PolCa - Effect of weightlessness on the distribution of calcium in the statocytes of Rapeseed roots (Brassica napus)
Cell and Molecular Biology
Plant Biology and Physiology
ISS Increment 19
- (2009) ISS Increment 20
V. Legué (1), J. Gerard (1), G. Gasset (2), D. Chaput (3)
|(1)||UMR INRA/UHP 1137|
Faculty of Sciences
UHP - University Nancy I - H. Poincare
54506 Vandoeuvre Cedex
|Tel: ||0033 (0) 3 83684232|
|Fax: ||0033 (0) 3 83684292|
|(2)||Faculté de Médecine|
Université Paul Sabatier
GSBMS - Groupement Scientifique en Biologie et Médecine Spatiales
|||V. Legué, E. Blancaflor, C. Wymer, G. Perbal, D. Fantin, S. Gilroy, (1997), "Cytoplasmic free Ca2+ in Arabidopsis roots changes in response to touch but not gravity", Plant Physiology, 114, pp. 789-800.|
PolCa & GRAVIGEN are complementary studies, using the same conditions & biological material but analysing different aspects of the gravi-response.
To understand the signal transduction mechanism of Brassica napus root gravitropism during change of polarity of the root statocyte.
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.
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.
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.
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
Experiment Concept Diagram
The experiment will examine the repolarisation of statocytes in the roots of Brassica napus seedlings. Seedlings will grown either in microgravity or 1g conditions, then subject to a brief exposure of either micro-g or 1g to observe the redistribution of the amayloplasts.
Fig. 2: Experiment Concept Diagram.
Fig. 3: Mission Concept Diagram.
Brassica napus seeds, germinated inflight.
– Loading of dry seeds & all reagents into experiment cassette at L-9 days in scientist home laboratory.
– Transport of assembled ECs to Baikonour in condition temperature stowage (6°C preferred, minimum 4°C, maximum 8°C).
– Soyuz Taxi flight launch: Seeds launched dry at ambient temperature (+4°C to +30°C).
– Experiment activation on ISS by hydration of seeds.
– 6 Experimental steps after launch (incubation at 22°C, min 20°C, maximum 25°C) preferable 22 ±1°C.
– Hydration, incubation, cassette exchange between centrifuge & static, incubation fixation & wash step.
– Number of replicate seeds per condition = 8 seeds (TBC after tests with hardware similar to flight model).
– During Soyuz download phase the experiment can be maintained at ambient temperature (+4°C to 30°C range) for at least 26h (already tested) to 30h (to be tested).
– Transport of samples from landing site to scientists laboratory at +6°C (6°C preferred, minimum 4°C, maximum 8°C).
GENERAL EXPERIMENT PROCEDURE
– 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, 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.
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.
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.
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.
|Fig. 1: Polarisation of the statocyte.|
|Fig. 2: Experiment Concept Diagram.|
|Fig. 3: Mission Concept Diagram.|
|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.|
Jason Hatton (e-mail: email@example.com)