Biology: Plant biology

Plant Biology and Physiology

ISS Increment 19

Further missions:
- (2009) ISS Increment 20

2009

A. Graziana ⁽¹⁾, C. Mazars ⁽¹⁾, R. Rangeva ⁽¹⁾, G. Gasset ⁽²⁾, D. Chaput ⁽³⁾

Kubik

AIM
To determine how gene expression in germinating Brassica napus seedlings is modified by microgravity & hence elucidate the mechanisms of gravity perception and transduction in plant roots.

SPECIFIC GOALS
- Quantify the expression of genes known to be sensitive to gravity in 2 d old Brassica napus seedlings in microgravity and compare this to gene expression in 1g.
- Identify new clusters of genes which are sensitive to microgravity in germinating Brassica napus seedlings.

GENERAL DESCRIPTION
Growth and development in plants are influenced by the environment. The effects of some environmental factors such as light or temperature are easy to evaluate in normal laboratory conditions but it is more difficult to evaluate the effects caused by the modulation or absence of gravity.

To study the effects of gravity on Earth it is possible either to increase the value of the gravity vector, using centrifugation or to constantly change the direction of the gravity vector (from the plant's perspective) using a "Clinostat" to simulate some effects of weightlessness. Experiments performed in actual microgravity are nevertheless required to understand the biological process and to validate the various models.

It has been already demonstrated that the expression of several genes is modified by changes in the value of the gravity vector. GRAVIGEN will evaluate the expression of genes known to be modulated by modification of the gravity vector. It will then be possible to identify genes whose expression is modulated either by gravity or only by hyper or micro gravity.

The result will help us to understand how gravity is percieved and how this morphological information is transducted in plants.

Spaceflight relevence
The experiment examines the gene expression of Brassica napsus seedlings in microgravity and compared to 1g conditions, The required duration of microgravity is only available during an orbiting spaceflight.

Previous Space Flight
GRAVIGEN Experiment in IBIS on FOTON-M1, lost during launch failure.

POLCA & GRAVIGEN are complementary studies, using the same conditions & biological material but analysing different aspects of the gravi-response.

The experiment will examine how gene expression in germinating Brassica napus seedlings is modified by microgravity. Seedling will grown either in microgravity or 1g conditions and the expression of some genes between the two samples compared.

Fig. 1: Mission Concept.

Biological Samples
Brassica napus seeds, germinated inflight

Experiment protocol
- Loading of dry seeds & all reagents into experiment cassette at L-9 days in scientist home laboratory.
- Transport of assembled ECs to Baiknour in condition temperature stowage (~22°C).
- Soyuz Taxi flight launch: Seeds launched dry at ambient temperature (+10°C to + 30°C range).
- Alternative launch scenario: Progress upload in passive condition T°C stowage (+ 10°C to +30°C):

• Stowage between 2 and 4 months at ambient temperature until experiment starts (Fixative RNAlater can be stored at ambient temperature starting from delivery to laboratories).
• Prior to activation, the experiment must be allowed to reach 22°C (min 20°C, max 25°C) for 2 hours prior to start of experiment.

- Experiment activation on ISS by hydration of seeds.
- 2 Experimental steps after launch (incubation at 22°C, min 20°C, maximum 25°C) preferable 22 ±1°C:

• Hydration & fixation.
• Samples maintained at ambient temperature after fixation (in case of Soyuz scenario) for at least 24h to 96h at 22°C (already tested) but this period has to be minimised.

- Number of replicate seeds per condition = 8 seeds.
- In case of Progress upload, fixation should occur no more than 14 days prior to return. Fixed samples should be maintained at 6°C +/- 2°C until return.
- During Soyuz download phase the experiment can be maintained at ambient temperature (+4°C to +30°C range) for a maximum of 48h. Stowage at low temperature is preferable.
- Transport of samples from landing site to scientists’ laboratory at +6°C ±2°C (temperature range +2°C to +10°C is acceptable but duration and temperature history before this transport has to be taken into account).

GENERAL EXPERIMENT PROCEDURE
Parameters measured
Inflight parameters measured:
- Temperature profile from delivery before flight until delivery to PI after the mission
- Time of experiment activation (hydration), fixation, centrifuge on/off times.

Ground reference experiment(s)
Ground control experiment will be done in investigators home laboratory.
4 EU should be used for ground experiment done in parallel to flight experiment with a short delay (delay TBD).
4 EU should be used after flight in order to perform a ground experiment done with real temperature profile recorded during the mission.

Fig. 2: Detailed Experiment Timeline and associated Functional Objectives.

Fig. 3: Flow Diagram with Functional Objective indicated.

Fig. 4: Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline.

Science deliverables
- Temperature of experiment samples during flight (10 minute intervals, 0.5°C accuracy).
- Time of experiment activation (hydration), fixation, centrifuge on/off times.
- Brassica Napus seedling samples fixed after completion of flight experiment protocol, in RNAlater solution.

Planned analysis
On Earth, gravity appears as a permanent stimulus perceived by plants throughout their evolution. It is necessary for the regulation of their growth and development for correctly orientating roots downwards and leaves upwards (gravitropism). Moreover to survive on land, plants have had to resist to the gravitational force.
On Earth, experiments were done on Brassica napus seedlings grown either at 1g (control) , or < 1g on clinostat or at 5g on centrifuge. The results obtained in these ground conditions have clearly pointed out that the expression of some genes was modulated by gravity conditions. Particularly genes* involved in the formation of cell wall components, cytoskeletal proteins, auxin-related events and calcium ATPase were mainly modulated by our experimental conditions.

EXPECTED RESULTS
GRAVIGEN should help to quantify the level of expression of these genes* in space-microgravity conditions and compare these expression levels with those attained in 1g space-conditions (centrifuge) or 1g (ground conditions).
GRAVIGEN experiment in space, is absolutely necessary to validate the previous results obtained on ground conditions and to indicate if the experiments performed on ground are reliable enough and useful to be done to plan and to model the behaviour of plants in the forthcoming space flights.

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Jason Hatton (e-mail: jason.hatton@esa.int)