[Cell Wall]
Reverse genetic approach to exploring genes responsible for cell-wall dynamics in supporting tissues of Arabidopsis under gravity conditions

RESEARCH OVERVIEW

• Reverse Genetic Approach to Exploring Genes Responsible for Cell Wall Dynamics in Supporting Tissues of Arabidopsis Under Microgravity Conditions (Cell Wall) examined the genes responsible for the construction of the cell wall (rigid outermost layer) in Arabidopsis thaliana (a small plant of the mustard family) in 1G (terrestrial gravity) and microgravity conditions. The cell wall of plants plays a crucial role in forming supportive tissues.
• Upon return to Earth, the properties of harvested plant stem material were evaluated by scientists to analyze the effects of 1G and microgravity on the cell wall and supporting tissues.
• Experience with plant growth related to microgravity environments is basic research that is relevant to planning plant cultivation studies concerning future long-duration space exploration missions.

DESCRIPTION

The Cell Wall investigation explored the molecular mechanism by which the cell wall (rigid outermost layer) construction of supporting tissues in Arabidopsis thaliana was regulated by gravity. The goal of Cell Wall was to determine if the supporting tissue in A. thaliana was achieved by coordinated actions of a defined set of cell wall related genes and to determine if their gene expressions were regulated by gravity to adapt to the terrestrial atmospheric environment under differing gravity conditions.

Cell Wall focused on the genes’ encoding proteins which are involved in construction and restructuring of the plant cell wall. Ground studies have indicated that a defined subset of cell wall genes was specifically responsible for the formation of supporting tissues in terrestrial plants on Earth; and that plants have acquired the molecular machinery by which the load on the shoot due to gravity can be monitored to regulate precisely the actions of these genes to construct supporting tissue. The Cell Wall experiment utilized the stems of A. thaliana to identify sets of genes, which play a central role in the cell wall construction and restructuring processes of supporting tissues in the shoot.

Under hypergravity conditions, the unfavorable phenotypes were intensified and exhibited a low viability. However, it was highly expected that the defects of such mutants would be rescued and could grow and develop normally as wild types in microgravity, where formation of the tough cell wall is not required. To confirm this expectation, mutant strains were cultivated under microgravity and at 1G conditions on the ISS up to the reproductive stage. Phenotypes concerning growth and development were compared using video images. Also, using plant materials frozen on orbit and on Earth, analysis was performed to assess changes in expression of genes involved in formation of microtubules, plasma membrane, cell walls, and levels of related cellular components.

The Cell Wall investigation tested four strains of A. thaliana; the wild type (normal function of microtubule-associated proteins), lefty (showed disordered growth patterns on Earth), hmg1 (defective in formation of cortical microtubules) and gene modified wild-type (Promoter GUS (glucuronidase) introduced).

Cell Wall operations occurred in the European Modular Cultivation System (EMCS) facility; a multi-user experiment facility for biological investigations under microgravity. EMCS allows cultivation, stimulation and crew assisted operation of biological experiments under well controlled conditions. The Plant Cultivation Chamber (PCC), which was developed for the Multigen experiment, was installed in standard Experiment Containers (EC) of the EMCS and referred to as the Cell Wall Flower Pot. The Cell Wall Flower Pot is a chamber where plant seeds grow to mature plants and produce legumes with new seeds. Cell Wall is a cooperative investigation between the European Space Agency (ESA), Japan Aerospace Exploration Agency (JAXA) and National Aeronautics and Space Administration (NASA).

Once the experiment was complete, the samples were returned to Earth and analyzed for changes in expression of genes involved in the formation of microtubules, plasma membrane, cell walls, and levels of related cellular components.

SPACE APPLICATIONS

The Cell Wall experiment aimed to explore the molecular mechanism by which the cell-wall construction of supporting tissues in land plants is regulated via gravity signal, which can benefit space explorers.

EARTH APPLICATIONS

Data gathered from the Cell Wall study will further the understanding of how plant growth and development at a molecular level can lead to significant advancements in agricultural production on Earth.

OPERATIONAL REQUIREMENTS AND PROTOCOLS

Cell Wall was designed to operate using pre-existing Experimental Unique Equipment (EUE) from the Multigen experiment, referred to as the Cell Wall Flower Pot. Eight Cell Wall Flower Pots required installation into EMCS; 4 on rotor A which rotated at 1G and 4 on rotor B, which did not rotate. The Cell Wall flower pots required watering and automated image acquisition daily, which was provided by the EMCS. The crewmembers were required to provide on-orbit support by checking the EMCS daily for nominal operations. Cultivation of these plants lasted approximately 33 to 53 days at 23 degrees Celsius requiring 16 hours of light and 8 hours of dark daily. The EMCS contained 60 percent humidity with 0.03 percent carbon dioxide. After the plants reached a length of approximately 10 cm, the Cell Wall Flower Pots were removed from the EMCS and placed inside the Microgravity Sciences Glovebox (MSG), to prepare the plant material for harvesting. Crewmembers were required to divide, preserve and store the samples per a cue card provided by the Cell Wall investigators.
The crew provided on-orbit support by checking the EMCS daily for nominal operations. Crewmembers divided, fixed and stored the samples using the Kennedy Space Center Fixation Tube (KFT); a system designed to contain plant or other small biological samples during flight and to chemically fix and stain tissue samples.

The KFTs provided three levels of containment for the chemical fixative during stowage and operations. After cultivation, all plant stems were harvested and inserted into KFTs and Ziploc bags. Samples were stored in KFTs containing either RNAlaterTM (aqueous, non-toxic tissue and cell collection reagent that stabilizes and protects cellular RNA in intact) and stowed at -35 degrees Celsius; or KFTs containing formaldehyde and stowed at +2 degrees Celsius. The remaining stems were stored in Ziploc bags and placed inside the Minus Eighty Laboratory Freezer for ISS (MELFI) at -95 degrees Celsius. Once the experiment was completed, the samples were returned to Earth for analysis.