Understanding HKT transporters: Essential components of cereal salt

tolerance - a commentary
Ravikiran KT1, Jogendra Singh2, Vijayata Singh2, Vineeth TV3
1
ICAR-Central Soil Salinity Research Institute, Regional Research Station, Lucknow – 226 0002,
Uttar Pradesh, India
2
ICAR-Central Soil Salinity Research Institute, Karnal – 132 001, Haryana, India
3
ICAR-Central Soil Salinity Research Institute, Regional Research Station, Bharuch – 392012,
Gujarat, India
Corresponding email: Ravi.KT@icar.gov.in

Introduction

It is widely accepted that when plants are exposed to salt, they may experience

two types of responses depending on the duration of exposure - osmotic stress in the

early stages and ion toxicity in the later stages (Munns 1993; Munns and Tester 2008).

This pattern is observed in almost all plant species. The primary goal of any glycophyte

(salt-sensitive plant) is to maintain a higher K +/Na+ ratio to ensure proper cellular

functioning. During osmotic stress, the plant tries to prevent the entry and transit of

excess Na+ within the xylem through various mechanisms, commonly known as salt

exclusion. The ions that manage to pass through these checkpoints eventually enter the

cytoplasm, leading to ion toxicity. To deal with the toxicity and the subsequent decrease

in osmotic potential, the plant can either sequester these ions in the vacuoles or

accumulate compatible solutes. These mechanisms are most effective in dealing with

ionic stress rather than osmotic stress.

Considering that the xylem acts as the primary conduit for water and nutrients
throughout the plant, it is paramount to maintain an unimpeded flow free from Na+ ions
to prevent their accumulation in leaves and other economically significant tissues (Fig.
1). This is achieved through the selective removal of excess Na + ions from the xylem,
facilitated by a family of transporters known as high-affinity K + transporters (HKT).
These transporters have been widely recognized as a fundamental mechanism for salt
tolerance in Arabidopsis, rice, and wheat (Hamamoto et al., 2015). The xylem is responsible
for transporting water and nutrients throughout the plant, making it critical to ensure that the

flow remains unobstructed by Na+ ions. The accumulation of these ions in leaves and other

important tissues can cause significant damage. To prevent this, excess Na+ ions are selectively

removed from the xylem with the help of high-affinity K + transporters (HKT), which are a family

of transporters (Fig. 1). These transporters play a vital role in salt tolerance in Arabidopsis, rice,

and wheat (Hamamoto et al., 2015).

Figure 1. High-affinity K+ Transporters (HKT)

Types and role of HKTs

The HKT1 gene was the first HKT gene discovered in wheat, and scientists

discovered that it acts as a Na +-K+ transporter in specific areas of roots and leaves

(Schachtman and Schroeder, 1994). Later studies revealed two types of HKT

transporters: class I and class II. Class I HKT transporters (HKT1s) are primarily

responsible for the selective transport of Na+ and are present in both monocots and

dicots. On the other hand, class II HKT transporters (HKT2s) mainly mediate Na +/K+ co-

transport and are mostly found in monocots (Hauser and Horie, 2010; Ismail and Horie,

2017). These HKT transporters belong to the HKT/Trk/Ktr-type K+ transporter

superfamily, which is found in microorganisms, plants, and parasites (Yamaguchi et al.,

2013). The difference in transport behaviour between the two types of HKT transporters

is due to specific amino acid residues called the 'selectivity filter' motif (Mäser et al.,
2002). Class I HKT transporters contain Ser-Gly-Gly-Gly residues, while class II HKT

transporters have Gly-Gly-Gly-Gly residues. A single Ser residue alters the transport

behavior, making it exclusive to Na+ ions, while transporters with glycine residues prefer

Na+ and K+ ions (Platten et al., 2006; Kronzucker and Britto, 2011). Substantial

quantitative trait loci (QTLs) have been identified in wheat and rice for salinity tolerance,

and these have been linked to HKT transporter-encoding genes, indicating their critical

role in enhancing crop salinity tolerance.

HKTs in Cereal crops: case studies

In rice, a major quantitative trait locus (QTL), Saltol, explaining 43–70% of

phenotypic variation, was mapped on chromosome 1 using a population derived from a
cross between IR29 and Pokkali (Gregorio, 1997; Bonilla et al., 2002). Within this genomic
region, another significant QTL governing shoot K+ concentration, qSKC1, explaining
40.1% of the variation, was identified using a different donor, Nonabokra (Lin et al.,
2004). This QTL was subsequently fine-mapped, leading to the cloning of the candidate
gene OsHKT1;5, which encodes a sodium transporter responsible for maintaining K +
homeostasis (Ren et al., 2005). Similarly, in wheat, Kna1 was the first major QTL
identified for salt tolerance (associated with a high K +/Na+ ratio in leaves) and was
mapped on chromosome 4DL, with the underlying gene later identified as TaHKT1;5-D
(Dvořák and Gorham, 1992; Byrt et al., 2014). Two major genes, Nax1 and Nax2 (Na+
eXclusion), were found to confer salinity tolerance in durum wheat (Munns, 2003). Nax1
is located on chromosome 2A, while Nax2 was found on chromosome 5A (Lindsay et al.,
2004; Byrt et al., 2007). These two genes govern different functions – Nax1 removes Na+
from the root xylem, lower parts of leaves, and leaf sheaths, whereas Nax2 acts
exclusively in roots (James et al., 2006). Subsequently, the candidate genes underlying
these putative loci were identified as TmHKT1;4-A2 for Nax1 and TmHKT1;5-A for Nax2
(Huang et al., 2006; Tounsi et al., 2016).

The roles of three HKT transporters have been particularly well elucidated in rice:
OsHKT1;4, OsHKT1;5 (SKC1), and OsHKT1;1. It has been demonstrated that OsHKT1;4 is
Na+ exclusive (for xylem unloading), with its expression predominantly observed in leaf
sheaths, as well as in the stem and peduncle during the reproductive stage (Suzuki et al.,
2016). Furthermore, OsHKT1;4 has been found to exhibit a significant association with
salt tolerance in a genome-wide association study (GWAS) (Liu et al., 2019). Two
orthologs of OsHKT1;4 in wheat, TdHKT1;4-1 and TdHKT1;4-2, demonstrated a high
affinity for Na+ when expressed in Xenopus oocytes, outperforming other monovalent
cations (Ben Amar et al., 2013). Among these, TmHKT1;4-A2 stands out as a potential
candidate for the Nax1 locus of durum wheat. Both TmHKT1;4-A1 and TmHKT1;4-A2
exhibit equal expression in leaves, but TmHKT1;4-A2's expression is more pronounced in
roots, resembling the function ascribed to the Nax1 locus, making it the most probable
candidate (Tounsi et al., 2016). Furthermore, the promoter region of TmHKT1;4-A2
contains cis-acting elements related to somatic, heavy metal, oxidative, and hormonal
stresses, in addition to salt stress (Tounsi et al., 2021). Similarly, OsHKT1;5, previously
associated with maintaining a high K+/Na+ ratio, is involved in unloading Na+ in the
plasma membrane of xylem parenchyma cells in leaf sheaths and roots, as well as in
phloem parenchyma cells in the vascular bundles of basal nodes (Kobayashi et al., 2017).
A correlation has been observed between the alleles of OsHKT1;5 and the tolerance of
respective genotypes or species of origin (Platten et al., 2013). Moreover, OsHKT1;5
appears to be under combinatorial control of DNA methylation (OsSUVH7), chaperoning
(OsBAG4), protein structure (amino acid residues), and transcriptional regulation
(OsMYB106, OsGrx_C7) (Shohan et al., 2019; Wang et al., 2020; Verma et al., 2021).

TaHKT1;5‐D, the wheat orthologue of OsHKT1;5 and the underlying gene of the
Kna1 locus, is a Na+ selective transporter governing xylem Na+ efflux in the stele of
wheat roots (Byrt et al., 2014). Consequently, a two-stage Na+ exclusion model has been
proposed in rice, involving OsHKT1;5 and OsHKT1;4 (Cotsatftis et al., 2012). OsHKT1;5
primarily unloads Na+ from the xylem in roots, while the same function is carried out by
OsHKT1;4 in the stem and leaf sheath, particularly in young leaves, which receive a more
abundant share of appropriately spliced mRNA than older leaves. The expression of
OsHKT1;1 was mainly observed in the phloem of leaf blades and is regulated by the
OsMYBc transcription factor (Wang et al., 2015). The differential roles of different
variants (V1 to V8) of Pokkali rice OsHKT1;1 have been established in rice salt tolerance
(Imran et al., 2020). Additionally, class II HKT transporters, such as OsHKT2;1/2 and
OsHKT2;4, have been reported in rice, although there is limited insight into their roles.
However, consistent with their classification, it is generally agreed that they co-transport
both Na+ and K+ and contribute significantly to the uptake of Na +, even in salt-tolerant
genotypes like Nonabokra (Suzuki et al., 2016). This phenomenon should be viewed as a
mechanism adopted by the plant to balance its osmolarity with the external
environment, rather than as a weakness.

Limited reports exist regarding the role of maize HKT transporters. ZmHKT2 has
been identified as the underlying gene for a quantitative trait locus (QTL) related to
shoot K+ content and salt tolerance in maize. This transporter extracts K + from the xylem,
reducing shoot K+ content. Its deficiency enhances the shoot K+/Na+ ratio and improves
salt tolerance (Cao et al., 2019). Similarly, ZmHKT1 and ZmHAK4 have been identified as
underlying genes for QTLs (ZmNC1 and ZmNC2), encoding a Na+-specific transporter in
the plasma membrane of the root stele. They govern shoot Na + concentration, and their
loss of function increases Na+ levels in the shoot, rendering plants more salt-sensitive
(Zhang et al., 2017). The role of ZmHKT1, localized in roots, is further supported by the
effects of its promoter variants on root diameter at the seedling stage in maize (Li et al.,
2018). Two splice variants have been reported for the ZmHKT1;1 gene – ZmHKT1;1a and
ZmHKT1;1b, both belonging to class I HKT transporters. Both variants improve salt
tolerance when expressed in tobacco, albeit by invoking slightly different stress-related
gene responses (Ren et al., 2015). Allelic variants of ZmHKT1;5 were explored in a
candidate-gene-based genome-wide association study (GWAS), demonstrating variation
in tolerance response corresponding to allelic variants. Furthermore, its role in
maintaining the Na+/K+ ratio was confirmed by expression in tobacco (Jiang et al., 2018).
Thus, these HKT transporters represent valuable candidates for breeding salt-tolerant
crop varieties.

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