A novel tonoplast Na + /H + antiporter gene from date palm (PdNHX6) confers enhanced salt tolerance response in Arabidopsis

Abstract

Key message:
A sodium hydrogen exchanger (NHX) gene from the date palm enhances tolerance to salinity in Arabidopsis plants. Plant sodium hydrogen exchangers/antiporters (NHXs) are pivotal regulators of intracellular Na+/K+ and pH homeostasis, which is essential for salt stress adaptation. In this study, a novel orthologue of Na+/H+ antiporter was isolated from date palm (PdNHX6) and functionally characterized in mutant yeast cells and Arabidopsis plants to assess the behavior of the transgenic organisms in response to salinity. Genetically transformed yeast cells with PdNHX6 were sensitive to salt stress when compared to the empty vector (EV) yeast cells. Besides, the acidity value of the vacuoles of the transformant yeast cells has significantly (p ≤ 0.05) increased, as indicated by the calibrated fluorescence intensity measurements and the fluorescence imagining analyses. This observation supports the notion that PdNHX6 might regulate proton pumping into the vacuole, a crucial salt tolerance mechanism in the plants. Consistently, the transient overexpression and subcellular localization revealed the accumulation of PdNHX6 in the tonoplast surrounding the central vacuole of Nicotiana benthamiana leaf epidermal cells. Stable overexpression of PdNHX6 in Arabidopsis plants enhanced tolerance to salt stress and retained significantly higher chlorophyll, water contents, and increased seed germination under salinity when compared to the wild-type plants. Despite the significant increase of Na+, transgenic Arabidopsis lines maintained a balanced Na+/K+ ratio under salt stress conditions. Together, the results obtained from this study imply that PdNHX6 is involved in the salt tolerance mechanism in plants by controlling K+ and pH homeostasis of the vacuoles.

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Plant Cell Reports
https://doi.org/10.1007/s00299-020-02549-5
ORIGINAL ARTICLE
A novel tonoplast Na+/H+ antiporter gene fromdate palm (PdNHX6)
confers enhanced salt tolerance response inArabidopsis
IbtisamAl‑Harrasi1· GerryAplangJana1· HimanshuV.Patankar1· RashidAl‑Yahyai2· SivamathiniRajappa3·
PrakashP.Kumar3· MahmoudW.Yaish1
Received: 30 March 2020 / Accepted: 25 April 2020
© Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Key message A sodium hydrogen exchanger (NHX) gene from the date palm enhances tolerance to salinity in Arabi-
dopsis plants.
Abstract Plant sodium hydrogen exchangers/antiporters (NHXs) are pivotal regulators of intracellular Na+/K+ and pH
homeostasis, which is essential for salt stress adaptation. In this study, a novel orthologue of Na+/H+ antiporter was isolated
from date palm (PdNHX6) and functionally characterized in mutant yeast cells and Arabidopsis plants to assess the behavior
of the transgenic organisms in response to salinity. Genetically transformed yeast cells with PdNHX6 were sensitive to salt
stress when compared to the empty vector (EV) yeast cells. Besides, the acidity value of the vacuoles of the transformant
yeast cells has significantly (p ≤ 0.05) increased, as indicated by the calibrated fluorescence intensity measurements and
the fluorescence imagining analyses. This observation supports the notion that PdNHX6 might regulate proton pumping
into the vacuole, a crucial salt tolerance mechanism in the plants. Consistently, the transient overexpression and subcellular
localization revealed the accumulation of PdNHX6 in the tonoplast surrounding the central vacuole of Nicotiana bentha-
miana leaf epidermal cells. Stable overexpression of PdNHX6 in Arabidopsis plants enhanced tolerance to salt stress and
retained significantly higher chlorophyll, water contents, and increased seed germination under salinity when compared to
the wild-type plants. Despite the significant increase of Na+, transgenic Arabidopsis lines maintained a balanced Na+/K+
ratio under salt stress conditions. Together, the results obtained from this study imply that PdNHX6 is involved in the salt
tolerance mechanism in plants by controlling K+ and pH homeostasis of the vacuoles.
Keywords NHXs· Date palm· Abiotic stress· Salinity· pH regulation
Introduction
Soil salinization is one of the main abiotic stresses that limits
the survival and productivity of plants (Munns and Gilliham
2015). Various plant species respond differently to the saline
environment, and their stress tolerance mechanisms depend
on the overall genetic makeup. Therefore, plants are clas-
sified as either halophytes or glycophytes. Halophytes are
salt-tolerant plants; whereas, glycophytes are salt-sensitive
plants (Himabindu etal. 2016; Mishra and Tanna 2017).
In glycophytes, a high cytosolic K+/Na+ ratio is essential
for optimal metabolic functions under salt stress conditions
(Shabala and Pottosin 2014). Hence, the K+/Na+ balance is
achieved by reducing the noxious accumulation of sodium
ions (Na+) and by enhancing the cellular uptake of potas-
sium ions (K+) (Munns and Tester 2008). Non-selective
cation channels and Na+–K+ transporters are the primary
Communicated by Neal Stewart.
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0029 9-020-02549 -5) contains
supplementary material, which is available to authorized users.
* Mahmoud W. Yaish
myaish@squ.edu.om; mwyaish@gmail.com
1 Department ofBiology, College ofSciences, Sultan Qaboos
University, P.O. Box36, 123Muscat, Oman
2 Department ofCrop Sciences, College ofAgricultural
andMarine Sciences, Sultan Qaboos University, P.O. Box34,
123Muscat, Oman
3 Department ofBiological Sciences, Faculty ofScience,
National University ofSingapore, Singapore117543,
Singapore

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regulators of the intracellular Na+ and K+ homeostasis
(Tester and Davenport 2003).
Plant sodium hydrogen exchangers/antiporters (NHXs)
are closely related to the NHE family of mammalian Na+/
K+ exchangers (Bassil etal. 2012). The NHX/NHE family
has been classified into two groups based on their subcel-
lular localization: plasma membrane and intracellular (Brett
etal. 2005). Furthermore, all the plant intra-cellular NHX
have been subdivided into two subgroups, namely class-I
and class-II; class-I proteins localize in the vacuolar mem-
brane, whereas class-II proteins localize in the pre-vacuolar
compartments (PVC) and other non-vacuolar endosomal
compartments (Jiang etal. 2010). NHX gene family has been
extensively studied in different plant species under salinity
stress conditions (Ford etal. 2012; Gong etal. 2014; Guan
etal. 2011; Li etal. 2011; Liu etal. 2012; Mishra etal. 2014;
Xu etal. 2013). The intracellular transmembrane NHXs play
a vital role in pH, Na+ and K+ homeostasis (Barragán etal.
2012; Reguera etal. 2015). Based on the subcellular loca-
tion, they contribute to two main salt stress tolerance strate-
gies; vacuolar sequestration of Na+ (Bonales-Alatorre etal.
2013) and/or extrusion of cytosolic Na+ to the extracellular
space (Hamam etal. 2016). Besides, the NHX proteins are
involved in stomatal function, growth, and development,
as well as in protein and vesicle trafficking (Barragán etal.
2012; Reguera etal. 2015).
NHX6 is a concomitant isoform of NHX5 localized in the
endosomal membranes of the Golgi, trans-Golgi network
(TGN), and PVC (Jiang etal. 2010; Reguera etal. 2015).
These two endosomal NHXs were previously demonstrated
to regulate intracellular processes of cell expansion, vacu-
olar protein biogenesis, trafficking, pH, and osmotic pressure
regulation (Li etal. 2011; Reguera etal. 2015). Apart from
this, the molecular characterization of AtNHX5 and AtNHX6
genes in nhx5 and nhx6 double mutant Arabidopsis lines
enhanced the root growth, facilitated K+ transport and pH
homeostasis in transgenic plants (Wang etal. 2015).
The date palm has evolved as a relatively abiotic stress-
tolerant tree; hence, this plant has recently attracted con-
siderable attention for being a good source of information
regarding the salt adaptation mechanisms from physiological
(Al Kharusi etal. 2017, 2019; Jana etal. 2019; Yaish 2015),
and molecular aspects (Al-Harrasi etal. 2018; Patankar etal.
2016, 2018, 2019b; Yaish etal. 2015, 2017; Zaid and De
Wet 1999).
As a primary investigation step, researchers apply a
common strategy in which channel and transporter cod-
ing genes are functionally characterized in model plants
and are consequently used to improve salinity tolerance in
various crops. For example, overexpression of the Arabi-
dopsis NHX5 (AtNHX5) enhanced salt- and drought toler-
ance in paper mulberry by promoting the accumulation of
osmolytes, which counteracts the osmotic stress resulting
from abiotic stresses (Li etal. 2011). Similarly, overex-
pression of the Arabidopsis AtNHX1 gene in tomato plants
enhanced their tolerance to salt stress by retaining high intra-
cellular K+ level and increasing proline and sugar accumula-
tion in the cytosol (Leidi etal. 2010). Another report showed
that overexpression of a vacuolar NHX1 gene from mung
bean (VrNHX1) in Arabidopsis increased salt tolerance by
increasing the K+/Na+ ratio, enhancing proline accumula-
tion and by reducing the accumulation of malondialdehyde
(MDA) (Mishra etal. 2014).
A previous RNA sequence analysis showed that PdNHX6
gene was induced in the leaf tissues of date palm seedlings
when subjected to salinity (Yaish etal. 2017). Therefore, we
conducted molecular and functional characterization of this
gene in yeast cells and the Arabidopsis model plant to gain
a better understanding of the role played by PdNHX6 in salt
tolerance mechanisms. Our results showed that overexpres-
sion of PdNHX6 in a mutant yeast strain (BYT458) impaired
the growth under salinity stress and led to a reduction in the
vacuolar pH value. However, transgenic Arabidopsis plants
overexpressing PdNHX6 showed enhanced tolerance to salt
stress, which was associated with a higher chlorophyll accu-
mulation, relative water content (RWC), and seed germina-
tion rates when compared to non-transgenic control plants.
Additionally, transgenic Arabidopsis plants were able to
neutralize the increased Na+ concentration under salt stress
and maintained a balanced intracellular K+ level.
Materials andmethods
In silico analysis ofprotein sequence
Deduced amino acid sequences of the NHX family of the
date palm and NHX6 of other plant species were retrieved
from the National Center of Biotechnology Information
(NCBI) database. The retrieved sequences were aligned
using Clusta1W (Feng and Doolittle 1987), and the phylo-
genetic trees were constructed using the Neighbor-Joining
(N-J) algorithm, with 1000 bootstrap replicates, imple-
mented within the MEGA 7 program (Kumar etal. 2016).
NCBI conserved domain database (CDD) was used to assign
the conserved domains to the NHX6 family of different spe-
cies (Marchler-Bauer etal. 2014). The theoretical isoelectric
point (pI) and molecular weight (Mw) of the PdNHX6 were
predicted through the ExPASy (ProtScale) tool (Gasteiger
etal. 2003). A putative promoter sequence of 2000-bp
upstream PdNHX6 was analyzed for identifying the tran-
scription factor binding sites (TFBSs) using the PlantPAN
2.0 database (https ://Plant PAN2.itps.ncku.edu.tw/) (Chow
etal. 2015). The distribution of TFBSs involved in abiotic
stress within this region compared with other general TFBSs
is represented in a pie diagram. Protein transmembrane

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topology and signal peptides were predicted from amino
acid sequences using Protter database (https ://wlab.ethz.ch/
prott er/start /) (Omasits etal. 2013).
Plant materials andgrowth conditions
Date palm (cv. Khalas) seeds were thoroughly washed with
tap water, followed by surface sterilization using 70% etha-
nol. The seeds were further washed several times with sterile
distilled water and then soaked in water overnight at 37°C.
The next day, the seeds were mixed with sterilized moist
vermiculite and incubated at 37°C in the dark for one week.
The germinated seeds were transferred to 2-L pots contain-
ing peat moss and sand mixture (1:1, v/v). The plants were
grown under the controlled environmental conditions of
30°C, 16h light/8h dark photoperiod cycle, and watered to
field capacity. The salt stress treatment was applied using a
300-mM NaCl solution, as previously described (Al Harrasi
etal. 2017).
Quantitative PCR (qPCR) analysis andmolecular
cloning ofPdNHX6 gene
Total RNA was extracted from the roots and leaves of the
date palm seedlings, treated with DNAse I, and used to
synthesize cDNA. The qPCR gene expression analysis of
PdNHX6 was carried out using a primer pair (NHX6FA
and NHX6RA) (Supplementary TableS1), as previously
described [32]. Briefly, the qPCR reaction mixture contained
5-μL SsoAdvanced Universal SYBR® Green Supermix
(Bio-Rad, USA), 0.1 μL of each primer (10pmol), 2 μL of
20-fold diluted cDNA and 2.8 μL of nuclease-free water in a
CFX96 Touch Real-Time PCR Detection System (Bio-Rad,
USA) at 95°C for 20s followed by 40 cycles of 95°C for
3s, and 60°C for 30s. The cDNA of PdNHX6 was amplified
from salt-treated root samples by PCR and the primer pair:
NHX6FB and NHX6RB (Supplementary TableS1). The
resultant PCR product was cloned into pGEM-T Easy vec-
tor (Promega, Madison, USA) and the DNA construct was
confirmed by DNA sequencing. The positive clone was sub-
sequently amplified using NHX6FC and NHX6RC primer
pair, including the attB1 and attB2 DNA sites. The PCR
product was subsequently cloned into an entry pDONR™/
Zeo vector (Invitrogen, Carlsbad, USA), which was then
used to introduce PdNHX6 into different expression vec-
tors for functional analysis using gateway cloning approach
(Thermo Fisher Scientific, USA).
Transient expression ofPdNHX6 inNicotiana
benthamiana leaf epidermal cells
The cDNA of PdNHX6 was cloned in-frame along with
the N-terminal yellow fluorescent protein (YFP), into
pSITE-nEYFP-C1 expression vector (TAIR stock ID: CD3-
1648). The construct was verified for in-frame fusion by
DNA sequencing, and the plasmid was further amplified in
E. coli cells, followed by plasmid extracted using GeneJET
plasmid miniprep kit (Thermo Scientific, Germany), as per
the manufacturer’ protocol.
Leaves of Nicotiana benthamiana (3–4weeks old) were
co-infiltrated with Agrobacterium harboring pSITE-nEYFP-
C1:: PdNHX6 as well as a tonoplast marker plasmid, VAC-
RK (TAIR stock ID: CD3-975). The epidermal cells were
observed three days after infiltration under a confocal laser
scanning microscope (FV3000 Olympus) with FV3000 soft-
ware for image processing. The expression of PdNHX6 and
the tonoplast marker were detected using YFP filter set (Ex:
514nm/Em: 520–560nm) and RFP filter set (Ex: 561nm/
Em: 600–650nm), respectively.
Heterologous expression of PdNHX6 inyeast
PdNHX6 cDNA was cloned into the yeast plasmid pYES-
DEST52 (Thermo Fisher Scientific, USA), under the control
of galactose inducible GAL1 promoter. The function of the
recombinant vector (PdNHX6) compared to the empty vector
(EV) was assayed in Saccharomyces cerevisiae mutant strain
(BYT458; genotype: BY4741; ena1-5Δ::loxP nha1Δ::loxP
vnx1Δ::loxP) (Petrezselyova etal. 2013), which was kindly
provided by Professor Hana Sychrova, Czech Republic.
Both PdNHX6 and EV were introduced into the yeast cells
by PEG-lithium acetate method and using Yeastmaker™
yeast transformation system 2 (Clonetech Laboratories Inc.,
Mountain View, California, USA) as per the manufacturer’
instructions. The genetically transformed yeast cells were
selected based on the auxotrophic selectable marker URA3
gene, which enhances the growth of transformed colonies on
solid synthetic medium (SSM) lacking uracil. The salt stress
tolerance ability of PdNHX6 yeast cells compared to EV was
tested on medium supplemented with 100mM NaCl using
yeast spot assay (Patankar etal. 2018).
Similarly, the behavior of the PdNHX6 and EV yeast cells
was tested in SD liquid culture supplemented with 50-mM
NaCl. The intracellular Na+ and K+ levels in yeast cells were
measured as previously described (Patankar etal. 2019a). In
this experiment, three independent yeast colonies were used
to assess the response of PdNHX6 and EV to the salinity
stress condition.
Estimation ofvacuolar pH oftheyeast cells
harboring PdNHX6
Vacuolar pH of the transformed yeast cells was meas-
ured and compared with EV control cells using
2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein

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(BCECF-AM) (Molecular Probes, Eugene, Oregon, USA),
to understand the role of PdNHX6 in salinity tolerance
mechanism. A vacuolar pH calibration curve was prepared
as previously described (Ali etal. 2004), and used to calcu-
late the vacuolar pH of EV and PdNHX6 transformed yeast
cells. Briefly, yeast cells (six replicates each) were cultured
in SD medium (pH 5.0) for two days, then pelleted and
washed twice with distilled water. The cells were incubated
with 50-mM BCECF-AM at 30 ºC for 40min and resus-
pended in the same medium after three washes and immedi-
ately read at 490nm in Epoch Microplate Spectrophotometer
(BioTek, Winooski, USA) supported by Gen5 software (ver-
sion 2.07). Fluorescence intensity (FI) was measured at 490-
nm excitation wavelength, and then the background values of
BCECF-free culture were subtracted from FI and normalized
to cell densities to calculate the vacuolar pH of PdNHX6 and
EV. For the vacuolar pH imaging, slides were coated with
0.1-mg/ml concanavalin A (Sigma-Aldrich, St. Louis, MO,
USA), incubated overnight at 30 ºC, and then washed twice
to remove excess concanavalin A. For the yeast cultures, the
EV and PdNHX6 labeled with 50-mM BCECF were spotted
on concanavalin A spot, incubated at 30 ºC for 10min and
washed twice with distilled water. A drop of SD medium
was added to yeast cells and then visualized under the fluo-
rescence microscope.
Heterologous expression of PdNHX6 inArabidopsis
PdNHX6 cDNA was cloned into the binary plant expres-
sion vector pEarleyGate 203 (TAIR stock ID: CD3-689),
under the control of cauliflower mosaic virus CaMV 35S
promoter. A sequence confirmed clone of EarleyGate 203::
PdNHX6 was introduced into Agrobacterium tumefaciens
LBA4404strain (Invitrogen, Carlsbad, USA) using elec-
troporation. The resultant construct was confirmed by PCR
using a gene-specific and plasmid-specific primer (Sup-
plementary Table1). Subsequently, the construct was used
to genetically transform Arabidopsis thaliana (ecotype
ColumbiaCol-0) through the floral dip method (Clough and
Bent 1998). The floral dip was repeated after two weeks
to increase the transformation efficiency. Seeds were col-
lected after maturation (T0) and replanted for selection
using 0.01% glufosinate ammonium solution (BASTA®)
(Bayer, Bielefeld, Germany). The survived plantlets (T1)
were genotyped for the presence of the transgene by PCR
using the 35S forward primer, a gene-specific reverse primer
(NHX6RA), gene-specific forward (NHX6FA) and a OCS
terminator reverse primers (Supplementary TableS1). T2
Seeds were collected and grown on half-strength Murashige
and Skoog (MS) medium plates supplemented with 15-mg/
ml BASTA® and only the transgenic lines, which showed
the 3:1 Mendelian genetic segregation ratio were selected
and planted in soil. The seeds of T3 selected lines were
further tested for 100% survival on MS medium plates,
supplemented with 15-mg/ml BASTA representing the
independent homozygous transgenic lines. In this study,
three independent homozygous lines were considered for
the forthcoming experiments. To confirm the expression of
PdNHX6 in Arabidopsis transgenic lines, a semi-quantitative
RT-PCR was carried out. Total RNA was extracted from
the WT and transgenic Arabidopsis plants (T3 generation)
using a RNeasy Plant Mini Kit (Qiagen, Germantown,
USA), as per the manufacturer’s instructions. After DNAse
I treatment (Qiagen, USA) of the isolated RNA, cDNA was
synthesized using a SuperScript IV First-Strand Synthesis
System (Invitrogen, Carlsbad, USA), as per the manufac-
turer’s instructions. Semi-quantitative RT-PCR of PdNHX6
was performed using a primer pair NHX6FA and NHX6RA
(Supplementary TableS1). The Arabidopsis actin 2 (acces-
sion number AT3G18780) was used as a reference gene,
which was amplified by a primer pair ACT2F and ACT2R
(Supplementary TableS1).
In vitro assay ofroot length, fresh anddry weights
Cold-stratified seeds from WT and three transgenic lines
were germinated on half-strength MS-agar plates for four
days. The seedlings were then transferred to half-strength
MS-agar plates (control) or MS-agar plates supplemented
with 100-mM NaCl (salt stress) for two weeks. Thereafter,
the root length, fresh and dry weights of the WT and trans-
genic lines were measured. The measurement was carried
out for four biological replicates, each of which had four
technical replicates.
In vitro seed germination rate test
Surface-sterilized seeds from WT and three transgenic lines
were sown on MS-agar plates (control) and MS-agar plates
supplemented with 100-mM NaCl (salt stress). The plates
were kept under the controlled growth conditions; 22°C
temperature, 60% relative humidity, and 16-h light/8-h dark
photoperiod cycle. The germinated seeds were counted each
day for seven days. The germination was considered by vis-
ible radical emergence. Four replicates and 60 seeds of each
line per replicate were used in the experiment.
Growth ofPdNHX6 transgenic lines grown
undersalinity stress
The WT and PdNHX6 transgenic seeds were germinated on
half MS plates for four days; thereafter, the plantlets were
transferred to the potting mixtures in 0.5-L pots. The plant-
lets were incubated under the controlled growth conditions,
which were mentioned previously. During the first three weeks
of the experiment, all plants were watered to field capacity

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with distilled water. After that, the plants were watered with a
200-mM NaCl solution (salinity stress treatment) every four
days for two weeks. However, plants of the control treatment
were continually watered with distilled water. Each treatment
group included four technical replicates and consisted of three
biological replicates. After the elapsed period of the salinity
stress treatment, the plants recovered from their respective
stress by watering them with distilled water to determine their
recovery ability. The soil electrical conductivity (EC) of each
treatment group was measured using an Em50 Digital Data
Logger (Decagon Devices, WA, USA).
Assaying physiological parameters of PdNHX6
transgenic lines
The response of the PdNHX6 transgenic plants to salinity
was compared with the WT plants in terms of phenotypic
changes as well as physiological parameters. The physiologi-
cal parameters, including relative water content (RWC), total
chlorophyll, Na+, and K+ concentration were analyzed. RWC
of the leaves was measured using the following equation: Leaf
RWC (%) = [(fresh weight − dry weight)/(turgid weight − dry
weight)] × 100 (Mullan and Pietragalla 2012). Chlorophyll was
extracted from fresh leaf tissues using 80% acetone; then, the
concentration was calculated as previously described (Arnon
1949).
The concentration of Na+ and K+ ions was measured from
14-day-old Arabidopsis plants grown on half-strength MS
medium (control) or in plates supplemented with 100-mM
NaCl (salt stress). Furthermore, the plants were dried, and
Na+ and K+ ions were extracted using 0.5-M nitric acid fol-
lowed by two days incubation on a room temperature-shaker
at 100rpm. The samples were filtered, and the concentrations
of the soluble Na+ and K+ ions were measured using a Sys-
tronics flame photometer 128 (Systronics, India), following
a previously published protocol (Munns etal. 2010). Three
biological replicates, each with four technical replicates, were
used in this analysis.
Statistical analysis
Statistical analysis was performed using IBM SPSS Statis-
tics 21 package. One-way analysis of variance (ANOVA) test
was used to compare the differences between the mean. The
significance (p < 0.05) between means of the tested variables
was determined by Duncan’s Multiple Range Test (DMRT).
Results
NHX2 andNHX6 are thepredominant members
ofNHX family indate palm genome
In silico analysis, based on the available information in Gen-
Bank, had identified 11 members of NHX family in the date
palm genome (Fig.1a). The analysis showed that date palm
genome encodes four isoforms each of NHX2 and NHX6
genes, while there was only one isoform each for NHX1,
NHX4, and NHX8. The analysis also showed that the NHX1,
NHX2, and NHX6 members were similar, as they have clus-
tered together within the same branch on the phylogenetic
tree, while the NHX6 isoforms and NHX8 have clustered
separately, away from the groups. In this report, we present
PdNHX6 (Accession number XM_008812982.2) as the first
gene which was successfully cloned among this family. The
Fig. 1 The phylogenetic analysis of the date palm sodium hydrogen
exchangers (NHX) protein family (a), and the phylogenetic relation-
ship between PdNHX6 (red font) and its isoforms from different plant
species (b). The phylogenetic trees were constructed using the neigh-
bor-joining algorithm. The numbers shown on the nodes represent the
percentage of 1000 replicates in the bootstrap analysis (colour figure
online)

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coding sequence of this gene consists of 1521bp, which
codes for 506-amino acid protein (55.43kDa) with an aver-
age isoelectric point (pI) of 5.65. Phylogenetic analysis
of NHX6 protein sequences from different plant species
revealed that PdNHX6 was closely related to the African
oil palm (Elaeis guineensis) and pineapple (Ananas como-
sus) (Fig.1b).
Prediction of PdNHX6 transmembrane topology dis-
played 11 transmembrane domains (Fig.2a), a region where
PdNHX6 shares high similarities with other orthologs
(Supplementary Fig. S1). This region is commonly con-
served among NHX family and known as sodium/hydrogen
exchanger 3. Apart from this, the conserved region consists
of three highly conserved acidic residues at D132, E156, and
D161 (Fig.2b), which was previously proven to be essential
for K+ transport (Wang etal. 2015). In addition to the trans-
membrane domains, PdNHX6 encodes signal peptides at the
N-terminus of the protein sequence, which may mark the
Fig. 2 The putative topology of 11 transmembrane domains and sig-
nal peptide predicted in PdNHX6 using Protter database (a). Multi-
ple sequence alignment of the deduced amino acid sequence of date
palm (PdNHX6) and seven NHX6 isoforms from different plant spe-
cies (b). Yellow boxes within the Na+/H+ exchanger three conserved
domains (red box), are the conserved acidic residues of PdNHX6;
D132, E156, and D161. These residues were predicted based on
alignment with AtNHX6, which had been demonstrated to be essen-
tial for K+ and pH homeostasis. Blue arrows indicate differences in
protein sequence among the conserved regions (colour figure online)

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protein for secretion. Therefore, this protein might exist as a
membranous or/and soluble protein. The protein sequence
of PdNHX6 has 78% identity with the Arabidopsis NHX6,
AtNHX6 (NP_178079.2), where it showed differences in
protein sequence along the conserved region (Fig.2b, blue
arrows). Interestingly, when the topology of the AtNHX6
was investigated, the result showed the presence of 12 trans-
membrane domains however, unlike PdNHX6, no signal
peptide was predicted (Supplementary Fig. S2). This obser-
vation may imply a variation in function between PdNHX6
and AtNHX6.
The putative promoter ofPdNHX6 codes forvarious
TFBSs associated withabiotic stresses
A putative promoter region of 2000bp located upstream of
PdNHX6 coding sequence was computationally searched for
the presence of the TFBSs. Among the 1583 putative TFBSs
revealed by the analysis, 38% TFBSs were previously known
to be abiotic stress-responsive, such as the bHLH, bZIP,
AP2/ERF, WRKY, MYB, trihelix, NAC, and ZF-HD (Sup-
plementary Fig. S3). These TFBSs were previously known
for their role in the regulation of the expression of drought
and salinity responsive genes (Dai etal. 2007; Krishnamur-
thy etal. 2019; Liu etal. 2014; Xu etal. 2018; Yaish etal.
2010; Zhang etal. 2009).
Expression ofPdNHX6 wasupregulated inleaf tissue
whenexposed tosalinity
The expression level of PdNHX6 was detected in root and
leaf tissues of date palm seedlings under control and salin-
ity stress conditions (Fig.3). While PdNHX6 was not sig-
nificantly (p < 0.05) affected in the roots when exposed to
salt stress, it was noticed that PdNHX6 was significantly
upregulated by almost 17-fold in the leaf tissues under the
same conditions.
Expression ofPdNHX6 inyeast impaired growth
undercontrol andsalinity stress conditions
The effect of PdNHX6 was tested in yeast mutant strain
cells (BYT458), which contains nonfunctional vacuolar
and plasma membrane transporter system (Petrezselyova
etal. 2013). Yeast cells overexpressing PdNHX6, as well as
empty vector (EV) transformant cells (negative control) were
grown on SSM medium (Control), or SSM supplemented
with 100-mM NaCl (Fig.4b). Under both control and salt
stress conditions, the PdNHX6 cells showed impaired growth
compared to the EV cells (Fig.4b).
Similarly, the growth rates of PdNHX6 transgenic yeast
were significantly (p < 0.05) reduced in liquid cultures using
LSM or LSM supplemented with 50-mM NaCl, when com-
pared with the EV cells (Fig.4c, d). The 50-mM NaCl was
used as salt stress because a concentration of NaCl had dras-
tically inhibited the growth of these mutant yeast cells.
Determining the intracellular accumulation of Na+ and
K+ ions of EV and transgenic yeast cells could partially help
predict the modulated response to the salt stress upon the
transgene expression. The concentration of ions was meas-
ured in yeast cells grown in LSM, supplemented with 0-mM
or 25-mM NaCl (Fig.5). The 25-mM NaCl was selected
because it is the highest non-lethal concentration for the
mutant yeast cells. Although both EV and transgenic yeast
grew at similar rates, the level of Na+ ions was significantly
(p < 0.05) higher in the transgenic yeast compared to EV
cells under salt stress conditions (25-mM NaCl) (Fig.5a).
The concentration of K+ ions was also measured, and it was
found that the accumulation of K+ in transgenic yeast was
significantly (p < 0.05) higher than the EV under both con-
trol and salinity conditions (Fig.5b). These observations
may support the hypothesis that PdNHX6 regulates Na+ and
K+ ions uptake in yeast under salt stress.
PdNHX6 isinvolved inhomeostatic vacuolar
regulation ofpH inyeast cells
It was previously shown that NHX6 is involved in vacu-
olar protein trafficking and pH regulation (Li etal. 2011;
Reguera etal. 2015). To test this ability of PdNHX6, the
Fig. 3 Quantitative PCR (qPCR) expression analysis of PdNHX6 in
the roots and leaves of date palm seedlings under control and salinity
stress conditions. Bars represent the mean ± SE of three independent
biological replicates. Different letters on top of the bars represent a
significant difference at p < 0.05

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vacuolar pH for both EV and PdNHX6 transformed yeast
was measured using a calibration curve (Supplementary
Fig. S4). Acidic vacuolar pH was detected for EV and
PdNHX6 yeast cells. However, a significantly (p < 0.05)
lower pH was detected in the vacuole of PdNHX6 trans-
formed yeast when compared to the EV yeast (Fig.6a).
This observation supports the notion that PdNHX6 acti-
vates H+ pumping into the vacuole and hence involved
in pH regulation under salinity stress. Likewise, fluores-
cence images showed lower acidic vacuolar pH values for
PdNHX6 yeast compared to the EV, as indicated by pH
scale bar (Fig.6b).
PdNHX6 was transiently expressed in Nicotiana
benthamiana leaves. Subcellular localization results
showed that PdNHX6 was accumulated in tonoplast
(Fig.7bI). This was confirmed by the co-localization with
tonoplast-mCherry marker (Fig.7bII). The PdNHX6-YFP
fluorescence showed excellent overlap with mCherry
fluorescence signal in the merged image (Fig.7bIII).
Fig. 4 Overexpression of PdNHX6 in a salt-sensitive yeast strain
(BYT458). Schematic presentation of the yeast expression vector
construct (a). The transgenic PdNHX6 and the empty vector (EV)
yeast cells were spotted on solid media SSM (control), and SSM
supplemented with 100-mM NaCl (salt stress) to assay the response
of the transgenic yeast (b). The EV was used as a negative control
in the experiment. The growth response of transgenic PdNHX6 and
EV yeast cells in a liquid medium, LSM (control) (c), and LSM
supplemented with 50 mM NaCl (d). Each OD value represents the
mean ± SE of three independent biological replicates and the statisti-
cal significance at p < 0.05 is indicated by an asterisk (*)
Fig. 5 Intracellular Na+ (a) and K+ (b) concentration in PdNHX6
and EV yeast cells grown in LSM (control) and LSM supplemented
with 25mM NaCl. The bars represent the mean concentration ± SE of
three independent biological replicates. Different alphabets on top of
the bars represent a significant difference at p < 0.05

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This serves as additional support for the involvement of
PdNHX6 in vacuolar pH regulation in the plant cells.
Expression of PdNHX6 inArabidopsis plants confers
tolerance tosalinity stress
Transgenic Arabidopsis plants were genotyped using PCR to
confirm the genetic transformation and stability of PdNHX6
(Supplementary Fig. S5). Transgenic Arabidopsis lines were
selected for further studies only if their T2 progeny segre-
gated on a 3:1 ratio, to ensure the presence of only a single
transgene within the genome. Subsequently, three independ-
ent homozygous lines were obtained (T3), and the expres-
sion of PdNHX6 was verified by semi-quantitative RT-PCR
(Fig.8b, Supplementary Fig. S6).
The growth of the transgenic lines was assayed on MS
medium, along with WT plants, to evaluate the effect of
PdNHX6 expression on root length, fresh and dry weights
under control (Supplementary Fig. S7a) and salinity treat-
ment (Supplementary Fig. S7b) conditions. There were
insignificant differences between the root lengths of the
transgenic and WT plants when grown under control and
NaCl treatment, however, the dry weight of the transgenic
seedlings was slightly higher than those of WT under both
treatments, and the fresh weight of the transgenic lines TL1
Fig. 6 Measurement of vacuolar pH of PdNHX6 and EV yeast cells
using fluorescent BCECF-AM dye based on the calibration curve.
The fluorescence intensity (FI) was measured at 490-nm excitation
wavelengths, and the background values of BCECF-free culture was
subtracted from FI and normalized to cell densities to calculate the
vacuolar pH of PdNHX6 and EV (a), Calculated pH represents the
mean ± SE of six independent biological replicates. The p value on
top of the bars represents a significant difference between the pH of
PdNHX6 and EV. Accumulation of BCECF-AM dye in the vacuoles
of PdNHX6 and EV yeast cultured (b). Bright-field (top) and fluo-
rescent (bottom) images at an excitation wavelength of 440nm were
captured at 40X magnification. Scale bar = 100μm, and pH scale bar
on the right side of images shows color-based pH range
Fig. 7 Subcellular localization of PdNHX6 in Nicotiana benthamiana
leaf epidermal cells. Schematic representation of the plasmid con-
struct (pSITE-nEYFP-C1), consisting of the nEYFP fused PdNHX6,
expressed under the control of a constitutive CaMV 35S promoter (a).
The accumulation of nEYFP-PdNHX6, as well as mCherry [tonoplast
marker (vac-rk CD3‐975)], in the leaf epidermal cells, were observed
under a confocal laser scanning microscope (b). PdNHX6 expression
was detected in the tonoplast membrane indicated in white arrows.
Images were captured under YFP channel (Ex: 514 nm/Em: 520–
560nm) (I), RFP channel (Ex: 561nm/Em: 600–650nm) (II) and the
merged channel of both (III). Scale bar = 10µm

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and TL2 was also marginally higher but only under the salt
stress condition (Supplementary Fig. S5b).
K+ and Na+ concentrations were measured in the WT
and transgenic plants subjected to the control and salt treat-
ments (Fig.8c, d), to study the effect of PdNHX6 on ion
homeostasis under salt stress. The Na+ concentration was
significantly (p < 0.05) increased in the WT and transgenic
plants in response to salinity (Fig.8c), however, a significant
(p < 0.05) reduction in K+ concentration under salt stress
was detected only in the WT plants (Fig.8d). Neverthe-
less, the decrease in K+ concentration of transgenic plants
was insignificant compared to the control condition. This
observation may reflect the ability of the transgenic plants
to maintain the ionic balance under salinity stress, even with
the increased level of Na+ (Fig.8c).
For further evaluation of the role of PdNHX6, Arabi-
dopsis plants of three independent homozygous transgenic
lines, together with WT, were exposed to salinity on soil
(Fig.11a, b). At the end of the treatment, the EC of the con-
trol and salt-treated soils were 0.90 ± 0.45 (mean ± SD) and
44.9 ± 1.2 dS/m, respectively. Transgenic plants treated with
salinity showed better viability in comparison to the WT
(Fig.9a), and the transgenic lines were able to withstand the
salt stress, especially TL2 plants, which remained vigorous
and intact until the end of the treatment (after two weeks)
(Fig.9b). The total chlorophyll content was measured and
compared with the WT plants to evaluate the photosyn-
thetic efficiency of the transgenic plants. The transgenic
lines showed a higher chlorophyll level than the WT under
control and salinity treatment conditions (Fig.9c); while, the
increase in the chlorophyll content was significant (p < 0.05)
in two transgenic lines (TL1 and TL2) when exposed to the
salt stress (Fig.9c). Similarly, TL1 and TL2 showed a signif-
icantly (p < 0.05) higher RWC, compared to the WT plants,
under the salinity condition (Fig.9d).
The effect of salinity treatment was also monitored for
individual plants until the maturation stage (Fig.10). The
PdNHX6 transgenic lines showed enhanced tolerance to
salinity stress compared to the WT (Fig.10a, b). Although
these transgenic plants showed a higher salinity tolerance in
comparison with WT, these plants could not recover after
two weeks of salinity exposure (Fig.10c, d).
The germination rate ofthetransgenic line seeds
changed undersalinity
To evaluate the effect of salinity treatment on seed quality,
the germination rate of WT and transgenic lines seeds was
tested on plain MS plates (control) as well as on MS plate
supplemented with 100-mM NaCl (Fig.11a, b). The seeds
were incubated on the medium for seven days, and the ger-
mination rate was calculated each day.
Most of the WT and the transgenic seeds were able to
germinate on control plates but showed different germina-
tion rates based on the genotype. For example, after 24h
from stratification, the germination rate of TL1 seeds was
significantly (p < 0.05) higher than the wild type. Never-
theless, a significant (p < 0.05) increase in the germination
rate of TL1 and TL2 seeds, in comparison to the WT, was
Fig. 8 Generation and molecular characterization of PdNHX6 trans-
genic Arabidopsis lines. Schematic representation of the plasmid
construct overexpressing PdNHX6 fused with the Myc epitope (a).
Expression analysis of three independent homozygous PdNHX6
transgenic Arabidopsis lines (TL1, TL2, and TL3) and the WT using
semi-quantitative RT-PCR and shown on an ethidium bromide-
stained 1% agarose gel. Amplification products of Arabidopsis actin
gene (AtACT2) (AT3G18780) was used as the internal control (b) The
effect of PdNHX6 on intracellular Na+ (c) and K + (d) homeostasis
in Arabidopsis plantlets grown on MS-agar plates (shaded bars) and
MS-agar plates supplemented with 100-mM NaCl (dotted bars). The
bars represent the mean concentration ± SE of three independent bio-
logical replicates. The bars with different letters are significantly dif-
ferent at p < 0.05

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detected on the second and third days (Fig.11c). However,
most of the seeds used in this experiment have germinated
by the sixth day of the experiment (Fig.11b, c). Seeds
grown under salt treatment conditions showed different
germination patterns (Fig.11a, b). While the WT seeds
started germination only after 24h, the transgenic seeds
germinated earlier (Fig.11d). For example, 12% of TL2
seeds were germinated within 24h compared with 0% of
the WT seeds. Besides, the germination rate of TL1 seeds
was significantly (p < 0.05) higher than WT seeds on day
five, and both TL1 and TL2 seeds showed the highest ger-
mination rate until the seventh day.
Discussion
Plant tolerance to salt stress can be achieved by three major
strategies; Na+ or Cl− exclusion and secretion, Na+ or
Cl− compartmentalization in the tissues, and osmotic toler-
ance (Munns and Tester 2008). Transport of water and ions
(primarily Na+, K+, and Cl−) into and throughout the plants
is the fundamental action to these mechanisms (Craig Plett
and Møller 2010), where the antiport activity of Na+/H+and
K+/H+exchangers in the tonoplast vesicles is considered
as the first model of secondary active transport procedure
in plants (Rodríguez-Rosales etal. 2009). In this study, we
functionally characterized PdNHX6 as the first gene from
the date palm NHX family.
In silico analysis revealed that PdNHX6 shared the
Na+/H+exchanger 3 domain, which was characterized as
monovalent cation: proton antiporter-1 (CPA1), with other
members of NHX6 plant orthologs. This domain can cata-
lyze the electroneutral exchange of monovalent Na+ for H+
(Ma etal. 2015). Furthermore, computational analysis of
the PdNHX6 gene sequence revealed the existence of vari-
ous putative TFBSs recognized as abiotic stress-responsive
elements within the promoter region. Among these TFBSs,
bHLH is abundantly distributed within the putative PdNHX6
promoter region. A recent study indicated that under salin-
ity, the expression of the Arabidopsis AtNHX1 and AtNHX6
genes is regulated by two bHLH TFs (AtMYC2 and Atb-
HLH122) (Krishnamurthy etal. 2019). Additionally, binding
of AST1 transcription factor to a trihelix site in the promoter
regions of AtNHX2, AtNHX3, and AtNHX6 enhanced salinity
tolerance in Arabidopsis (Xu etal. 2018). The trihelix site
was also identified in this study within the putative PdNHX6
promoter. This finding may suggest a similar mode of inter-
action between PdNHX6 and the functional NHX previously
characterized in other plant species, upon exposure to the
saline environment. The expression level of this gene, as
determined by qPCR, significantly increased in the leaf tis-
sues under salt stress; however, this expression was insig-
nificantly altered in the root tissues when grown under the
same environmental conditions. This finding may suggest
that PdNHX6 is extruding Na+ from the leaf tissues via the
phloem. However, this proposed mechanism requires further
experimental justifications.
Overexpression of PdNHX6 in Arabidopsis plants
enhanced tolerance to salt stress. Transgenic lines exposed to
salinity showed higher photosynthetic efficiency in terms of
retaining more chlorophyll, as well as higher RWC than the
Fig. 9 Assaying the salinity tolerance ability of PdNHX6 transgenic
Arabidopsis lines in vivo. The soil-grown transgenic PdNHX6 lines
exposed to salinity (200-mM NaCl) after 7 (a) and 14 (b) days of
treatment. The chlorophyll content (c) and relative water content (d)
of WT and three homozygous PdNHX6 transgenic Arabidopsis lines,
exposed to salinity stress

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control WT plants. Similar results were obtained when other
date palm genes of salinity tolerance enhancement capabili-
ties such as the aquaporin gene PdPIP1;2 (Patankar etal.
2019a) and the metallothionein 2A (PdMT2A) (Patankar
etal. 2019b) were introduced into Arabidopsis.
The subcellular localization results showed that PdNHX6
was accumulated in the tonoplast membrane. While previ-
ous studies in different plant species showed that the NHX6
is accumulated in the endosomal membranes of the Golgi,
TGN, and PVC (Bassil etal. 2012; Jiang etal. 2010; Reguera
etal. 2015; Wang etal. 2015), this is the first study to show
that NHX6 can be a tonoplast-located antiporter. In fact,
Na+ influx has increased under salt stress conditions in both
PdNHX6 transgenic and WT plants.
Nevertheless, the transgenic plants could maintain a
balanced K+ level compared to the WT, which showed a
reduced intracellular K+ level under salt stress conditions. A
previous study showed that there are three conserved acidic
residues (D165, E189, and D194) in AtNHX6, which are
responsible for K+ transport and plant growth (Wang etal.
2015). Computational amino acid sequence analysis showed
that these residues are also present within the PdNHX6
sequence, which may perform a similar role in date palm
salt tolerance mechanism.
Overexpression of PdNHX6 in yeast mutant strain did
not improve yeast cell growth under both control and salt
stress. Conversely, a previous study showed that over-
expression of the Arabidopsis AtNHX6 in AXT3 mutant
yeast strain had enhanced the growth under salinity stress
conditions (Wang etal. 2015). Nevertheless, our data
revealed the involvement of PdNHX6 in Na+/K+ and pH
regulation in the yeast cells. Similar to the results obtained
from the analysis of the transgenic Arabidopsis, the trans-
genic yeast cells have accumulated higher Na+ levels than
the EV strain under salt stress conditions. However, the K+
accumulation was significantly higher than the EV under
Fig. 10 Assaying the salinity tolerance ability of PdNHX6 transgenic
Arabidopsis lines in vivo. The soil-grown transgenic PdNHX6 lines
and the WT exposed to salinity (200-mM NaCl) after 7 (a) and 14
(b) days of treatment. The transgenic lines showed tolerance to salt
stress and remained green even after 2 weeks of salt stress treatment.
To compare the recovery ability of the transgenic and the WT plants
after salt treatment, the plants were irrigated with distilled water for
an additional 2 weeks (c, d). After the first week of recovery period
(c), TL1 and TL2 plants showed better survival than the WT

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both control and salinity stress conditions. This notion is
consistent with the commonly known function of NHXs
by which this protein usually regulates the K+ transport
under normal growth conditions (Jiang etal. 2010); while,
it is involved in Na+ and K+ transport under salt stress
(Barragán etal. 2012; Reguera etal. 2015). Given the
notion that PdNHX6 regulates the vacuolar pH in yeast
cells, suggests that PdNHX6 might facilitate the exchange
of Na+/H+ across the vacuolar membranes. While these
NHX6 proteins are known for endosomal pH regulation
function by preventing acidification through the H+ leak-
age out of these compartments (Qiu 2016), the increase in
acidity of the vacuole of the transgenic yeast indicated by
our data could be more related to the vacuolar K+ trans-
portation, which was confirmed by the increase in the K+
level in the transgenic yeast. The increased level of H+ in
the vacuole generates a proton gradient that might be used
for K+ exchange through other tonoplast channels, such
as two-pore K+ channels (TPK1) (Latz etal. 2013). This
retains a higher cytosolic K+ level, a mechanism which is
essential for the salinity tolerance in plants. In addition,
the pH homeostasis is required for proper protein traffick-
ing to the vacuole as well as proteolytic processing of stor-
age proteins (Reguera etal. 2015). Therefore, this might
be another mechanism by which PdNHX6 may enhance
salinity tolerance in date palm. A previous study revealed
Fig. 11 Seed germination
assay of PdNHX6 transgenic
Arabidopsis lines and WT.
Seed germination on control
MS plates and MS plates with
100mM NaCl (salt stress) on
day 3 (a) and day 6 (b). The
same number of seeds were
used in each treatment and the
germination rate of seeds grown
under normal growth condition
(c) and salinity stress condition
(d) over 7 days was calculated.
The results shown are the
mean ± SE of four independent
biological replicates and the sta-
tistical significance at p < 0.05 is
indicated by asterisks (*)

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that NHX5 and NHX6 play a crucial role in the process-
ing of seed storage proteins (Qiu 2016), a mechanism that
leads to a reduction in the nitrogen and amino acid require-
ments during the germination process and early growth of
the seedlings (Dragwidge etal. 2018). Consistently, the
results obtained from this study showed that the overex-
pression of the PdNHX6 resulted in earlier germination of
the transgenic seeds. In addition, NHX5 and NHX6 can
regulate plant development through an auxin-dependent
mechanism (Dragwidge etal. 2018; Fan etal. 2018), a
mechanism, which may affect seed development and ger-
mination under stress.
In conclusion, the molecular and functional charac-
terization of the PdNHX6 conducted in this study high-
lighted the importance of this gene in salinity tolerance
response of the date palm. Heterologous expression of
PdNHX6 enhanced salinity tolerance through the K+ and
vacuolar pH homeostasis. The positive effect of these cel-
lular adjustments was apparent on the transgenic plants as
there was a significant increase in the chlorophyll, water
contents, and seed germination rates. Collectively, our data
suggest that this gene is a good candidate to explore the
development of salt-tolerant date palm tree…
Author contribution statement IA-H conceived, designed,
performed the experiments, analyze data, and wrote the
manuscript; GAJ performed the experiments, HVP revised
the manuscript, RA-Y revised the manuscript, SR and PPK
performed the subcellular localization of PdNHX6 and
revised the manuscript, and MWY designed the experiment,
supervised the work, wrote the manuscript, and contributed
reagents/materials/analysis tools.
Acknowledgements The authors would like to thank Professor Hana
Sychrova, Institute of Physiology Academy of Sciences of the Czech
Republic, Prague, Czech Republic, for donating the salt-sensitive
mutant S. cerevisiae BYT458 strain, which was used in this study.
Funding This study is supported by the generous grant number RC/
RG-SCI/BIOL/18/01 from the research council (TRC), Oman to MWY.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest. All authors revised and approved the final manuscript.
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