PLANTS IN SPACE:Identification of Gravisensing-related Genes
Gravisensing
Evolution of the Biosphere 23.8.-3.9.2010Oriane MaugerElena Ilka RensenDeborah SchieranoDaniel Tamarit
What is Microgravity? Objects in Space Station appear to be “weightless” (zero
gravity) Actual gravity is 1x10-6 g (where 1 g = 9.8 m/s2 =
acceleration due to gravity on earth)
Where is Microgravity?
Parabolic flight
Spaceflight
International Space Station
Free fall
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
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
Identifying gravisensing-related genes
Introduction
Extraterrestrial plant-based life support systems
Ferl et al. 2002
Identifying gravisensing-related genes
Introduction
Plant TropismsDifferential growth responses that reorient plant organs in response to direction of physical stimuli
Identifying gravisensing-related genes
Introduction
Gravitropism
Identifying gravisensing-related genes
Introduction
Identifying gravisensing-related genes
Introduction
Ceratopteris richardii as a model
g
What genes are implicated in gravisensing in the fern Ceratopteris richardii ?
Identifying gravisensing-related genes
Objective
Salmi and Roux., 2008
Identifying gravisensing-related genes
Material and methods
mRNA and protein levels
mRNAs
qPCR
Proteins
Western Blot
If the results are not concordant, we could reduce the cluster of candidate genes for gravisensing.
Identifying gravisensing-related genes
Material and methods
Agrobacterium : ability to transfer DNA between itself and plants
Interest gene downstream a strong promotor
Overexpression
Interest antisens gene
No or underexpression
RNA interference
Create a transgenic plant
Identifying gravisensing-related genes
Results and discussion
Rhizoid growth
Graviperception No graviperception
g
Mesures
EMCS
Identifying gravisensing-related genes
Material and methods
αrhizoid
Vector gravity
Identifying gravisensing-related genes
Material and methods
Experiment
µg 1 g 2 g 1 g 2 g
Wild type
Knocked-out
Over-expressed
Space (EMCS*) Ground (ERM*)*European Modullar Cultivation System
*Experiment Reference Model
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
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.
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
Identifying gravisensing-related genes
Material and methods
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).
TCS +18°C - +40°C selectable
ACS[CO
2 ]
0 – 5.5 % [O
2] 15 – 22 % selectable
ROTOR
Two identical rotors (programable) providing 0.001 to 2 g or μg level when not rotation.
4 ECs on each rotor in radial orientation
Umidity controlled individually for each EC
Central reservoir for fresh and waste water (250 ml)
Common illumiantion (white or IR light) for all ECs
One video camera for 2 EC's each – resolution camera 5x
EXPERIMENT CONTAINER (EC)
EC provides a safe enviroment for the biological sample, isolating them from the external enviroment and vice-versa .
Internal volume: 60x60x160 mm
Transparent cover (observation/ illumination)
Sensor for: temperature, umidity, pressure,
Supplied with controlled atmosphere and water
Connected power and data handling system, 4 digital i/o channels
EXPERIMENT REFERENCE MODEL (ERM)
The ERM is designed for ground reference experiments and provides an enviroment to the Ecs identical to the Flight Model.
Identifying gravisensing-related genes
Results and discussion
What do we expect?Gravisensing
NO SENSING SENSING SENSING
NO SENSING NO SENSING NO SENSING
μg - control 1 g - control 2 g - control
μg - knocked-out 1 g - knocked-out 2 g - knocked-out
NO SENSING SENSING SENSING
μg - over-expressed 1 g - over-expressed 2 g - over-expressed
Identifying gravisensing-related genes
Results and discussion
Negative results:
- Gene not involved in graviperception
Identifying gravisensing-related genes
Results and discussion
Negative results:
- Gene not involved in graviperception
- Epistatic effects
Gravisensing
Identifying gravisensing-related genes
Results and discussion
Negative results:
- Gene not involved in graviperception
- Epistatic effects
Gravisensing
Identifying gravisensing-related genes
Results and discussion
Negative results:
- Gene not involved in graviperception
- Epistatic effects
Gravisensing
Identifying gravisensing-related genes
Results and discussion
Negative results:
- Gene not involved in graviperception
- Epistatic effects
GravisensingGravisensing
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.
Identifying gravisensing-related genes
Conclusion
Applications:
- Understanding graviperception
- Improving growth in microgravity conditions Extraterrestrial Plant-based Life Support Systems
Identifying gravisensing-related genes
ReferencesFerl, 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”.
Identifying gravisensing-related genes
References
Thank you for your attention!
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