Light has a principal role in the Arabidopsis transcriptomic response to the spaceflight environment.

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.

Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight.

BACKGROUND: 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. RESULTS: To provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate. CONCLUSIONS: FENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.

Spaceflight impacts xyloglucan oligosaccharide abundance in Arabidopsis thaliana root cell walls.

Over the course of more than a decade, space biology investigations have consistently indicated that cell wall remodeling occurs in a variety of spaceflight-grown plants. Here, we describe a mass spectrometric method to study the fundamental composition of xyloglucan, the most abundant hemicellulose in dicot cell walls, in space-grown plants. Four representative Arabidopsis root samples, from a previously conducted spaceflight experiment - Advanced Plant EXperiment - 04 (APEX-04), were used to investigate changes in xyloglucan oligosaccharides abundances in spaceflight-grown plants compared to ground controls. In situ localized enzymatic digestions and surface sampling mass spectrometry analysis provided spatial resolution of the changes in xyloglucan oligosaccharides abundances. Overall, the results showed that oligosaccharide XXLG/XLXG and XXFG branching patterns were more abundant in the lateral roots of spaceflight-grown plants, while XXXG, XLFG, and XLFG/XLFG were more abundant in the lateral roots of ground control plants. In the primary roots, XXFG had a higher abundance in ground controls than in spaceflight plants. This methodology of analyzing the basic components of the cell wall in this paper highlights two important findings. First, that are differences in the composition of xyloglucan oligosaccharides in spaceflight root cell walls compared to ground controls and, second, most of these differences are observed in the lateral roots. Thus, the methodology described in this paper provides insights into spaceflight cell wall modifications for future investigations.

Integrative transcriptomics and proteomics profiling of Arabidopsis thaliana elucidates novel mechanisms underlying spaceflight adaptation.

Spaceflight presents a unique environment with complex stressors, including microgravity and radiation, that can influence plant physiology at molecular levels. Combining transcriptomics and proteomics approaches, this research gives insights into the coordination of transcriptome and proteome in Arabidopsis' molecular and physiological responses to Spaceflight environmental stress. Arabidopsis seedlings were germinated and grown in microgravity (µg) aboard the International Space Station (ISS) in NASA Biological Research in Canisters - Light Emitting Diode (BRIC LED) hardware, with the ground control established on Earth. At 10 days old, seedlings were frozen in RNA-later and returned to Earth. RNA-seq transcriptomics and TMT-labeled LC-MS/MS proteomic analysis of cellular fractionates from the plant tissues suggest the alteration of the photosynthetic machinery (PSII and PSI) in spaceflight, with the plant shifting photosystem core-regulatory proteins in an organ-specific manner to adapt to the microgravity environment. An overview of the ribosome, spliceosome, and proteasome activities in spaceflight revealed a significant abundance of transcripts and proteins involved in protease binding, nuclease activities, and mRNA binding in spaceflight, while those involved in tRNA binding, exoribonuclease activity, and RNA helicase activity were less abundant in spaceflight. CELLULOSE SYNTHASES (CESA1, CESA3, CESA5, CESA7) and CELLULOSE-LIKE PROTEINS (CSLE1, CSLG3), involved in cellulose deposition and TUBULIN COFACTOR B (TFCB) had reduced abundance in spaceflight. This contrasts with the increased expression of UDP-ARABINOPYRANOSE MUTASEs, involved in the biosynthesis of cell wall non-cellulosic polysaccharides, in spaceflight. Both transcripts and proteome suggested an altered polar auxin redistribution, lipid, and ionic intracellular transportation in spaceflight. Analyses also suggest an increased metabolic energy requirement for plants in Space than on Earth, hence, the activation of several shunt metabolic pathways. This study provides novel insights, based on integrated RNA and protein data, on how plants adapt to the spaceflight environment and it is a step further at achieving sustainable crop production in Space.

Transcriptomic dynamics in the transition from ground to space are revealed by Virgin Galactic human-tended suborbital spaceflight.

The Virgin Galactic Unity 22 mission conducted the first astronaut-manipulated suborbital spaceflight experiment. The experiment examined the operationalization of Kennedy Space Center Fixation Tubes (KFTs) as a generalizable approach to preserving biology at various phases of suborbital flight. The biology chosen for this experiment was Arabidopsis thaliana, ecotype Col-0, because of the plant history of spaceflight experimentation within KFTs and wealth of comparative data from orbital experiments. KFTs were deployed as a wearable device, a leg pouch attached to the astronaut, which proved to be operationally effective during the course of the flight. Data from the inflight samples indicated that the microgravity period of the flight elicited the strongest transcriptomic responses as measured by the number of genes showing differential expression. Genes related to reactive oxygen species and stress, as well as genes associated with orbital spaceflight, were highly represented among the suborbital gene expression profile. In addition, gene families largely unaffected in orbital spaceflight were diversely regulated in suborbital flight, including stress-responsive transcription factors. The human-tended suborbital experiment demonstrated the operational effectiveness of the KFTs in suborbital flight and suggests that rapid transcriptomic responses are a part of the temporal dynamics at the beginning of physiological adaptation to spaceflight.

Advancing the automation of plant nucleic acid extraction for rapid diagnosis of plant diseases in space.

Human space exploration missions will continue the development of sustainable plant cultivation in what are obviously novel habitat settings. Effective pathology mitigation strategies are needed to cope with plant disease outbreaks in any space-based plant growth system. However, few technologies currently exist for space-based diagnosis of plant pathogens. Therefore, we developed a method of extracting plant nucleic acid that will facilitate the rapid diagnosis of plant diseases for future spaceflight applications. The microHomogenizer™ from Claremont BioSolutions, originally designed for bacterial and animal tissue samples, was evaluated for plant-microbial nucleic acid extractions. The microHomogenizer™ is an appealing device in that it provides automation and containment capabilities that would be required in spaceflight applications. Three different plant pathosystems were used to assess the versatility of the extraction process. Tomato, lettuce, and pepper plants were respectively inoculated with a fungal plant pathogen, an oomycete pathogen, and a plant viral pathogen. The microHomogenizer™, along with the developed protocols, proved to be an effective mechanism for producing DNA from all three pathosystems, in that PCR and sequencing of the resulting samples demonstrated clear DNA-based diagnoses. Thus, this investigation advances the efforts to automate nucleic acid extraction for future plant disease diagnosis in space.

Utilizing the KSC Fixation Tube to Conduct Human-Tended Plant Biology Experiments on a Suborbital Spaceflight.

Suborbital spaceflights now enable human-tended research investigating short-term gravitational effects in biological systems, eliminating the need for complex automation. Here, we discuss a method utilizing KSC Fixation Tubes (KFTs) to both carry biology to suborbital space as well as fix that biology at certain stages of flight. Plants on support media were inserted into the sample side of KFTs preloaded with RNAlater in the fixation chamber. The KFTs were activated at various stages of a simulated flight to fix the plants. RNA-seq analysis conducted on tissue samples housed in KFTs, showed that plants behaved consistently in KFTs when compared to petri-plates. Over the time course, roots adjusted to hypoxia and leaves adjusted to changes in photosynthesis. These responses were due in part to the environment imposed by the encased triple containment of the KFTs, which is a requirement for flight in human spacecraft. While plants exhibited expected reproducible transcriptomic alteration over time in the KFTs, responses to clinorotation during the simulated flight suggest that transcriptomic responses to suborbital spaceflight can be examined using this approach.

Plants grown in Apollo lunar regolith present stress-associated transcriptomes that inform prospects for lunar exploration.

The extent to which plants can enhance human life support on other worlds depends on the ability of plants to thrive in extraterrestrial environments using in-situ resources. Using samples from Apollo 11, 12, and 17, we show that the terrestrial plant Arabidopsis thaliana germinates and grows in diverse lunar regoliths. However, our results show that growth is challenging; the lunar regolith plants were slow to develop and many showed severe stress morphologies. Moreover, all plants grown in lunar soils differentially expressed genes indicating ionic stresses, similar to plant reactions to salt, metal and reactive oxygen species. Therefore, although in situ lunar regoliths can be useful for plant production in lunar habitats, they are not benign substrates. The interaction between plants and lunar regolith will need to be further elucidated, and likely mitigated, to best enable efficient use of lunar regolith for life support within lunar stations.

Epigenomic Regulators Elongator Complex Subunit 2 and Methyltransferase 1 Differentially Condition the Spaceflight Response in Arabidopsis.

Background: Plants subjected to the novel environment of spaceflight show transcriptomic changes that resemble aspects of several terrestrial abiotic stress responses. Under investigation here is whether epigenetic modulations, similar to those that occur in terrestrial stress responses, have a functional role in spaceflight physiological adaptation. The Advanced Plant Experiment-04 - Epigenetic Expression experiment examined the role of cytosine methylation in spaceflight adaptation. The experiment was conducted onboard the International Space Station, and 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)] along with a wild-type Col-0 control. Results: The elp2-5 plants suffered in their physiological adaptation to spaceflight in that their roots failed to extend away from the seed and the overall development of the plants was greatly impaired in space. The met1-7 plants suffered less, with their morphology affected by spaceflight in a manner similar to that of the Col-0 controls. The differentially expressed genes (DEGs) in spaceflight were dramatically different in the elp2-5 and met1-7 plants compared to Col-0, indicating that the disruptions in these mutants resulted in a reprogramming of their spaceflight responses, especially in elp2-5. Many of the genes comprising the spaceflight transcriptome of each genotype were differentially methylated in spaceflight. In Col-0 the majority of the DEGs were representative of the now familiar spaceflight response, which includes genes associated with cell wall remodeling, pathogen responses and ROS signaling. However, the spaceflight transcriptomes of met1-7 and elp2-5 each presented patterns of DEGs that are almost completely different than Col-0, and to each other. Further, the DEGs of the mutant genotypes suggest a more severe spaceflight stress response in the mutants, particularly in elp2-5. Conclusion: Arabidopsis physiological adaptation to spaceflight results in differential DNA methylation in an organ-specific manner. Disruption of Met1 methyltransferase function does not dramatically affect spaceflight growth or morphology, yet met1-7 reprograms the spaceflight transcriptomic response in a unique manner. Disruption of elp2-5 results in poor development in spaceflight grown plants, together with a diminished, dramatically reprogrammed transcriptomic response.

NDVI imaging within space exploration plant growth modules - A case study from EDEN ISS Antarctica.

The concept of using informative wavelength imagery to monitor plant health and ecosystem stability from space is derived from the deployment of Landsat and the development of the Normalized Difference Vegetative Index, or NDVI. NDVI presents the relative reflectance of the Near IR from plant leaves as a measure of relative plant health in terrestrial habitats and landscapes. However, the use of NDVI and NDVI-like imagery is rapidly evolving toward higher spatial resolution and more localized assessments of plant health, such as the use of drone imagery to monitor outdoor farms, and the use of mounted cameras within indoor growing facilities. With the advancement of plant growth systems in support of human space exploration, especially to the moon and Mars, remote assessment of plant health within exploration habitats becomes a critical element for development. This project examines the deployment of NDVI-like capabilities within a planetary analog greenhouse on the Antarctic ice shelf. The EDEN ISS Antarctica project provides a case study on the practical use of specific wavelength imagery to monitor plant health within space exploration environments. GoPro cameras, modified to dual bandpass capabilities, provided Single Image NDVI analyses for a year within the EDEN ISS Future Exploration Greenhouse at the Neumayer Station III in Antarctica. Images were acquired on site, analyzed remotely, and archived for the entire duration of the deployment through a combination of back-room science activities and operational communications with the Neumayer Station III. The results provide insights into the potential use of specific imaging wavelengths to enhance crop production in space exploration.