PLANTS IN SPACE: Identification of Gravisensing-related Genes

1. PLANTS IN SPACE: Identification of Gravisensing-related Genes Evolution of the Biosphere 23.8.-3.9.2010 Oriane Mauger Elena Ilka Rensen Deborah Schierano Daniel Tamarit Gravisensing

2. Identifying gravisensing-related genes Introduction What is Microgravity? • Objects in Space Station appear to be “weightless” (zero gravity) • Actual gravity is 1x10-6g (where 1 g = 9.8 m/s2 = acceleration due to gravity on earth) Parabolic flight Spaceflight International Space Station Free fall Where is Microgravity?

3. Identifying gravisensing-related genes Introduction “Plant research on the ISS fulfills two major goals: • Increase basic knowledge of how these organisms sense and respond to their environment, especially gravity-related phenomena • Provide the underpinning for enabling sustained human habitation in space.” NASA, “Life and Physical Sciences Research for a New Era of Space Exploration: An Interim Report”, 2010

4. Identifying gravisensing-related genes Introduction Life Support Systems • maintain suitable living conditions for humans outside Earth's atmosphere • Via physical and chemical means • transport of O2, H2O & food • cleaning & recycling of air and water • Future: Plant-based Life-Support Systems

5. Identifying gravisensing-related genes Introduction Extraterrestrial plant-based life support systems Ferl et al. 2002

6. Identifying gravisensing-related genes Introduction Plant Tropisms Differential growthresponses that reorient plant organs in response to direction of physical stimuli

7. Identifying gravisensing-related genes Introduction Gravitropism

8. Identifying gravisensing-related genes Introduction Ceratopteris richardii as a model g

9. Identifying gravisensing-related genes Objective What genes are implicated in gravisensing in the fern Ceratopteris richardii ? Salmi and Roux., 2008

10. Identifying gravisensing-related genes Material and methods mRNA and protein levels Proteins mRNAs qPCR Western Blot If the results are not concordant, we could reduce the cluster of candidate genes for gravisensing.

11. Identifying gravisensing-related genes Material and methods Create a transgenic plant Agrobacterium : ability to transfer DNA between itself and plants Interest gene downstream a strong promotor Interest antisens gene RNA interference No or underexpression Overexpression

12. Identifying gravisensing-related genes Results and discussion Rhizoid growth Graviperception No graviperception g

13. Identifying gravisensing-related genes Material and methods Mesures EMCS rhizoid α Vector gravity

14. Identifying gravisensing-related genes Material and methods Experiment Space (EMCS*) Ground (ERM*) *European Modullar Cultivation System *Experiment Reference Model

15. Identifying gravisensing-related genes Material and methods Biological conditions • 7 days before the flight : • soak in sterile water • Darkness • 29 ⁰C • 1 day before the flight : • -remove the water • -put in petri dishes • -put in nutritive medium • 2nd day in orbit : • -ambient light

16. Identifying gravisensing-related genes Material and methods The ISS serves as a research laboratory that has a microgravity environment in which crews conduct experiments in biology, chemistry, human biology, physics, astronomy and meteorology Destiny is the primary research laboratory for U.S. payloads, supporting a wide range of experiments and studies contributing to health, safety and quality of life for people all over the world.

17. Identifying gravisensing-related genes Material and methods ESA's contribution to the ISS for experiments with plants will be the European Modular Cultivation System (EMCS) in the US Destiny Laboratory

18. The EMCS consists of a gas tight incubator containing two centrifuges with space for 4 Experiment Container on each rotor. INCUBATOR The incubator provides a gas tight, thermally controlle denvironment to the Experiment Containers (EC’s). ROTOR Two identical rotors (programable) providing 0.001 to 2 g or μg level when not rotation. EXPERIMENT CONTAINER (EC) EC provides a safe enviroment for the biological sample, isolating them from the external enviroment and vice-versa . EXPERIMENT REFERENCE MODEL (ERM) The ERM is designed for ground reference experiments and provides an enviroment to the Ecs identical to the Flight Model.

19. Identifying gravisensing-related genes Results and discussion What do we expect? Gravisensing μg - control 1 g - control 2 g - control NO SENSING SENSING SENSING μg - knocked-out 1 g - knocked-out 2 g - knocked-out NO SENSING NO SENSING NO SENSING μg - over-expressed 1 g - over-expressed 2 g - over-expressed NO SENSING SENSING SENSING

20. Identifying gravisensing-related genes Results and discussion Negative results: • - Gene not involved in graviperception

21. Identifying gravisensing-related genes Results and discussion Negative results: • - Gene not involved in graviperception • - Epistatic effects Gravisensing

22. Identifying gravisensing-related genes Results and discussion Negative results: • - Gene not involved in graviperception • - Epistatic effects Gravisensing

23. Identifying gravisensing-related genes Results and discussion Negative results: • - Gene not involved in graviperception • - Epistatic effects Gravisensing

24. Identifying gravisensing-related genes Results and discussion Negative results: • - Gene not involved in graviperception • - Epistatic effects Gravisensing Gravisensing

25. Identifying gravisensing-related genes Results and discussion Positive results: further studies • - Role of the protein(s) involved in graviperception. • - Molecular and systems biology of gravisensing. • - Mechanism of gene regulation. • - Extend the study to more plant species.

26. Identifying gravisensing-related genes Conclusion Applications: - Understanding graviperception - Improving growth in microgravity conditions Extraterrestrial Plant-based Life Support Systems

27. Identifying gravisensing-related genes References Ferl, R., Wheeler, R., Levine, H. G., Paul, A-L. (2002). “Plants in space”, Current Opinion in Plant Biology, 5: 258–263 Kimbrough, J. M., Salinas-Mondragon, R., Boss, W. E., Brown, C. S., Sederoff, H. W. (2004). “The fast and transient transcriptional network of gravity and mechanical stimulation in the Arabidopsis root”. Apex. Plant Physiology. 136: 2790-2805. Martzivanou, M., Hampp, R. (2003). “Hyper-gravity effects on the Arabidopsis transcriptome”. Physiologia plantarum, 118: 221-231 Palme, K., Dovchenko, A., Ditengou, F. A. (2006). “Auxin transport and gravitational research: perspectives”. Protoplasma. 229: 175-181. Roux, S. J., Chatterjee A. , Hillier S., and Cannon, T. (2003). “Early development of fern gametophytes in microgravity”. Adv. Space Res., 31: 215-220. Rutherford, G., Tanurdzic, M., Hasebe, M., Banks, A. (2004). “A systemic gene silencing method suitable for high throughput, reverse genetic analyses of gene function in fern gametophytes”. BMC Plant Biology, 4: 6. Salmi, M. L., Roux, S. J. (2008). “Gene expression changes induced by space flight in single-cells of the fern Ceratopteris richardii”. Planta. 229: 151-159. Wolverton, C., Ishikawa, H., Evans, M. L.‚ “The kinetics of root gravitropism: dual motors and sensors“, Journal of Plant Growth Regulation, 21: 102-112 Committee for the Decadal Survey on Biological and Physical Sciences in Space , National Research Council, (2010) “Life and physical sciences research for a new rra of space exploration: an interim report”. NASA, (2009) “International Space Station ECLSS Technical Task Agreement Summary Report”.

28. Identifying gravisensing-related genes References Thank you for your attention!