EXPERIMENT RECORD N° 9416
GRAVI2 - Threshold Acceleration for Gravisensing, Part 2: Amyloplast displacement and calcium signalling in root gravisensing
  1. 2013 • ISS Increment 37-38
  2. 2014 • ISS Increment 39-40
Life Sciences:
  • Plant Biology and Physiology
EMCS
Jason Hatton
jason.hatton@esa.int
V. Legué (1), J. Gérard (1), V. Pereda-Campos (2)
(1)  
Université Henri Poincaré
UMR INRA/UHP 1136 - Interactions Arbres/Micro-organismes
IFR 110 - Génomique, Ecophysiologie et Ecologie Fonctionnelles
BP 239
54506 Vandoeuvre Cedex
FRANCE
Tel:  
+33(0)3.83.68.42.3231
Fax:  
+33(0)3.83.39.40.13
e-mail:  
Valerie.Legue@scbiol.uhp-nancy.fr
jgerard@scbiol.uhp-nancy.fr
(2)  
Toulouse University
GSBMS
FRANCE
e-mail:  
pereda@cict.fr

BACKGROUND
When a seedling is growing in the vertical (normal) position and is placed in the horizontal position, its extremities (root, shoot) start bending in order to recover their normal orientation with respect to the gravity vector. The primary root is most often studied, since in this organ the site of stimulus perception is distinct from the zone of response, the root curvature. The former is located in the root cap, whereas the latter is situated behind the root tip at the level of both the distal root meristem and the proximal elongation zone.

Three parameters of the gravitropic response should be known to better understand the perception and the transduction of the gravistimulus. The first one is the presentation dose (minimum quantity of stimulation to provoke a significant curvature), the second one is the threshold acceleration for gravisensing (minimum acceleration which can be perceived), and the last one is the minimum deviation from the gravity vector, which leads to a re-orientation of the root.

Upon a gravistimulation a change was observed in the polarity of cell perceiving gravity signal with an amyloplast sedimentation on the peripheral side. Transduction pathways were immediately activated with changes of calcium-dependant pathways. In parallel, many studies have demonstrated that cytoplasmic free Ca2+ concentration ([Ca2+] cyt) is affected by environmental stimuli. The regulation of this homeostasis involves a series of transduction events such as the synthesis and activation of calcium binding and targeted proteins, like calmodulin. From results obtained in GRAVI 1 experiment (D. Driss-Ecole et al., 2008), the threshold of acceleration perceived by the lentil root has been estimated to be 1.37 x 10-5 g showing that roots are strongly sensitive to gravistimulus.

AIM
To study the implication of amyloplasts displacement and calcium signalling in root gravisensing and, thus to understand cellular signalling mechanisms involved during the threshold acceleration.

Specific goals

1. To subject lentil seedling roots to centrifugal acceleration levels from 10E-2g to 2g and in microgravity
2. To determine the movement of amyloplasts under the influence of the stimulation.
3. To analyse free calcium distribution and calcium binding- and targeted-proteins


The GRAVI2 experiment is a follow-on of GRAVI 1: Video observation and recording of Lentil (Lens culinaris) seedlings roots after gravistimulation.

Related research:
Threshold Acceleration for Gravisensing (GRAVI 1)
ISS 13S (Soyuz TMA-9) + Increment 14 - 2006

Lens culinaris seeds are launched and installed in the EMCS facility. Germination will be induced by hydration of seeds. Different samples will be exposed to different g-levels or at the same g-levels for different durations to determine the movement of amyloplasts under the influence of the stimulation and to analyse free calcium distribution and calcium binding- and targeted-proteins.

General Experiment Procedure
The time period of growth is 30 h from hydration for all samples as follows:
Run #1
(Comparison with results of GRAVI-1 experiment, examination of cell ultrastructure, Ca2+ localisation & gene expression): For the first run, half of the samples are hydrated and allowed to germinate in μg conditions for 30 hours, the other half are exposed to 21h of μg after hydration and then centrifuged at 0.01g for 9 hours.

Run #2 (Kinetics of Ca2+ redistribution with gravistimulation & downstream gene expression): For the second run, after almost 30 hours of germination in microgravity, half of the samples are exposed for 15 minutes at 2g and the other half for 5 minutes at 2g.

The total duration of both runs (run 1 and run 2) must be identical (with one hour maximum difference).
At the end of the experiment, all samples are fixed on the centrifuge and then stowed at 4ºC (for detailed margins, see below in the attachments: Functional Objectives table). Fixed samples must stay on centrifuge between 1h and 3h in order to ensure complete fixation.

Ground reference experiment(s):
The ground reference experiment shall consist of a continuous nominal 1g cultivation of 4 EUEs. If a science model is available, the ground reference experiment shall be performed in parallel to the flight. If not, the ground reference experiment shall be performed 3-4 weeks later.

Biological Samples:
Lens culinaris seeds.

Number of samples per condition / g-level:
Preferred configuration
- 24 lentil seeds per CC. Seedlings shall have a preferred root length of 10 mm (minimum 9 mm) at the time of fixation.
- 2 CCs per EUE (48 seeds)

- 4 fixatives with different science objectives for analysis:
Fix #1 = Glutaraldehyde mix. Amyloplast redistribution & cell ultrastructure by electron transmission microscopy, include endoplasmic reticulum (a key Ca2+ reserve). This is very important cell ultrastructure data for comparison to Gravi-1.
Fix #2 = Paraformaldehyde mix. Immunolocalisation using light and electron microscopy. Poorer resolution of cell ultrastruture than for Fix #1 samples.
Fix #3 = Paraformaldehyde mix + potassium pyroantimonate. Localisation of intracellular Ca2+
Fix #4 = RNAlater. Gene expression

  • Run #1:
    - 4 EUEs (192 seeds) centrifuged at 0.01g for 9 hours (see Functional Objectives table for time margins).
    - 4 EUEs in microgravity for 30 hours. (see Functional Objectives table for time margins).
              + Per g-level treatment one EC each of Fix#1, Fix #2, Fix #3 & Fix #4
  • Run #2:
    - 4 EUEs centrifuged for 15 minutes at 2g (see Functional Objectives table for time margins).
    - 4 EUEs centrifuged for 5 minutes at 2g. (see Functional Objectives table for time margins).
              + Per g-level treatment;
                      1x EC Fix #2
                      1x EC Fix #3
                      2x EC Fix #4
    - A 100% germination rate is expected. 60% of germination is sufficient to achieve the scientific objectives.
    Reduced EC configurations: see section 4.4 for trade-offs & science impact in case of the need to reduce the number of EC’s

Planned analyses
Post-flight parameters measured

- Angle of curvature (from video images)
- Amyloplasts distribution from microscopy images
- Calcium-binding and calcium-targeted proteins localisation
- Free calcium distribution in statocytes
- Expression of calcium-regulated genes in root cap

Expected results

- Change in calcium localization (2g samples)
- Translocation of calcium-targeted protein
- Increased curvature in 2g
- Modified gene expression

[1]  
D. Driss-Ecole, V. Legué, E. Carnero-Diaz, G. Perbal, (2008), "Gravisensitivity and automorphogenesis of lentil seedling roots grown on board the International Space Station", Physiologia Plantarum, 134, pp. 191-201.
[2]  
E.B. Blancaflor, (2002), "The cytoskeleton and gravitropism in higher plants", Journal of Plant Growth Regulation, 21, pp. 120-136.
[3]  
D. Driss-Ecole, V. Legué, E. Carnero-Diaz, G. Perbal, (2008), "Gravisensitivity and automorphogenesis of lentil seedling roots grown on board the International Space Station", Physiologia Plantarum, 134, pp. 191-201.
[4]  
D. Driss-Ecole, A. Lefranc, G. Perbal, (2003), "A polarised cell: the root statocyte", Physiologia Plantarum, 118, pp. 305-312.
[5]  
J.M. Fasano, S.J. Swanson, E.B. Blancaflor, P.E. Dowd, T. Kao, S. Gilroy, (2001), "Changes in root cap pH are required for the gravity response of the Arabidopsis root", The Plant Cell, 13, 4, doi: http:/​/​dx.​doi.​org/​10.​1105/​tpc.​13.​4.​907, pp. 907-921.
[6]  
V. Legué, E.B. 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.
[7]  
E. McCormack, Y.C. Tsai, J. Braam, (2005), "Handling calcium signaling: Arabidopsis CaMs and CMLs", Trends in Plant Science, 10, pp. 383-387.
[8]  
G. Perbal, D. Driss-Ecole, (2003), "Mechanotransduction in gravisensing cells", Trends in Plant Science, 8, pp. 498-504.
[9]  
G. Perbal, D. Driss-Ecole, (1994), "Sensitivity to gravistimulus of lentil seedling roots grown in space during the IML 1 Mission of Spacelab", Physiologia Plantarum, 90, 2, pp. 313-318.
[10]  
G. Perbal, D. Driss-Ecole, (1989), "Polarity of statocytes in lentil seedlings roots grwn in space (Spacelab D1 Mission)", Physiologia Plantarum, 75, pp. 518-524.
[11]  
C. Plieth, A.J. Trewavas, (2002), "Reorientation of seedlings in the earth’s gravitational field induces calcium transients", Plant Physiology, 129, pp. 786-796.
[12]  
J. Zhao, H.Y. Yang, E.M. Lord, (2004), "Calcium levels increase in the lily stylar transmitting tract after pollination", Sexual Plant Reproduction, 16, 6, pp. 259-263.
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Figure 1: Lentil seeds will be flown on the ISS and watered in order to induce germination in microgravity. During a 30 hours germination period, different gravity levels will be applied to the samples for different durations in order to better understand the parameters of the gravitropic response. The experiment will investigate two gravity levels (0.01g and 2g). However, because of the position of the seeds on the centrifuge, there will be a gravity-gradient within each culture chamber, the lowest g-value perceived by the seeds being 0.005g, the highest being 2g (as illustrated above).

Figure 2: Overview Experiment Timeline and associated Functional Objectives Note: The duration of each run is 30 (+/-5) hours. Both runs must have the same duration (1 hour maximum difference). Note #2: The flow diagram only indicates the general sequence of operations. Refer to the Functional Objectives table for actual time margins and inter-relationship between steps.

Figure 3: Functional Objective Table #1/3 (Temperatures and Durations).

Figure 4: Functional Objective Table: #2/3 (Humidity, gas and gravity).

Figure 5: Functional Objective Table #3/3 (Additional requirements).
 
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