Molecular Biology of Plant Development in the Space Flight Environment (CARA) - 09.05.18

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ISS Science for Everyone

Science Objectives for Everyone
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.
This will take some tiny pruning shears. Because plants have no experience in adapting to microgravity, they may respond with improper changes in gene expression. This study showed an altered pattern of gene expression in the root tip cells of plants in microgravity relative to ground controls, suggesting that these genes are important to the physiological adaptation of plants to space. Notably, while some plant varieties expressed more genes to adjust, some varieties expressed less. These findings suggest that it may be possible to eliminate certain unnecessary environmental responses by genetic modification to produce plant varieties better adapted for growth in microgravity (Paul, 2017).

The following content was provided by Anna-Lisa Paul, Ph.D., and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: Petri Plants

Principal Investigator(s)
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), Melbourne, FL, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Information Pending

ISS Expedition Duration
March 2014 - September 2014

Expeditions Assigned

Previous 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.

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Experiment Description

Research Overview

  • 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.


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Space Applications
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.

Earth Applications

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.

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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

Operational Protocols []:  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. 

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Decadal Survey Recommendations

Information Pending

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Results/More Information

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 have shown that space grown plants display the normal ground “vertical” distribution of auxin in the primary root but a different distribution of cytokinin relative to ground controls. These results suggest that the establishment of the auxin-gradient system, the primary guide for gravity signaling in the root, is not affected by weightlessness and 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 appear to change in expression during spaceflight.  Having cell-specific visual markers is important to identify where in the root spaceflight auxin-regulated changes occur.

New entry: 
Plants physiologically adapt to a changing environment by regulating patterns of gene expression. This “transcriptome” (the segments of DNA copied into RNA) creates a map of the response, which can then be compared among varieties, and between treatments – like between plants grown in space, and those grown on the ground. Plants did not evolve in an environment without gravity, so they do not have an evolutionary history of how to metabolically deal with such a stimulus. Because plants have not developed adaptive strategies to cope with microgravity, it raises the possibility that signals in this novel environment inappropriately activate non-adaptive responses. This study investigated whether inappropriate, or non-adaptive responses to microgravity could be identified by comparing the spaceflight transcriptomes of genetically similar cultivars of Arabidopsis thaliana (Arabidopsis), and also by comparing the spaceflight transcriptomes of plants differing by only a single gene. RNA sequencing (RNAseq) was used to identify the differentially expressed genes in root tips of plants grown on the ISS in microgravity, and in comparable plants on the ground. RNAseq provided a quantitative measure of how variations in genetic background and lighting impacted the expression of genes in the spaceflight environment. Results showed that all plants grown in microgravity in this study expressed genes differently relative to their ground controls, indicating that changes in gene expression are required for physiological adaptation to spaceflight. The relative impact of the spaceflight environment was inferred by the number of genes that were differentially expressed. In other words, a plant genotype that required the differential expression of a large number of genes was thought to struggle more to adjust to spaceflight than a genotype that expressed fewer genes. The genotype of the cultivar WS required far fewer genes to physiologically adapt to spaceflight than the genotype of cultivar Col-0. This study then asked whether manipulating the genotype of the Col-0 plant could alter the plant’s response to microgravity. It was found that disabling just one gene in Col-0 could reduce the number of genes needed by Col-0 to physiologically adapt to spaceflight. These results suggest that a strategy of genome manipulation could be used to improve the adaptability of plants to spaceflight by reducing the transcriptome cost of physiological adaptation. In addition, the observation that of the hundreds of genes that are differentially expressed in each genotype, only about a dozen are held in common to all three, thus hundreds of differentially expressed genes are likely not to be truly necessary for physiological adaptation to spaceflight. Taken together, these results suggest that responses to truly novel environments may include responses from signals that are incorrectly activated, and further suggests that these incorrect responses can be genetically removed to lighten the metabolic load on the adaptive process. In conclusion, these data suggest that genetic manipulation can produce varieties that are better adapted for growth in microgravity through the elimination of unnecessary environmental responses (Paul, 2017)

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Results Publications

    Paul AL, Sng NJ, Zupanska AK, Krishnamurthy A, Schultz ER, Ferl RJ.  Genetic dissection of the Arabidopsis spaceflight transcriptome: Are some responses dispensable for the physiological adaptation of plants to spaceflight?. PLOS ONE. 2017 June 29; 12(6): e0180186. DOI: 10.1371/journal.pone.0180186. PMID: 28662188.

    Ferl RJ, Paul AL.  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.

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Ground Based Results Publications

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ISS Patents

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Related Publications

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Related Websites
University of Florida - Horticultural Science Department
Glowing Plants Helps Biological Studies on ISS

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image NASA Image: ISS039E017696 - View of Characterizing Arabidopsis Root Attractions (CARA) during light intensity measurements.
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image NASA Image: ISS039E018809 - View of Characterizing Arabidopsis Root Attractions (CARA).
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image NASA Image: ISS039E020814 - View of Characterizing Arabidopsis Root Attractions (CARA).
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image NASA Image: ISS039E020887 - NASA astronaut Steve Swanson harvests plant specimens from Characterizing Arabidopsis Root Attractions (CARA).
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