1. Mary A. Bisson Plant Physiologist/Cell Biologist

2. Chara Alga most closely related to higher plants Large internodal cells Species with different salt tolerance

3. Summary of Research Topics Ion transport and salt tolerance Sodium transport Turgor regulation Ligand-gated channels Gravitropic responses

4. Ion transport and salt tolerance Two closely related species of algae Salt sensitive: Chara australis Salt tolerant: Chara longifolia

5. Sodium transport Keep cytoplasmic sodium low Transport options Prevent entry (low permeability) Sequester in vacuole Export from cytoplasm

6. Sodium transport Keep cytoplasmic sodium low Transport options Prevent entry (low permeability) Sequester in vacuole Export from cytoplasm

7. Sodium transport Keep cytoplasmic sodium low Transport options Prevent entry (low permeability) Sequester in vacuole Export from cytoplasm

8. Sodium fluxes: comparison between species Use radioactive isotope to measure influx, efflux, compartmentation of Na + Results Influx similar in two species Sequestration in vacuole low in both species Efflux differs

9. Cytoplasmic sodium export: comparison between species Export higher in C. longifolia (salt-tolerant) than in C. australis (salt-sensitive) Export higher when C. longifolia adapted to salt water

10. Possible mechanisms of sodium export Na + /H + export pH sensitivity Inhibitor studies Different in salt-adapted and freshwater cells Other transport systems? ATPase?

11. Possible mechanisms of sodium export Na + /H + export pH sensitivity Inhibitor studies Different in salt-adapted and freshwater cells Other transport systems? ATPase?

12. Research opportunities Electrophysiology Ion fluxes Molecular biology (in collaboration with M. Hollingsworth)

13. Summary of Research Topics Ion transport and salt tolerance Sodium transport Turgor regulation Ligand-gated channels Gravitropic responses

14. Turgor regulation Need for turgor regulation Turgor = hydrostatic pressure difference between cell and external medium Provides structure Driving force for growth

15. Turgor regulation Hypo- and hypertonic stresses Hypertonic: increase salt, decrease turgor, “wilt” Hypotonic: decrease salt, increase turgor, burst Measure electrical responses, pressure Model for mechanism of turgor responses

16. Model for turgor regulation Turgor Error signal Pre-set Turgor Osmotic pressure Membrane potential K + channel activity K + conc. Ca 2+ channel activity Cytoplasmic Ca 2+ activity Release from Internal store Cl - channel activity Cl - conc. ? ? ?

17. Research opportunities Electrophysiology and turgor probe Patch clamp

18. Summary of Research Topics Ion transport and salt tolerance Sodium transport Turgor regulation Ligand-gated channels Gravitropic responses

19. Channel activity Looking for channels to test model Characterize new channel Cl - channel on the vacuolar membrane Gated by acetylcholine and nicotine Affects action potential (?)

20. Research Opportunities Patch clamp Physiologic effects--action potential? Bioinformatic studies--putative channels? Extend to higher plants

21. Summary of Research Topics Ion transport and salt tolerance Sodium transport Turgor regulation Ligand-gated channels Gravitropic responses

22. Why study Chara? Single colorless cell 0-1 h: Statoliths aggregate, sediment 2-24 h: Asymmetric growth 24 h: Complete re-orientation

23. Statoliths do not sediment in vertical rhizoids Vacuole Nucleus Statoliths: suspended in actin. In constant, random motion.

24. Why should statoliths sediment in gravistimulated rhizoids? Actin disintegrates? Not seen in micrographs (Braun and Wasteneys) Actin network distorts to move statoliths lower? Ambiguous in micrographs Statoliths detach from actin, fall straight down? Natural cycle of releasing and reattaching to actin shifts in favor of release?

25. Measure statolith movement Measure statolith movement continuously after a change in orientation

26. Quantify statolith movement:

27. What steps precede statolith sedimentation? Dependent on the number of statoliths Rate of settling, gravitropism slows with few statoliths

28. What steps precede statolith sedimentation? Dependent on the number of statoliths Dependent on Ca 2+ A number of Ca 2+ antagonists inhibit statolith movement and/or gravitropism

29. Altered gravitropism: Ca 2+ antagonists Why? Cytoskeletal involvement? How?

30. Research opportunities: Cell biology Determining role of actin: microscopy, inhibitor, etc. Looking at cytoplasmic Ca 2+ : cell imaging