Molecular Biology of Plant Development in the Space Flight Environment (Characterizing Arabidopsis Root Attractions (CARA)) - 03.04.14
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The Molecular Biology of Plant Development in the Space Flight Environment (Characterizing Arabidopsis Root Attractions (CARA)) investigation focuses on the growth and development of Arabidopsis thaliana seedlings in the space flight environment, with a specific focus on how a root knows which direction to grow in when gravity is absent. Plants are grown in agar, a nutrient solution with a gelatin-like consistency, and exposed to light or dark conditions. Plants are harvested on orbit, preserved with a chemical preservative, and returned to the ground for postflight evaluation.
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OpNom Petri Plants
Center for the Advancement of Science in Space (CASIS), Rockledge, FL, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
National Laboratory (NL)
ISS Expedition Duration
March 2014 - September 2014
Previous ISS Missions
Characterizing Arabidopsis Root Attractions (CARA) builds upon the previously flown Plant Growth Investigations in Microgravity (PGIM) experiment which flew on STS-93, the Biological Research in Canisters (BRIC)-16 experiment which flew on STS-131, and the Transgenic Arabidopsis Gene Expression System (TAGES) experiment which flew in the Advanced Biological Research System (ABRS) during Increments 19-24.
Plants experiencing space flight are quite normal in appearance but can exhibit growth habits distinctly different from plants on earth. Molecular Biology of Plant Development in the Space Flight Environment (Characterizing Arabidopsis Root Attractions (CARA) explores the molecular biology guiding the altered growth of plants, specifically roots, in space flight.
Characterizing Arabidopsis Root Attractions (CARA) specifically addresses the signaling mechanisms that influence root growth in Arabidopsis plants grown without gravity, and how that changes with or without light. By using molecular and genetic tools, fundamental questions regarding the signals that affect how a root knows to grow toward gravity and/or in the opposite direction of the shoot may be answered.
Characterizing Arabidopsis Root Attractions (CARA) advances the fundamental understanding of the molecular biological responses to extraterrestrial environments. This understanding further defines the impacts of space flight on biological systems to better enable the United States’ future space exploration goals.
The space flight environment addresses key gravity-related biological issues using a simple, robust operational approach that has been successfully demonstrated with previous Space Shuttle and International Space Station (ISS) flights. Plants experiencing space flight mount an adaptive response that can be measured in terms of patterns of gene expression and morphological (shape) changes in growth and development. During previous space flight experiments a remarkable number of gene expression changes during space flight that are associated with cell wall restructuring and altered root growth were observed. Differences in the way two distinct ecotypes (genetically distinct population) of Arabidopsis responded to the space flight environment, particularly with respect to inherent root-growth patterns were also observed. This project is designed to tie these observations together.
Previous space flight results indicate that removing the effects of gravity can reveal novel root responses that are not observed on earth. In the absence of gravity but the presence of directional light, Arabidopsis roots are strongly negatively phototrophic and grow in the opposite direction of shoot growth; however, ecotype Wassilewskija (WS) and Columbia (Col-0) display two distinct, marked differences in their growth patterns. Root growth in WS slant or “skew” strongly to the right on the surface of agar plates on orbit, while Col-0 grows with little deviation away from the light source. In the absence of light, the roots of Arabidopsis ecotype Landsberg also demonstrate an inherent skew to the right, while Col-0 appears to grow randomly on orbit. Until now, skewing and waving were thought to be gravity-dependent phenomena, but the APEX-TAGES experiments demonstrated that these characteristic patterns of growth are both independent of gravity and influenced by light.
Among the genes that differ between WS and Col-0, one major gene of interest is phytochrome D (phyD) which encodes a protein involved in light-mediated signaling, especially shade avoidance. The phyD gene in WS contains a deletion which results in the translation of a faulty protein. However, although WS lacks phyD, it does contain normal levels of phyA, phyB and phyC. There is considerable overlap in the roles of the members of the phytochrome family, and it is difficult to tease out the specific role of each. However, a mutation in phyD in a Col-0 background mimics some of the phenotypic features of the WS ecotype. Phytochromes are also of interest as they have a demonstrated role in phototropism in roots. These effects can be difficult to evaluate in a unit gravity environment, but microgravity experiments have indeed demonstrated that at least two members of the family, phyA and phyB, play a role in positive phototropism in roots.
The hypothesis to be tested is that the differences between the WS and Col-0 will reveal key genes involved in the morphology of root growth on orbit. Further, it is hypothesized that phyD contributes to the light-mediated signal transduction that influences the tropic direction of root growth on orbit, and that Col-0 plants deficient in this gene will mimic the negatively phototrophic patterns of WS roots on orbit. Two tools are used for analyses: whole genome transcriptome analyses and morphometric analysis. The results anticipated include the identification of a number of differentially expressed genes that help define gravity-independent responses unique to each ecotype, and insight into the role of the phyD gene in root growth. The fundamental scientific relevance of this experiment is that it provides insight into the signal transduction pathways that control tropism and adaptive physiology in plants. The experiment also showcases how the unique research environment of the ISS provides insight into fundamental and widely applicable biological questions that cannot be answered on earth where gravity would mask many of the underlying phenomena.
The project uses very few resources and has a flexible strategy that makes it an ideal system for ISS and for a variety of launch vehicles. The petri plates containing the plants launch passively in an ambient soft stowage bag and transfer to ISS ambient stowage. The experiment is activated using ambient ISS light. Following an 11 day growth period, the plants are harvested to KSC Fixation Tubes (KFTs) containing an RNALater chemical preservative. KFTs are transferred to MELFI at -80°C 8 to 24 hours after harvest. KFTs return at either -20°C (preferred) or at ambient. The petri plates and Harvest Kit are not returned.
Previous investigations by this research team pioneered the use of RNALater as an on-orbit fixative for gene expression analysis and calibrated Arabidopsis RNA extraction procedures as related to spaceflight fixation in RNALater. Development of an enhanced reporter gene technique to thoroughly understand a plant’s response to a new environment (spaceflight, Mars and Lunar habitats) could lead to subsequent generations of plants that could be genetically modified to better support human efforts to explore and colonize those environments. Additionally, the focus of this experiment builds on previous flight data and tests hypotheses that arose directly from earlier ISS experiments. This cycle of experimentation and hypothesis-driven science is precisely the model on which CASIS and NASA see the utilization of the ISS being built as a scientific platform.
This investigation provides a deeper understanding of the biological response of an organism to a novel environment. Novel environments provide unique insight to how plants can adapt or be adapted to challenging environments on earth, including marginal or reclaimed lands and lands that are recovering from extreme environmental assault such as mining or industrial intrusion.
This investigation also provides an increased understanding of how other plants, such as crops, may respond to the new environmental changes occurring on our planet-like rising levels of CO2 in the atmosphere, as spaceflight vehicles (including the ISS) are often high CO2 environments.
Finally, cell growth patterns in plants are influenced by the lack of gravity in space. Analysis of such growth patterns enables understanding of how plants, especially plant roots, guide their movements through soil in search of nutrients and water. This in turn provides information for crop breeders to more rapidly select varieties adapted to various soil conditions that would normally compromise plant health.
Time between turnover and launch: plated seeds stable for up to 30 days at ambient; refrigeration at +4°C extends shelf life
- Plates can withstand up to 30°C for short periods; if sustained temperatures >25°C, need thermal insulation
- On-orbit: Ambient until harvest
- Blackout cloth removed from all plates and plates exposed to ISS ambient light for between 1-12 hours to activate experiment
Re-wrap 10 plates with blackout cloth and return to stowage
- Remaining 10 plates exposed to ISS ambient light for 11 days
- Photography requirements for light exposed plates only-photos at 3, 6 and 9 days after initial light exposure
11 days following activation:
- Dark grown petri plates unwrapped
- Photographs required of all petri plates
- Each pair of plates (1 light grown, 1 dark grown) harvested to 1 KFT; request live video of harvest operation if possible
- KFTs transferred to -80°C MELFI 8-24 hours after harvest
- Petri plates and Harvest Kit trashed
KFTs return in Cold Bag at -20°C (preferred) or at ambient
- KFTs cannot be at ambient for more than 7 days following MELFI removal
- Frozen KFTs (-80°C) can remain in MELFI on ISS for up to a year if downmass not available
Operational Protocols [nasa.gov]: Twenty petri plates, ten KFTs and one Harvest Kit are transferred from ambient stowage on the ascent vehicle to ISS. To activate the experiment, blackout cloths wrapped around each petri plate are removed and petri plates are attached to a wall (using Velcro) to be exposed to ambient light. After 1 to 12 hours of light exposure, ten petri plates are re-wrapped in blackout cloth and returned to ambient stowage. The remaining ten petri plates remain attached to a wall for the duration of the experiment run. After an experiment duration of 11 days, all of the petri plates, KFTs and the Harvest Kit are transferred to the Maintenance Work Area for the harvest activity. The ABRS Photogrid, currently on ISS, is the preferred method to secure the petri plate during harvest and to provide a black background for photography. Each petri plate is photographed, one photo with the lid on and one with the lid off, and harvested. The KFTs have a mesh divider so that one dark grown and one light grown petri plate can be harvested into a single KFT. The harvest activity requires the crewmember to use forceps from the Harvest Kit to pull the plants from the agar surface of the petri plate. Once the plants are in a KFT, the KFT are actuated to deliver the RNALater chemical preservative to the plants. The harvest procedure is completed for all twenty petri plates. Upon completion of the harvest, the petri plates and Harvest Kit are trashed. Eight to twenty-four hours following the harvest, the ten KFTs are transferred to MELFI at -80°C. The KFTs return in a Cold Bag at -20°C.
University of Florida - Horticultural Science Department
University of Florida
Glowing Plants Helps Biological Studies on ISS
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