Epigenomics in an extraterrestrial environment: organ-specific alteration of DNA methylation and gene expression elicited by spaceflight in Arabidopsis thaliana.

BACKGROUND: Plants adapted to diverse environments on Earth throughout their evolutionary history, and developed mechanisms to thrive in a variety of terrestrial habitats. When plants are grown in the novel environment of spaceflight aboard the International Space Station (ISS), an environment completely outside their evolutionary history, they respond with unique alterations to their gene expression profile. Identifying the genes important for physiological adaptation to spaceflight and dissecting the biological processes and pathways engaged by plants during spaceflight has helped reveal spaceflight adaptation, and has furthered understanding of terrestrial growth processes. However, the underlying regulatory mechanisms responsible for these changes in gene expression patterns are just beginning to be explored. Epigenetic modifications, such as DNA methylation at position five in cytosine, has been shown to play a role in the physiological adaptation to adverse terrestrial environments, and may play a role in spaceflight as well. RESULTS: Whole Genome Bisulfite Sequencing of DNA of Arabidopsis grown on the ISS from seed revealed organ-specific patterns of differential methylation compared to ground controls. The overall levels of methylation in CG, CHG, and CHH contexts were similar between flight and ground DNA, however, thousands of specifically differentially methylated cytosines were discovered, and there were clear organ-specific differences in methylation patterns. Spaceflight leaves had higher methylation levels in CHG and CHH contexts within protein-coding genes in spaceflight; about a fifth of the leaf genes were also differentially regulated in spaceflight, almost half of which were associated with reactive oxygen signaling. CONCLUSIONS: The physiological adaptation of plants to spaceflight is likely nuanced by epigenomic modification. This is the first examination of differential genomic methylation from plants grown completely in the spaceflight environment of the ISS in plant growth hardware developed for informing exploration life support strategies. Yet even in this optimized plant habitat, plants respond as if stressed. These data suggest that gene expression associated with physiological adaptation to spaceflight is regulated in part by methylation strategies similar to those engaged with familiar terrestrial stress responses. The differential methylation maps generated here provide a useful reference for elucidating the layers of regulation of spaceflight responses.

HSFA2 Functions in the Physiological Adaptation of Undifferentiated Plant Cells to Spaceflight.

Heat Shock Factor A2 (HsfA2) is part of the Heat Shock Factor (HSF) network, and plays an essential role beyond heat shock in environmental stress responses and cellular homeostatic control. Arabidopsis thaliana cell cultures derived from wild type (WT) ecotype Col-0 and a knockout line deficient in the gene encoding HSFA2 (HSFA2 KO) were grown aboard the International Space Station (ISS) to ascertain whether the HSF network functions in the adaptation to the novel environment of spaceflight. Microarray gene expression data were analyzed using a two-part comparative approach. First, genes differentially expressed between the two environments (spaceflight to ground) were identified within the same genotype, which represented physiological adaptation to spaceflight. Second, gene expression profiles were compared between the two genotypes (HSFA2 KO to WT) within the same environment, which defined genes uniquely required by each genotype on the ground and in spaceflight-adapted states. Results showed that the endoplasmic reticulum (ER) stress and unfolded protein response (UPR) define the HSFA2 KO cells' physiological state irrespective of the environment, and likely resulted from a deficiency in the chaperone-mediated protein folding machinery in the mutant. Results further suggested that additional to its universal stress response role, HsfA2 also has specific roles in the physiological adaptation to spaceflight through cell wall remodeling, signal perception and transduction, and starch biosynthesis. Disabling HsfA2 altered the physiological state of the cells, and impacted the mechanisms induced to adapt to spaceflight, and identified HsfA2-dependent genes that are important to the adaption of wild type cells to spaceflight. Collectively these data indicate a non-thermal role for the HSF network in spaceflight adaptation.

Skewing in Arabidopsis roots involves disparate environmental signaling pathways.

BACKGROUND: Skewing root patterns provide key insights into root growth strategies and mechanisms that produce root architectures. Roots exhibit skewing and waving when grown on a tilted, impenetrable surface. The genetics guiding these morphologies have been examined, revealing that some Arabidopsis ecotypes skew and wave (e.g. WS), while others skew insignificantly but still wave (e.g. Col-0). The underlying molecular mechanisms of skewing and waving remain unclear. In this study, transcriptome data were derived from two Arabidopsis ecotypes, WS and Col-0, under three tilted growth conditions in order to identify candidate genes involved in skewing. RESULTS: This work identifies a number of genes that are likely involved in skewing, using growth conditions that differentially affect skewing and waving. Comparing the gene expression profiles of WS and Col-0 in different tilted growth conditions identified 11 candidate genes as potentially involved in the control of skewing. These 11 genes are involved in several different cellular processes, including sugar transport, salt signaling, cell wall organization, and hormone signaling. CONCLUSIONS: This study identified 11 genes whose change in expression level is associated with root skewing behavior. These genes are involved in signaling and perception, rather than the physical restructuring of root. Future work is needed to elucidate the potential role of these candidate genes during root skewing.

Phosphorylation-related modification at the dimer interface of 14-3-3ω dramatically alters monomer interaction dynamics.

14-3-3 proteins are generally believed to function as dimers in a broad range of eukaryotic signaling pathways. The consequences of altering dimer stability are not fully understood. Phosphorylation at Ser58 in the dimer interface of mammalian 14-3-3 isoforms has been reported to destabilise dimers. An equivalent residue, Ser62, is present across most Arabidopsis isoforms but the effects of phosphorylation have not been studied in plants. Here, we assessed the effects of phosphorylation at the dimer interface of Arabidopsis 14-3-3ω. Protein kinase A phosphorylated 14-3-3ω at Ser62 and also at a previously unreported residue, Ser67, resulting in a monomer-sized band on native-PAGE. Phosphorylation at Ser62 alone, or with additional Ser67 phosphorylation, was investigated using phosphomimetic versions of 14-3-3ω. In electrophoretic and chromatographic analyses, these mutants showed mobilities intermediate between dimers and monomers. Mobility was increased by detergents, by reducing protein concentration, or by increasing pH or temperature. Urea gradient gels showed complex structural transitions associated with alterations of dimer stability, including a previously unreported 14-3-3 aggregation phenomenon. Overall, our analyses showed that dimer interface modifications such as phosphorylation reduce dimer stability, dramatically affecting the monomer-dimer equilibrium and denaturation trajectory. These findings may have dramatic implications for 14-3-3 structure and function in vivo.

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.

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.

14-3-3 proteins in plant physiology.

Plant 14-3-3 isoforms, like their highly conserved homologues in mammals, function by binding to phosphorylated client proteins to modulate their function. Through the regulation of a diverse range of proteins including kinases, transcription factors, structural proteins, ion channels and pathogen defense-related proteins, they are being implicated in an expanding catalogue of physiological functions in plants. 14-3-3s themselves are affected, both transcriptionally and functionally, by the extracellular and intracellular environment of the plant. They can modulate signaling pathways that transduce inputs from the environment and also the downstream proteins that elicit the physiological response. This review covers some of the key emerging roles for plant 14-3-3s including their role in the response to the plant extracellular environment, particularly environmental stress, pathogens and light conditions. We also address potential key roles in primary metabolism, hormone signaling, growth and cell division.

Organ-specific remodeling of the Arabidopsis transcriptome in response to spaceflight.

BACKGROUND: Spaceflight presents a novel environment that is outside the evolutionary experience of terrestrial organisms. Full activation of the International Space Station as a science platform complete with sophisticated plant growth chambers, laboratory benches, and procedures for effective sample return, has enabled a new level of research capability and hypothesis testing in this unique environment. The opportunity to examine the strategies of environmental sensing in spaceflight, which includes the absence of unit gravity, provides a unique insight into the balance of influence among abiotic cues directing plant growth and development: including gravity, light, and touch. The data presented here correlate morphological and transcriptome data from replicated spaceflight experiments. RESULTS: The transcriptome of Arabidopsis thaliana demonstrated organ-specific changes in response to spaceflight, with 480 genes showing significant changes in expression in spaceflight plants compared with ground controls by at least 1.9-fold, and 58 by more than 7-fold. Leaves, hypocotyls, and roots each displayed unique patterns of response, yet many gene functions within the responses are related. Particularly represented across the dataset were genes associated with cell architecture and growth hormone signaling; processes that would not be anticipated to be altered in microgravity yet may correlate with morphological changes observed in spaceflight plants. As examples, differential expression of genes involved with touch, cell wall remodeling, root hairs, and cell expansion may correlate with spaceflight-associated root skewing, while differential expression of auxin-related and other gravity-signaling genes seemingly correlates with the microgravity of spaceflight. Although functionally related genes were differentially represented in leaves, hypocotyls, and roots, the expression of individual genes varied substantially across organ types, indicating that there is no single response to spaceflight. Rather, each organ employed its own response tactics within a shared strategy, largely involving cell wall architecture. CONCLUSIONS: Spaceflight appears to initiate cellular remodeling throughout the plant, yet specific strategies of the response are distinct among specific organs of the plant. Further, these data illustrate that in the absence of gravity plants rely on other environmental cues to initiate the morphological responses essential to successful growth and development, and that the basis for that engagement lies in the differential expression of genes in an organ-specific manner that maximizes the utilization of these signals--such as the up-regulation of genes associated with light-sensing in roots.

Spaceflight engages heat shock protein and other molecular chaperone genes in tissue culture cells of Arabidopsis thaliana.

PREMISE OF THE STUDY: Gravity has been a major force throughout the evolution of terrestrial organisms, and plants have developed exquisitely sensitive, regulated tropisms and growth patterns that are based on the gravity vector. The nullified gravity during spaceflight allows direct assessment of gravity roles. The microgravity environments provided by the Space Shuttle and International Space Station have made it possible to seek novel insights into gravity perception at the organismal, tissue, and cellular levels. Cell cultures of Arabidopsis thaliana perceive and respond to spaceflight, even though they lack the specialized cell structures normally associated with gravity perception in intact plants; in particular, genes for a specific subset of heat shock proteins (HSPs) and factors (HSFs) are induced. Here we ask if similar changes in HSP gene expression occur during nonspaceflight changes in gravity stimulation. METHODS: Quantitative RT-qPCR was used to evaluate mRNA levels for Hsp17.6A and Hsp101 in cell cultures exposed to four conditions: spaceflight (mission STS-131), hypergravity (centrifugation at 3 g or 16 g), sustained two-dimensional clinorotation, and transient milligravity achieved on parabolic flights. KEY RESULTS: We showed that HSP genes were induced in cells only in response to sustained clinorotation. Transient microgravity intervals in parabolic flight and various hypergravity conditions failed to induce HSP genes. CONCLUSIONS: We conclude that nondifferentiated cells do indeed sense their gravity environment and HSP genes are induced only in response to prolonged microgravity or simulated microgravity conditions. We hypothesize that HSP induction upon microgravity indicates a role for HSP-related proteins in maintaining cytoskeletal architecture and cell shape signaling.

Fundamental plant biology enabled by the space shuttle.

The relationship between fundamental plant biology and space biology was especially synergistic in the era of the Space Shuttle. While all terrestrial organisms are influenced by gravity, the impact of gravity as a tropic stimulus in plants has been a topic of formal study for more than a century. And while plants were parts of early space biology payloads, it was not until the advent of the Space Shuttle that the science of plant space biology enjoyed expansion that truly enabled controlled, fundamental experiments that removed gravity from the equation. The Space Shuttle presented a science platform that provided regular science flights with dedicated plant growth hardware and crew trained in inflight plant manipulations. Part of the impetus for plant biology experiments in space was the realization that plants could be important parts of bioregenerative life support on long missions, recycling water, air, and nutrients for the human crew. However, a large part of the impetus was that the Space Shuttle enabled fundamental plant science essentially in a microgravity environment. Experiments during the Space Shuttle era produced key science insights on biological adaptation to spaceflight and especially plant growth and tropisms. In this review, we present an overview of plant science in the Space Shuttle era with an emphasis on experiments dealing with fundamental plant growth in microgravity. This review discusses general conclusions from the study of plant spaceflight biology enabled by the Space Shuttle by providing historical context and reviews of select experiments that exemplify plant space biology science.

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.

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.

An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis.

Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.

Plant growth strategies are remodeled by spaceflight.

BACKGROUND: Arabidopsis plants were grown on the International Space Station within specialized hardware that combined a plant growth habitat with a camera system that can capture images at regular intervals of growth. The Imaging hardware delivers telemetric data from the ISS, specifically images received in real-time from experiments on orbit, providing science without sample return. Comparable Ground Controls were grown in a sister unit that is maintained in the Orbital Environment Simulator at Kennedy Space Center. One of many types of biological data that can be analyzed in this fashion is root morphology. Arabidopsis seeds were geminated on orbit on nutrient gel Petri plates in a configuration that encouraged growth along the surface of the gel. Photos were taken every six hours for the 15 days of the experiment. RESULTS: In the absence of gravity, but the presence of directional light, spaceflight roots remained strongly negatively phototropic and grew in the opposite direction of the shoot growth; however, cultivars WS and Col-0 displayed two distinct, marked differences in their growth patterns. First, cultivar WS skewed strongly to the right on orbit, while cultivar Col-0 grew with little deviation away from the light source. Second, the Spaceflight environment also impacted the rate of growth in Arabidopsis. The size of the Flight plants (as measured by primary root and hypocotyl length) was uniformly smaller than comparably aged Ground Control plants in both cultivars. CONCLUSIONS: Skewing and waving, thought to be gravity dependent phenomena, occur in spaceflight plants. In the presence of an orienting light source, phenotypic trends in skewing are gravity independent, and the general patterns of directional root growth typified by a given genotype in unit gravity are recapitulated on orbit, although overall growth patterns on orbit are less uniform. Skewing appears independent of axial orientation on the ISS - suggesting that other tropisms (such as for oxygen and temperature) do not influence skewing. An aspect of the spaceflight environment also retards the rate of early Arabidopsis growth.

The 14-3-3 proteins of Arabidopsis regulate root growth and chloroplast development as components of the photosensory system.

The 14-3-3 proteins specifically bind a number of client proteins to influence important pathways, including flowering timing via the photosensory system. For instance, 14-3-3 proteins influence the photosensory system through interactions with Constans (CO) protein. 14-3-3 associations with the photosensory system were further studied in this investigation using 14-3-3 T-DNA insertion mutants to study root and chloroplast development. The 14-3-3 µ T-DNA insertion mutant, 14-3-3µ-1, had shorter roots than the wild type and the difference in root length could be influenced by light intensity. The 14-3-3 ν T-DNA insertion mutants also had shorter roots, but only when grown under narrow-bandwidth red light. Five-day-old 14-3-3 T-DNA insertion and co mutants all had increased root greening compared with the wild type, which was influenced by light wavelength and intensity. However, beyond 10 d of growth, 14-3-3µ-1 roots did not increase in greening as much as wild-type roots. This study reveals new developmental roles of 14-3-3 proteins in roots and chloroplasts, probably via association with the photosensory system.

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.

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.

Root Skewing-Associated Genes Impact the Spaceflight Response of Arabidopsis thaliana.

The observation that plant roots skew in microgravity recently refuted the long-held conviction that skewing was a gravity-dependent phenomenon. Further, spaceflight root skewing suggests that specific root morphologies and cell wall remodeling systems may be important aspects of spaceflight physiological adaptation. However, connections between skewing, cell wall modification and spaceflight physiology are currently based on inferences rather than direct tests. Therefore, the Advanced Plant Experiments-03-2 (APEX-03-2) spaceflight study was designed to elucidate the contribution of two skewing- and cell wall-associated genes in Arabidopsis to root behavior and gene expression patterns in spaceflight, to assess whether interruptions of different skewing pathways affect the overall spaceflight-associated process. SPIRAL1 is a skewing-related protein implicated in directional cell expansion, and functions by regulating cortical microtubule dynamics. SKU5 is skewing-related glycosylphosphatidylinositol-anchored protein of the plasma membrane and cell wall implicated in stress response signaling. These two genes function in different cellular pathways that affect skewing on the Earth, and enable a test of the relevance of skewing pathways to spaceflight physiological adaptation. In this study, both sku5 and spr1 mutants showed different skewing behavior and markedly different patterns of gene expression in the spaceflight environment. The spr1 mutant showed fewer differentially expressed genes than its Col-0 wild-type, whereas sku5 showed considerably more than its WS wild-type. Developmental age played a substantial role in spaceflight acclimation in all genotypes, but particularly in sku5 plants, where spaceflight 4d seedlings had almost 10-times as many highly differentially expressed genes as the 8d seedlings. These differences demonstrated that the two skewing pathways represented by SKU5 and SPR1 have unique and opposite contributions to physiological adaptation to spaceflight. The spr1 response is less intense than wild type, suggesting that the loss of SPR1 positively impacts spaceflight adaptation. Conversely, the intensity of the sku5 responses suggests that the loss of SKU5 initiates a much more complex, deeper and more stress related response to spaceflight. This suggests that proper SKU5 function is important to spaceflight adaptation.