Molecular Biology of Plant Development in the Space Flight Environment (CARA) - 08.18.16
The Characterizing Arabidopsis Root Attractions (CARA) experiment looks at mechanisms at the molecular and genetic level that influence the growth of a plant’s roots in the absence of gravity, and how those change with or without light. Researchers expose one set of seedlings to light, keep another set in the dark, and then examine how each environment influences the patterns of root growth. Some of the plants are also imaged with the Light Microscopy Module on orbit, and at the end of the experiment, all plants are harvested by the astronaut, and preserved for their return to Earth in order to evaluate genes associated with plant responses on orbit. Science Results for Everyone
Scientists are getting to the root of plant growth in space. Auxin is a plant growth hormone that helps guide the direction of root growth. Two experiments on the International Space Station found normal distribution of auxin in the roots of space-grown plants, suggesting weightlessness does not affect this system. However, space-grown plants show differences in root-tip distribution of cytokinin, a plant hormone that often works in concert with auxin to regulate cell division and tissue growth. The effect of weightlessness on distribution of cytokinin in roots suggests that some of the spaceflight-induced features of root growth may be cytokinin-related. Spaceflight also causes changes in the expression of many genes that are regulated by auxin and other plant hormones, as well as genes that regulate the size and shapes of cells that influence root growth patterns. These results provided cell-specific visual markers of where spaceflight-related auxin and cytokinin signaling occur, and how these signals may help to guide root growth in an environment without gravity. Experiment Details
OpNom: Petri Plants
Anna-Lisa Paul, Ph.D., University of Florida, Gainesville, FL, United States
Robert J. Ferl, Ph.D., University of Florida, Gainesville, FL, United States
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
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.
CARA provides a deeper understanding of the fundamental mechanisms behind the cell growth patterns of roots growing in a novel environment that, one which lacks the normal stimulus of gravity. This knowledge can contribute to the development of plants that are better adapted to spaceflight and other altered gravity environments – like the moon and Mars. The ability to grow healthier plants in microgravity will make it possible to use them more effectively in future efforts to explore and colonize space. Additionally, the focus of this experiment builds on previous flight data and tests hypotheses that arose directly from earlier ISS experiments of this research team. 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 laboratory platform.
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.
Operational Requirements and Protocols
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
Decadal Survey Recommendations
Information Pending^ back to top
On Earth, plant growth is primarily guided by light and gravity. Indeed, it has long been thought that for many of the growth patterns exhibited by plant roots, gravity was required. Research on the ISS has shown that many of these processes are in fact independent of gravity, but scientists are still learning about how plants “know” how to grow without it. Part of this research is investigating how well-characterized plant hormones that function in cell elongation and division – like auxin and cytokinin – work to guide plants in an environment without gravity to act as a cue for where these hormones should be synthesized or repressed. On Earth, auxin (which promotes cell elongation) plays a major role in guiding roots to maintain growth in the direction of the pull of gravity. When a root is growing perfectly vertical (with the tip “down” on Earth) auxin is distributed uniformly through the central region of the root tip, and cell elongation occurs evenly and the root continues its downward growth. However, if the gravity vector is disrupted, as by turning a vertically gown plant on its side, the distribution of auxin shifts so that cell elongation is promoted on the side of the root facing away from the pull of gravity. Those cells elongate until the tip is again pointing down, and the gradient of auxin is again uniformly vertical. Meanwhile, cytokinin is promoting cell division to keep renewing the cells of the root tip as it grows. This scenario describes what happens to the distribution of auxin when the direction of the gravity stimulus is changed on Earth, but what happens if the gravity stimulus is removed all together? Scientists were able to watch the distribution of auxin and cytokinin in roots grown on the ISS by following the distribution of fluorescent markers in real time, the objective being to determine whether gravity plays a direct role in establishing the auxin-mediated gravity-sensing system in primary roots. The current results are from two independent experiments: CARA (Characterizing Arabidopsis Root Attractions) and APEX-03-2 (Advanced Plant Experiment 03-2), which were each completed at different times aboard the International Space Station (ISS). The fluorescent markers were created by making a “reporter gene” composed of a green glowing gene from a jellyfish linked to either an auxin or cytokinin sensor, and then inserting the reporters into plants. Any cells which were actively using auxin or cytokinin would glow green. Scientists were able to view live the distribution of auxin and cytokinin in growing Arabidopsis thaliana plants on the ISS with the Light Microscopy Module (LMM), a specialized fluorescent microscope on the ISS built specifically to work with samples in microgravity. The images from the plant on orbit were compared with control plants imaged on the ground. In addition, spaceflight-grown plants and their ground controls were preserved and also examined post flight. Results showed that space grown plants displayed the normal ground “vertical” distribution of auxin in the primary root. These data suggest that the establishment of the auxin-gradient system, the primary guide for gravity signaling in the root, is not affected by weightlessness, but rather that auxin gradients in the primary root are fundamentally developmental, and those developmental auxin gradients were then coopted to be sensitive to gravity responses. However, the cytokinin distribution in the root tip differs between spaceflight and the ground controls, suggesting spaceflight-induced features of root growth may be cytokinin related. Thus, spaceflight appears benign to auxin and its role in the development of the primary root tip, whereas spaceflight may influence cytokinin-associated processes. The role of auxin in structures other than the root tip bears investigation, as auxin-regulated genes are seen to change in expression during spaceflight, when examining the root as a whole, and root appearance is also influenced by spaceflight. The value of the present study is in having cell-specific visual markers to identify where in the root potential spaceflight auxin-regulated changes do occur.^ back to top
Ferl RJ, Paul A. The effect of spaceflight on the gravity-sensing auxin gradient of roots: GFP reporter gene microscopy on orbit. npj Microgravity. 2016 January 21; 2: 15023. DOI: 10.1038/npjmgrav.2015.23.
Ground Based Results Publications
University of Florida - Horticultural Science Department
Glowing Plants Helps Biological Studies on ISS
NASA Image: ISS039E017696 - View of Characterizing Arabidopsis Root Attractions (CARA) during light intensity measurements.
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NASA Image: ISS039E018809 - View of Characterizing Arabidopsis Root Attractions (CARA).
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NASA Image: ISS039E020814 - View of Characterizing Arabidopsis Root Attractions (CARA).
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NASA Image: ISS039E020887 - NASA astronaut Steve Swanson harvests plant specimens from Characterizing Arabidopsis Root Attractions (CARA).
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