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

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


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


Introduction

Extraterrestrial plant-based life support systems

Ferl et al. 2002


Introduction

Plant TropismsDifferential growth responses that reorient plant organs in response to direction of physical stimuli


Introduction

Gravitropism


Introduction


Introduction

Ceratopteris richardii as a model

g

What genes are implicated in gravisensing in the fern Ceratopteris richardii ?


Objective

Salmi and Roux., 2008


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.



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


Results and discussion

Rhizoid growth

Graviperception No graviperception

g

Mesures

EMCS



αrhizoid

Vector gravity



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



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.



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, 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.



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



Negative results:

- Gene not involved in graviperception



Negative results:


- Epistatic effects

Gravisensing



Negative results:


- Epistatic effects

Gravisensing



Negative results:


- Epistatic effects

Gravisensing



Negative results:


- Epistatic effects

GravisensingGravisensing



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.


Conclusion

Applications:

- Understanding graviperception

- Improving growth in microgravity conditions Extraterrestrial Plant-based Life Support Systems


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”.


References

Thank you for your attention!

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