The Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes. This data set includes single-molecule profiling in user-defined targets using Flap-Enabled Next-Generation Capture (FENGC) on Arabidopsis root tissues to reveal precise modification of DNA methylation regulated by Elongator Complex Subunit 2 during spaceflight.

On Earth plants are constantly exposed to a gravitational field of 1G. Gravity affects a plant in every step of its development. Germinating seedlings orient their radicle and hypocotyl and growing plants position organs at a specific Gravitropic Set-point Angle dictated by the asymmetric distribution of auxin depending on the gravity vector. Hence gravitropism is one of the fundamental growth responses in plants. For any experiment studying the effects of gravity on plants the ultimate control is the microgravity in space. In this study Arabidopsis seeds were flown to the International Space Station and allowed to germinate and grow for 3 days in microgravity. Arabidopsis Wild Type Col-0 seeds were plated onto twenty-two 60mm Petri plates loaded into PDFUs and inserted 4 Biological Research in Canisters (BRICs). Approximately 800 seeds were sterilized plated on each 60mm Petri plates and cold stratified for 16 hours followed by 2 hours of white light treatment. The BRICs were maintained at 4C until spaceflight to ensure seed germination in microgravity. After 3 days of germination and growth the seedlings were fixed by injecting RNAlater into the chamber. They were kept at ambient temperature for 12 hours followed by freezing at -80C. An additional 22 plates were used as ground controls. After the spaceflight tissue from five plates was pooled to make each of three replicates. Both membrane and soluble proteins were extracted from the pooled seedlings. Proteins were trypsin digested labelled with iTRAQ and identified using tandem mass spectrometry.

On Earth plants are constantly exposed to a gravitational field of 1g. Gravity affects a plant in every step of its development. Germinating seedlings orient their radicle and hypocotyl and growing plants position organs at a specific Gravitropic Set-point Angle dictated by the asymmetric distribution of auxin depending on the gravity vector. Hence gravitropism is one of the fundamental growth responses in plants. For any experiment studying the effects of gravity on plants the ultimate control is the microgravity in space. In this study Arabidopsis seeds were flown to the International Space Station and allowed to germinate and grow for 3 days in microgravity. Arabidopsis Wild Type Col-0 seeds were plated onto twenty-two 60mm Petri plates loaded into PDFUs and inserted 4 Biological Research in Canisters (BRICs). Approximately 800 seeds were sterilized plated on each 60mm Petri plates and cold stratified for 16 hours followed by 2 hours of white light treatment. The BRICs were maintained at 4C until spaceflight to ensure seed germination in microgravity. After 3 days of germination and growth the seedlings were fixed by injecting RNAlater into the chamber. They were kept at ambient temperature for 12 hours followed by freezing at -80C. An additional 22 plates were used as ground controls. After the spaceflight tissue from five plates was pooled to make each of three replicates. Both membrane and soluble proteins were extracted from the pooled seedlings. Proteins were trypsin digested labelled with iTRAQ and identified using tandem mass spectrometry.

When germinated and grown on-board the ISS (International Space Station), plant do not exhibit abnormal structures but they do have altered growth habits and this project aims to investigate the molecular mechanisms that provide the foundation for the altered growth habits observed in orbit. APEX03-2 (Advanced Plant Experiment 03-2), also known as TAGES-ISA (Transgenic Arabidopsis Gene Expression System-Intracellular Signaling Architecture) specifically addresses the growth and molecular changes that occur in Arabidopsis thaliana plants during spaceflight by using molecular and genetic tools, and by asking fundamental questions regarding root structure, growth and cell wall remodeling may be answered. This investigation advances the fundamental understanding of the molecular biological responses to extraterrestrial environments. This understanding helps to further define the impacts of spaceflight on biological systems to better enable NASA's future space exploration goals.

We address a key baseline question of whether gene expression changes are induced by the orbital environment and then we ask whether undifferentiated cells cells presumably lacking the typical gravity response mechanisms perceive spaceflight. Arabidopsis seedlings and undifferentiated cultured Arabidopsis cells were launched in April 2010 as part of the BRIC-16 flight experiment on STS-131. Biologically replicated DNA microarray and averaged RNA digital transcript profiling revealed several hundred genes in seedlings and cell cultures that were significantly affected by launch and spaceflight. The response was moderate in seedlings; only a few genes were induced by more than 7-fold and the overall intrinsic expression level for most differentially expressed genes was low. In contrast cell cultures displayed a more dramatic response with dozens of genes showing this level of differential expression a list comprised primarily of heat shock-related and stress-related genes. This baseline transcriptome profiling of seedlings and cultured cells confirms the fundamental hypothesis that survival of the spaceflight environment requires adaptive changes that are both governed and displayed by alterations in gene expression. The comparison of intact plants with cultures of undifferentiated cells confirms a second hypothesis: undifferentiated cells can detect spaceflight in the absence of specialized tissue or organized developmental structures known to detect gravity.

Understanding the molecular mechanisms by which plants sense and adapt to changes in the space environment is essential for generating plants that are better adapted to withstand space flight microgravity and other adverse conditions encountered in space. The objective of our spaceflight experiment x93Plant Signaling in Microgravity x94 (carried out on the International Space Station ISS) was to compare transcript profiles of wild type and transgenic InsP 5-ptase plants with compromised InsP3 signaling. The transgenic Arabidopsis plants constitutively express the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase) an enzyme that specifically hydrolyzes the lipid-derived second messenger inositol 1,4,5-trisphosphate (InsP3). These transgenic plants exhibit normal growth and morphology; however their responses to environmental stimuli including gravity and drought are altered. Seedlings were grown for 5 days under continuous light in experimental containers placed in the European Modular Cultivation system (EMCS) onboard the ISS. The EMCS consists of two rotors within a controlled chamber allowing for a x931g x94 control in space. After sample retrieval from the ISS RNA was isolated from shoot and root tissue and subjected to RNA sequencing. Two-way comparisons of micro g versus x931 x94g have uncovered regulatory mechanisms that are both conserved and altered between the wild type and transgenic seedlings.

Understanding the molecular mechanisms by which plants sense and adapt to changes in the space environment is essential for generating plants that are better adapted to withstand space flight, microgravity, and other adverse conditions encountered in space. The objective of our spaceflight experiment “Plant Signaling in Microgravity” (carried out on the International Space Station, ISS), was to compare transcript profiles of wild type and transgenic InsP 5-ptase plants with compromised InsP3 signaling. The transgenic Arabidopsis plants constitutively express the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes the lipid-derived second messenger inositol 1,4,5-trisphosphate (InsP3). These transgenic plants exhibit normal growth and morphology; however, their responses to environmental stimuli including gravity and drought are altered. Seedlings were grown for 5 days under continuous light in experimental containers placed in the European Modular Cultivation system (EMCS) onboard the ISS. The EMCS consists of two rotors within a controlled chamber, allowing for a “1g” control in space. After sample retrieval from the ISS, RNA was isolated from shoot and root tissue and subjected to RNA sequencing. Two-way comparisons of micro g versus “1”g have uncovered regulatory mechanisms that are both conserved and altered between the wild type and transgenic seedlings.

The project focuses on understanding the transcriptional and post-transcriptional mechanisms that regulate early seedling development in spaceflight and microgravity. One of the goals of the PRR experiment was to study the role of small regulatory RNAs in plant response to the space environment using Arabidopsis thaliana.

On Earth plants are constantly exposed to a gravitational field of 1G. Gravity affects a plant in every step of its development. Germinating seedlings orient their radicle and hypocotyl and growing plants position organs at a specific Gravitropic Set-point Angle dictated by the asymmetric distribution of auxin depending on the gravity vector. Hence gravitropism is one of the fundamental growth responses in plants. For any experiment studying the effects of gravity on plants, the ultimate control is the microgravity in space. In this study, Arabidopsis seeds were flown to the International Space Station and allowed to germinate and grow for 3 days in microgravity. Arabidopsis Wild Type Col-0 seeds were plated onto twenty-two 60mm Petri plates, loaded into PDFUs and inserted 4 Biological Research in Canisters (BRICs). Approximately 800 seeds were sterilized, plated on each 60mm Petri plates and cold stratified for 16 hours followed by 2 hours of white light treatment. The BRICs were maintained at 4C until spaceflight to ensure seed germination in microgravity. After 3 days of germination and growth, the seedlings were fixed by injecting RNAlater into the chamber. They were kept at ambient temperature for 12 hours followed by freezing at -80C. An additional 22 plates were used as ground controls. After the spaceflight, tissue from five plates was pooled to make each of three replicates. Both membrane and soluble proteins were extracted from the pooled seedlings. Proteins were trypsin digested, labelled with iTRAQ and identified using tandem mass spectrometry.

Epigenetic changes in the DNA methylome are increasingly shown to play an integral role in regulating gene expression necessary for plants adaption to environmental stressors. Plants subjected to the novel environment of spaceflight onboard the International Space Station (ISS), show stress-related transcriptomic changes most notably associated with pathogen stress response. Here, we investigate how known terrestrial stress associated epigenetic modulations might play a role in spaceflight adaptation. To examine the role of 5mCyt in spaceflight adaptation, the APEX04-EPEX experiment conducted onboard the ISS evaluated the spaceflight altered genome wide methylation profiles of two methylation regulating gene mutants (methyltransferase 1 (met1-7) and elongator complex subunit 2 (elp2-5) that are involved in pathogen defense response, along with a wild type Col-0 control. MethylSeq and RNAseq analyses were performed on both spaceflight grown samples and ground grown controls. In addition, the epigenetics effects that may contribute to the differential gene expression patterns observed between leaf and root tissues were also investigated in an organ-specific manner.