Using Brachypodium distachyon to Investigate Monocot Plant Adaptation to Spaceflight (APEX-06) - 06.13.18

Overview | Description | Applications | Operations | Results | Publications | Imagery

ISS Science for Everyone

Science Objectives for Everyone
The Using Brachypodium distachyon to Investigate Monocot Plant Adaptation to Spaceflight (APEX-06) experiment investigates the growth of the common grass species Brachypodium distachyon in the microgravity environment of space. The grasses grow from seedlings aboard the International Space Station (ISS), and are returned as frozen samples to Earth-based labs for detailed analysis and comparison with Earth based control groups. APEX-06 aims to compare the growth and gene-expression patterns of Brachypodium distachyon with those of the dicotyledonous model plant Arabidopsis thaliana, which has been extensively studied in space and whose behavior in microgravity is better understood.
Science Results for Everyone
Information Pending

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

OpNom: APEX-06

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

Co-Investigator(s)/Collaborator(s)
Shih-Seng Su, Ph.D., University of Wisconsin - Madison, Madison, WI, United States

Developer(s)
NASA Kennedy Space Center, Cape Canaveral, FL, United States
University of Wisconsin-Madison, Madison, WI, 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 - August 2018

Expeditions Assigned
53/54,55/56

Previous Missions
Information Pending

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Experiment Description

Research Overview

  • The Using Brachypodium distachyon to Investigate Monocot Plant Adaptation to Spaceflight (APEX-06) investigation focuses on investigating the growth, development, and transcriptome profiles of Brachypodium distachyon seedlings under spaceflight.
  • Most major cereal grain crops are monocots. However, most investigations of plant molecular adaptation to the spaceflight environment were carried out on the dicotyledonous model plant Arabidopsis thaliana. It remains unknown whether the conclusions from such studies can be extrapolated to monocotyledonous plants. Therefore, to fill this knowledge gap, the research team plans to investigate the growth, development, and transcriptome profiles of Brachypodium distachyon seedlings under spaceflight conditions, and compare these adaptive responses between accessions found to display distinct sensitivity to gravity in ground-based experiments.
  • Candidate genes identified as being differentially expressed between microgravity-exposed and ground-based controls are targeted by genome editing to investigate their function in plant development and ultimately, evaluate their contribution to plant growth and development under microgravity.

Description

The goal of Using Brachypodium distachyon to Investigate Monocot Plant Adaptation to Spaceflight (APEX-06) is to investigate the morphological and molecular adaptations of Brachypodium distachyon seedlings to the microgravity environment encountered on the International Space Station (ISS). Three Brachypodium distachyon accessions (Bd21, Bd21-3 and Gaz8) are germinated and grown in novel growth devices within the Veggie facility on the ISS. Each growth device is composed of a Magenta jar containing a block of Oasis LC-2 foam surrounded by layers of gauze and Nitex. The foam is positioned on top of a nutrient injection platform connected to an easily accessible spout that protrudes outside of the jar. Surface-sterilized, dehusked seeds are inserted embryo side up within the dry foam before the launch of the cargo vehicle, and maintained in a dry state until transfer into Veggie on ISS. Each growth device carries 24 pre-planted dry seeds from one accession. Each accession is represented by four devices (four biological repeats).
 
Upon arrival at the ISS, the growth devices are transferred into Veggie, and germination is triggered by injection of growth medium into the foam through the spout and exposure to red light (low setting) for 24 hours. Following germination, seedlings are grown in light (red and blue LEDs set at low; green LED on) for four more days. During this growth period temperature and humidity levels are recorded using HOBO Data Loggers. Seedlings are then photographed on their growth support, harvested, and transferred into Kennedy Space Center Fixation Tubes (KFTs), fixed in RNALater (Ambion) for 24 to36 hours at room temperature, and then stored in MELFI at -80°C until return on a SpaceX Dragon cargo vehicle for post-flight analysis. A ground-based control (GC) using the same experimental setup is duplicated at the Kennedy Space Center (KSC) in a Veggie facility, itself enclosed within a controlled chamber that is programmed to precisely recapitulate the temperature, humidity, and CO2 profiles that were recorded on ISS, with a 48-hour delay. Seedlings are handled in the same way for both the ground-based control and the ISS experiment.
 
A second ground-based experiment is also carried out at KSC, aimed at evaluating the early transcriptomic responses of Brachypodium seedlings to plant reorientation within the gravity field (gravi-stimulation). Seeds from the Bd21 reference accession are planted in growth devices, germinated, and grown in the Veggie facility at KSC under the growth conditions described above. At the end of a five-day growth period, half of the devices are rotated by 180° for gravi-stimulation (GC GS), whereas the other half are rotated by 360° as a mechano-stimulus control (GC MS). After five minutes of stimulation, seedlings are photographed and dissected to separate shoots and roots. Each tissue sample is fixed in RNALater within KFTs for 24 to 36 hours at room temperature, and then frozen for storage at -80°C.
 
After all samples are collected, fixed in RNALater within KFTs, and returned to KSC, the frozen, fixed materials are transferred into Corning tubes at KSC, and then transported to the Research team’s laboratory at the University of Wisconsin-Madison for subsequent molecular analysis.
 
In the laboratory, fixed samples are thawed, and the roots and shoots are dissected. RNA is then extracted from the dissected tissues using the Direct-Zol RNA extraction kit (Zymo Research). After quality control verification, RNAs extracted from the Bd21 samples are submitted to the University of Wisconsin-Madison Gene Expression Center (Biotech Center) for cDNA library construction and next generation sequencing.
 
After sequencing, the RNA-seq reads are trimmed, filtered, and aligned to the Brachypodium reference genome (Bd21). CLC’s normalization tool is used to define the numbers of reads per kilobase of exon model per million mapped reads (RPKM) for genes and isoforms in each analyzed sample. Transcriptome assemblies for the tested conditions are merged with Cuffmerge, and differentially expressed transcripts are identified using DESeq and EdgeR. The following Bd21 samples are compared for differential expression profiles:
  • ISS root vs GC root
  • ISS shoot vs GC shoot
  • GC GS root vs GC MS root
  • GC GS shoot vs GC MS shoot
  • GC GS root vs GC root
  • GC MS root vs GC root
  • GC GS shoot vs GC shoot
  • GC MS shoot vs GC shoot
These comparisons allow for the identification of differentially expressed genes between these conditions. Differential expression is verified in Bd21, and tested in Bd21-3 and Gaz-8, using RT-qPCR approaches. After verification of differential expression, candidate genes are functionally characterized using a combination of insertion mutagenesis, CRISPR/Cas9-based genome editing, transgenic over-expression, and cell biology. While beyond the scope of this project, these functional analyses generate mutants and overexpressing plants that could ultimately be tested for improved or altered adaptation to the spaceflight conditions, or modified responses to gravi-stimulation under 1g, or hypergravity, conditions.
 
The morphology of the plants is also analyzed using the pictures taken at the end of each experiment. Each digitized image is subjected to a morphometric analysis of the seedlings, using the Image J analysis software. Parameters that are to be quantified include shoot and root length, straightness, and tip curvature. Comparisons between treatments are supported by statistical analysis (t- and F-tests; ANOVA). These studies are projected to uncover a potential impact of the microgravity environment of ISS, and of gravi- and/or mechano-stimulation, on the morphology of the plants.

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Applications

Space Applications
APEX-06 expands the understanding of plant growth in space. Detailed understanding of how different plants grow in space can provide for better life support system design and resource planning for long term space missions. This investigation also provides a better comparative understanding of how different plant groups adapt and cope under microgravity, and other unique conditions of space.

Earth Applications
APEX-06 provides fundamental information about plant biology. A better understanding of grass and cereal crop stress response systems can serve in agriculture, habitat restoration, and natural resource management. Results from this experiment also advance comparative understanding of how plants use genetic and biomolecular systems to protect themselves under stressful conditions.

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Operations

Operational Requirements and Protocols

APEX-06 hardware consists of 12 growth devices which contain the Brachypodium distachyon seeds, 12 RNAlater KFTs, two data loggers, nutrient solution, harvest kit, and support fixtures. The investigation begins with the addition of a water-based nutrient solution to each of the growth devices, which are then placed inside the Veggie facility currently on orbit. The initial Veggie light setting requires only the red lights at the low setting for a period of 24 hours. After 24 hours, the Veggie light settings are revised with blue at low, green on, and red remaining at low. This initiates the growth of the Brachypodium distachyon seeds and continues for four days. After the growth period, each growth device is disassembled, which allows the plants to be removed from the assembly, photographed, and harvested into RNALater KFTs. These KFTs remain at ambient temperature on the ISS for 24 hours to allow sufficient perfusion of the RNAlater into the plant material. The KFTs are then placed in the Minus Eighty Laboratory Freezer for ISS (MELFI) at -80°C until return on a SpaceX Dragon cargo vehicle for post-flight analysis.

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Decadal Survey Recommendations

CategoryReference
Plant and Microbial Biology P2

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Results/More Information

Information Pending

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Related Websites

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Imagery