The objective of the GRAVI experiment: an analysis of the gravisensing mechanisms in roots
The scientific results highlight the effect on amyloplast movement on the calcium-dependent pathways.
In this first part of GRAVI experiment (GRAVI-1), the threshold acceleration and the presentation dose of the gravitropic reaction of lentil (Lens culinaris L. cv. Anicia) seedlings grown in microgravity (Driss-Ecole et al., 2008 ) were determined. Following a period of growth in microgravity conditions, seedlings were subjected to low accelerations for several hours.
During root growth, the root orientation and curvatures were followed by time-lapse photography combined with still images downlinked in near real time to ground. The position of the root tip and the root curvature were analysed as a function of time.
Based on this, the GRAVI-1 team observed that the embryonic root curved strongly away from the cotyledons (automorphogenesis) in microgravityand then from 17 hours to 30 hours slowly straightened out (autotropism). Under these conditions the threshold acceleration perceived by the roots was found to be around 2×10 -3 g. The threshold acceleration was estimated using a hyperbolic module to be 1.4 x10-5g (Driss-Ecole et al., 2008 ).
Considering the results obtained from GRAVI-1, GRAVI-2 aimed to investigate the response of the lentil root curvature as a compromise using threshold levels between 0.01g and 2g. Since calcium is considered to be an important second messenger involved in root gravisensing, the Ca2+ localisation in statocytes will be in focus and related to the positioning of the amyloplasts. The regulation of calcium-downstream gene expression will be analysed.
The experiment specific goals are addressed in the design of the runs experiment:
- Run #1 = Comparison with results of GRAVI-1 experiment, examination of cell ultrastructure, Ca2+ localisation and gene expression
- Run #2 = Ca2+ redistribution with gravistimulationand downstream gene expression.
Four fixatives with different science objectives for analysis are used:
- 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