Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in setting up the spaceflight responses are being identified; their role in spaceflight physiological adaptation are increasingly understood and the fact that different genotypes adapt differently is recognized. However the basic question of whether these spaceflight responses are required for survival has yet to be posed and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses of two Arabidopsis ecotypes Col-0 and WS as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS phyD displayed a significantly reduced spaceflight transcriptome response compared to Col-0 suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed an even simpler spaceflight transcriptome response in the ambient light of the ISS more broadly indicating that the plant genotype can be manipulated to reduce the transcriptome cost of plant physiological adaptation to spaceflight and suggesting that genetic manipulation might further reduce or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS the WS genotype actually mounted a larger transcriptome response than Col-0 suggesting that the in-space light environment affects physiological adaptation which further implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in setting up the spaceflight responses are being identified; their role in spaceflight physiological adaptation are increasingly understood and the fact that different genotypes adapt differently is recognized. However the basic question of whether these spaceflight responses are required for survival has yet to be posed and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses of two Arabidopsis ecotypes Col-0 and WS as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS phyD displayed a significantly reduced spaceflight transcriptome response compared to Col-0 suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed and even simpler spaceflight transcriptome response in the ambient light of the ISS more broadly indicating that the plant genotype can be manipulated to reduce the transcriptome cost of plant physiological adaptation to spaceflight and suggesting that genetic manipulation might further reduce or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS the WS genotype actually mounted a larger transcriptome response than Col-0 suggesting that the in-space light environment affects physiological adaptation which further implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation. CARA Seed Lines and Planting: Three seed lines Wild-Type Wassilewskija (Ws) Columbia-0 (Col-0) and Col-0 PhyD (phyD) Mutants were tested for viability sterility and ability to maintain dormancy before the launch. Tested batches of seeds were planted on phytagel plates as one genotype per plate for gene expression analysis in replicates of three. One set was planted for the flight and one for ground control. The plates were wrapped such that every surface of the plate was covered by two layers of Duvetyn Black-Out cloth (Seattle Fabrics) (Sng et al 2014). The plates were stored 4 xb0 C until launch and was then launched in a cold-stow bag to maintain the plates at 4 xb0 C until integration and activation on the ISS. On Orbit Operations and harvest: The dormant plates were activated on station by removing the Black-Out cloth wrapping 12 days after launch. The plates were then placed on a fabric that was mounted in the US Laboratory module on the wall adjoining the MELFI freezer and secured using Velcro. The plants were allowed to grow on orbit for 11 days; some in the ambient light of ISS and some in the dark. The dark-grown plates were first activated by exposing the seeds to light for 4 hours and then re-wrapped in Black-Out cloth for the duration of the growth period. A corresponding set of seedlings were grown as ground control in KSC. At 11 days seedlings were photographed harvested into KFT containing RNAlater solutions and returned for post-flight analysis. All plates were harvested into KFTs with their counterpart (e.g. Light 1 was harvested with Dark 1). Once the plants were placed in the KFTs the KFT was actuated with RNAlater to preserve the sample. At 24 hours post-harvest KFTs were then transferred to MELFI the -32 xb0C freezer. Following SpaceX-3 splashdown in the Pacific Ocean the KFTs transferred to the Cold Stowage charter plane at

['Plants are primary producers of food and oxygen on Earth and will likewise be indispensable to the establishment of large-scale sustainable ecosystems and human survival in space. To contribute to the understanding of how plants respond to spaceflight stresses, we examined the relevance of the unfolded protein response (UPR), a conserved signaling cascade that responds to a number of unfavorable environmental stresses, in the model plant species Arabidopsis thaliana. To do so, we compared the transcriptional responses of wild type and UPR-defective seedlings to spaceflight during the SpaceX-CRS12 mission to the International Space Station. We established that orbital culture substantially altered the expression of hundreds of stress related genes compared to ground control conditions. Although many of these genes were differentially regulated in the UPR mutants in the ground control conditions compared to wild type, their expression was largely equalized in all genotypes by flight. Our results have yielded new information on how plants respond to growth in orbit and support the hypothesis that spaceflight induces the activation of signaling pathways that compensate for the loss of UPR regulators in the control of downstream transcriptional regulatory networks.']

Space environment is suspected to generate reactive oxygen species (ROS) and induce oxidative stress in plants however little is known about the gene expression of ROS gene network in plants grown in long-term space flight. RNA-Seq was used to define the large-scale gene expression profiles of Mizuna harvested after 27 days cultivation in the international space station to understand the molecular response and adaptation to space environment.Results: Total reads of transcripts from the Mizuna grown in the international space station as well as on the ground by RNA-Seq using next generation sequencing technology showed 8,258 and 14,170 transcripts up- and down-regulated in the space-grown Mizuna respectively when compared with those from the ground-grown Mizuna. A total of 20 in 32 ROS oxidative marker genes were up-regulated including high expression of 4 hallmarks and preferentially expressed gene associated with ROS-scavenging genes was thioredoxin glutaredoxin and alternative oxidase genes. In the transcription factors of ROS gene network MEKK1-MKK4-MPK3 OXI1-MKK4-MPK3 and OXI1-MPK3 of MAP cascades induction of WRKY22 by MEKK1-MKK4-MPK3 cascade induction of WRKY25 and repression of ZAT7 by Zat12 were suggested. RbohD and RbohF genes were up-regulated preferentially in NADPH oxidase genes which produce ROS.Conclusions: Our large-scale transcriptome analysis demonstrated that the space environment induced oxidative stress and ROS gene network was activated in the space-grown Mizuna some of which were common genes up-regulated by abiotic and biotic stress and were preferentially up-regulated genes by the space environment even though Mizuna grew in the space as well as on the ground showing that plants could acclimate to the space environment by reprograming the expression of ROS gene network.

Space environment is suspected to generate reactive oxygen species (ROS) and induce oxidative stress in plants however little is known about the gene expression of ROS gene network in plants grown in long-term space flight. RNA-Seq was used to define the large-scale gene expression profiles of Mizuna harvested after 27 days cultivation in the international space station to understand the molecular response and adaptation to space environment.Results: Total reads of transcripts from the Mizuna grown in the international space station as well as on the ground by RNA-Seq using next generation sequencing technology showed 8,258 and 14,170 transcripts up- and down-regulated in the space-grown Mizuna respectively when compared with those from the ground-grown Mizuna. A total of 20 in 32 ROS oxidative marker genes were up-regulated including high expression of 4 hallmarks and preferentially expressed gene associated with ROS-scavenging genes was thioredoxin glutaredoxin and alternative oxidase genes. In the transcription factors of ROS gene network MEKK1-MKK4-MPK3 OXI1-MKK4-MPK3 and OXI1-MPK3 of MAP cascades induction of WRKY22 by MEKK1-MKK4-MPK3 cascade induction of WRKY25 and repression of ZAT7 by Zat12 were suggested. RbohD and RbohF genes were up-regulated preferentially in NADPH oxidase genes which produce ROS.Conclusions: Our large-scale transcriptome analysis demonstrated that the space environment induced oxidative stress and ROS gene network was activated in the space-grown Mizuna some of which were common genes up-regulated by abiotic and biotic stress and were preferentially up-regulated genes by the space environment even though Mizuna grew in the space as well as on the ground showing that plants could acclimate to the space environment by reprograming the expression of ROS gene network.

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.

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.

Experimentation on the International Space Station has reached the stage where repeated and nuanced transcriptome studies are beginning to illuminate the structural and metabolic differences between plants grown in space compared to plants on the Earth. Genes that are important in establishing the spaceflight responses are being identified, their roles in spaceflight physiological adaptation are increasingly understood, and the fact that different genotypes adapt differently is recognized. However, the basic question of whether these spaceflight responses are actually required for survival has yet to be posed, and the fundamental notion that spaceflight responses may be non-adaptive has yet to be explored. Therefore the experiments presented here were designed to ask if portions of the plant spaceflight response can be genetically removed without causing loss of spaceflight survival and without causing increased stress responses. The CARA experiment compared the spaceflight transcriptome responses in the root tips of two Arabidopsis ecotypes, Col-0 and WS, as well as that of a PhyD mutant of Col-0. When grown with the ambient light of the ISS, phyD plants displayed a significantly reduced spaceflight transcriptome response compared to Col-0, suggesting that altering the activity of a single gene can actually improve spaceflight adaptation by reducing the transcriptome cost of physiological adaptation. The WS genotype showed an even simpler spaceflight transcriptome response in the ambient light of the ISS, more broadly indicating that the plant genotype can be manipulated to reduce the cost of spaceflight adaptation, as measured by transcriptional response. These differential genotypic responses suggest that genetic manipulation could further reduce, or perhaps eliminate the metabolic cost of spaceflight adaptation. When plants were germinated and then left in the dark on the ISS, the WS genotype actually mounted a larger transcriptome response than Col-0, suggesting that the in-space light environment affects physiological adaptation, which implies that manipulating the local habitat can also substantially impact the metabolic cost of spaceflight adaptation.

The Characterizing Arabidopsis Root Attractions (CARA) spaceflight experiment provides comparative transcriptome analyses of plants grown in both light and dark conditions within the same spaceflight. CARA compared three genotypes of Arabidopsis grown in ambient light and in the dark on board the International Space Station (ISS); Col-0, Ws, and phyD, a phytochrome D mutant in the Col-0 background. In all genotypes, leaves responded to spaceflight with a higher number of differentially expressed genes (DEGs) than root tips, and each genotype displayed distinct light / dark transcriptomic patterns that were unique to the spaceflight environment. The Col-0 leaves exhibited a substantial dichotomy, with ten-times as many spaceflight DEGs exhibited in light-grown plants versus dark-grown plants. Although the total number of DEGs in phyD leaves is not very different from Col-0, phyD altered the manner in which light-grown leaves respond to spaceflight, and many genes associated with the physiological adaptation of Col-0 to spaceflight were not represented. This result is in contrast to root tips, where a previous CARA study showed that phyD substantially reduced the number of DEGs. There were few DEGs, but a series of space-altered gene categories, common to genotypes and lighting conditions. This commonality indicates that key spaceflight genes are associated with signal transduction for light, defense, and oxidative stress responses. However, these key signaling pathways enriched from DEGs showed opposite regulatory direction in response to spaceflight under light and dark conditions, suggesting a complex interaction between light as a signal, and light-signaling genes in acclimation to spaceflight.

This study's objective was to develop an understanding of the biological effects of space radiation on plants with the goal of producing fresh food during long duration space missions to support astronauts' nutritional and psychological needs.10-day-old Arabidopsis seedlings were exposed to simulated Galactic Cosmic Rays (GCR) and assessed for transcriptomic changes. The simulated GCR irradiation was carried out in the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Lab (BNL). The exposures were conducted acutely for two dose points at 40 cGy or 80 cGy, with sequential delivery of proton, helium, oxygen, silicon, and iron ions. Control and irradiated seedlings were then harvested and stabilized in RNAlater at 3 hrs. post irradiation. Total RNA was isolated for transcriptomic analyses using RNAseq. The data revealed that the transcriptomic responses were dose-dependent, with significant upregulation of DNA repair pathways and downregulation of glucosinolate biosynthetic pathways. Glucosinolates are important for plant pathogen defense and for the taste of a plant, which are both relevant to growing plants for spaceflight. These findings fill in knowledge gaps of how plants respond to radiation in beyond-Earth environments.