EXPERIMENT RECORD N° 9544
Seedling Growth 3
  1. 2016 • ISS Increments 49-50
  2. 2017 • ISS Increments 51-52
Life Sciences:
  • Plant Biology and Physiology
EMCS
Jason Hatton
jason.hatton@esa.int
F.J. Medina (1), R. Herranz (1), E. Carnero-Diaz (2), E. Boucheron-Dubuisson (2), J. Saez-Vasquez (3), J.Z. Kiss (4), R. Edelmann (5)
(1)  
CSIC - Centro de Investigaciones Biológicas
Ramiro de Maeztu 9
28040 Madrid
SPAIN
Tel:  
+34(0)91.8373112
e-mail:  
fjmedina@cib.csic.es
rherranz@cib.csic.es
(2)  
UMR 7205-ISYEB-CNRS-MNHN-UPMC-EPHE
Muséum National d´Histoire Naturelle
DSE - Département de Systématique et Evolution
CP 39
12, rue Buffon
75005 Paris
FRANCE
e-mail:  
eugenie.carnero_diaz@upmc.fr
elodie.boucheron_dubuisson@upmc.fr
(3)  
CNRS-IRD-Université de Perpignan via Domitia
52 avenue Paul Alduy
Perpignan Cedex 9
FRANCE
e-mail:  
saez@univ-perp.fr
(4)  
Dean of the Graduate School
The University of Mississippi
100 Graduate House
University, MS 38677
USA
e-mail:  
jzkiss@olemiss.edu
(5)  
Department of Botany
Miami University
316 Pearson
Oxford, OH
USA
e-mail:  
edelmare@muohio.edu
Combined implementation of:
Project ID: ILSRA-2009-0932 "Effects of red light stimulation on cell growth and proliferation under spaceflight conditions in Arabidopsis thaliana (LICEA)"
and
Project ID: ILSRA-2009-1177 "Novel explorations into the interactions between light and gravity sensing in plants"

The ILSRA-2009-0932 LICEA experiment has complimentary objectives and requirements to ILSRA-2009-1177 project, with a common team of scientific co-investigators between the two projects. To optimise the science gain and minimise the ISS resource requirements of the two experiments, the two studies have been combined.


LICEA - Effects of red light stimulation on cell growth and proliferation under spaceflight conditions in Arabidopsis thaliana

BACKGROUND

This project builds on our previous spaceflight and ground-based experiments by the science team using the model plant Arabidopsis thaliana. In the ROOT and GENARA-A experiments (performed on the ISS) and in the ROOT CELL PROLIFERATION (approached up until now on ground-based facilities), results indicated that cell growth and proliferation in root meristems are altered by changing the effective gravity, including the expression of relevant cell cycle genes. Furthermore, the distribution of auxin is altered, resulting in modifications of the pattern of primary and secondary root growth.

On the other hand, in the TROPI experiment in the EMCS on the ISS, the phototropic response of seedlings grown in microgravity was studied and phytochrome photoreceptors were identified; then, growth, development, and phototropic curvature of plants in response to varying qualities of light were analysed, as well as global gene expression changes using DNA microarrays.

OVERVIEW
The contribution of light stimuli to the regulation of cell growth and proliferation will be evaluated by using red-light treatments, known to stimulate the rates of cell proliferation and ribosome biogenesis, on auxin mutants, on ribosome-biogenesis mutants as well as on a transformed line containing the GFP (Green Fluorescent Protein) reporter gene coupled with an auxin-responsive element. Microscopical, immunocytochemical, RT-PCR and proteomic post-flight analyses will be performed using these strains and also wild-type samples.

Furthermore, digital images downlinked from all these samples throughout their growth will be analysed in order to detect and identify alterations in the phototropic response.

Figure 1
summarises the working hypothesis of the interaction between light and gravity which will be explored in this experiment.

AIM

The experiment aims to understand the combined influence of light and gravity on plant development through the identification of changes in the mechanisms and regulation of essential cellular functions. Proliferation and growth of root meristematic cells are the functions to be mechanistically studied, and auxin transport and perception will be analysed as a regulatory process of these cellular functions, also affecting the developmental pattern of the plant.

The Seedling Growth investigation was divided into three parts:

SPECIFIC GOALS
1) To determine the modifications of cellular processes and mechanisms caused by red light stimulation under environmental conditions of microgravity or fractional gravity in space. Our interest is focused on root meristematic cell growth and proliferation, two basic and essential cell processes which are fundamental for the plant developmental programme. Specifically, we want to study:
a) The effect of phytochrome activation by red light, as a potentially proliferative stimulus, on the compensation of the alterations in cell cycle and ribosome biogenesis caused by the gravitational stress;
b) The concerted action of light and gravity in controlling plant growth and development through effects on cell proliferation, especially focused in the root development.
2) To investigate the effects of red-light-stimulation in the space environment on the alteration of auxin transport and perception, and of auxin distribution in the root.
Auxins are regulators and coordinators of meristematic cell growth and proliferation and are key factors affecting plant development.

3) To know the alterations in the phototropic behavior after red light stimulation, under different gravity conditions, of plants defective in essential components of the apparatus of auxin transport and/or perception, or lacking essential factors of cell growth and proliferation.

4) To detect gravity thresholds of the cellular effects of the gravitational stress, by means of the investigation of the relationship between light and gravity in fractional gravity. This investigation may also serve to approach a simulation of the gravity conditions in Moon and Mars.

Novel explorations into the interactions between light and gravity sensing in plants

OVERVIEW

The major goals of this project are to determine how gravity and light responses influence each other in plants and to better understand the cellular signaling mechanisms involved in plant tropisms. This proposed project builds on our previous spaceflight experiments on the ISS with the EMCS using the model plant Arabidopsis
thaliana
. In this new proposed project, we plan to confirm and extend our discovery during the recent ISS experiment of a novel red-light-based positive phototropic response in plant shoots.

The EMCS, an existing and successfully flown hardware, is an automated facility and can be operated largely with telemetry. The experiment can be accomplished with telemetric science and therefore fit with the extremely limited down mass capabilities of current spaceflight opportunities.
Experimental containers can be launched with any of the currently available spacecraft, and during an experimental run, images of seedling growth responses can be downlinked to Earth.

This project is also relevant to the recent emphasis at NASA to study plant growth and development at fractional g-levels such as those found on the Moon and Mars. Improved knowledge of the basic mechanistic processes that will be the focus of this project is vital to develop ways to use plants in extraterrestrial bioregenerative life support systems.

AIM
The aim is to investigate fundamental interactions among red-light signaling pathways and the gravity sensing mechanisms of the plant Arabidopsis thaliana.

SPECIFIC GOALS
The specific objectives of this proposed research are:
1. To confirm and characterise the novel red light-dependent phototropic response in flowering plants;
2. To investigate the relationship between light and gravity by measuring thresholds in fractional gravity;
3. To determine whether the red-light-effect on blue-light-based phototropism is a direct or indirect effect;

Justification for the Need of Space Experiment
The ISS is the only laboratory facility where it is possible to reliably and simultaneously vary light and gravity stimuli for the proposed experiments.

HYPOTHESIS

Our hypothesis is that positive red-light-sensing, which was known in older plant lineages, is masked by normal 1-g conditions in more recently evolved lineages.


RELATED RESEARCH

GENARA-A Gravity Regulated Genes in Arabidopsis thaliana
ISS Increment 23-24 - 2010
The experiment has been divided into two parts. Part A was performed on the ISS. Proteomic study of seedlings grown in space in search for products of gravity-regulated genes. Part B has been incorporated into the new experiment “Plant Development” for future execution on the ISS.

ROOT - Effects of the space environment on the nuclear structure and function of plant root meristematic cells grown in microgravity
ISS 7S (Soyuz TMA-3) Spanish "Cervantes" Mission - 2003
Seedlings germinated and grown in space were analysed for parameters of cell proliferation and ribosome biogenesis. These processes appeared altered.

TROPI Experiments:
Performed on the ISS in 2006 and 2010. Study of the phototropic response of seedlings in microgravity. Finding and characterisation of a new phototropism of hypocotyls in response to red light.

GraPhoBox - Study into interaction of effect of light and gravity on the growth processes of plants

ISS 8S (Soyuz TMA-4) Dutch "Delta" Mission - 2004
All equipment was launched to the ISS on SpaceX-11 mission on 3 June 2017. The FixBoxes were inserted into Biolab TCUs on 5 June and remained there during the entire SpX-11 docking phase, except for when in use.

The Seedling Growth 3 spaceflight experiment, led by ESA and supported by NASA was performed in the EMCS on-board ISS between 12 and 27 June 2017. Run 1 was performed between 12 and 19 June, Run 2 from 20 to 26 June. 
The past two experiments in the Seedling Growth series returned frozen samples only. A new feature of the Seedling Growth 3 experiment was chemical fixation of some of the samples. This fixation was important to allow for the specific objectives of this experiment.

Crew members Jack Fischer and Peggy Whitson performed the sample processing after both runs. For Run 1, all samples were fixed in FixBoxes, while for Run 2 half of the samples were fixed, and the other half frozen. During the sample processing for Run 1, one of the FixBoxes was not closed properly, and got stuck. The samples were then not fixed, and the FixBox was frozen to preserve the samples. The remaining sample processing went according to plan. All samples were downloaded with SpaceX-11 on 3 July 2017.

PLANNED PROCEDURE
The seeds will be stored dry until the start of the experiment on orbit. The experiments will be activated by the hydration of seeds. The samples will be maintained in EMCS at a constant temperature of 22º C, different g-levels (microgravity, 1 g, 0.1 g, 0.3 g, 0.5 g and 0.8 g) controlled humidity and illuminated with white, red and/or blue light according to the protocol.
The experiment is split into 4 distinct parts, each part consisting of a different number of runs. The experiment runs will be performed with different g-levels and total run durations, summarised as follows;


during ISS Increment 39/40
Part 1 - Seedling Growth-1 (SG-1): Phototropism

- Run 1: 6 days duration, microgravity
- Run 2: 6 days duration, 0.1 g
- Run 3: 6 days duration, 0.3 g
- Run 4: 6 days duration, 1 g
during ISS Increment 41/42
Part 2 - Seedling Growth-2 (SG-2): Tropisms, Auxin and Cell Cycle

- Run 1: 6 days duration, 0.5 g
- Run 2: 6 days duration, 0.8 g
- Run 3: 6 days duration, microgravity and 1 g
during ISS Increment 47/48
Part 3 - Seedling Growth-3 (SG-3): Proliferating Cell Structure

- Run 1: 6 days duration, microgravity and 1g
- Run 2: 6 days duration, 0.3 g
(The use of a dedicated hardware (Fixation Box, FixBox for short) providing 3 LoCs is foreseen during the Seedling Growth-3 part to achieve the PFA (Paraformaldehyde) and GA (Glutaraldehyde) chemical fixation of the samples.)

Part 4 - Seedling Growth-4 (SG-4): Synthesis/experiment contingency
The runs will be defined based on the outcome of the results of proceeding parts of the experiment.

Data on germination, growth and tropic responses (e.g. curvature of shoots/roots) will be obtained from imaging of the culture containers.
At the end of each run, samples will be either frozen or chemically fixed according to the programme (PFA - Paraformaldehyde, GA - Glutaraldehyde, or Deep Freezing) and cold stored until post-flight retrieval.

Ground reference experiment
Post-flight run
as ground control duplicating the experiment conditions in flight (timeline, temperature etc.) under 1 g conditions, as well as under simulated microgravity, in a random positioning machine.

Biological Samples
Biological sample / Specimen type
Species: Arabidopsis thaliana
Ecotype: Columbia
Wild type (Col0)
Mutants:

eir 1.1: mutant for auxin transport (efflux)
aux 1.7: mutant for auxin transport (influx)
tir1: mutant for auxin perception
Atnuc1(Δ90) and Atnuc2:
mutants for the nucleolin-like protein AtNUC-L1

Genetic constructions with Green Fluorescent Protein (GFP):
DR5:GFP (DR5 is an auxin responsive element)
Ecotype: Landsberg erecta
Wild type (Ler)
Phytochrome mutants:
PhyB
PhyA

Treatments / conditions (e.g. activators, drugs, tracers, fixatives)
Plant culture medium:
Whatman #17 filter paper soaked in half strength Murashige and Skoog salts (Duchefa Cat. No. M0221;
Physiol. Plant. (2003) 15, 473-497) with 1% (w/v) sucrose buffered with 1mM MES (pH 5.5) and allowed to dry.

Seeds are affixed with 1% (w/v) gum guar onto a black gridded membrane made of mixed cellulose esters.

Distilled water for hydration.

Experiment termination by either Deep Freezing (<-68°C) or
Chemical Fixation with the following fixatives:
Fixative #1: 5 % Paraformaldehyde (PFA) in Phosphate Buffered Saline (PBS)
Fixative #2: 4.5 % Glutaraldehyde (GA) + 1.5% Paraformaldehyde in PBS

Required g-levels
0 g
0.1 g
0.3 g
0.5 g
0.8 g
1 g

Number of samples required for each g-level / condition
5 cassettes per EC.
At least 14 seeds per cassette.

Planned analyses
Post-flight parameters measured:

From downlinked images:
- Rate of germination,
- Seedling and root length,
- Seedling tropistic curvatures
- Number and distribution of secondary roots,

From frozen / fixed / preserved samples:
1. Light and electron microscopy (parameters related to root meristematic cell growth and proliferation):
- Immunodetectionof nucleolin, RNA pol1 and cyclinB1
- Rate of local cell production
- Nucleolar size
- Nucleolar ultrastructure-proportion of granular component
- Number of cells per mm in the root meristem

2. Molecular Biology (RNA and Protein extraction)
- Global gene expression: microarray technique
- Expression of relevant genes (nucleolin, cyclins, auxin responsive elements): RT-PCR or qPCR
- Protein analysis: two-dimensional electrophoresis, proteomic techniques
Expected results:
- Definition with a precision higher than presently available of the factors involved, either as causes or effects, in the uncoupling of cell proliferation and cell growth which has previously described in root meristematic cells from plants grown in a microgravity environment.
- Determination of the role of the auxin polar transport in the cellular alterations caused by the altered gravity stress.
- Evaluation of the effect of a pulse of red light on counteracting the alterations in cell growth and proliferation caused by the gravitational stress.
- Estimation of the effects of fractional gravity on altering cell growth and proliferation parameters.

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[2]  
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click on items to display

Figure 1: Schematic representation of the effects of gravity and light on the roots of developing plants and, specifically, on root meristematic cells, and the pending questions which are approached in this experiment. The main hypothesis to test in the project is whether or not the combined stimuli of altered gravity (microgravity) and modulated light (red light) produce effects on the auxin distribution in roots which may result in alterations of essential functions of meristematic cells, such as cell cycle and ribosome biogenesis.

Summary Overview Flow Diagrams: Seedling Growth-1

Seedling Growth-1 - Run #1

Seedling Growth-1 - Run #2

Seedling Growth-1 - Run #3

Seedling Growth-1 - Run #4

Summary Overview Flow Diagrams: Seedling Growth-2

Seedling Growth-2 - Run #1

Seedling Growth-2 - Run #2

Seedling Growth-2 - Run #3

Summary Overview Flow Diagrams: Seedling Growth-3

Seedling Growth-3 - Run #1

Seedling Growth-3 - Run #2
 
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