Experiment

Aniso Tubule Experiment Details

Background

After the aquatic ancestors of terrestrial plants first came ashore more than 500 million years ago, they developed a series of adaptations to survive under 1 g conditions. The development of a rigid plant body may have been one of the critical responses required for plants to survive under 1 g conditions. We have termed this phenomenon ‘gravity resistance’ and have analyzed the nature and mechanisms of gravity resistance using hypergravity conditions produced by centrifugation. As a result, we found that plant body becomes shorter and thicker in proportion to the logarithm of the magnitude of gravity (Figure 1).

The body shape of a plant depends predominantly on the shape of individual cells and the direction of cell expansion. Cortical microtubules, a characteristic structure in interphase cells of plants, are assumed to be responsible for anisotropic expansion of plant cells by directing the orientation of cellulose microfibrils (Figure 2). In proportion to the logarithm of the magnitude of gravity, the percentage of cells with transverse cortical microtubules decreased, and that with longitudinal cortical microtubules increased (Figure 1). Microtubule-associated proteins (MAPs) play a variety of roles in the organization of microtubules. The MAP65 family proteins form cross-bridges between adjacent microtubules and are required for the bundling of microtubules and the maintenance of transverse microtubule orientation. The expression of MAP65-1 was down-regulated in proportion to the logarithm of the magnitude of gravity. These results indicate that reorientation of cortical microtubules that is caused by the action of MAPs may be involved in the development of a short and thick body in response to gravity.

Objective

Based on the results of ground-based experiments that used centrifugal hypergravity, it is hypothesized that plants would develop a long and thin body by increasing the number of cells with transverse cortical microtubules under microgravity conditions in space. Also, the microgravity-induced reorientation of cortical microtubules may be caused by the action of MAPs. To confirm this hypothesis, we will examine growth modifications of Arabidopsis hypocotyls in space. We also analyze the changes in dynamics of cortical microtubules and MAPs by observing Arabidopsis hypocotyls expressing GFP-fused tubulins and MAPs with the fluorescence microscope in the Kibo Module on the ISS. In particular, it is meaningful to analyze the modifications to dynamics of cortical microtubules and MAPs under microgravity conditions on-site in real time by observing GFP-expressing lines.

Outline of the Experiment

The Aniso Tubule experiment consists of 10 runs. Seeds of GFP-expressing Arabidopsis lines are surface sterilized by ethanol, and dried on sterilized filter paper. Dry sterilized seeds are sown in the cultivation/ observation chamber. The chambers for first five runs were launched by HTV-4 and next runs will be launched by SpX-4. At the beginning of each run, distilled water is injected with a syringe into the chamber. And then, the chamber is kept in the Minus Eighty Laboratory Freezer for ISS (MELFI) at 2°C for 96 h, and exposed to white light for 6 h to induce germination. Germinated plants are grown in the dark at 22°C for 70 h using the Cell Biology Experiment Facility (CBEF). After the incubation, the chamber is taken out of CBEF, and distilled water is injected with a syringe into the chamber for microscopic observation. And then, the chamber is set on the fluorescence microscope in the Kibo Module. Growth modification in epidermal cells of hypocotyl is observed. Dynamics of both cortical microtubules and MAPs in epidermal cells of hypocotyl, labeled with GFP, are also observed. All operation is controlled from ground. Micrographs are saved and down-linked to earth in real time or within a minimum delay. After acquisition of micrographs, seedlings grown in space are trashed. Ground-control experiment is performed after space flight by accurately reproducing the conditions of incubation in the space experiment.

This is the Point!

This experiment aims to reveal the mechanism of gravity resistance, which is an important gravitational response for plants, by using microgravity conditions in space. If the mechanism of gravity resistance can be clarified, it will enable the controlling of plant forms on Earth. Furthermore, it will be useful for plant cultivation in space, in the future.