Understanding water pathways in leaves…

1. Light and temperature induced variable leaf symplasmic hydraulic conductances in Juglans regia L.Are aquaporins involved?H Cochard1, S Sakr1, A Nardini2cochard@clermont.inra.fr1 UMR PIAF, INRA France 2 Trieste University, Italy

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

3. Mesophyll cell walls Xylem resistance Stomatal resistance ~ Mesophyll cell symplasm Symplasmic Apoplasmic Gaseous Apoplasmic Understanding water pathways in leaves… ~ ?

4. 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 Why should symplasmic resistances be variable? Because water molecules cross cell membranes through a Nobel prize winning molecule :

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

6. Objectives of this study : • 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

7. Experimental Setup

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

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

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

11. 106 Can stomatal movement be responsible? NO! Light Dark

12. Effect of light intensity on leaf hydraulic conductance

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

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

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

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

17. Do JrPIP2 transcripts accumulate with light ? 1 2 3 4 T Dark Light Dark JrPIP2,1 JrPIP2,2 JrAct 3 Transcript of: 4 2 1 YES

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

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

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

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

24. Light and temperature induced variable leaf symplasmic hydraulic conductances in Juglans regia L.Are aquaporins involved?H Cochard1, S Sakr1, A Nardini2cochard@clermont.inra.fr1 UMR PIAF, INRA France 2 Trieste University, Italy