Lecture of Significance of Salt Stress

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



Control with stress

Transgenic 1 with stressTransgenic 2 with stress

Transgenic 3 with stress

Transgenic 1 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

Documents

Lecture of Significance of Salt Stress