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ScienceDirect

HKT transporters mediate salt stress resistance in

plants: from structure and function to the field
Shin Hamamoto1, Tomoaki Horie2, Felix Hauser3,
Ulrich Deinlein3, Julian I Schroeder3 and Nobuyuki Uozumi1

Plant cells are sensitive to salinity stress and do not require inhibition of cell expansion, cell division and nutrient
sodium as an essential element for their growth and balance. While high salt concentrations inhibit plant
development. Saline soils reduce crop yields and limit available growth, plants are endowed to some degree with a toler-
land. Research shows that HKT transporters provide a potent ance against salt accumulation. Plants alleviate the toxic
mechanism for mediating salt tolerance in plants. Knowledge of effect of Na+ by excluding Na+ from leaf tissue [4] and by
the molecular ion transport and regulation mechanisms and the sequestration of Na+ into vacuoles. The precise control of
control of HKT gene expression are crucial for understanding Na+/K+ selective accumulation in shoot and root tissues is
the mechanisms by which HKT transporters enhance crop an essential task to maintain cellular cation homeostasis in
performance under salinity stress. This review focuses on HKT the presence of imposed high salt concentration and high
transporters in monocot plants and in Arabidopsis as a dicot external osmolarity [5–7].
plant, as a guide to efforts toward improving salt tolerance of
plants for increasing the production of crops and bioenergy Initial studies in the model plant Arabidopsis thaliana
feedstocks. identified the AtHKT1;1 transporter as a largely Na+
Addresses selective transporter [8] and found that the AtHKT1;1
1
Department of Biomolecular Engineering, Graduate School of gene provides a key mechanism for protecting leaves
Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, from Na+ over-accumulation and salt stress (Table 1)
Japan
2 [4,9,10]. Diverse approaches for the isolation of strong
Division of Applied Biology, Faculty of Textile Science and Technology,
Shinshu University, Nagano 386-8567, Japan salt tolerance genes using a combination of conventional
3
Division of Biological Sciences, Cell and Developmental Biology and novel molecular genetic approaches have been per-
Section, Food and Fuel for the 21st Century Center, University of formed with the aim to improve salt tolerance of crop
California San Diego, La Jolla, CA 92093-0116, USA species and to make saline soils accessible for agriculture
Corresponding author: Uozumi, Nobuyuki
[11,12].
([email protected])
The initial ‘class II’ HKT transporter family member was
first isolated as a K+ uptake transporter in wheat, which
Current Opinion in Biotechnology 2015, 32:113–120 exhibited Na+/K+ co-transport activity [13,14]. Later stu-
This review comes from a themed issue on Plant biotechnology dies showed that indeed two classes of HKT transporters
Edited by Inge Broer and George N Skaracis are found in plants, several of the class II HKT trans-
porters (‘HKT2s’) mediate Na+/K+ transport activity,
For a complete overview see the Issue and the Editorial
whereas the class I HKT transporters (‘HKT1s’) usually
Available online 18th December 2014
mediate relatively Na+ selective transport [8,15]. Further
http://dx.doi.org/10.1016/j.copbio.2014.11.025 evidence on HKTs in various plants has proven the
0958-1669/# 2014 Elsevier Ltd. All rights reserved. physiological significance of HKTs in saline environ-
ments [16–18]. The family of HKTs belongs to the
HKT/Trk/Ktr-type K+ transporter superfamily found in
microorganisms and plants [19], which indicates that
HKT/Trk/Ktr-type K+ transporters may be crucial for
their growth and development.
Introduction
Potassium (K+) and sodium (Na+) are alkali metals and Fundamental structure properties of HKTs
have similar chemical properties and content ratio in non- affecting their function
saline soils. Nevertheless, physiological impacts of these Knowledge of the structure and function relationship and
elements on the metabolism and growth of plants are regulation of HKTs is a key to modifying HKT-linked
quite different. K+ is known to be an essential macro- traits. HKTs belong to a class of K+ transporters [20].
nutrient in plants [1,2] and maintaining high K+/Na+ AtHKT1;1 consists of four repeated transmembrane
ratios in shoots (or leaves) has been suggested to be a domain-pore loop-transmembrane domain motifs, similar
major strategy for glycophyte plants to cope with salinity to ion conducting pore-forming units of K+ channels
stress [3]. The osmotic effect of salt stress can lead to (Figure 1) [21]. Homologous bacterial KtrB form a dimer
various physiological and morphological changes, such as recruiting KtrA octamer subunits at the cytosolic side

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114 Plant biotechnology

Table 1

Expression profiles of HKT genes of Arabidopsis thaliana and Oryza sativa. Information of OsHKT genes is extracted from the Rice
Expression Profile Database (RiceXPro; http://ricexpro.dna.affrc.go.jp/)

Gene name Locus ID Primary tissues expressed Timing of expression

AtHKT1;1 At4g10310 Root Germinating period to maturity
Leaf petiole
Leaf veins
OsHKT1;1 LOC_Os04g51820 Leaf blade Vegetative
Os04g0607500 Leaf sheath Reproductive
Anther
Lemma
Palea
OsHKT1;3 LOC_Os02g07830 Leaf blade Vegetative
Os02g0175000
OsHKT1;4 LOC_Os04g51830 Stem Reproductive
Os04g0607600 Ripening
OsHKT1;5 LOC_Os01g20160 Leaf sheath Reproductive
Os01g0307500 Root Ripening
Stem
OsHKT2;1 LOC_Os06g48810 Leaf sheath Vegetative
Os06g0701700 Root Reproductive
Lemma
Palea
OsHKT2;3 LOC_Os01g34850 Leaf blade Vegetative
Os01g0532600 Leaf sheath Reproductive
Root
OsHKT2;4 LOC_Os06g48800 Leaf blade Vegetative
Os06g0701600 Leaf sheath Reproductive
Ripening

(Figure 2a) [22]. Most pore loops of HKT/Trk/Ktr-type

K+ transporters contain a hallmark Gly residue, which
corresponds to the first Gly of a ‘GYG’ signature selec-
Figure 1
tivity sequence of K+ channels [23]. The replacement of
glycine by serine in loop 1 of AtHKT1;1 and Vibrio
M1BPBM2B
D2
M1DPDM2D
D4
alginolyticus KtrB results in Na+ selectivity of the trans-
M1APAM2A M1CPCM2C
D1 D3 Y
porters rather than in a K+ uptake system (Figure 1)
N [15,24]. These findings support the proposed evolution-
S G G G ary correlation between the three classes of K+ transport
G G G systems, HKT/Trk/Ktr-type K+ transporters, K+ channels
R and Kdp-type K+ pumps which are present in prokaryotic
cells [25]. The X-ray crystal structures of TrkH from V.
parahaemolyticus and KtrB from Bacillus subtilis support
N
C the related ion permeation mechanism of these transpor-
ATP/ADP ters (Figure 2) [26,27]. Considering the analogy of HKT/
Current Opinion in Biotechnology Trk/Ktr-type K+ transporters with K+ channels and Kdp-
type K+ pumps, the Na+ transport property of HKT found
Schematic image of HKT/Trk/Ktr transport system consisting of ion in AtHKT1;1 may be evolutionarily acquired from a
translocating membranous proteins and cytosolic subunits based on K+ selective prototype. Note that a distinct class of
combined data on these transporters in bacteria and plants. Cytosolic
K+ transporters found in plants, the ‘Kup/HAK/KT’ K+
ocatameric ATP-binding regulatory subunits are found in bacteria, but
missing in plant HKTs. Yellow circles represents ATP/ADP. Left panel, transporters differ from the above transporters [28].
Red circles indicate the positions involving the Na+–K+ selectivity filter,
the gating and the regulation of the transport activity (see text). Y TaHKT2;1 isolated from wheat possesses Na+-coupled
shows an N-linked glycosylation site found in AtHKT1;1 in in vitro K+ transport activity [13,28,29]. Consistently, the related
expression analysis. The C-terminal end of the transmembrane domain
KtrB mediates the dimeric stability of KtrB. Right panel, the counter-
cyanobacterial Ktr system shows Na+ activation of K+
clockwise arrangements of the 4-fold MPM motif and octameric uptake [30]. This suggested that even K+-selective HKT/
cytosolic subunits. Trk/Ktr-type transporters likely respond to external

Current Opinion in Biotechnology 2015, 32:113–120 www.sciencedirect.com

 HKT transporters mediate salt tolerance in plants Hamamoto et al. 115

Figure 2

(a)

KtrB

KtrA

(b)

AtHKT1;1

(c)

TrkH

TrkA 100
50
3
Current Opinion in Biotechnology

Sequence similarity of plant HKT and the potassium transporters from B. subtilis (KtrAB) and V. parahaemolyticus (TrkHA). (a) Crystal structure of
the membrane spanning KtrB transporter subunit in side view (left) and top view (right). The box shows the complete multimeric KtrB transport
protein structure (green) with the cytoplasmic regulatory subunit KtrA (blue). (b) Approximated structural model of AtHKT1 based on KtrB in side
view (left) or top view (right). The model was obtained from ModBase (uniprot: Q84TI7; Template PDB Code: 4j7cI). (c) Side view (left) and top
view (right) of the membrane-spanning TrkH transporter subunit. The box shows the complete multimeric TrkH structure (green) with the
cytoplasmic regulatory subunit TrkA (blue). In all panels the purple sphere depicts a potassium ion. The structures are colored according to the
sequence conservation with blue being the highest and red being the least conserved sequence. The MAFFT alignment to determine the degree of
conservation comprises the sequences from the structure and the sequences used in Hauser and Horie [3].

increased Na+ concentrations. However not every HKT/ the eighth and last transmembrane domain of HKT/Trk/
Trk/Ktr-type K+ transporter possesses Na+ activation Ktr-type transporters, which is not present in K+ channels,
properties including an ortholog from Trypanosoma, which is crucial for classifying it as a ‘transporter’ (Figure 1)
is insensitive to Na+ for its K+ transport activity [31]. [32]. The reconstitution of VpTrkH in liposome mem-
branes without the cytosolic regulatory subunit TrkA
Several regions have been identified to be involved in the confirmed permeation of Rb+, a K+ analog, through
regulation and gating of HKT/Trk/Ktr-type transporters. the transporter (Figure 2) [26]. An intramembrane loop
The conserved single positive residue in the middle of in the sixth transmembrane domain functions as gate that

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116 Plant biotechnology

controls KtrB activity in V. alginolyticus [33,34]. An intra- Two transcription factors, ABI4 and AtZIP24 have been
membrane domain loop and the conserved positive resi- reported as negative regulators of AtHKT1;1 expression.
due in the eighth and last transmembrane domain was The loss of function mutant of ABI4, an ABA responsive
positioned beneath of the ion selectivity filter to prevent transcription factor, acquired salt tolerance with
ion permeation in the closed state of TrkH/KtrB [26,27]. decreased Na+ accumulation and increased AtHKT1;1
gene expression levels (Figure 3). ABI4-overexpressor
The bacterial TrkA/KtrA subunit termed KTN/RCK or plants exhibited suppressed AtHKT1;1 expression leading
KtrA/TrkA, contains the nucleotide binding sites for to decreased salt tolerance [45]. These findings are
ADP/ATP in the cytosol, which alters the gating of interesting as other responses to ABA can enhance salt
TrkH/KtrB-mediated K+ permeation (Figures 1 and 2) tolerance [46], whereas surprisingly ABA could enhance
[24,30,35]. The tertiary structure of TrkA/KtrA supported salt sensitivity via AtHKT1;1 regulation [45]. Further
the notion that the nucleotide binding altered the con- research into these opposing ABA responses will be of
formation of TrkA/KtrA itself as well as of TrkH/KtrB interest. AtZIP24 belongs to a group of bZIP-type tran-
[27,36,37]. A counterpart to the regulatory subunit TrkA/ scription factors that are induced by salt stress [47]. In
KtrA has not been identified in plant genomes. AtZIP24 RNAi lines transcript levels of AtHKT1;1 were
increased, suggesting that AtZIP24 represses AtHKT1;1
Regulatory mechanisms of AtHKT1;1 gene expression under salt stress.
expression
Arabidopsis HKT1;1 null mutant plants show an accumu- Positively regulating transcription factors that induce
lation of Na+ in shoots when cultivated on medium AtHKT1;1 expression remain unknown. The AtHKT1;1
supplemented with NaCl, which indicated that AtHKT1;1 promoter sequence contains a small RNA binding region
is a part of the salt tolerance mechanism [4,9,38]. Tissue 2.6 kb upstream of the AtHKT1;1 start codon, which was
specific expression analyses of AtHKT1;1 showed expres- found to be more strongly methylated in leaves compared
sion in the root stele and leaf vasculature [4]. Further to roots (Figure 3a). Differences in methylation may
biochemical immuno-localization analyses found that contribute to higher level of AtHKT1;1 expression in roots
AtHKT1;1 protein is targeted to the plasma membrane [48]. As another transcriptional cis regulatory element in
of xylem parenchyma cells and suggested that AtHKT1;1 the AtHKT1;1 promoter, a direct repeat sequence located
functions in the removal of Na+ from the xylem sap [39]. 5.3 kb to 3.9 kb upstream of the transcriptional start
An alternate model reported expression of AtHKT1;1 in codon of AtHKT1;1 was found through the comparative
the phloem and that AtHKT1;1 activity in the phloem approach on salt sensitivity in several Arabidopsis cultivars
might contribute to circulation of Na+ in the whole plant (Figure 3) [41,48]. The repeat sequence is relevant for the
(Figure 3, Table 1) [9]. A soil microbe, B. subtilis GB03, root specific expression of AtHKT1;1, thereby preventing
enhanced Na+-tolerance of Arabidopsis seedlings via the an increase of Na+ content in shoots. A grafting approach
induction of the expression of AtHKT1;1 in shoots and the demonstrated that the AtHKT1;1 activity in roots con-
repression of the expression in roots [40]. In contrast to tributed to regulate Na+ concentrations in the shoot.
the aforementioned phenomenon, reduced expression of Furthermore the Na+ distribution found in enhancer trap
AtHKT1;1 in roots of certain Arabidopsis accessions con- lines, which were engineered to overexpress AtHKT1;1
ferred Na+ tolerance [41,42]. Time-dependent and tis- only in stelar root cells, supports this role of AtHKT1;1 in
sue-specific expression of AtHKT1;1 likely optimizes roots [42]. Patch-clamp analyses revealed a Nernstian
Na+ flux across the plasma membrane of xylem parench- behavior of AtHKT1;1-mediated channel-like currents
yma cells and thus contributes to the Na+ distribution in with a strong selectivity for Na+ over K+ in vivo [49].
whole plants.
Physiological significance of HKT
Cytokinin (CK) signaling, which is mediated by a two- transporters in salinity resistance in monocots
component system, plays a significant role as a negative Seven functional OsHKT genes were found to exist in a
regulator in the resistance against salt stress. CK appli- japonica rice (Oryza sativa) cultivar (Table 1) and similar
cation conferred repression of AtHKT1;1 in shoots and composition of the HKT gene family was observed in
roots through both type-B response regulator, ARR1 and wheat and barley [50,51]. OsHKT2;1/2 and OsHKT2;2,
ARR2, resulting in an increase in sodium contents in the are similar class II transporters from salt tolerant indica
shoot but a decrease in roots of wild-type Arabidopsis rice varieties Nona Bokra and Pokkali, respectively [52].
plants, analogous to athkt1;1 mutant lines (Figure 3) [43]. These were suggested to contribute to the salt tolerant
Independently it was reported that the expression level of phenotype due to maintenance of K+ acquisition through
AtHKT1;1 in whole plants increased in a mutant defective their K+–Na+ co-transport under salinity stress [53].
in the CK synthetic enzymes, ipt1,3,5,7 [44]. These The OsHKT2;2 gene in a japonica cultivar is a pseudo-
studies together imply that CK can enhance the sodium gene [50]. The OsHKT2;1 transporter functions as a
accumulation in shoots by down-regulating expression unique class II Na+ transporter [52,54]. A primary role of
levels of AtHKT1;1. OsHKT2;1 was found to be mediation of nutritional Na+

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 HKT transporters mediate salt tolerance in plants Hamamoto et al. 117

Figure 3

(a) tandem repeat ATG

methylation ABE
At4g10310
AtHKT1;1
–5.3kb –3.9kb –2.6kb –1.1kb –0.7kb

(b)
Na+

shoot
root Na+

phloem xylem
xylem phloem
xylem phloem

Na+ Na+
Na+
AtHKT1;1
nucleus
H+
AtHKT1;1
ABI4 ARR1,12
Na+ bZIP24 Na+
Na+

xylem parenchyma
cytokinin
Current Opinion in Biotechnology

(a) Diagram of upstream sequence of ATHKT1;1 gene. The tandem repeat sequence, methylation site and ABI4 binding element (ABE) are found
as cis elements [41,45,48]. (b) Schematic model illustrating AtHKT1;1 and the DNA binding proteins, ABI4, bZIP24, ARR1 and ARR12, which all
have been reported as negative regulators of AtHKT1;1 expression [43,45,47].

absorption and Na+ uptake into K+-starved rice plants to (Nax2) and TaHKT1;5-D (Kna1) were found to be Na+
compensate K+ deficiency [54]. selective transporters and expressed in root stelar cells
[61,62]. These findings indicate that AtHKT1;1 in Ara-
QTL analyses of salt tolerant rice and wheat varieties have bidopsis and HKT1;5 in rice and wheat share a similar
led to the identification of class I HKT transporters that are physiological role in protecting leaves from salinity stress
salt tolerance determinants mediating Na+ exclusion from by mediating xylem Na+ unloading in roots [16,18]. In
shoots or leaves [55,56,57]. The HKT1;5 locus from Nona wheat, another important QTL named Nax1 contributes to
Bokra plants was narrowed down as a QTL that maintains the retrieval of Na+ from root xylem and Na+ sequestration
higher K+ contents in shoots under salinity stress, but was in leaf sheaths during salinity stress [60,63]. The
found to encode a Na+ selective transporter preferentially TmHKT1;4-A2 gene from T. monococcum was found to
expressed in xylem parenchyma cells in roots [55]. The be a likely candidate for the Nax1 locus [56]. The rice
HKT1;5 loci from the wheat relative (Triticum monococcum) OsHKT1;4 gene has also been proposed to play an import-
and bread wheat (Triticum aestivum), which were named ant role in Na+ sequestration in leaf sheaths [64] but the
Nax2 and Kna1 locus, respectively [57–60], have been detailed physiological function remains to be elucidated.
demonstrated to be the salt tolerance determinant loci In contrast to the case of rice and wheat, relatively low
in wheat [61,62]. In particular, the introgression of the dependence of restricted Na+ loading into xylem upon
Nax2 locus into a commercial durum wheat cultivar sig- overall salinity tolerance of barley plants has been reported
nificantly enhanced salt tolerance, resulting in a 25% [65,66] Salt tolerant barley varieties exhibited higher K+/
increases in grain yield compared to control lines in salt- Na+ ratios in the xylem sap by maintaining efficient K+
impacted soil in the field [61]. Products of TmHKT1;5-A loading into xylem in roots [65].

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118 Plant biotechnology

In conclusion, soil salinity is becoming an increasingly 12. Shavrukov Y, Bovill J, Afzal I, Hayes JE, Roy SJ, Tester M,
Collins NC: HVP10 encoding V-PPase is a prime candidate for
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 Yoshida K, Bakker EP, Shinmyo A, Oiki S et al.: Glycine residues
Acknowledgements in potassium channel-like selectivity filters determine
This work was supported by the Japan Society for the Promotion of Science potassium selectivity in four-loop-per-subunit HKT
24580135, 26102711 to S.H., and 24246045, 24658090, 25292055 to N.U., transporters from plants. Proc Natl Acad Sci U S A 2002,
and the Ministry of Education, Culture, Sports, Science and Technology, 99:6428-6433.
Japan 25119709 to T.H. Research in J.I.S.’ laboratory on HKT transporters The substitution of Ser for Gly at the first selective filter in HKT trans-
was funded by the U.S. Department of Energy Office of Science, Division porters is responsible for enhanced Na+ translocation.
of Chemical, Geo, and Biosciences, Office of Basic Energy Sciences by
16. Schroeder JI, Delhaize E, Frommer WB, Guerinot ML, Harrison MJ,
Award Number DE-FG02-03ER15449 and by a DAAD Fellowship to U.D. Herrera-Estrella L, Horie T, Kochian LV, Munns R, Nishizawa NK
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