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.

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.

Effects of oxygen fertilization on damage reduction in flooded snap bean (Phaseolus vulgaris L.).

Flooding is one of the major abiotic stresses for vegetable production in Florida. Hydroponic and pot trials were conducted with snap bean to evaluate the effects of oxygen fertilization on the biochemical and physiological status of flooded snap bean plants. There were three treatments in the hydroponic trials were: (1) flooded (control), (2) bubble aeration with ambient air, and (3) hydrogen peroxide (H2O2) applied at the beginning of the trial. Plant health was evaluated by determining nitrogen (N) and phosphorus (P) uptake rates. The greenhouse pot trials were used to quantify the effects of three different application rates of solid oxygen fertilizers as calcium peroxide (CaO2) and magnesium peroxide (MgO2). The results showed that plant N and P uptake rates were significantly greater (p < 0.05) with H2O2 than without H2O2. The N uptake rates with H2O2 were like that of those with bubbling. The uptake rate of NH4+ was significantly greater than that of NO3- with the bubbling and H2O2 conditions, but the uptake rate of NO3- was significantly greater than that of NH4+ in the flooding condition. The plant height, leaf greenness, shoot biomass, and yield were all significantly greater with CaO2 or MgO2 than without either solid oxygen fertilizer. The minimum damage of flooded snap bean was found with 2 g CaO2 or 4 g MgO2 per pot. These results indicated that oxygen fertilization may potentially improve yield of flooded snap bean plants.

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.

The Plant Health Monitoring System of the EDEN ISS Space Greenhouse in Antarctica During the 2018 Experiment Phase.

The EDEN ISS project has the objective to test key technologies and processes for higher plant cultivation with a focus on their application to long duration spaceflight. A mobile plant production facility was designed and constructed by an international consortium and deployed to the German Antarctic Neumayer Station III. Future astronaut crews, even if well-trained and provided with detailed procedures, cannot be expected to have the competencies needed to deal with all situations that will arise during a mission. Future space crews, as they are today, will be supported by expert backrooms on the ground. For future space-based greenhouses, monitoring the crops and the plant growth system increases system reliability and decreases the crew time required to maintain them. The EDEN ISS greenhouse incorporates a Plant Health Monitoring System to provide remote support for plant status assessment and early detection of plant stress or disease. The EDEN ISS greenhouse has the capability to automatically capture and distribute images from its suite of 32 high-definition color cameras. Collected images are transferred over a satellite link to the EDEN ISS Mission Control Center in Bremen and to project participants worldwide. Upon reception, automatic processing software analyzes the images for anomalies, evaluates crop performance, and predicts the days remaining until harvest of each crop tray. If anomalies or sub-optimal performance is detected, the image analysis system generates automatic warnings to the agronomist team who then discuss, communicate, or implement countermeasure options. A select number of Dual Wavelength Spectral Imagers have also been integrated into the facility for plant health monitoring to detect potential plant stress before it can be seen on the images taken by the high-definition color cameras. These imagers and processing approaches are derived from traditional space-based imaging techniques but permit new discoveries to be made in a facility like the EDEN ISS greenhouse in which, essentially, every photon of input and output can be controlled and studied. This paper presents a description of the EDEN ISS Plant Health Monitoring System, basic image analyses, and a summary of the results from the initial year of Antarctic operations.

Spaceflight-induced alternative splicing during seedling development in Arabidopsis thaliana.

Plants grown in spaceflight experience novel environmental signals, including those associated with microgravity and ionizing radiation. Spaceflight triggers a response involving transcriptional re-programming and altered cell morphology, though many aspects of this response remain uncharacterized. We analyzed the spaceflight-induced transcriptome with a focus on genes that undergo alternative splicing to examine differential splicing associated with spaceflight-an unstudied characteristic of the molecular response to spaceflight exposure. RNA sequence data obtained during the APEX03 spaceflight experiment that was collected from two Arabidopsis thaliana ecotypes at two seedling stages grown onboard the International Space Station, or as ground controls at Kennedy Space Center, were re-examined to detect alternative splicing differences induced by spaceflight. Presence/absence variation analysis was used to identify putative expression-level differences in alternatively spliced isoforms between spaceflight and ground controls and was followed by analysis of significant differential alternative splicing. This study provides the first evidence of a role for alternative splicing in the molecular processes of physiological adaptation to the spaceflight environment.

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.

A member of the CONSTANS-Like protein family is a putative regulator of reactive oxygen species homeostasis and spaceflight physiological adaptation.

A feature of the physiological adaptation to spaceflight in Arabidopsis thaliana (Arabidopsis) is the induction of reactive oxygen species (ROS)-associated gene expression. The patterns of ROS-associated gene expression vary among Arabidopsis ecotypes, and the role of ROS signalling in spaceflight acclimation is unknown. What could differences in ROS gene regulation between ecotypes on orbit reveal about physiological adaptation to novel environments? Analyses of ecotype-dependent responses to spaceflight resulted in the elucidation of a previously uncharacterized gene (OMG1) as being ROS-associated. The OMG1 5' flanking region is an active promoter in cells where ROS activity is commonly observed, such as in pollen tubes, root hairs, and in other tissues upon wounding. qRT-PCR analyses revealed that upon wounding on Earth, OMG1 is an apparent transcriptional regulator of MYB77 and GRX480, which are associated with the ROS pathway. Fluorescence-based ROS assays show that OMG1 affects ROS production. Phylogenetic analysis of OMG1 and closely related homologs suggests that OMG1 is a distant, unrecognized member of the CONSTANS-Like protein family, a member that arose via gene duplication early in the angiosperm lineage and subsequently lost its first DNA-binding B-box1 domain. These data illustrate that members of the rapidly evolving COL protein family play a role in regulating ROS pathway functions, and their differential regulation on orbit suggests a role for ROS signalling in spaceflight physiological adaptation.

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.

Utilization of single-image normalized difference vegetation index (SI-NDVI) for early plant stress detection.

PREMISE OF THE STUDY: An imaging system was refined to monitor the health of vegetation grown in controlled conditions using spectral reflectance patterns. To measure plant health, the single-image normalized difference vegetation index (SI-NDVI) compares leaf reflectance in visible and near-infrared light spectrums. METHODS AND RESULTS: The SI-NDVI imaging system was characterized to assess plant responses to stress before visual detection during controlled stress assays. Images were analyzed using Fiji image processing software and Microsoft Excel to create qualitative false color images and quantitative graphs to detect plant stress. CONCLUSIONS: Stress was detected in Arabidopsis thaliana seedlings within 15 min of salinity application using SI-NDVI analysis, before stress was visible. Stress was also observed during ammonium nitrate treatment of Eruca sativa plants before visual detection. Early detection of plant stress is possible using SI-NDVI imaging, which is both simpler to use and more cost efficient than traditional dual-image NDVI or hyper-spectral imaging.

Comparing RNA-Seq and microarray gene expression data in two zones of the Arabidopsis root apex relevant to spaceflight.

PREMISE OF THE STUDY: The root apex is an important region involved in environmental sensing, but comprises a very small part of the root. Obtaining root apex transcriptomes is therefore challenging when the samples are limited. The feasibility of using tiny root sections for transcriptome analysis was examined, comparing RNA sequencing (RNA-Seq) to microarrays in characterizing genes that are relevant to spaceflight. METHODS: Arabidopsis thaliana Columbia ecotype (Col-0) roots were sectioned into Zone 1 (0.5 mm; root cap and meristematic zone) and Zone 2 (1.5 mm; transition, elongation, and growth-terminating zone). Differential gene expression in each was compared. RESULTS: Both microarrays and RNA-Seq proved applicable to the small samples. A total of 4180 genes were differentially expressed (with fold changes of 2 or greater) between Zone 1 and Zone 2. In addition, 771 unique genes and 19 novel transcriptionally active regions were identified by RNA-Seq that were not detected in microarrays. However, microarrays detected spaceflight-relevant genes that were missed in RNA-Seq. DISCUSSION: Single root tip subsections can be used for transcriptome analysis using either RNA-Seq or microarrays. Both RNA-Seq and microarrays provided novel information. These data suggest that techniques for dealing with small, rare samples from spaceflight can be further enhanced, and that RNA-Seq may miss some spaceflight-relevant changes in gene expression.

Phenotypic characterization of an Arabidopsis T-DNA insertion line SALK_063500.

In this article we report the identification of a homozygous lethal T-DNA (transfer DNA) line within the coding region of the At1G05290 gene in the genome of Arabidopsis thaliana (Arabidopsis) line, SALK_063500. The T-DNA insertion is found within exon one of the AT1G05290 gene, however a homozygous T-DNA allele is unattainable. In the heterozygous T-DNA allele the expression levels of AT1G05290 were compared to wild type Arabidopsis (Col-0, Columbia). Further analyses revealed an aberrant silique phenotype found in the heterozygous SALK_063500 plants that is attributed to the reduced rate of pollen tube germination. These data are original and have not been published elsewhere.

ARG1 Functions in the Physiological Adaptation of Undifferentiated Plant Cells to Spaceflight.

Scientific access to spaceflight and especially the International Space Station has revealed that physiological adaptation to spaceflight is accompanied or enabled by changes in gene expression that significantly alter the transcriptome of cells in spaceflight. A wide range of experiments have shown that plant physiological adaptation to spaceflight involves gene expression changes that alter cell wall and other metabolisms. However, while transcriptome profiling aptly illuminates changes in gene expression that accompany spaceflight adaptation, mutation analysis is required to illuminate key elements required for that adaptation. Here we report how transcriptome profiling was used to gain insight into the spaceflight adaptation role of Altered response to gravity 1 (Arg1), a gene known to affect gravity responses in plants on Earth. The study compared expression profiles of cultured lines of Arabidopsis thaliana derived from wild-type (WT) cultivar Col-0 to profiles from a knock-out line deficient in the gene encoding ARG1 (ARG1 KO), both on the ground and in space. The cell lines were launched on SpaceX CRS-2 as part of the Cellular Expression Logic (CEL) experiment of the BRIC-17 spaceflight mission. The cultured cell lines were grown within 60 mm Petri plates in Petri Dish Fixation Units (PDFUs) that were housed within the Biological Research In Canisters (BRIC) hardware. Spaceflight samples were fixed on orbit. Differentially expressed genes were identified between the two environments (spaceflight and comparable ground controls) and the two genotypes (WT and ARG1 KO). Each genotype engaged unique genes during physiological adaptation to the spaceflight environment, with little overlap. Most of the genes altered in expression in spaceflight in WT cells were found to be Arg1-dependent, suggesting a major role for that gene in the physiological adaptation of undifferentiated cells to spaceflight. Key Words: ARG1-Spaceflight-Gene expression-Physiological adaptation-BRIC. Astrobiology 17, 1077-1111.