Analysis of Sodium and Potassium Transporters

and their Association in Salt Stress Tolerance

Prof. P. B. Kavi Kishor


Department of Genetics
Osmania University
Hyderabad 500 007

There is a need for Understanding Molecular Mechanisms of
Salt Stress Since the Problem of Salinity is Severe

World population has steeped up to 6.663 billion and if this feature of
growth continues, world population would reach up to 9.2 billion by
2050.

Out of 14 billion ha of available land on earth for farming, arid and
semi-arid regions compose 6.5 billion ha. There are 1200 million hectares
of land that is affected by soil salinity.

With the increase of salinity in the soils over the years, it is expected
that by 2050, more than 50% of the available land for agriculture will be
lost because of severe salinity.

The exhaustion of essential resources such as fresh water has imposed
serious constraints on the cultivation of food crops.

Hence, there is every need to understand molecular mechanisms
underlying salt stress tolerance in plants.
Outline of the Cardinal Responses of Plants to Salt and
Drought Stresses
Salinization of the medium

Depression of the external
w

Perception of stress by the plant

Uptake of ions

Little Much

Synthesis of organic solutes High internal salt concentration

Little Much Toleration Susceptibility

Low tissue concentration, Osmotic Damage to membranes
Low turgor adjustment organelles, enzymes
Salt Stress Mostly Affects Photosynthesis
Salt stress affects the closure of stomata

Salt stress influences the photosynthetic rate by affecting the rate of
CO
2
entry through the stomata

Photosynthesis decreases as a result of stomatal closure

Stomatal closure during salt stress reduces CO
2
concentration at the
photosynthetic site thereby limiting both assimilation and the
utilization of photochemically derived ATP and NADPH
2

Bleaching of pigments occurs in chloroplasts during salt stress

Photosynthetic apparatus would become susceptible to
photoinhibition and photooxidation

Photorespiration is reduced at lower water potential which may limit
the plants ability to deal with surplus energy
There are three Distinct Types of Plant Responses or
Tolerance to Salt
The mechanisms of salt tolerance fall into three categories :
1. Tolerance to Osmotic Stress :

The osmotic stress immediately reduces cell expansion in root tips and
young leaves, and causes stomatal closure.

Plants synthesize and accumulate osmotic agents such as proline and
glycine betaine in cytoplasm, but accumulate sugars, and K
+
in
vacuoles and other compatible solutes to adjust osmotic strength.

A reduced response to the osmotic stress would result in greater leaf
growth and stomatal conductance, but the resulting increased leaf area
would benefit only plants that have sufficient soil water.

Greater leaf area expansion would be productive when a supply of
water is ensured such as in irrigated food production systems, but
could be undesirable in water limited systems, and cause the soil water
to be used up before the grain is fully matured.
Mechanisms of Salt Tolerance are Three Types
2. Na
+
exclusion from root plasma membrane (by SOS pathway) and
leaf blades (through salt glands) :

Na
+
exclusion by roots ensures that Na
+
does not accumulate to toxic
concentrations within leaves.

A failure in Na
+
exclusion manifests its toxic effect after days or
weeks, depending on the species, and causes premature death of older
leaves.
3. Tissue tolerance to Na
+
and Cl
-
:
Tolerance requires compartmentalization of Na
+
and Cl
-
at the
cellular and intracellular level to avoid toxic concentrations within
the cytoplasm, especially in mesophyll cells in the leaf.

Toxicity occurs with time, after leaf Na
+
increases to high
concentrations in the older leaves.
Na
+
and K
+
Uptake Systems in Plants is Complicated
Na
+
is toxic at high millimolar concentrations.

Plants require high K
+
compared to Na
+
.

K
+
is an essential macronutrient and the most abundant cation in
plants.

K
+
contributes up to 10% of total plant dry weight and plays an
important role in the activation of enzymatic reactions, photosynthesis,
protein synthesis, maintenance of cellular osmolarity, regulation of turgor,
stomatal movement and cell elongation.

K
+
uptake in plants is mediated by two mechanisms, a low affinity
system (KIRCs, KORCs) that functions when extracellular K
+

concentration is high (> 200 M to mM) and a high affinity system (KUP-
HAK) that functions at low extracellular K
+
concentrations.

The ratio of K
+
/Na
+
in plant cells will depend on the concerted action of
transport systems located at plasma and vacuolar membranes and
probably involves K
+
selective, Na
+
selective and non-selective pathways.



Sodium Uptake

Under typical physiological conditions, plants maintain a high
cytosolic K
+
/Na
+
ratio.

Given the negative membrane potential difference at the plasma
membrane (- 140 mV), a rise in extracellular Na
+
concentration
will establish a large electrochemical gradient favouring the
passive transport of Na
+
into the cells (through non-selective cation
channels, i.e. NSCC).

Na
+
ions can be transported into the cell through K
+
transporters.
Plants use low and high affinity transporters to take up K
+
from
the extracellular medium.





Sodium Efflux at the Plasma Membrane is Through
Salt Overly Sensitive Protein (SOS1)
In plants, the main mechanism for Na
+
efflux is mediated by the
plasma membrane H
+
-ATPase.

The H
+
-ATPase uses the energy of ATP hydrolysis to pump H
+

out of the cell, generating an electrochemical H
+
gradient.

This proton motive force generated by the H
+
-ATPase operates
the Na
+
/H
+
antiporters, which couple the movement of H
+
into
the cell along its electrochemical gradient to the extrusion of Na
+

against its electrochemical gradient.

It is the SOS1 (or sodium proton antiporter, NHX1) protein
located at the plasma membrane in concert with SOS2 and SOS3
complex helps in exclusion of Na
+
ions out of the cell.


Regulation of Na
+
and K
+
Homeostasis by the SOS pathway
High Na
+
H
+
V-ATPase
P Pase
SOS3

SOS2
Vacuole


NHX
HKT1
?
SOS3
Transcriptional and
posttranscriptional
gene regulation
SOS1
SOS2
K
+
Ca
2+
H
2
O
?
Ca
2+
H
+
Na
+
Na
+
H
+
Na
+
pH 5.5
pH 7.5
H
+
Methodology Adapted in the Present Study
Genetic transformation in tomato and tobacco using Agrobacterium
NHX1 gene isolation from sorghum
Transfer into pTZ57R/T intermediate vector
Construction of pCAMBIA1300 vector using SOS1 or NHX1
Functional validation of transgene integration
I n silico analysis of NHX and HAK homologs
Transfer of NHX1 into the vector PRT100

NHX1 Gene specific primers for partial
gene were designed based on homology of
the gene isolated from different species
using NCBI, CLUSTALW and IDT tools.

Partial NHX1 gene sequence was used
to localize full length NHX1 gene using the
blastpar-simple.html tool with complete
Sorghum genome sequence and the
position of gene was retrieved, which is on
chromosome 9.


Genbank accession number for this gene
is EU482408 and protein ID is ACD64982.
NHX1 (SOS1) Gene is Located on Chromosome 9 in Sorghum
1.5 kb
M 1 2

pCAMBIA1300
vector
2.3 kb
M 1
NHX1 Gene is 1473 bp in Length in Sorghum and has 10 Exons and 9
Introns
cloning and characterization of full length NHX1 gene


Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10
Arrows represent the exons (10) while bars represent the intron regions (9)
3' UTR
CODING REGION 1473 bp

Diagrammatic representation of full length NHX1 cDNA
5' UTR
252 bp 320 bp
MGLDLGALLKSGALSVSDYDAIVSINIFIALLCSCIVIGHLLEGNRWVNESITALVMGLIT
GGVILLVTNGTNSRILVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIILFGAVGTLI
SFVIISLGAMGLFKKLDVGPLELGDYLAIGAIFSATDSVCTLQVLNQDETPLLYSLVFGEG
VVNDATSVVLFNTIENLDIANFDAIVLLNFVGKFLYLFFTSTILGVATGLLSAYIIKKLCF
ARHTFATLSFIAEIFLFLYVGMDALDIEKWKLASSSPKKPIALSAIILGLVMVGRAAFVFP
LSFLSNLSKKEARPKISFKQQVIIWWAGLMRGAVSIALAYNKFTSSGHTEVRVNAIMITST
VIVVLFSTMVFGLLTKPLLSLLIPPRTGLNTSSLLSSQSILDPLLTSMVGSDFDVGQINSP
QYNLQFILTAPTRSVHRLWRKFDDRFMRPMFGGRGFVPFVPGSPVERSVPEPHLGTVTEAE
HS

NHX1 has 490 Amino Acids and 8 Transmembrane Segments
The complete amino acid sequence was subjected to MEME online analysis tool with following parameters: a) width of motifs from 6 to 50,
b) maximum number of motifs (10), c) distribution of single motif among the sequence is one per sequence. Red color- Amelioride binding
site, Blue and Orange NHX signature sequence

8 transmembrane segments are present in NHX1 gene with a hydrophobicity
plot of 0.68. In the graph red color peaks are the transmembrane regions.


Different stages of regeneration and hardening of NHX1
transgenic tomato plants
Explants on selection medium Multiple shoots Rooting
Transgenic plants in the net house
Shoot initiation Shoot elongation Development of roots
Different stages of regeneration and hardening of NHX1 transgenic
tobacco plants
Transgenic plants in the net house
Explants on selection medium
Plant, Fruit and Seed Sizes are Reduced in Transgenics Tomatoes
control transgenic
control Transgenic
After transformation with NHX1 gene, morphological changes were
observed in fruit size. Transgenic plants produced small sized fruits with
more yield compared to untransformed control plants.
PCR and RT-PCR amplification in control and transgenic plants. M =
Molecular weight marker; +C = positive control, C = non-transformed control;
Lanes 1-4 = transgenic plants
NHX1 PCR GFP PCR
hptII PCR
PCR Amplification of NHX1 was Observed in Transgenics but not in Controls
750 bp
776 bp
752 bp
776 bp
752 bp
750 bp
M +C C 1 2 3 4
RT-PCR Showed Transcript Levels only in Transgenics
M +C C 1 2 3 4
M +C C 1 2 3 4 M +C C 1 2 3 4
M +C C 1 2 3 4
M +C C 1 2 3 4
Southern hybridization of genomic DNA samples of transgenic

plants using NHX1 gene specific probe +C = vector DNA; C =
un-transformed control. Lanes 1 4 = transgenic plants.
Southern Blot Revealed Insertion of NHX1 Gene in Transgenics
NHX1 gene integration was confirmed by digesting the genomic DNA from
transgenic plants with HindIII restriction enzyme and probed with gene
specific probe. The autoradiogram generated after exposing the blot to the
film showed positive hybridization at the 776 bp region for each putative
transgenic plant samples except in untransformed control plants
+C C 1 2 3 4
NHX1 Protein is Localized at the Plasma Membrane Level
Localization of NHX1 protein at plasma membrane level using Leica
confocal microscope. T.S. of tomato transgenic leaf with GFP in green color
and chlorophyll pigment in red color

To localize the NHX1 protein at membrane level, GFP peak was measured at 510 nm. Since
plants contain many auto florescent compounds including lignin, GFP was masked at the same
wavelength. To minimize this problem, auto fluorescence compounds were given red color but
GFP with green color at the same wave length.
Transgenics are Confirmed with GFP using Flow Cytometer
In the above graph, green peak is
untransformed control plant with
autofluorescence and black peak is
transgenic with GFP reporter gene
With 150 mM NaCl Treatment, Untransformed Plants
Wilted and Turned Brown, But not Transgenics
Transgenic control
Control Transgenic
One month old untransformed control and T
1
transgenic seedlings were
watered with 150 mM NaCl for 72 h. Control plants wilted within 5 days,
while the transgenic seedlings were not affected and grew normally
Fluorescence Lifetime Imaging Revealed Less Sodium
Green Fluorescence
in Transgenic Roots and Stems Compared to Controls
9-day-old seedlings treated with 150 mM NaCl. Root and stem segments were cut
from the mature zone and incubated in 10 mM of Sodium Green solution. After 1
h of incubation, samples were examined using confocal microscopy. Na
+
content
in each cell compartment is proportional to the intensity of Sodium Green
uorescence
Transgenic Root Control root
Transgenic stem Control stem
Type of
Sample
Type of
explant

With stress (% by mass/PPM)

Without stress (% by mass/PPM)
Ions Ca
2+
K
+
Na
+
Cl
-
Ca
2+
K
+
Na
+
Cl
-

Untransformed
Control
Stem 2.38%
23800
3.75%
37500
1.29%
12900
0.38%
3800
0.14%
1400
0.20%
2000
0.04%
400
0.25%
2500
Leaf 0.44 %
4400
0.39%
3900
0.06%
600
0.09%
900
0.30%
3000
0.40%
4000
0.03%
300
0.55%
5500
Root 0.09%
900
0.01%
100
0.10%
1000
0.20%
2000
3.53%
35300
0.28%
2800
0.60%
6000
1.19%
1900
Transgenic-I Stem 0.96%
9600
0.57%
5700
0.58%
5800
0.57 %
5700
1.52 %
15200
0.06%
600
0.35%
3500
0.98%
9800
Leaf 0.50%
5000
0.20%
2000
0.04%
400
0.15%
1500
0.79%
7900
0.20 %
2000
0.02 %
200
0.07 %
700
Root 0.21%
2100
0.002%
200
0.05%
500
0.12%
1200
0.22%
2200
0.06%
600
0.11%
1100
0.06%
600
Transgenic-II Stem 0.60%
6000
0.29%
2900
0.53%
5300
0.49%
4900
1.43 %
14300
0.08%
800
0.35%
3500
0.89%
9800
Leaf 0.40%
4000
0.13%
1300
0.032%
320
0.09%
900
0.74%
7400
0.25 %
2500
0.03 %
300
0.78 %
780
Root 1.68%
16800
0.27%
2700
0.041%
410
0.82%
8200
0.31%
3100
0.05%
500
0.17%
1700
0.67%
670
Ion Analysis by ICP-OES Revealed Less Na
+
, But
more K
+
and Ca
2+
in Transgenics

Compared to Controls
Data indicate average of 4 samples taken from two independent transgenic lines. Ion analysis
(% by mass/PPM) of control and transgenic plants (I and II) with and without stress using
inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) instrument.
Chlorophyll Fluorescence Varied Significantly Between
Transgenics and Untransformed Controls
Control without stress
Transgenic 1 without stress
Transgenic 2 without stress
Transgenic 3 without stress
Control with stress
Transgenic 1 with stress
Transgenic 2 with stress
Transgenic 3 with stress
Transgenic 1 after recovery
Transgenic 2 after recovery
Transgenic 3 after recovery
Fluorescence variation was observed under normal and saline (150 mM NaCl)
conditions. Transgenics showed more tolerance and recovered after stress , whereas
untransformed control plants eventually died after 10 days
Chlorophyll fluorescence of control and transgenic plants were measured using
Handy Pea instrument under dark conditions with 3 parameters (without stress,
150mM NaCl for 72 h and after recovery from stress)
Comparative studies have been carried out in order to characterize the
Na
+
and K
+
transporter genes that determine morphological and
functional similarities of the grasses

The idea behind this study is that the knowledge gained in one species
can be easily transferred to other grass species which will be of great
interest for genes controlling salt stress and K
+
nutrition

Comparative studies can also result in an increase in our
understanding of the evolutionary mechanisms that have led to the
current structure of grass genomes

Cation proton antiporter (CPA1) family is a large family comprising of
3665 genes with 46 unique families. Of these, many genes with unknown
function are present. NHX1 and HAK1 also belong to this family
Genome-wide I n silico Analysis of Cation (Na
+
and K
+
) Transporters
is Necessary to Know the Number of Genes and Tissue Localizations
Comparative map of NHX1 homologs among 3 species
NHX Transporters are not Present on Chromosomes 4, 6 and 7 in Sorghum,
3, 5 and 9 in Maize, 3 and 4 in Rice
Features in red have correspondances, red line denotes automated name based correspondance
Top hits of Na
+
transporters were collected from sorghum, rice and maize and uploaded
into cMAP tool using PERL script. In each plant, NHX1 and NHX3 genes are highly
conserved and present on same chromosome, likewise NHX2, NHX4, NHX5 and NHX6
genes are also present on same chromosome regions.
Synteny Analysis for NHX1 Revealed more Block Duplication Events
than Tandem Duplication Events
Duplication type Count Percentage
No Duplication
event
16 61.54 %
Tandem
duplication event
3 11.54 %
Block duplication
event
9 34.62 %
Tandem & block
duplication event
2 7.69 %
Gene duplication type venn diagram and table
Synteny map of NHX1 gene
Tandem duplication

Block duplication
NHX Homologs are Highly Conserved, but Evolutionarily Diverged
NHX homologs are highly conserved but are evolutionarily diverged about maximum due to
genome duplications 700 million years ago among monocots. NHX1 gene isolated from Sorghum
bicolor is closely related to NHX3 gene of Oryza sativa.
Distance matrix histogram showing the functional diversity of Na
+

transporters using DIVEIN tool as a tree based method
Not Much Functional Divergence of NHX Transporters
Has Been Observed Using DIVEIN Tool
Protein-Protein Interaction Map of NHX1
NHX proteins have produced
20 interactors in yeast
and Arabidopsis
Gene
name
Sorghum Maize Rice
Chr a.a ORF E TM L chr a.a ORF E TM L chr a.a ORF E TM L
NHX1 9 490 1473 12 9 V 10 304 915 7 5 P 5 454 1365 8 8 V
NHX2 2 784 2355 15 8 P 7 518 1557 13 10 Cy 7 458 1377 10 10 P
NHX3 9 823 2472 17 8 P 10 539 1620 12 10 Cy 5 544 1635 13 10 V
NHX4 2 696 2091 16 4 P 7 518 1557 13 10 Cy 7 458 1377 10 10 P
NHX5 2 696 2091 15 4 P 7 518 1557 13 10 Cy 7 479 1440 11 9 P
NHX6 2 696 2091 15 5 P 7 518 1557 13 10 Cy 7 479 1440 12 9 P
NHX7 4 558 1677 3 0 N 6 612 1839 9 1 N
NHX8 8 720 2163 6 0 Ch 1 829 2490 14 0 N 12 1025 3078 16 6 Cy
NHE
isoform1
8 597 1794 5 0 Ch 8 884 2655 12 0 N 11 806 2421 8 0 N
NHE
isoform2
3 1127 3384 2 0 Cy 10 819 2460 10 0 N 2 817 2451 7 0 N
NHE
isoform3
3 726 2181 4 0 N 7 1278 3837 2 0 P 10 733 2202 1 0 M
NHE
isoform4
10 1150 3453 7 0 N 6 1007 3024 5 0 N 5 1189 3570 8 0 Ch
NHE
isoform5
5 955 2868 3 2 P 7 748 2247 4 0 N 1 812 2439 1 1 N
NHE
isoform6
2 699 2100 3 0 N 7 765 2298 8 0 N 8 810 2433 7 0 N
NHE
isoform7
1 686 2061 1 0 N 6 909 2730 3 0 Cy 2 630 1890 1 1 P
NHE
isoform8
1 908 2727 8 1 V 8 955 2868 6 0 Ch 5 561 1686 5 6 P
NHE
isoform9
2 596 1788 3 0 Ch 4 1033 3099 5 0 Cy 9 595 1788 3 0 Ch
Chr-chromosome number from which the gene sequence is retrieved, a.a-amino acid sequence, ORF-open reading frame, E-number of exons, TM-number of transmembrane segments. L-
predicted cellular location of gene. Ch-chloroplast. Cy-cytosol. M-mitochondria. N-nuclear. P-plasmamembrane. V-vacuolar membrane.

NHX Transporters Comparison Table Among Sorghum, Maize and Rice
Comparative Map of HAK Transporters Revealed that HAK1
and KUP1 Have Evolutionary Lineage
Top hits of K
+
transporters were collected from sorghum, rice and
maize and uploaded into cMAP tool using PERL script.
Synteny Map of HAK1 Gene Among the Three Different Species
Duplication type Count Percentage
No Duplication event 22 52.38%
Tandem duplication
event
7 16.67%
Block duplication
event
14 33.33%
Tandem & block
duplication event
1 2.38%
Tandem duplication

Block duplication
More Block Duplication Events were Observed
Compared to Tandem Duplications also for HAK1 Gene
HAK Homologs are Conserved but Diverged Evolutionarily
K
+
homologs are highly conserved but are evolutionarily diverged about maximum
due to genome duplications, 700 million years ago among monocots. HAK1 gene
isolated from Sorghum bicolor is closely related to KUP1 gene
Distance matrix histogram showing the functional diversity of K
+

transporters using DIVEIN tool as a tree based method
Unlike NHX Transporters, HAK Transporters are
Functionally Diverged as Indicated by DIVEIN
Protein-Protein Interaction Map of K
+
Transporters
22 Interactors with KUP
Protein Have Been
Observed with Arabidopsis
Complex Network of Sodium and Potassium Transporters
SOS1 is Interacting
with RCD1 (radical induced
Cell death1), potassium
transporters, calcium
exchangers and NHX proteins
Full length NHX1 gene was isolated from Sorghum bicolor and
cloned into pCAMBIA1300 binary vector and transformed into
Agrobacterium tumefaciens LBA4404 strain using freeze thaw
method.

NHX1 gene characterization showed that it has 8
transmembrane segments with 10 exons and 9 introns.
Genetic transformation of tomato and tobacco plants was
carried out and putative transgenics were selected on selection
media.

Preliminary screening of T
0
and T
1
transgenics for gene
integration was confirmed by molecular methods like PCR, RT-
PCR and Southern blotting.

I n silico analysis of cation transporters (Na
+
and K
+
) was done.
cMAPS and synteny plots at genome level for 3 grass species were
generated.

Conclusions
SOS1: shows sodium proton antiporter activity

Located in: integral to membrane

Expressed in: 23 different plant tissues (when PLAZA tool was
used)

Expressed during: 13 growth stages (using PLAZA
bioinformatics tool)

The NHX genes were shown to have produced 20 interactors in
yeast and Arabidopsis. All genes excepting NHX1 and NHX3 were
found to have orthologs while NHX5 alone is known to have nine
interacting partners in Arabidopsis
Phylogenetic analysis reveals that NHX1 and NHX2 originated via
evolutionary lineage-specific events

The interesting fact found with above tools is that NHX gene
interacts with Radical induced Cell Death (RCD1), a regulator of
oxidative stress and also expresses in plant structures during
different stages
Conclusions
Functions of HAK: Potassium uptake, membrane transport, inward
and outward rectifier potassium activity, cyclic nucleotide binding,
root hair elongation etc.

Located in: integral to membrane

Expressed in: 26 plant structures (or tissues when PLAZA tool was
used)

Expressed during: 15 growth stages (using PLAZA bioinformatics
tool)
Phylogenetic analysis reveals that HAK1 and KUP1 originated via
an evolutionary lineage-specific events


Conclusions