Assessing Radish Health during Space Cultivation by Gene Transcription.

During the Advanced Plant Habitat experiment 2, radish plants were grown in two successive grow-outs on the International Space Station (ISS) for 27 days each. On days 10, 18, and 24, leaf punch (LP) samples were collected and frozen. At harvest, bulb tissue was sampled with oligo-dT functionalized Solid Phase Gene Extraction (SPGE) probes. The space samples were compared with samples from ground controls (GC) grown at the Kennedy Space Center (KSC) under the same conditions as on the ISS, with notably elevated CO2 (about 2500 ppm), and from lab plants grown under atmospheric CO2 but with light and temperature conditions similar to the KSC control. Genes corresponding to peroxidase (RPP), glucosinolate biosynthesis (GIS), protein binding (CBP), myrosinase (RMA), napin (RSN), and ubiquitin (UBQ) were measured by qPCR. LP from day 24 and bulb samples collected at harvest were compared with RNA-seq data from material that was harvested, frozen, and analyzed after return to Earth. The results showed stable transcription in LP samples in GC but decreasing values in ISS samples during both grow-outs, possibly indicative of stress. SPGE results were similar between GC and ISS samples. However, the RNA-seq analyses showed different transcription profiles than SPGE or LP results, possibly related to localized sampling. RNA-seq of leaf samples showed greater variety than LP data, possibly because of different sampling times. RSN and RPP showed the lowest transcription regardless of method. Temporal analyses showed relatively small changes during plant development in space and in ground controls. This is the first study that compares developmental changes in space-grown plants with ground controls based on a comparison between RNA-seq and qPCR analyses.

Spaceflight studies identify a gene encoding an intermediate filament involved in tropism pathways.

We performed a series of experiments to study the interaction between phototropism and gravitropism in Arabidopsis thaliana as part of the Seedling Growth Project on the International Space Station. Red-light-based and blue-light-based phototropism were examined in microgravity and at 1g, a control that was produced by an on-board centrifuge. At the end of the experiments, seedlings were frozen and brought back to Earth for gene profiling studies via RNASeq methods. In this paper, we focus on five genes identified in these space studies by their differential expression in space: one involved in auxin transport and four others encoding genes for: a methyltransferase subunit, a transmembrane protein, a transcription factor for endodermis formation, and a cytoskeletal element (an intermediate filament protein). Time course studies using mutant strains of these five genes were performed for blue-light and red-light phototropism studies as well as for gravitropism assays on ground. Interestingly, all five of the genes had some effects on all the tropisms under the conditions studied. In addition, RT-PCR analyses examined expression of the five genes in wild-type seedlings during blue-light-based phototropism. Previous studies have supported a role of both microfilaments and microtubules in tropism pathways. However, the most interesting finding of the present space studies is that NFL, a gene encoding an intermediate filament protein, plays a role in phototropism and gravitropism, which opens the possibility that this cytoskeletal element modulates signal transduction in plants.

NASA GeneLab RNA-seq consensus pipeline: standardized processing of short-read RNA-seq data.

With the development of transcriptomic technologies, we are able to quantify precise changes in gene expression profiles from astronauts and other organisms exposed to spaceflight. Members of NASA GeneLab and GeneLab-associated analysis working groups (AWGs) have developed a consensus pipeline for analyzing short-read RNA-sequencing data from spaceflight-associated experiments. The pipeline includes quality control, read trimming, mapping, and gene quantification steps, culminating in the detection of differentially expressed genes. This data analysis pipeline and the results of its execution using data submitted to GeneLab are now all publicly available through the GeneLab database. We present here the full details and rationale for the construction of this pipeline in order to promote transparency, reproducibility, and reusability of pipeline data; to provide a template for data processing of future spaceflight-relevant datasets; and to encourage cross-analysis of data from other databases with the data available in GeneLab.

From Spaceflight to Mars g-Levels: Adaptive Response of A. Thaliana Seedlings in a Reduced Gravity Environment Is Enhanced by Red-Light Photostimulation.

The response of plants to the spaceflight environment and microgravity is still not well understood, although research has increased in this area. Even less is known about plants' response to partial or reduced gravity levels. In the absence of the directional cues provided by the gravity vector, the plant is especially perceptive to other cues such as light. Here, we investigate the response of Arabidopsis thaliana 6-day-old seedlings to microgravity and the Mars partial gravity level during spaceflight, as well as the effects of red-light photostimulation by determining meristematic cell growth and proliferation. These experiments involve microscopic techniques together with transcriptomic studies. We demonstrate that microgravity and partial gravity trigger differential responses. The microgravity environment activates hormonal routes responsible for proliferation/growth and upregulates plastid/mitochondrial-encoded transcripts, even in the dark. In contrast, the Mars gravity level inhibits these routes and activates responses to stress factors to restore cell growth parameters only when red photostimulation is provided. This response is accompanied by upregulation of numerous transcription factors such as the environmental acclimation-related WRKY-domain family. In the long term, these discoveries can be applied in the design of bioregenerative life support systems and space farming.

RNAseq Analysis of the Response of Arabidopsis thaliana to Fractional Gravity Under Blue-Light Stimulation During Spaceflight.

Introduction: Traveling to nearby extraterrestrial objects having a reduced gravity level (partial gravity) compared to Earth's gravity is becoming a realistic objective for space agencies. The use of plants as part of life support systems will require a better understanding of the interactions among plant growth responses including tropisms, under partial gravity conditions. Materials and Methods: Here, we present results from our latest space experiments on the ISS, in which seeds of Arabidopsis thaliana were germinated, and seedlings grew for six days under different gravity levels, namely micro-g, several intermediate partial-g levels, and 1g, and were subjected to irradiation with blue light for the last 48 h. RNA was extracted from 20 samples for subsequent RNAseq analysis. Transcriptomic analysis was performed using the HISAT2-Stringtie-DESeq pipeline. Differentially expressed genes were further characterized for global responses using the GEDI tool, gene networks and for Gene Ontology (GO) enrichment. Results: Differential gene expression analysis revealed only one differentially expressed gene (AT4G21560, VPS28-1 a vacuolar protein) across all gravity conditions using FDR correction (q < 0.05). However, the same 14 genes appeared differentially expressed when comparing either micro-g, low-g level (< 0.1g) or the Moon g-level with 1g control conditions. Apart from these 14-shared genes, the number of differentially expressed genes was similar in microgravity and the Moon g-level and increased in the intermediate g-level (< 0.1g), but it was then progressively reduced as the difference with the Earth gravity became smaller. The GO groups were differentially affected at each g-level: light and photosynthesis GO under microgravity, genes belonged to general stress, chemical and hormone responses under low-g, and a response related to cell wall and membrane structure and function under the Moon g-level. Discussion: Transcriptional analyses of plants under blue light stimulation suggests that root blue-light phototropism may be enough to reduce the gravitational stress response caused by the lack of gravitropism in microgravity. Competition among tropisms induces an intense perturbation at the micro-g level, which shows an extensive stress response that is progressively attenuated. Our results show a major effect on cell wall/membrane remodeling (detected at the interval from the Moon to Mars gravity), which can be potentially related to graviresistance mechanisms.

RNA-seq analyses of Arabidopsis thaliana seedlings after exposure to blue-light phototropic stimuli in microgravity.

PREMISE: Plants synthesize information from multiple environmental stimuli when determining their direction of growth. Gravity, being ubiquitous on Earth, plays a major role in determining the direction of growth and overall architecture of the plant. Here, we utilized the microgravity environment on board the International Space Station (ISS) to identify genes involved influencing growth and development of phototropically stimulated seedlings of Arabidopsis thaliana. METHODS: Seedlings were grown on the ISS, and RNA was extracted from 7 samples (pools of 10-15 plants) grown in microgravity (µg) or Earth gravity conditions (1-g). Transcriptomic analyses via RNA sequencing (RNA-seq) of differential gene expression was performed using the HISAT2-Stringtie-DESeq2 RNASeq pipeline. Differentially expressed genes were further characterized by using Pathway Analysis and enrichment for Gene Ontology classifications. RESULTS: For 296 genes that were found significantly differentially expressed between plants in microgravity compared to 1-g controls, Pathway Analysis identified eight molecular pathways that were significantly affected by reduced gravity conditions. Specifically, light-associated pathways (e.g., photosynthesis-antenna proteins, photosynthesis, porphyrin, and chlorophyll metabolism) were significantly downregulated in microgravity. CONCLUSIONS: Gene expression in A. thaliana seedlings grown in microgravity was significantly altered compared to that of the 1-g control. Understanding how plants grow in conditions of microgravity not only aids in our understanding of how plants grow and respond to the environment but will also help to efficiently grow plants during long-range space missions.

Plant responses to gravity.

Tropisms are directed growth-mediated plant movements which allow plants to respond to their environment. Gravitropism is the ability of plants to perceive and respond to the gravity vector and orient themselves accordingly. The gravitropic pathway can be divided into three main components: perception, biochemical signaling, and differential growth. Perception of the gravity signal occurs through the movement/sedimentation of starch-filled plastids (termed statoliths) in gravity sensing cells. Once perceived, proteins interact with the settling statoliths to set a cascade of plant hormones to the elongation zones in the roots or shoots. Plant growth regulators that play a role in gravitropism include auxin, ethylene, gibberellic acid, jasmonic acid, among others. Differential growth on opposing sides of the root or shoot allow for the plant to grow relative to the direction of the perceived gravity vector. In this review, we detail how plants perceive gravity and respond biochemically in response to gravity as well as synthesize the recent literature on this important topic in plant biology. Keywords: auxin, gravitropism, gravity perception, plant growth regulators, space biology, statolith.

Preparation of a Spaceflight Experiment to Study Tropisms in Arabidopsis Seedlings on the International Space Station.

Utilization of orbiting spacecraft allows for studying biological processes in conditions of microgravity. Centrifuges on board these platforms also allow for the creation of partial gravity vectors (to simulate the Moon or Mars levels of gravity) as well as onboard 1-g controls for the space experiments. Thus, the mechanisms of gravity and light perception in plants can be analyzed in a unique environment which can give insights into fundamental processes in biology. Here, we describe the methods for preparation of a plant biology experiment with seedlings of Arabidopsis thaliana utilizing the European Modular Cultivation System (EMCS) on the International Space Station (ISS). The procedures outlined in this paper have been successfully used in several of our recent spaceflight experiments, which have given unique insights into the basic mechanisms of tropisms.

The combined effects of real or simulated microgravity and red-light photoactivation on plant root meristematic cells.

MAIN CONCLUSION: Red light is able to compensate for deleterious effects of microgravity on root cell growth and proliferation. Partial gravity combined with red light produces differential signals during the early plant development. Light and gravity are environmental cues used by plants throughout evolution to guide their development. We have investigated the cross-talk between phototropism and gravitropism under altered gravity in space. The focus was on the effects on the meristematic balance between cell growth and proliferation, which is disrupted under microgravity in the dark. In our spaceflight experiments, seedlings of three Arabidopsis thaliana genotypes, namely the wild type and mutants of phytochrome A and B, were grown for 6 days, including red-light photoactivation for the last 2 days. Apart from the microgravity and the 1g on-board control conditions, fractional gravity (nominally 0.1g, 0.3g, and 0.5g) was created with on-board centrifuges. In addition, a simulated microgravity (random positioning machine, RPM) experiment was performed on ground, including both dark-grown and photostimulated samples. Photoactivated samples in spaceflight and RPM experiments showed an increase in the root length consistent with phototropic response to red light, but, as gravity increased, a gradual decrease in this response was observed. Uncoupling of cell growth and proliferation was detected under microgravity in darkness by transcriptomic and microscopic methods, but red-light photoactivation produced a significant reversion. In contrast, the combination of red light and partial gravity produced small but consistent variations in the molecular markers of cell growth and proliferation, suggesting an antagonistic effect between light and gravity signals at the early plant development. Understanding these parameters of plant growth and development in microgravity will be important as bioregenerative life support systems for the colonization of the Moon and Mars.

A novel blue-light phototropic response is revealed in roots of Arabidopsis thaliana in microgravity.

MAIN CONCLUSION: Blue-light positive phototropism in roots is masked by gravity and revealed in conditions of microgravity. In addition, the magnitude of red-light positive phototropic curvature is correlated to the magnitude of gravity. Due to their sessile nature, plants utilize environmental cues to grow and respond to their surroundings. Two of these cues, light and gravity, play a substantial role in plant orientation and directed growth movements (tropisms). However, very little is currently known about the interaction between light- (phototropic) and gravity (gravitropic)-mediated growth responses. Utilizing the European Modular Cultivation System on board the International Space Station, we investigated the interaction between phototropic and gravitropic responses in three Arabidopsis thaliana genotypes, Landsberg wild type, as well as mutants of phytochrome A and phytochrome B. Onboard centrifuges were used to create a fractional gravity gradient ranging from reduced gravity up to 1g. A novel positive blue-light phototropic response of roots was observed during conditions of microgravity, and this response was attenuated at 0.1g. In addition, a red-light pretreatment of plants enhanced the magnitude of positive phototropic curvature of roots in response to blue illumination. In addition, a positive phototropic response of roots was observed when exposed to red light, and a decrease in response was gradual and correlated with the increase in gravity. The positive red-light phototropic curvature of hypocotyls when exposed to red light was also confirmed. Both red-light and blue-light phototropic responses were also shown to be affected by directional light intensity. To our knowledge, this is the first characterization of a positive blue-light phototropic response in Arabidopsis roots, as well as the first description of the relationship between these phototropic responses in fractional or reduced gravities.

Space, the final frontier: A critical review of recent experiments performed in microgravity.

Space biology provides an opportunity to study plant physiology and development in a unique microgravity environment. Recent space studies with plants have provided interesting insights into plant biology, including discovering that plants can grow seed-to-seed in microgravity, as well as identifying novel responses to light. However, spaceflight experiments are not without their challenges, including limited space, limited access, and stressors such as lack of convection and cosmic radiation. Therefore, it is important to design experiments in a way to maximize the scientific return from research conducted on orbiting platforms such as the International Space Station. Here, we provide a critical review of recent spaceflight experiments and suggest ways in which future experiments can be designed to improve the value and applicability of the results generated. These potential improvements include: utilizing in-flight controls to delineate microgravity versus other spaceflight effects, increasing scientific return via next-generation sequencing technologies, and utilizing multiple genotypes to ensure results are not unique to one genetic background. Space experiments have given us new insights into plant biology. However, to move forward, special care should be given to maximize science return in understanding both microgravity itself as well as the combinatorial effects of living in space.

Light and gravity signals synergize in modulating plant development.

Tropisms are growth-mediated plant movements that help plants to respond to changes in environmental stimuli. The availability of water and light, as well as the presence of a constant gravity vector, are all environmental stimuli that plants sense and respond to via directed growth movements (tropisms). The plant response to gravity (gravitropism) and the response to unidirectional light (phototropism) have long been shown to be interconnected growth phenomena. Here, we discuss the similarities in these two processes, as well as the known molecular mechanisms behind the tropistic responses. We also highlight research done in a microgravity environment in order to decouple two tropisms through experiments carried out in the absence of a significant unilateral gravity vector. In addition, alteration of gravity, especially the microgravity environment, and light irradiation produce important effects on meristematic cells, the undifferentiated, highly proliferating, totipotent cells which sustain plant development. Microgravity produces the disruption of meristematic competence, i.e., the decoupling of cell proliferation and cell growth, affecting the regulation of the cell cycle and ribosome biogenesis. Light irradiation, especially red light, mediated by phytochromes, has an activating effect on these processes. Phytohormones, particularly auxin, also are key mediators in these alterations. Upcoming experiments on the International Space Station will clarify some of the mechanisms and molecular players of the plant responses to these environmental signals involved in tropisms and the cell cycle.

Turning heads: the biology of solar tracking in sunflower.

Solar tracking in the common sunflower, Helianthus annuus, is a dramatic example of a diurnal rhythm in plants. During the day, the shoot apex continuously reorients, following the sun's relative position so that the developing heads track from east to west. At night, the reverse happens, and the heads return and face east in anticipation of dawn. This daily cycle dampens and eventually stops at anthesis, after which the sunflower head maintains an easterly orientation. Although shoot apical heliotropism has long been the subject of physiological studies in sunflower, the underlying developmental, cellular, and molecular mechanisms that drive the directional growth and curvature of the stem in response to extrinsic and perhaps intrinsic cues are not known. Furthermore, the ecological functions of solar tracking and the easterly orientation of mature heads have been the subject of significant but unresolved speculation. In this review, we discuss the current state of knowledge about this complex, dynamic trait. Candidate mechanisms that may contribute to daytime and nighttime movement are highlighted, including light signaling, hormonal action, and circadian regulation of growth pathways. The merits of the diverse hypotheses advanced to explain the adaptive significance of heliotropism in sunflower are also considered.

Identification of bioconversion quantitative trait loci in the interspecific cross Sorghum bicolor × Sorghum propinquum.

For lignocellulosic bioenergy to be economically viable, genetic improvements must be made in feedstock quality including both biomass total yield and conversion efficiency. Toward this goal, multiple studies have considered candidate genes and discovered quantitative trait loci (QTL) associated with total biomass accumulation and/or grain production in bioenergy grass species including maize and sorghum. However, very little research has been focused on genes associated with increased biomass conversion efficiency. In this study, Trichoderma viride fungal cellulase hydrolysis activity was measured for lignocellulosic biomass (leaf and stem tissue) obtained from individuals in a F5 recombinant inbred Sorghum bicolor × Sorghum propinquum mapping population. A total of 49 QTLs (20 leaf, 29 stem) were associated with enzymatic conversion efficiency. Interestingly, six high-density QTL regions were identified in which four or more QTLs overlapped. In addition to enzymatic conversion efficiency QTLs, two QTLs were identified for biomass crystallinity index, a trait which has been shown to be inversely correlated with conversion efficiency in bioenergy grasses. The identification of these QTLs provides an important step toward identifying specific genes relevant to increasing conversion efficiency of bioenergy feedstocks. DNA markers linked to these QTLs could be useful in marker-assisted breeding programs aimed at increasing overall bioenergy yields concomitant with selection of high total biomass genotypes.

Bioenergy grass feedstock: current options and prospects for trait improvement using emerging genetic, genomic, and systems biology toolkits.

For lignocellulosic bioenergy to become a viable alternative to traditional energy production methods, rapid increases in conversion efficiency and biomass yield must be achieved. Increased productivity in bioenergy production can be achieved through concomitant gains in processing efficiency as well as genetic improvement of feedstock that have the potential for bioenergy production at an industrial scale. The purpose of this review is to explore the genetic and genomic resource landscape for the improvement of a specific bioenergy feedstock group, the C4 bioenergy grasses. First, bioenergy grass feedstock traits relevant to biochemical conversion are examined. Then we outline genetic resources available bioenergy grasses for mapping bioenergy traits to DNA markers and genes. This is followed by a discussion of genomic tools and how they can be applied to understanding bioenergy grass feedstock trait genetic mechanisms leading to further improvement opportunities.