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
Identifying gravisensing-related genes
Introduction
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)
Parabolic flight
Where is
Microgravity?
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 gravityrelated 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 Tropisms
Differential growth responses that reorient
plant organs in response to direction of
physical stimuli
Perception
Transduction
cascade
(amplifying
signal)
Response
(differential
growth)
Identifying gravisensing-related genes
Introduction
Gravitropism
Identifying gravisensing-related genes
Introduction
Ceratopteris richardii as a model
g
Identifying gravisensing-related genes
Objective
What genes are implicated in
gravisensing in the fern Ceratopteris richardii ?
Salmi and Roux., 2008
Identifying gravisensing-related genes
Material and methods
mRNA and protein levels
mRNAs
Proteins
qPCR
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
Create a transgenic plant
Agrobacterium :
ability to transfer DNA
between itself and plants
Interest gene downstream
a strong promotor
Interest antisens gene
RNA interference
Overexpression
No or underexpression
Identifying gravisensing-related genes
Results and discussion
Rhizoid growth
Graviperception
No graviperception
g
Identifying gravisensing-related genes
Material and methods
Mesures
EMCS
rhizoid
α
Vector gravity
Identifying gravisensing-related genes
Material and methods
Experiment
µg
1g
2g
1g
2g
Wild type
Knockedout
Overexpressed
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
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.
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
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,
ROTOR
thermally controlle denvironment
to
theidentical
Experiment
Containers
Two
rotors
(programable)
(EC’s).
providing 0.001 to 2 g or μg level
EXPERIMENT CONTAINER (EC)
when not rotation.
EC provides a safe enviroment for the biological sample, isolating them
4 ECs on each rotor in radial orientation
from
enviroment
andselectable
vice-versa
.
EXPERIMENT
REFERENCE
MODEL (ERM)
TCSthe external
+18°C
- +40°C
Umidity controlled individually for each EC
The
ERMvolume:
is
designed
for
ground
Internal
60x60x160
mm reference experiments and provides an
[CO
]
0
–
5.5
%
2
ACS
Central
reservoir
fresh
and waste
water Model.
(250 ml)
enviroment
to ]the for
Ecs
identical
toselectable
the Flight
[O
15
–
22
%
Transparent 2cover (observation/ illumination)
Sensor
for:
temperature,
umidity,
Common
illumiantion
(white
or IRpressure,
light) for all ECs
Supplied with controlled atmosphere and water
One video camera for 2 EC's each – resolution camera 5x
Connected power and data handling system, 4 digital i/o channels
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
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
Gravisensing
Gravisensing
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
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”.
Identifying gravisensing-related genes
References
Thank you for your
attention!