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

AIM
To understand the signal transduction mechanism of Brassica napus root gravitropism during change of polarity of the root statocyte.

SPECIFIC GOALS
1. Determine calcium distribution in statocytes in 1g, microgravity and during 1g → microgravity or microgravity → 1g transition.

2. Determine calmodulin localisation in statocytes in 1g, microgravity and during 1g → microgravity or microgravity → 1g transition.

GENERAL DESCRIPTION
The gravity on Earth is a permanent factor in our environment which drives the growth of plants and particularly of their roots. This phenomenon is called gravitropism. Some dedicated cells, the statocytes, located at the cap of the roots, perceive the gravity signal. It has already been shown that the polarity of the statocytes, induced, for instance by an alteration of the gravitational signal, plays a major role in gravity signaling. The polarisation of the statocyte is illustrated in Figure 1.

Fig. 1: Polarisation of the statocyte.

Calcium ions also play a role in transduction mechanisms of gravity. In response to a signal, the concentration of free calcium in the cytoplasm increases, follows by a cascades of transduction events conducting to a regulation a calcium homeostasie. Calcium binding proteins like calmodulins, are an important role in this event.
The goal of PolCa is to understand the transduction mechanism while the polarity of the statocytes is changing. During the experiment, the seeds are hydrated and then chemically fixed according to the experimental protocol. The distribution of free calcium and the localisation of calmodulin will be analysed using cellular approaches.

Spaceflight relevance
The experiment examines the growth of Brassica seedlings in microgravity and compared to 1g conditions, as well as the effect of transitions between microgravity/1g conditions. Furthermore, contact between intracellular structures can only be achieved under low gravity conditions due to the effect of sedimentation under terrestrial gravity. The required duration of microgravity is only available during an orbiting space flight.

PRECURSOR FLIGHTS
Transmission of gravistimulus in the statocyte of lentil roots grown in space (1992)
Mission: STS-42, IML-1 Experimenter(s): G. Perbal, D. Driss-Ecole, J. Raffin

Effects of microgravity on statocyte polarity and starch metabolism (1996)
Mission: STS-76, Spacehab (Shuttle-to-MIR mission 03: S/MM 03).
Experimenter(s): G. Perbal, D. Driss-Ecole

GRAVI 1 - Threshold Acceleration for Gravisensing (2007)
Mission: ISS 13S (Soyuz TMA-9) + Increment 14
Experimenter(s): G. Perbal, D. Driss-Ecole and V. Legué

• Temperature profile from delivery before flight until delivery to PI after the mission
• Time of experiment activation (hydration), fixation, centrifuge on/off times, translocation of containers centrifuge <--> static racks

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. 4: Detailed Experiment Timeline and associated Funtional Objectives (FO).

Fig. 5: Flow Diagram with Functional Objectives (FO) indicated.

Fig. 6: 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). Temperature profile is required from delivery before flight until delivery to PI after the mission.
Time of experiment activation (hydration), fixation, centrifuge on/off times, translocation of containers centrifuge <-> static racks.
Brassica Napus seedling samples fixed after completion of flight experiment protocol, in wash buffer.

Planned analyses
The purpose of the PolCa experiment is to study the effect of the change in statocyte polarity on calcium-dependant pathways like the distribution of cytoplasmic free Ca²⁺ and the calmodulin localisation.
The analysis of subcellular localisation calcium distribution in plant cells has been investigated using in vivo approaches (Legué et al., 1997). Nevertheless this technique cannot use in space conditions, only in situ techniques (after fixation) can be developed. In this project, we will analyse the localisation of free calcium after chemical fixation and calcium precipitation using potassium pyroantimonate. This compound has the advantage to have a high specificity with free calcium ions, it can reveal calcium at low concentration after observation with electron transmission microscopy, both in animal and plant cells (Zhao et al., 2004). During these last years, we improved this method with our plant material.
We suppose that if a calcium distribution occurs in some conditions, it will be accompanied by an increase of calmodulin proteins (CaM). Calmodulin localisation will be analysed using antibodies against this family of proteins. The specificity of used antibodies has been already confirmed and our prelimary studies have allowed visualising CaM in statocytes.

EXPECTED RESULTS
PolCa and GRAVIGEN experiments allow us to dissect the effect of change in amyloplasts-ER interactions on the Calcium dependant pathways and will be taken new data concerning the transduction signal of gravity.
We will expect that the return of amyloplasts-ER contact will induce a change in Calcium distribution and CaM localisation.