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8/12/2019 Lecture of Significance of Salt Stress
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Analysis of Sodium and Potassium Transporters
and their Association in Salt Stress Tolerance
Prof. P. B. Kavi Kishor
Department of Genetics
Osmania UniversityHyderabad 500 007
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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 hectaresof 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.
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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
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Salt Stress Mostly Affects Photosynthesis
Salt stress affects the closure of stomata
Salt stress influences the photosynthetic rate by affecting the rate ofCO2entry through the stomata
Photosynthesis decreases as a result of stomatal closure
Stomatal closure during salt stress reduces CO2 concentration at thephotosynthetic site thereby limiting both assimilation and theutilization of photochemically derived ATP and NADPH2
Bleaching of pigments occurs in chloroplasts during salt stress
Photosynthetic apparatus would become susceptible tophotoinhibition and photooxidation
Photorespiration is reduced at lower water potential which may limitthe plantsability to deal with surplus energy
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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.
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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 orweeks, 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 withinthe cytoplasm, especially in mesophyll cells in the leaf.
Toxicity occurs with time, after leaf Na+ increases to high
concentrations in the older leaves.
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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.
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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 thepassive 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
+
fromthe extracellular medium.
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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.
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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
+
Ca2+
H2O
?
Ca2+
H+
Na+
Na+
H+
Na+
pH 5.5
pH 7.5
H+
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Methodology Adapted in the Present Study
Genetic transformation in tomato and tobacco using Agrobacterium
NHX1gene isolation from sorghum
Transfer into pTZ57R/T intermediate vector
Construction of pCAMBIA1300 vector usingSOS1 orNHX1
Functional validation of transgene integration
I n sili coanalysis of NHX and HAK homologs
Transfer of NHX1into the vector PRT100
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NHX1Gene specific primers for partialgene 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 NHX1gene 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 geneis EU482408and protein ID is ACD64982.
NHX1(SOS1) Gene is Located on Chromosome 9 in Sorghum
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1.5 kb
M 1 2
pCAMBIA1300vector
2.3 kb
M 1
NHX1Gene is 1473 bp in Length in Sorghum and has 10 Exons and 9
Intronscloning 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 cDNA5' UTR
252 bp 320 bp
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MGLDLGALLKSGALSVSDYDAIVSINIFIALLCSCIVIGHLLEGNRWVNESITALVMGLIT
GGVILLVTNGTNSRILVFSEDLFFIYLLPPIIFNAGFQVKKKQFFRNFITIILFGAVGTLI
SFVIISLGAMGLFKKLDVGPLELGDYLAIGAIFSATDSVCTLQVLNQDETPLLYSLVFGEGVVNDATSVVLFNTIENLDIANFDAIVLLNFVGKFLYLFFTSTILGVATGLLSAYIIKKLCF
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.
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Different stages of regeneration and hardening of NHX1
transgenic tomato plants
Explants on selection medium Multiple shoots Rooting
Transgenic plants in the net house
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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
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Plant, Fruit and Seed Sizes are Reduced in Transgenics Tomatoes
control transgenic
control Transgenic
After transformation with NHX1gene, morphological changes were
observed in fruit size. Transgenic plants produced small sized fruits with
more yield compared to untransformed control plants.
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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
NHX1PCR GFP PCR hptIIPCR
PCR Amplification of NHX1was Observed in Transgenics but not in Controls
750 bp776 bp 752 bp
776 bp
752 bp
750 bp
M +C C 1 2 3 4RT-PCR Showed Transcript Levels only in Transgenics
M +C C 1 2 3 4M +C C 1 2 3 4M +C C 1 2 3 4
M +C C 1 2 3 4M +C C 1 2 3 4
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Southern hybridization of genomic DNA samples of transgenicplants 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
NHX1gene integration was confirmed by digesting the genomic DNA from
transgenic plants with HindIII restriction enzyme and probed with genespecific 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
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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.
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Transgenics are Confirmed with GFP using Flow Cytometer
In the above graph, green peak is
untransformed control plant withautofluorescence and black peak is
transgenic with GFP reporter gene
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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 cutfrom 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
fluorescence
Transgenic Root Control root Transgenic stem Control stem
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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 stressTransgenic 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)
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Comparative studies have been carried out in order to characterize theNa+ 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 greatinterest 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. NHX1and HAK1also belong to this family
Genome-wideI n sil ico Analysis of Cation (Na+and K+) Transporters
is Necessary to Know the Number of Genes and Tissue Localizations
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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, NHX1and NHX3genes are highly
conserved and present on same chromosome, likewise NHX2, NHX4,NHX5and NHX6
genes are also present on same chromosome regions.
Synteny Analysis for NHX1 Revealed more Block Duplication Events
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Synteny Analysis for NHX1Revealed 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 NHX1gene
Tandem duplication
Block duplication
NHX Homologs are Highly Conserved but Evolutionarily Diverged
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NHXHomologs 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. NHX1gene isolated from Sorghumbicoloris closely related to NHX3gene of Oryza sativa.
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Distance matrix histogram showing the functional diversity of Na+
transporters using DIVEIN tool as a tree based method
Not Much Functional Divergence of NHXTransporters
Has Been Observed Using DIVEIN Tool
P t i P t i I t ti M f NHX1
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Protein-Protein Interaction Map of NHX1
NHX proteins have produced
20 interactors in yeast
andArabidopsis
NHX Transporters Comparison Table Among Sorghum Maize and Rice
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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
NHEisoform5 5 955 2868 3 2 P 7748
22474
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
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Comparative Map of HAK Transporters Revealed that HAK1
and KUP1Have Evolutionary Lineage
Top hits of K+transporters were collected from sorghum, rice and
maize and uploaded into cMAP tool using PERL script.
S t M f HAK1 G A th Th Diff t S i
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Synteny Map of HAK1Gene Among the Three Different Species
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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
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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 geneisolated from Sor hum bicoloris closel related to KUP1 ene
U lik NHX T HAK T
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Distance matrix histogram showing the functional diversity of K+
transporters using DIVEIN tool as a tree based method
Unlike NHXTransporters, HAKTransporters are
Functionally Diverged as Indicated by DIVEIN
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Protein-Protein Interaction Map of K+Transporters
22 Interactors with KUP
Protein Have Been
Observed with Arabidopsis
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Complex Network of Sodium and Potassium Transporters
SOS1 is Interactingwith RCD1 (radical induced
Cell death1), potassium
transporters, calcium
exchangers and NHX proteins
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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 T0 and T1 transgenics for gene
integration was confirmed by molecular methods like PCR, RT-PCR and Southern blotting.
I n sil icoanalysis of cation transporters (Na+and K+) was done.
cMAPS and synteny plots at genome level for 3 grass species were
generated.
Conclusions
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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 andArabidopsis. All genes excepting NHX1 and NHX3were
found to have orthologs while NHX5alone is known to have nine
interacting partners in Arabidopsis
Phylogenetic analysis reveals that NHX1and NHX2originated viaevolutionary 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
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Functions of HAK: Potassium uptake, membrane transport, inwardand 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 wasused)
Expressed during: 15 growth stages (using PLAZA bioinformatics
tool)
Phylogenetic analysis reveals that HAK1and KUP1originated via
an evolutionary lineage-specific events
Conclusions