The analysis of the GRAVI-2 project

Part 1: Onboard picture analysis

Picture of the cultivation chambers containing the lentil seeds were taken during the whole duration of the experiment onboard 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.

iss_picture

Raw (top) and denoised (bottom) picture taken during the experiment onboard of the ISS. The 12 seeds of a cultivation chamber are visible, most of them showing distinguishable young roots growing in microgravity.

Part 2: Measurement of statolith displacement on root longitudinal sections

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.

semifine_picture

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).

 

Part 3: Involvement of intracellular calcium into gravity signal transduction

Intracellular 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 intracellular 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 intracellular 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 intracellular calcium into gravity signal transduction at the interface between various cellular structures.

tem_picture

Transmission electron micrscopy 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).

Part 4: Transcriptomic analysis

In order to identify which network of genes are overexpressed 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 last October 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 onboard.

 

 

 

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