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
Jason Hatton
V. Legué (1), J. Gérard (2), V. Pereda-Loth (3), M. Courtade-Said (3), B. Eche (3)
Clermont Université
Université Blaise Pascal and INRA
UMR INRA/Clermont Université 547 PIAF
Lorraine University
Toulouse University
GSBMS - University P. Sabatier

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.

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

The analysis of the GRAVI-2 project
(credit: January 2016 - GRAVI2 blog:
https://lensesinspace.wordpress.com/2016/01/14/the-analysis-of-the-gravi-2-project/ )

Part 1: On-board picture analysis
(compare: Figure 6)

Picture of the cultivation chambers containing the lentil seeds were taken during the whole duration of the experiment on-board of the ISS.

Apart from checking the successful conduct of the experiment and lentil seeds germination, these pictures offer a unique opportunity to test the ability of roots to detect a very low level of gravity (0.01g) and to induce a measurable curvature (gravitropism answer).

Recently, all 2468 pictures from the experiment were denoised and used to measure roots position and curvature each hour after germination (see pictures below). As observed and described during the first GRAVI-1 experiment, young lentil roots are bent and show an important straightening of the root at early stages of the root development (called autotropism, see video below). Ongoing analysis are dedicated to test the presence of an eventual gravitropism answer within the visible autotropism.

Part 2: Measurement of statolith displacement on root longitudinal sections (compare: Figure 7)

Some of the root apices that came back from the ISS in 2014 were imbedded in resin. During the last months, longitudinal sections of most of the samples were done in our laboratory (UMR PIAF INRA-UBP, Clermont-Ferrand). We are currently developing analysis tool to measure semi-automatically the position of the statolith in cells of the root columella (see pictures below). Statoliths are intracellular organelles which displacements are known to be involved into gravity perception. Differential positioning of the statoliths between microgravity, 0.01g condition and 2g gravistimulus will be used to quantify statolith displacement at 0.01g and check perception of the gravistimulus at 2g. In the coming months, we plan to go further on this point and to stack successive sections to obtain a 3D visualization of statoliths position in cells in microgravity.

Part 3: Involvement of intracellular calcium into gravity signal transduction (compare: Figure 8)

Intra-cellular calcium is a good candidate acting as a secondary messenger for gravity signal transduction. At the end of the GRAVI-2 experiment, some root apices were fixed with chemical reagent that precipitates the intra-cellular calcium. After tests, concerned samples were sent last week in a partner laboratory (CICS, Clermont-Ferrand) which will make ultra-fine longitudinal sectioning of the root apices (below 100nm thickness). Theses cuttings will be further used to quantify the number of intra-cellular calcium precipitates and compare their position to cell wall, to statolith location and to endoplasmic reticulum membranes (see picture below). Such data will help deciphering the involvement of intra-cellular calcium into gravity signal transduction at the interface between various cellular structures.

Part 4: Transcriptomic analysis

In order to identify which network of genes are over-expressed or repressed after a gravistimulus (2g 5min and 2g 15min), transcriptomic analyses will be performed on samples fixed with RNA preserving products. We are glad to announce that after one year and half storage, we successfully extracted October 2015 excellent quantity and quality of total RNA from all selected samples. Samples were sent to a bioinformatics platform (Genotoul, Toulouse) for RNA sequencing. We received the dataset this week. Those data will also allow us to identify candidate genes for future project concerning gravity perception, on ground or on-board.

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.
E.B. Blancaflor, (2002), "The cytoskeleton and gravitropism in higher plants", Journal of Plant Growth Regulation, 21, pp. 120-136.
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.
D. Driss-Ecole, A. Lefranc, G. Perbal, (2003), "A polarised cell: the root statocyte", Physiologia Plantarum, 118, pp. 305-312.
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.
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.
E. McCormack, Y.C. Tsai, J. Braam, (2005), "Handling calcium signaling: Arabidopsis CaMs and CMLs", Trends in Plant Science, 10, pp. 383-387.
G. Perbal, D. Driss-Ecole, (2003), "Mechanotransduction in gravisensing cells", Trends in Plant Science, 8, pp. 498-504.
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.
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.
C. Plieth, A.J. Trewavas, (2002), "Reorientation of seedlings in the earth’s gravitational field induces calcium transients", Plant Physiology, 129, pp. 786-796.
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).

Gravi-2 continues the research of its predecessor into how sensitive plants are to gravity. To find out, 768 lentil seeds were subjected to different levels of simulated gravity. Spinning them in centrifuges at different speeds on the International Space Station will recreate gravity, similar to how astronauts and fighter pilots are subjected to high g-forces in human centrifuges. The goal was to see at what gravity level the seedlings begin to show growth differences. Kept spinning for 31 hours at four different centrifuge speeds, the seedlings were observed as they grow.

more in-depth information can be found on the GRAVI2 blog: https://lensesinspace.wordpress.com/

Figure 6: Raw (top) and denoised (bottom) picture taken during the experiment on-board of the ISS. The 12 seeds of a cultivation chamber are visible, most of them showing distinguishable young roots growing in microgravity. credit: GRAVI2 team

Figure 7: Left: longitudinal section of a lentil root apex grown in microgravity colored with periodic acid and Schiff’s reagents (left). Right: image thresholding allow proper segmentation of cell walls and statoliths (black dots) into our zone of interest, the columella (green zone). credit: GRAVI2 team

Figure 8: Transmission electron microscopy view of a columella cell at different magnification (left: X7000; right: X15000). Several starch grains (sg) are visible within each statolith (st). Calcium (ca) are small black dots which position can be compared to statoliths, endoplasmic reticulum (er, visible at higher magnification) and cell wall (cw). credit: GRAVI2 team

Understanding the deeper mechanisms that cause a plant to grow in a particular direction has far-reaching possibilities for agriculture – as well as for astronauts who want to enjoy fresh vegetables on a long space mission. This image shows a lentil seedling root that grew on the International Space Station before being preserved in resin and cut along its length for analysis. The purple dots are starch-filled statoliths that usually drop towards gravity, but this plant grew in space and the statoliths are floating in the middle of their cells. In addition to these cross-sections, almost 2500 pictures charted the 768 seeds growing over 31 hours in microgravity and hypergravity in the European Modular Cultivation System on ESA’s Columbus space laboratory. “This research could not be done on Earth because gravity would get in the way of our readings,” explains Francois Bizet, who is analysing the images as part of a post-doctoral programme in France’s CNES space agency. Regular updates are posted on the experiment´s blog. https://lensesinspace.wordpress.com/ The results from the Gravi-2 experiment are showing what could be responsible for sending growth-direction signals to the plant’s cells. Gravi-2 continued an earlier experiment that examined the limits of how plants perceive gravity, with this second experiment looking in particular at how calcium is used by plants to regulate growth. On Earth, soluble calcium spreads to plant roots and is considered an important part of plant growth because they respond to environmental signals. Gravi-2 will also look at gene expression to highlight how intracellular calcium could be a second messenger for perceiving gravity. “We need to grow the seeds in different environments to compare results and work out which change is due to gravity,” says Valérie Legué from the Université Blaise Pascal in Clermont-Ferrand, France. “Understanding plant growth is the first step to adapting crops for more productive agriculture. If we could grow lentils vertically, for example, farmers could drastically increase crop yield per square metre.”

ESA´s webnews on the experiment, dated 22 April 2014.
© 2016 European Space Agency