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