Spaceflight-Induced Hypoxic-ROS Signaling (APEX-05) - 11.02.17

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ISS Science for Everyone

Science Objectives for Everyone
When grown in the confines of the International Space Station (ISS), plants do not seem to get enough air and as a result, exhibit a stress response in their genes and proteins. The Spaceflight-induced Hypoxic/ROS Signaling (APEX-05) experiment grows different wild and mutant varieties of Arabidopsis thaliana, in order to understand how their genetic and molecular stress response systems work in space. The plants grow from seeds in the Veggie plant growth facility aboard the ISS, are frozen, and returned to Earth for detailed laboratory analysis.
Science Results for Everyone
Information Pending

The following content was provided by Simon Gilroy, Ph.D., and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: APEX-05

Principal Investigator(s)
Simon Gilroy, Ph.D., University of Wisconsin-Madison, Madison, WI, United States

Sarah Swanson, Ph.D., University of Wisconsin-Madison, Madison, WI, United States
Richard Barker, Ph.D., University of Wisconsin-Madison, Madison, WI, United States
Won-Gyu Choi, Ph.D., University of Nevada-Reno, Reno, NV, United States

NASA Kennedy Space Center, Cape Canaveral, FL, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
NASA Research Office - Space Life and Physical Sciences (NASA Research-SLPS)

Research Benefits
Earth Benefits, Scientific Discovery

ISS Expedition Duration
September 2017 - February 2018

Expeditions Assigned

Previous Missions
Biological Research in Canisters (BRIC-17)

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Experiment Description

Research Overview

  • Plants are likely to make up a large part of future bio-regenerative life support systems used to sustain crew survival during future long-duration spaceflight missions. Spaceflight represents a stressful environment for biological systems. The molecular mechanisms behind spaceflight-induced stress responses in plants remains poorly defined at present. Understanding how plants are affected by spaceflight will be critical to using them in life support systems.
  • Additionally, the microgravity environment of spaceflight lacks the buoyancy-driven convection that helps mix gasses around biological systems on Earth. Intensely respiring tissues, such as plant roots, can use up the available oxygen locally, but on Earth this is replaced by convection-driven gas mixing with the surrounding oxygen-rich air. In space, oxygen used up by respiration is not rapidly replaced by convection-driven gas mixing. Local limits on oxygen availability trigger low oxygen, or hypoxic, stress in respiring tissues. Previous work has shown the gene CAX2 is important for resisting low oxygen stress on Earth. Analysis of previous spaceflight results also shows reactive oxygen species (ROS) may be important chemical signals in spaceflight stress responses. On Earth, low oxygen stress has also been linked to ROS response.
  • The Spaceflight-induced Hypoxic/ROS Signaling (APEX-05) Investigation compares wild-type plants grown on the International Space Station (ISS), with mutants defective in CAX2 and in the ROS producing gene RBOHD. Plants are grown in the Veggie facility onboard the ISS, chemically fixed, and then frozen for return to Earth. The levels of all of the plant genes are to be determined upon return, and compared to control samples grown on Earth.
  • The plants are also engineered with a visual reporter protein showing the cells in which the RBOHD gene is activated. This reporter is imaged in the samples that are returned to Earth, and compared with the whole-plant patterns of gene activation.
  • Results from this investigation provide further understanding of how plants change and adapt to the unique stresses of spaceflight. Results also show whether genetic engineering of plants, such as changing CAX2 gene activity, can be used to reduce some spaceflight-related stress.
  • Results from this investigation help provide additional information to better understand plant responses to naturally occurring low oxygen stress conditions on Earth, such as the hypoxic stress that plants experience due to events such as flooding.


In spaceflight, a complex set of interacting stimuli operating against the unique background of reduced gravity yields a currently poorly understood range of stress responses in plants. For example, the recently reported transcriptomics data from the plant Arabidopsis flown in a range of hardware all show transcriptional profiles indicating a broad stress response in both seedlings and cell cultures in space. Two prominent molecular fingerprints seen in this data are responses to oxidative stress and long-term reduction in oxygen (O2) availability (hypoxia). The development of hypoxic response may well be linked to the lack of buoyancy-driven convection of gasses in microgravity. On Earth, convection normally mixes gasses around respiring tissues (such as the roots of plants) resupplying O2 from the bulk air. Many stresses, including hypoxia, also trigger oxidative signaling and stress responses, such as through the production of Reactive Oxygen Species (ROS). The Spaceflight-induced Hypoxic/ROS Signaling (APEX-05) seeks to test 2 hypotheses related to these ideas: 1) plants growing on the International Space Station (ISS) exhibit hypoxia-related responses due to reduced access to O2; 2) plants growing on the ISS exhibit stress responses related to ROS and oxidative stress.
Previous research has provided two potential molecular elements in these responses. Mutants in a calcium (Ca2+) transporter named CAX2 are known to be resistant to low oxygen stress on Earth, likely due to disrupting the Ca2+ signals normally associated with signaling low O2 stress. Similarly, the ROS related to many stress responses in plants involves action of the enzyme RBOHD. Therefore, APEX-05 compares the responses of wild-type plants to mutants in both the CAX2 and RBOHD genes. The experiment consists of 24 Petri plates: 5 of wild-type (Col-0) Arabidopsis, 5 of the cax2-3 mutant; 5 of the cax2-2 mutant, and 5 of the rbohD mutant. One extra plate of each genotype is included to allow for unexpected issues with one plate for each type of plant.
Plants are germinated on orbit using the Veggie facility, grown with the Veggie’s LED lighting for 8 days (with photography at days 4 and 8), and then harvested into Kennedy Space Center Fixation Tubes (KFT) containing the chemical fixative RNAlater. After chemical fixation, the samples are frozen in the Minus Eighty Laboratory Freezer for ISS (MELFI) and stored until return to Earth. As the plants are to be germinated onboard the ISS, the seeds must be prevented from germinating prior to their arrival on orbit. To accomplish this, the seeds are planted on nutrient gel in 10 cm square Petri dishes prior to launch, but are then irradiated with far-red light (730 nm) for 10 minutes using a custom LED-based system and then wrapped in foil. The far-red light activates the dormancy program of the seeds, which prevents their germination until they are illuminated with red light (680 nm), which in this case is supplied by unwrapping the plates and placing them under the LED illumination of the Veggie facility. As an extra level of insurance against premature germination, low temperature storage is also used. Thus, the plates are stored post far-red irradiation at 4˚C, and transported to the ISS in a double cold bag also at 4˚C.
Once the plants have grown, been harvested and frozen, the samples are stable and are stored in the MELFI until return to Earth. For return, the samples are retrieved from the MELFI and transferred to a freezer bag. They are recovered (still frozen) after the SpaceX Dragon cargo vehicle splashes down, and then are transferred to Kennedy Space Center. Samples are partially thawed in their KFTs to the point where the frozen ice sample “plug” can be removed. This frozen sample is transported on dry ice to the university of Wisconsin-Madison for turnover to the Research team and subsequent analysis.
The plants used for this experiment are engineered to express Green Fluorescent Protein (GFP) driven by the RBOHD gene promoter. This GFP survives fixation in RNAlater and freezing. Therefore, once the samples are thawed at the University of Wisconsin-Madison, their GFP fluorescence is visualized using a confocal microscope. This GFP imaging allows the following of where the RBOHD gene was activated during flight down to specific cell types. The samples are then processed for RNA extraction, and the level all genes in their genomes assessed using the technique of RNAseq.
Parallel control samples to the flight materials are also set up with a 2-day delay to the flight. They are handled identically to the flight samples but instead of launch to the ISS, they are transferred to the International Space Station Environmental Simulator, where they are treated the same as the flight samples to provide paired ground controls.

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Space Applications
Plants are likely to be a major component of bio-regenerative life support strategies for long-term space missions, providing food, and contributing to air and water purification. Additionally, plants provide significant psychological support for crew on long duration missions, but exhibit a range of stress responses in spaceflight. These underlying stresses and the response systems, however, are still not fully understood. The APEX-05 investigation seeks to provide scientists with a better understanding of how plants change and adapt to the unique stresses of spaceflight. Results may also show whether genetic engineering of plants can be utilized to reduce some spaceflight-related stress responses.

Earth Applications
The APEX-05 Investigation addresses how low oxygen sensing, and other response systems, operate in plants in microgravity. Similar low oxygen stress occurs naturally on Earth during events, such as flooding or intense microbial blooms in the soil. These types of events can have profound effects on plant productivity, and can have important impacts on plants in both natural ecological systems and in agricultural settings. The results from this work can help provide a better understanding of how these events affect plant biology, and also aid in the development of countermeasures through plant breeding or genetic engineering.

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Operational Requirements and Protocols

Twenty four, foil-wrapped, 10 cm square Petri dishes are delivered to the ISS at 4˚C in a double cold bag. The plates consist of 5 plates containing seeds of the Arabidopsis thaliana Col-0 wild-type, 5 of the cax2-2 mutant, 5 of cax2-3 and 5 of the rbohD mutant. One spare plate of each genotype is also included as a backup for unexpected issues with any of the other samples. Each plate contains gel growth media and ~10 seeds. The seeds are previously irradiated with far-red light to induce dormancy, and so prevent premature germination prior to arrival at the ISS. The plates are unwrapped and placed in the Veggie facility, where the transfer from the dark to the LED illumination of the Veggie reverses the far-red effect and induces germination. Plants are grown for 8 days, with photography of each plate at day 4 and 8. All plants from each plate are then harvested into KFTs (one tube per plate) containing the chemical fixative RNAlater. After chemical fixation, the tubes are transferred to the MELFI and stored frozen until sample return. For sample return, tubes are transferred to a freezer bag and maintained frozen throughout the return process. The samples are recovered from the SpaceX Dragon cargo vehicle, transferred (frozen) to Kennedy Space Center, de-integrated, and turned over to the Research team for subsequent analysis.

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Decadal Survey Recommendations

Plant and Microbial Biology P2

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Results/More Information

Information Pending

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Related Websites

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