Na+/H+ transporters of the halophyte Mesembryanthemum crystallinum

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PhD thesis presentation of Cosentino Cristian.

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Na+/H+ transporters of the halophyte Mesembryanthemum crystallinum

  1. 1. Cosentino Cristian AG Thiel/Homann Na+/H+ transporters of the halophyte Mesembryanthemum crystallinum L. Damstadt, 31 July 2008
  2. 2. The problem of soil salinization Land affected by soil salinity Soil erosion and salinization 15% of total land area Soil salinity 1/3 of irrigated area (Regansamy (2006) J. Exp. Bot. 57, 1017-1023) Need of food production 60% precent of increase 50% 40% 30% 20% 10% Wild (2003), Cambridge University Press 0% 2000 2010 2020 2030 2040 2050
  3. 3. Na+ damages on cellular metabolism H 2O AA K+ Na+ Na+ Na+ Na+ H 2O K+ Na+ K+ Na+ Ψwaterin > Ψwaterout Osmotic imbalance Disruption of Disruption of the protein 1 and loose of water 2 enzymatic activities 3 synthesis process Necrosis of leaves Decrease of plant growth and yield
  4. 4. Most important mechanisms of Na+ tolerance Na+ extrusion to Na+ internal the apoplast 1 2 compartmentalization ↑ Na+out 400 mM Na+out pH 5.5out 20 mM Na+in pH 7.5in Up to 100 mM K+in 1000 mM Na+vac Na+cyt Chloroplast Na+cyt Na+vac Na+out Na+ cyt Vacuole pH 5.5vac Mesembryanthemum crystallinum as model plant 1. Switch C3  CAM 2. Halophyte salt includer
  5. 5. C3 vs CAM C3 CAM Vacuole Vacuole Malic acid CO2 Cytoplasm Cytoplasm CO2 CO2 Calvin Calvin cycle cycle Chloroplast Chloroplast Sugar Sugar ↓ Transpiration ↓ Photorespiration
  6. 6. Stress-triggered induction of CAM Malic Acid (1000 µmol m-2 sec-1 for 2 weeks) High light C3 CAM NaCl stress (400 mM NaCl for 2 weeks) Malic Acid and Na+ NaCl stressed plants shift to CAM in 2 weeks instead of >8 weeks
  7. 7. Double vacuolar function under salt stress-triggered CAM Dynamic function Static function CO2 ↑ DµH+ Malic Acid2- Vac Cyt H+ Na+ ↓ DµH+ H+ Cyt Vac Malic Acid2- Cyt Vac Malate turnover Na+ sequestration (metabolic purpose) (osmotic and detoxification purposes)
  8. 8. Where in the leaf mesophyll does Na+ accumulate?
  9. 9. Vacuolar pH distribution Neutral Acidic Mesophyll cells can have acidic as well as neutral vacuoles
  10. 10. Vacuolar Na+ distribution 200 µm 100 µm 100 µm into epidermal bladder cell around epidermal bladder cells around vascular tissues
  11. 11. Does Na+ accumulate in acidic or neutral vacuoles?
  12. 12. pH and Na+ vacuolar distributions 200 µm 300 µm pH staining Na+ staining pH Na+ More uniform distribution of Na+ accumulation over a wider range of vacuolar pH
  13. 13. Correlation of Na+ accumulation and vacuolar pH CoroNa Green Neutral Red (Na+) (pH) 100 µm Na+ accumulating vacuoles 80% 9 19 60% 14 40% 4 20% 0% Acidic Neutral Acidic Neutral acidic neutral acidic neutral C3 CAM CAM C3 Na+ preferentially accumulates into neutral vacuoles
  14. 14. 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 neutral vacuoles
  15. 15. How does Na+ enter the vacuole?
  16. 16. Na+/H+ antiporters mediate the Na+ transport H+ Na+out P-ATPase +++ 400 mM Na+out pH 5.5out ---- 20 mM Na+in pH 7.5in Up to ATP 100 mM K+in 1000 mM Na+vac Chloroplast NHX SOS1 H+ Na+vac Na+out Na+ Na+ Na+ cyt Na+ H+ cyt V-ATPase ATP ATP H+ Vacuole V-PPase H+ V-ATPaseH PPi + pH 5.5vac AtSOS1 mediates Na+ extrusion from the cell (Arabidopsis thaliana) AtNHX1 mediates Na+ accumulation into the vacuole (Arabidopsis thaliana)
  17. 17. Which Na+/H+ antiporters are expressed in Mesembryanthemum crystallinum?
  18. 18. The cloned antiporters Predicted Predicted Antiporter aa MW TMD localisation McSOS1 1115 127.3 12 Plasmamembrane McNhaD 577 61.5 13 Chloroplast McNHX1 549 60.8 10 Vacuole Vacuole/Pre- McNHX2 556 61.6 11 vacuole McNHX3 526 58.1 10 Endomembrane
  19. 19. Phylogenetic relationships Antiporters from halophyte plants
  20. 20. Are the cloned antiporters functional?
  21. 21. Functional complementation Functional Organism Mutation Selection complementation nha1 nhx1 ena1-4 200 mM NaCl McSOS1 and McNhaD Saccharomyces cerevisiae nhx1 Hygromycin B McNHX1 Escherichia coli nhaA nhaB 200 mM NaCl, 5 mM LiCl McNhaD
  22. 22. Is the expression of the cloned antiporters affected by salinity stress?
  23. 23. 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
  24. 24. 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
  25. 25. Expectd function for the cloned antiporters H+ P-ATPase +++ 400 mM Na+out Na+out pH 5.5out ---- 20 mM Na+in pH 7.5in Up to ATP 100 mM K+in 1000 mM Na+vac Chloroplast McNHX1 McSOS1 Na+ Na+ H+ Na+ Vacuole H+ H+ V-ATPase ATP ATP H+ McNHX2-3 McNhaD V-PPase H+ Na+ V-ATPaseH PPi + pH 5.5vac
  26. 26. How are the expression of McNhaD and Na+ accumulation correlated?
  27. 27. Na+ accumulates in chloroplasts M. crystallinum mesophyll cell Na+ is accumulated into chloroplasts
  28. 28. McNhaD localizes at the chloroplast envelope ChloroP 1.1 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 Plastidial envelope McNhaD:GFP localizes at the plastidial membrane
  29. 29. MaNhaD and Na+chl are correlated Correlation McNhaD Na+chl McNHaD 7.8 fold of increase Na+chl 9.7 fold of increase McNhaD is most likely involved in Na+ accumulation into chloroplasts
  30. 30. Na+ accumulates into Mesembryanthemum crystallinum chloroplasts McNhaD:GFP localizes at the plastidial membrane McNhaD most likely mediates the Na+ accumulation into the chloroplast
  31. 31. Na+ transport in Mesembryanthemum crystallinum ↑ Na+out Na+ McHAK1 McNHX2-3 Na+ McHKT1 McNHX1 Na+ H+ Na+ H+ H+ Na+ compartmentalization into the cell H+ McNhaD (rapid response to Na+ stress: Na+ McSOS1 Na+ McITR2 starting from day 3) myo-inositol Na+ Leaf Root Switch to CAM metabolism (slow response to dry conditions: McHAK1-4 starting from day 9) Na+ McSOS1 Na+ McHKTx H+ Na+ McITR1 Na+ myo-inositol Cloned Na+/H+ antiporters Xylem
  32. 32. The end I thank you for your attention!!!

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