PolCa - Effect of weightlessness on the distribution of calcium in the statocytes of Rapeseed roots (Brassica napus)
  1. 2008 • ISS Increment 18
Life Sciences:
  • Cell and Molecular Biology
  • Plant Biology and Physiology
Jason Hatton
V. Legué (1), J. Gerard (1), G. Gasset (2), D. Chaput (3)
UHP - University Nancy I - H. Poincare
Faculty of Sciences
BP 239
54506 Vandoeuvre Cedex
GSBMS - Groupement Scientifique en Biologie et Médecine Spatiales
Université Paul Sabatier
Faculté de Médecine
31032 Toulouse

PolCa & GRAVIGEN were 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.

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. Plants, particularly their roots, have the ability to sense and to re-orient their growth in response to gravity. 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.

Statocytes are the only cells that exhibit structural polarity with respect to gravity providing interactions with starch-containing plastids (amyloplasts) and the cortical endoplasmic reticulum (ER). Even if amyloplasts are widely considered as gravity sensor, there is no clear evidence that a change in amyloplasts-ER interactions could lead to a transduction gravity signal. Previous space experiments clearly showed that amyloplasts interactions with ER are not necessary to lead to a root re-orientation, suggesting that amyloplasts displacement mediate transduction events through cytoskeleton reorganisation and calcium-dependant pathways.

The polarisation of the statocyte is illustrated in Figure 1.

Figure 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 were hydrated and then chemically fixed according to the experimental protocol. The distribution of free calcium and the localisation of calmodulin was analysed using cellular approaches.

Spaceflight relevance
The experiment examined the growth of Brassica seedlings in microgravity and compared it to 1 g 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.

Figure 2: Experiment Concept Diagram.

Figure 3: Mission Concept Diagram.

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 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).

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, 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.

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

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

Figure 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.

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.

Space conditions provide a unique opportunity to provide a change of structural polarity in statocytes without a gravistimulation. The PolCa experiment has been conducted using Brassica napus seedlings, which submitted four different conditions: continuously on 1 g centrifuge or continuously in microgravity conditions. Some seedlings germinated on centrifuge for 28 h were transferred for 10 min into microgravity conditions, leading to a loss of amyloplast-ER interactions through amyloplasts displacement. Others seedlings germinated in microgravity conditions for 28 h were transferred to a centrifuge for 10 min.

100% of seed germination was obtained in all conditions. The analysis of amyloplast positioning (Figure 7) show clearly (A - left) a relocalisation of amyloplast in statocytes of seedlings grown microgravity conditions compared to those grown on 1g centrifuge; (A - right) a slight amyloplast displacement after 10 min of transfer. Surprisingly, despite the slight amyloplast displacement, a change in the number of calcium precipitation is revealed after each transfer. These results suggest that the calcium signalling seems to be affected by a slight amyloplast displacement. The presence of a gravi-receptor near the amyloplast is then hypothesised.
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.
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Figure 1: Polarisation of the statocyte.

Figure 2: Experiment Concept Diagram.

Figure 3: Mission Concept Diagram.

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

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

Figure 6: Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline.

Figure 7: Amyloplasts distribution (A) and number of calcium precipitates (B) in statocytes of seedling roots grown in microgravity condition (mg), on 1 g centrifuge in space (1 g, space control), in microgravity for 28 h and then transferred during 10 min on the centrifuge (mg + 1 g), or on 1 g centrifuge for 28 h and transferred to microgravity condition for 10 min (1g + mg). The relative distribution of amyloplasts is indicated by a color scale from yellow to red.
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