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PhD thesis presentation of Cosentino Cristian.
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Damstadt, 31 July 2008
Cosentino CristianAG Thiel/Homann
Na+/H+ transporters of the halophyte
Mesembryanthemum crystallinum L.
Need of food production
0%
10%
20%
30%
40%
50%
60%
2000 2010 2020 2030 2040 2050
precen
t of
increase
Wild (2003), Cambridge University Press
Land affected by soil salinity
The problem of soil salinization
Soil erosion and salinization15% of total land area
Soil salinity1/3 of irrigated area
(Regansamy (2006) J. Exp. Bot. 57, 1017-1023)
Na+ damages on cellular metabolism
Disruption of enzymatic activities
Na+
K+
K+
Na+
Na+
H2ONa+ Na+
H2O
Osmotic imbalance and loose of water
Ψwaterin > Ψwater
out
K+
Na+
AA
Disruption of the protein synthesis process1 2 3
Necrosis of leaves
Decrease of plant growth and yield
Up to1000 mM Na+
vac
20 mM Na+in
100 mM K+in
400 mM Na+out
pH 5.5vac
pH 5.5out
pH 7.5in
↑ Na+out
Chloroplast
Vacuole
Na+cyt
2. Halophyte salt includer
1. Switch C3 CAM
Mesembryanthemum crystallinum as model plant
Most important mechanisms of Na+ tolerance
Na+ internal compartmentalization
2Na+ extrusion to
the apoplast1
Na+vac
Na+out
Na+cyt
Na+cyt
Vacuole
Chloroplast
CO2
Sugar
Calvin cycle
Cytoplasm
C3 vs CAM
C3
CO2
Vacuole
Chloroplast
Malic acid
CO2
Sugar
Calvin cycle
Cytoplasm
CAM
↓ Transpiration↓ Photorespiration
C3 CAM
(1000 µmol m-2 sec-1 for 2 weeks)
High light
NaCl stressed plants shift to CAM in 2 weeks instead of >8 weeks
NaCl stress
(400 mM NaCl for 2 weeks)
Stress-triggered induction of CAM
Malic Acid
Malic Acid and Na+
Malate turnover
(metabolic purpose)Na+ sequestration
(osmotic and detoxification purposes)
Malic Acid2-
VacCyt
CO2
↑ DµH+
Malic Acid2-
VacCyt
↓ DµH+
VacCyt
H+
Na+
H+
Static functionDynamic function
Double vacuolar function under salt stress-triggered CAM
Where in the leaf mesophyll does Na+ accumulate?
Neutral Acidic
Vacuolar pH distribution
Mesophyll cells can have acidic as well as neutral vacuoles
into epidermal bladder cell around vascular tissuesaround epidermal bladder cells
Vacuolar Na+ distribution
100 µm100 µm200 µm
Does Na+ accumulate in acidic or neutral vacuoles?
pH and Na+ vacuolar distributions
More uniform distribution of Na+ accumulation over a wider range of vacuolar pH
pH Na+
pH staining Na+ staining
300 µm200 µm
Neutral Red (pH)
CoroNa Green (Na+)
0%
20%
40%
60%
80%
acidic neutral acidic neutral
CAM C3
14
19
4
9
NeutralAcidic NeutralAcidic
C3 CAM
Na+ accumulating vacuoles
Na+ preferentially accumulates into neutral vacuoles
Correlation of Na+ accumulation and vacuolar pH
100 µm
Mesophyll cells can have acidic as well as neutral vacuoles
Na+ accumulates mainly into and around epidermal bladder cells and vascular tissues
Na+ preferentailly accumulates in neutralvacuoles
How does Na+ enter the vacuole?
Na+/H+ antiporters mediate the Na+ transport
Up to1000 mM Na+
vac
20 mM Na+in
100 mM K+in
400 mM Na+out
pH 5.5vac
pH 5.5out
pH 7.5in
Na+out
H+
H+
ATP
ATP
P-ATPase
V-ATPase
PPi
H+V-PPase
ATP
V-ATPaseH+
+ + +
- - - -
Chloroplast
Vacuole
Na+vacNa+
H+Na+
H+SOS1NHX
AtSOS1 mediates Na+ extrusion from the cell (Arabidopsis thaliana)
AtNHX1 mediates Na+ accumulation into the vacuole (Arabidopsis thaliana)
Na+out
Na+cyt
Na+cyt
Which Na+/H+ antiporters are expressed in
Mesembryanthemum crystallinum?
The cloned antiporters
Antiporter aa MWPredicted
TMDPredicted
localisation
McSOS1 1115 127.3 12 Plasmamembrane
McNhaD 577 61.5 13 Chloroplast
McNHX1 549 60.8 10 Vacuole
McNHX2 556 61.6 11Vacuole/Pre-
vacuole
McNHX3 526 58.1 10 Endomembrane
Phylogenetic relationships
Antiporters from halophyte plants
Are the cloned antiporters functional?
Functional complementation
Organism Mutation SelectionFunctionalcomplementation
Saccharomycescerevisiae
nha1 nhx1 ena1-4 200 mM NaCl McSOS1 and McNhaD
nhx1 Hygromycin B McNHX1
Escherichia coli nhaA nhaB 200 mM NaCl, 5 mM LiCl McNhaD
Is the expression of the cloned antiporters affected
by salinity stress?
Na+-triggered expression of the Na+/H+ antiporters
Antiporter Leaf Root
McSOS1 1.8 ↑ Unchanged
McNhaD 7.8 ↑ Unchanged
McNHX1 3.6 ↑ Unchanged
McNHX2 Not detectable Not detectable
McNHX3 Unchanged Unchanged
Five antiporters belonging to different families have been cloned
The complementation experiments show that the cloned antiporter are functional
The expression of McSOS1, McNhaD and McNHX1 is induced in leaf by NaCl treatment
Up to1000 mM Na+
vac
20 mM Na+in
100 mM K+in
400 mM Na+out
H+
H+
ATP
ATP
pH 5.5vac
pH 5.5out
pH 7.5in
P-ATPase
V-ATPase
Na+out
PPi
H+V-PPase
ATP
V-ATPaseH+
Na+
H+
H+
Na+
H+
Na+
+ + +
- - - -
Chloroplast
Na+Vacuole
McSOS1
McNhaDMcNHX2-3
McNHX1
Expectd function for the cloned antiporters
How are the expression of McNhaD and Na+
accumulation correlated?
M. crystallinum mesophyll cell
Na+ accumulates in chloroplasts
Na+ is accumulated into chloroplasts
McNhaD:GFP localizes at the plastidial membrane
McNhaD localizes at the chloroplast envelope
Plastidial envelope
1 MASSLSSTSY YLSSSRPRKR PSLSWSPPYP VILRRASPAT VNSPSSLNTK PWGGKCRRYL
61 HRDGVIVRVE DKARGLSSSS PELEDLSSGE CDPLCSVDEI SSRDVEATYQ PKTDLVKALA
121 ILAAALTGTV AINHSWVAAN QDLALALLFG IGYAGIIFEE SLAFNKSGVG LLMAVSMWVI
181 RSIGAPSTEV AVAELQHATG EVSEIVFFLL GAMTIVEIID AHQGFKLVTE NITTRKPRTL
241 LWVVGFVTFF LSAILDNLTS TIVMVSLLRK LVPPSEYRKL LGAVVVIAAN AGGAWTPIGD
301 VTTTMLWIHG QLSTLPTMKD LIIPSAISLA VPLALMSLTS EANGKEESSS VMASEQMAPR
361 GQLVFAVGVG ALVSVPVFKS VTGLPPYMGM LLGLGVLWIL TDAIHYGESE RQRLKVPQAL
421 SRIDTQGALF FLGILLSVSS LESAGILREL ANYLDAHIPN IELIASAIGV VSAIVDNVPL
481 VAATMGMYDL TSFPKDSEFW QLVAFCAGTG GSMLIIGSAA GVAFMGMEKV DFFWYLKKVS
541 GFAFAGYAAG IAAYLAMHNL NFSLPTTLAH VPFISGS
ChloroP 1.1
McNhaD
Correlation
Na+chl
McNhaD is most likely involved in Na+ accumulation into chloroplasts
MaNhaD and Na+chl are correlated
McNHaD 7.8 fold of increase Na+chl 9.7 fold of increase
McNhaD:GFP localizes at the plastidial membrane
Na+ accumulates into Mesembryanthemum crystallinum chloroplasts
McNhaD most likely mediates the Na+ accumulation into the chloroplast
McNHX1Na+
H+
Na+
H+
McNhaD
Na+
H+
Na+
H+
Na+
H+
McNHX2-3
McSOS1
McSOS1
Na+
myo-inositol
McITR1
Na+
McHKT1
Na+
McHAK1
Na+
McHKTx
Na+
McHAK1-4
Root
Leaf
Xylem
McITR2
Na+myo-inositol
Na+ transport in Mesembryanthemum crystallinum
↑ Na+out
Na+ compartmentalization into the cell
(rapid response to Na+ stress: starting from day 3)
Switch to CAM metabolism(slow response to dry conditions:
starting from day 9)
Cloned Na+/H+ antiporters
I thank you for your attention!!!
The end