-injected X. laevisoocytes.

Supplemental Figure S4. Relative expression levels and phenotypic anal-yses of the HvHKT1;5 transgenic lines.

Supplemental Figure S5. Ca21 and Mg21 concentrations in the HvHKT1;5transgenic lines and the wild types under salt stress.

Supplemental Figure S6. Na1 uptake by roots under salt stress and Na1

and K1 concentrations in xylem sap in the HvHKT1;5 transgenic linesand the wild types.

Supplemental Table S1. The primers used in this study.

Supplemental Table S2. The information of HKT subfamily 1 members forthe phylogenetic analysis.

ACKNOWLEDGMENTS

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

Received October 22, 2019; accepted October 28, 2019; published November 5,2019.

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