The major goals of this project are to determine how gravity and light responses influence each other in plants and to better understand the cellular signaling mechanisms involved in plant tropisms. This proposed project builds on our previous spaceflight experiments on the ISS (TROPI-1, 2006; TROPI-2, 2010) with the EMCS using the model plant Arabidopsis. In this new proposed project, we plan to confirm and extend our discovery during the recent TROPI-2 ISS experiment of a novel red-light-based positive phototropic response in plant shoots.
The combined experiments will be divided into four flight payloads in order to maximize the science return from the original proposals. The experiments are to be carried out with 3+1 flights in series. The first three flight experiments are designed to accomplish specific aims enumerated in the two NASA and ESA approved proposals. The last flight experiment is intended to allow a final flight to confirm and/or extend the findings from the first three.
Thus, the specific objectives of this proposed research are: (1) To confirm and characterize the novel red light-dependent phototropic response in flowering plants. (2) To investigate the relationship between light and gravity by measuring thresholds in fractional gravity. (3) To determine whether the red light effect on blue-light-based phototropism is a direct or indirect effect.
Seedling Growth 1: Phototropism
Seedling Growth 2: Tropisms, Auxin, and Cell Culture
Seedling Growth 3: Proliferating Cell Structure
Seedling Growth 4: Synthesis/Experiment Contingency
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The Seedling Growth series of experiments (1, 2, 3 and possibly 4) is supported collaboratively by NASA and the European Space Agency (ESA). The experiment will be conducted in the European Modular Cultivation System (EMCS) on the International Space Station (ISS). The EMCS is operated by ESA and is located in the Columbus Module of the ISS.
These experiments can be accomplished with telemetric science and therefore fit with the extremely limited down mass capabilities of current spaceflight opportunities. The EMCS is an automated facility and can be operated largely with telemetry. Experimental containers will be launched via Space-X, and during an experimental run, images of seedling growth responses can be downlinked to Earth. Seedlings will be returned to Earth in the GLACIER freezer.
Project 1: Phototropic curvature
Utilizing the European Modular Cultivation System (EMCS) on board the International Space Station (ISS), we investigated the interaction between phototropic and gravitropic responses in three Arabidopsis thaliana genotypes, Landsberg wild type, as well as mutants of phytochrome A and phytochrome B. Onboard centrifuges were used to create a fractional gravity gradient ranging from reduced gravity up to 1 g. A novel positive blue-light phototropic response of roots was observed during conditions of microgravity, and this response was attenuated at 0.1 g. In addition, a red-light pretreatment of plants enhanced the magnitude of positive phototropic curvature of roots in response to blue illumination. Additionally, a positive phototropic response of roots was observed when exposed to red light, and a decrease in response was gradual and correlated with the increase in gravity. The positive red-light phototropic curvature of hypocotyls when exposed to red light was also confirmed. Both red-light and blue-light phototropic responses were also shown to be a product of directional light intensity. To our knowledge, this is the first characterization of a positive blue-light phototropic response in Arabidopsis roots, as well as the description of the relationship between these phototropic responses in fractional or reduced gravity.
Project 2: Gene Profiling
The transcriptome of seedlings were analyzed from experiments performed on the International Space Station (ISS) to study the interacting effects of light and gravity on plant tropisms. Seeds of Arabidopsis thaliana were germinated and grown in cassettes in the European Modular Cultivation System (EMCS) for 4 days at 1g then exposed to a range of gravitational accelerations (microgravity, 0.07g, 0.13g, 0.21g, 0.39g, 0.53g, and 1g) and light treatments (blue light with or without a 1 hour pretreatment with red light). At the end of the space experiments, the cassettes containing the seedlings were frozen in the Minus Eighty Laboratory Freezer (MELFI) and returned to Earth on space shuttle STS-131. The RNA was extracted from whole seedlings and used for the transcriptome analyses. A comparison of 1g-treated-spaceflight samples with ground controls identified 230 genes that were differentially regulated at least two-fold (p < 0.01). A further comparison of all spaceflight samples with ground controls identified approximately 280 genes that were differentially regulated at least two fold (p < 0.01). Of these genes, several were involved in regulating cell polarity (i.e., auxin, calcium, lipid metabolism), cell wall development, oxygen status, and cell defense or stress. However, when the transcriptome of the all g-treated spaceflight samples were compared with microgravity samples, only approx.130 genes were identified as being differently regulated (p < 0.01). Of these, only 27 were at least 2 fold differently regulated between microgravity and 1g samples and included putatitive/pseudo/undefined genes (n=14), transposable elements (n=5), an expansin (ATEXP24; At1g21240), a cell-wall kinase (WAK3; At1g21240), a laccase-like flavonoid oxidase (TT10;At5g48100), among others. Not surprisingly, there was a difference in the % of genes represented in individual gene categories when comparing spaceflight samples with ground controls and spaceflight microgravity versus spaceflight g-treated space samples. These results emphasize the need for tissue fixation on the centrifuge for an accurate spaceflight control. These results are one of the few reports of transcriptome analyses from a space experiment with plants that had gravity treatments using an on-board centrifuge.
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