1. Light and temperature induced variable leaf symplasmic hydraulic conductances in Juglans regia L. Are aquaporins involved? H Cochard 1, S Sakr 1, A Nardini 2 cochard@clermont.inra.fr 1 UMR PIAF, INRA France 2 Trieste University, Italy cochard@clermont.inra.fr XVII International Botanical Congress Vienna, Austria Center, 17 - 23 July 2005

2. Understanding water pathways in leaves… Apoplasmic Symplasmic Gaseous Mesopyll cell wall Mesopyll cell symplasm Evaporation in stomatal chambers Xylem conduits in veins

3. Understanding water pathways in leaves…  Apoplasmic Symplasmic Gaseous Xylem resistance Mesophyll cell walls Mesophyll cell symplasm Stomatal resistance Apoplasmic 

4. Why should symplasmic resistances be variable? Because water molecules cross cell membranes through a Nobel prize winning molecule : AQUAPORINS Tajkhorshid, E., Nollert, P., Jensen, M.O., Miercke, L.J., O'Connell, J., Stroud, R.M., and Schulten, K. (2002). Science 296, 525-530 http://nobelprize.org/chemistry/laureates/2003/chemanim1.mpg

5. Recent evidences for variable leaf hydraulic resistances Sack et al (2002) : light decreases leaf hydraulic resistance Tyree et al (2005) Leaf conductance

6. Confirm the light response Kleaf for walnut Confirm that stomatal movement was not involved Effect of light intensity Effect of temperature Molecular evidences for the implication of aquaporins in this light response Objectives of this study :

7. Experimental Setup

8. F1 F2 P Temp Bath air Air pressure tap Kleaf = Flow/LA / Pressure mmol s -1 MPa -1 m -2 1400 µmol m-2 s-1 Experimental Setup XYL’EM

9. A typical experiment Pressure Temperature Leaf conductance +400%

10. Can stomatal movement be responsible? Leaves are infiltrated with water The hydraulic conductance of the stomatal pore is also measured

11. Light Dark Can stomatal movement be responsible? NO! 10 6

12. Effect of light intensity on leaf hydraulic conductance

13. Effect of bath temperature on leaf conductance Little effect on dark conductance (except at 35°C) Strong effect on light conductances Maximum values kinetics

14. Light response of Kleaf is strongly temperature dependant Not explained by water fluidity under light conditions Effect of bath temperature on leaf conductance Behavior of a biological membrane Implication of aquaporins

15. flowers Bud Bark xylem leaf root JrPIP2-1 JrPIP2-2 ND D F M ARNr PIP Aquaporins in Walnut Sakr et al Plant Phys 2003

16. JrPIP2 Aquaporins is a water channel It increases the water permeability of a Xenopus ovocyte 0 20 40 60 80 100 120 140 160 180 200 220 240 Water JrPIP2-2 RD28 Pos ( m ms -1 ) JrPIP2-1 RD28 Water Sakr et al Plant Phys 2003

17. 1 3 2 4 1234 JrPIP2,1 JrPIP2,2 JrAct TDarkLightDark Transcript of: Do JrPIP2 transcripts accumulate with light ? YES

18. Can the gating of aquaporins alter leaf conductance? HgCl2 (200 µM) : known to inhibit (some) aquaporins among other proteins!

19. Strong and rapidly reversed inhibition of leaf conductance (minutes) DMSO dimethyl sulfoxide. Many effects, aquaporin inhibitor? Can the gating of aquaporins alter leaf conductance? control treated Osmotic effect? Effect on membrane polarity?

20. CHX (cycloheximide) : inhibits protein traduction in eukaryots Does leaf light response is caused by the activation or by the traduction (synthesis) of proteins ?

21. Does leaf light response is caused by the activation or by the traduction (synthesis) of proteins ? The light response is reversed in less than 1 hour with CHX The synthesis of at least one protein is required for the light response This protein has a relatively high turnover

22. Conclusions Leaf hydraulic resistance is strongly light and temperature dependant Variables leaf resistances are located in the symplasmic pathway Proteins (aquaporins) are involved in these responses The synthesis or the activation of these proteins seems finely regulated according to leaf microclimate WHY ?

23. Understanding water relations in leaves… Xylem resistance Mesophyll cell walls Mesophyll cell symplasm Stomatal resistance Leaf transpiration  xylem  leaf   Does variable symplasmic resistances buffer leaf water status and favor stomatal function ? Light Temperature Transpiration Resistance  buffered

24. Light and temperature induced variable leaf symplasmic hydraulic conductances in Juglans regia L. Are aquaporins involved? H Cochard 1, S Sakr 1, A Nardini 2 cochard@clermont.inra.fr 1 UMR PIAF, INRA France 2 Trieste University, Italy cochard@clermont.inra.fr XVII International Botanical Congress Vienna, Austria Center, 17 - 23 July 2005