EXPERIMENT RECORD N° 9178
WAICO 2 - Waving and Coiling of Arabidopsis Roots at Different g-levels
  1. 2010 • ISS Increments 23-24
Life Sciences:
  • Plant Biology and Physiology
BIOLAB
Jason Hatton
jason.hatton@esa.int
G. Scherer (1)
(1)  
University of Hannover
Institute of Floriculture, Abt. Ertragsphysiologie
Herrenhaeuser Strasse 2
30419 Hannover
GERMANY
Tel:  
+49(0)511.762.31534417
Fax:  
+49(0)511.7623608
e-mail:  
scherer@zier.uni-hannover.de

AIM
- To understand the interaction of circumnutation and gravitropism, by observing the waiving and coiling of Arabidopsis thaliana wild type and an agravitropic mutant (At1g61850 knockout). Specifically, verify that circumnutation of Arabidoposis roots is driven by an endogenous mechanism, that is independent of gravity as a cue.

This hypothesis was tested as follows:

- If the coiling of the roots increases as the g-level is decreased, then this indicates that as the gravitropic stimulus decreases the endogenous processes which drive coiling become dominant.

- If the roots grow straight as the gravity level decreases, then this suggests that the gravitropic stimulus provides an input to the coiling process. Hence in this case the coiling process is not endogenously driven, but dependant on gravity.

WAICO experiment had two experiment runs to compare effects on Arabidopsis at different g-levels; 1g for run#1 and 0.5g for run#2.

The run#1 (WAICO 1) had already been performed in the previous flight, however, the chemical fixation of the experiment was unsuccessful. Therefore, run#1 will be repeated instead of run#2 as WAICO 2 and the requirement of run#2 was withdrawn from the WAICO 1 experiment.

Specific goals

  1. Compare the waiving & coiling growth of Arabidopsis at different g-levels between 0 g & 1 g by timelapse imaging of the root growth over an approximately 2 week period.
  2. Compare the response of gravitropic wild type Arabidposis with the agravitropic AtPLA1 knockout Arabidopis mutant.
  3. Observe root ultrastructure (postflight fixed samples), in particularly microtubule orientation, to understand how the root structure contributes to the waving & coiling process.

Precursor flights: WAICO 1 (STS-122)

Figure 1: General experiment hardware layout in Biolab Experiment Container (EC).

Figure 2: General experiment concept.

RELATED RESEARCH
2-D/3-D Analysis of root development in microgravity studied on parabolic flights (ESA)
1st Cooperative Parabolic Flight Campaign - 2015

GRAMAT: Isolation of mRNA from microgravity and ground samples to identify gravity-related gene expression - SPARC: A specialized phospholipase A, and re-localization in auxin-transporting cells in micro-g
60th ESA Parabolic Flight Campaign - 2014

WAICO 1 - Waving and Coiling of Arabidopsis Roots at Different g-levels
ISS Increment 16/17 - 2007/2008

Arabidopsis Thaliana in Space: Perception of Gravity, Signal Transduction and Graviresponse in Higher Plants (AT-Space)
ISS Increment 16 - 2007

Perception Of Gravity, Signal Transduction And Graviresponse In Higher Plants using Innovative Genomic Technologies (AT-SPACE)
Foton-M1- 2002

Experiment protocol
Biological Samples: Arabidopsis thaliana, wild type (ecotype Wassilewkia) and strain (knockout line for gene At1g61850) used for experiment

Experiment protocol summary
– Seeds launched dry separated from agar, maintained at ambient during upload.
– Reagents stored on orbit at +4°C prior to the activation of the experiment for up to 7 months from time of hand-over.
– Seeds stored on orbit at +4°C prior to the activation of the experiment for up to 7 months from time of hand-over. Alternative storage is ambient, but in this case the seeds should be exposed to +4°C for at least 7 days (vernalisation) & vernalisation should occur within 1 month of the experiment activation.
– Cultivation for nominally 12 days at 22°C in ethylene free air with high humidity (80% or above) and continuous illumination (prefer 5,000-10,000 lux).
– Minimum 30 seeds per g-level and plant type (wild type and mutant).
– 1 cultivation bowl with 15 seeds each in each Experiment Container (EC).
– All seeds germinated at 1.g, with g-vector acting perpendicular to agar surface for approximately 3 days.
– Growing seedlings observed by video every day, samples briefly reorientated to permit perpendicular view of agar surface with growing seedlings.
– Samples reorientated at 45 degree to g-vector & cultivated for approximately 9 days.
– Samples photographed with high resolution stills camera at end of experiment run prior to fixation.
– Samples fixed, then washed twice at end of run.
– Refrigerated stowage of fixed samples after end of experiment run for up to 3 months (prefer download within 4-6 weeks after end of experiment run).
– Fixed samples downloaded in refrigerated stowage.

General Experiment Procedure
• Parameters measured:

– Inflight parameters measured;
• Daily video observation of root position
• High resolution photography of seedling at end of experiment run (before fixation)
– Postflight analysis
• Root structure (postflight analysis on fixed samples)

Figure 3: Detailed experiment timeline and associated functional objectives: Experiment run #1.

Figure 4: Flow Diagram with Functional Objectives indicated: Experiment run #1.

Figure 5: Detailed experiment flow for each Experiment Container (EC).

Table 1
List of Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline with reference to Figure 4 FO steps.

Ground reference experiment(s):
Postflight ground control run according to actual flight profile (sequence of actions, timeline, temperature profile).

OTHER experiment specific contraints
- At least one EC of each experimental condition should be downloaded.
- In case of off nominal growth or performance in individual EC’s priority should be given to the EC in each experimental condition which has highest quality samples. Negative selection criterior include the following:

- Poor germination
- Visible fungal or bacterial contamination
- Requirement for 4 EC’s to be downloaded in cold stowage
- Desirable to also download remaining 4 EC’s at ambient temperature

Science deliverables
- Daily images of experiment samples, of sufficent quality to show seedling size (green parts) and root size, shape & orientation
- High resolution images of seedlings at the end of the experiment run
- Formaldehyde fixed seedlings after completion of experimental protocol at two different g-levels for microscopy & structural analysis:

• Microtubule organisation
• Coiling structure of root wall

Planned analyses
- Root waiving & coiling behaviour determined from video data & digital stills image at four different g-levels.
- Microtubule organisation in cells of roots from fixed samples.
- Coiling structure of root wall in fixed samples, stem length and leaf sizes in fixed samples.
- Scanning & camera pictures of fixed seedlings in growth chambers (orbit and ground).
- Scanning pictures of fixed seedlings on ground, surface microscopy on fixed seedlings on ground.

Expected results

  • Understanding the contribution of g-forces to formation of root coils by coupling of root growth and root movement.
  • Improved understanding of root movements in soil for root anchoring and nurturing the plant body by salts.
click on items to display

Figure 1: General experiment hardware layout in Biolab Experiment Container (EC).

Figure 2: General experiment concept.

Figure 3: Detailed experiment timeline and associated functional objectives: Experiment run #1.
http://eea.spaceflight.es
a.int/attachments/spacest
ations/ID4c5c4c07ee206.xl
s

Figure 4: Flow Diagram with Functional Objectives indicated: Experiment run #1.

Figure 5: Detailed experiment flow for each Experiment Container (EC).

Table 1 List of Functional Objectives (FO) related to pre-flight/in-flight/post-flight timeline with reference to Fig. 4 FO steps.
 
© 2019 European Space Agency