1. Why are Spartina grasses so successful? Adaptations to anoxia and hydrogen sulfide Ray Lee and Brian Maricle School of Biological Sciences Washington State University

2. Spartina alterniflora and Spartina anglica Saltmarsh grasses native to the Eastern U.S. (S. alterniflora) and British Isles (S. anglica). Invasive species in Puget Sound and Willapa Bay in Washington State.

3. Why are physiological studies of Spartina relevant? Physiological processes are the link between environment and performance Environment Challenges opportunities Metabolic Structural adaptations Physiological processes Growth reproduction Performance

4. Spartina are physiologically resilient and vigorous Physiological tolerance –Wide range of salinities –Waterlogged soils Anoxia Hydrogen sulfide

5. Distribution of hydrogen sulfide in sediments Oxidized zone No hydrogen sulfide Anoxic zone Hydrogen sulfide-rich

6. Sulfide is a potent toxin to aerobic respiration µM levels inhibit mitochondrial cytochrome c oxidase Sulfide binds to hemoglobin forming sulfhemoglobin Sulfide spontaneously reacts with oxygen producing hypoxic/anoxic conditions Can be used as an energy source by sulfide- oxidizing bacteria

7. Chemoautotrophic symbiosis An adaptation to exploit sulfide-rich environments

8. Tolerating anoxic sediments Aerenchyma Anaerobic metabolism –Alcohol dehydrogenase Sulfide oxidation Spartina anglica root

9. Functions of aerenchyma Oxygen transport Reduce cellular oxygen demands

10. Root Ultrastructure 1 cm from root tip2 cm from root tip

11. Root Ultrastructure 4 cm from root tip6 cm from root tip

12. Root Ultrastructure 8 cm from root tip10 cm from root tip

13. The difference in root structure between treatments of Spartina alterniflora

14. A comparison of root structure between treatments of Spartina anglica

15. S. anglica respirometry experiments Use automated flow- through respirometry system Investigate oxygen transport

16. Flow-through respirometry

17. mitochondria O2O2 O2O2 O2O2 Root surface Root - high O 2 uptake High oxygen consumption and/or low aerenchyma supply

18. mitochondria O2O2 O2O2 O2O2 Root surface Root - low O 2 uptake Low oxygen consumption and/or high aerenchyma supply O2O2 O2O2

20. Oxygen transport is more effective in S. anglica compared with S. alterniflora

21. Checking for oxygen transport A plant can be sealed into a flask of N 2 - flushed water. An oxygen-sensing probe can be used to monitor the water--any increase in O 2 must have come through the plant.

22. Differences in oxygen transport between species Negative fluxes=uptake; positive fluxes=release; n=9, 11, 9, 9

23. mitochondria H2SH2S H2SH2S Root surface Sulfide volatilization Occurs in S. anglica but not S. alterniflora

24. Conclusions Function of increased aerenchyma appears to be to reduce oxygen demands NOT increase oxygen transport S. anglica has a highly effective oxygen AND sulfide transport system

25. Questions Can S. anglica grow better than S. alterniflora in anoxic/sulfidic conditions? Can sulfide levels ever be so high that plants cannot deal with it? What is the relationship between sulfide levels and effectiveness of eradication efforts?

26. Acknowledgements J. Doeller and D. Kraus (UAB) S. Hacker (WSU Vancouver) Kim Patten (WSU Long Beach) Miranda Wecker NSF, NOAA, WSU faculty seed grant

28. mitochondria O2O2 H2SH2S SO x O2O2 O2O2 Enzyme or Metal catalyst Root surface Sox mechanism

29. Spartina alterniflora roots catalyze the oxygenation of sulfide