On Earth plants are constantly exposed to a gravitational field of 1G. Gravity affects a plant in every step of its development. Germinating seedlings orient their radicle and hypocotyl and growing plants position organs at a specific Gravitropic Set-point Angle dictated by the asymmetric distribution of auxin depending on the gravity vector. Hence gravitropism is one of the fundamental growth responses in plants. For any experiment studying the effects of gravity on plants the ultimate control is the microgravity in space. In this study Arabidopsis seeds were flown to the International Space Station and allowed to germinate and grow for 3 days in microgravity. Arabidopsis Wild Type Col-0 seeds were plated onto twenty-two 60mm Petri plates loaded into PDFUs and inserted 4 Biological Research in Canisters (BRICs). Approximately 800 seeds were sterilized plated on each 60mm Petri plates and cold stratified for 16 hours followed by 2 hours of white light treatment. The BRICs were maintained at 4C until spaceflight to ensure seed germination in microgravity. After 3 days of germination and growth the seedlings were fixed by injecting RNAlater into the chamber. They were kept at ambient temperature for 12 hours followed by freezing at -80C. An additional 22 plates were used as ground controls. After the spaceflight tissue from five plates was pooled to make each of three replicates. Both membrane and soluble proteins were extracted from the pooled seedlings. Proteins were trypsin digested labelled with iTRAQ and identified using tandem mass spectrometry.

This study examined tissue-specific early transcriptional responses upon reorientation in wild type and starchless pgm-1 mutant seedlings of Arabidopsis. Seedlings were grown in vertical Petri dishes for four days. Treatment samples were reoriented 90 degrees for ten minutes while control plants remained vertical. All seedlings were flash frozen in liquid nitrogen and RNA was extracted from root tips, mature root, hypocotyl, and cotyledon fractions of the seedlings. RNA was sequenced via NovaSeq.

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.

Understanding the molecular mechanisms by which plants sense and adapt to changes in the space environment is essential for generating plants that are better adapted to withstand space flight, microgravity, and other adverse conditions encountered in space. The objective of our spaceflight experiment “Plant Signaling in Microgravity” (carried out on the International Space Station, ISS), was to compare transcript profiles of wild type and transgenic InsP 5-ptase plants with compromised InsP3 signaling. The transgenic Arabidopsis plants constitutively express the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes the lipid-derived second messenger inositol 1,4,5-trisphosphate (InsP3). These transgenic plants exhibit normal growth and morphology; however, their responses to environmental stimuli including gravity and drought are altered. Seedlings were grown for 5 days under continuous light in experimental containers placed in the European Modular Cultivation system (EMCS) onboard the ISS. The EMCS consists of two rotors within a controlled chamber, allowing for a “1g” control in space. After sample retrieval from the ISS, RNA was isolated from shoot and root tissue and subjected to RNA sequencing. Two-way comparisons of micro g versus “1”g have uncovered regulatory mechanisms that are both conserved and altered between the wild type and transgenic seedlings.

This study investigated plant responses to a range of gravity treatments. Altered gravity was achieved by growing seedlings on the ISS within the centrifuge of the European Modular Cultivation System (EMCS) and applying a specific rotation rate for a fixed time in the experiment. Seeds of wild-type (Col-0) and starchless (pgm-1) were used to determine the contribution of statoliths to gravity perception. Plants experienced an estimated 0.003 up to 1.0 g depending on the specific experimental run and position of the seed cassette on the centrifuge arm. Images were captured at various points during the experiment and at the conclusion of the 5-day growth period.

Physical forces greatly influence the growth and function of an organism. Altered gravity can perturb normal development and induce corresponding changes in gene expression. Understanding this relationship between the physical and biological realms is important for NASA's space travel goals. We use combined RNA-Seq and qRT-PCR to profile changes in early Drosophila melanogaster pupae exposed to chronic hypergravity (3 g, three times Earth's gravity) to highlight gravity-dependent pathways and gene products. Robust transcriptional response was evident among the pupae developed in a hypergravity environment compared to control. 1,513 genes showed significantly (p less than 0.05) altered gene expression in the 3 g samples. These findings were supported with qRT-PCR data. Major biological processes affected include ion transport, redox homeostasis, immune and humoral stress response, proteolysis, and cuticle development.

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. 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 hours. 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.

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

To reveal outcomes of microgravity on molecular processes within the cellular environment we have employed a mass-spectrometry based proteomics approach. Proteomics analysis based on mass spectrometry allows for the relative quantitation of a large number of proteins concurrently, and in a relatively unbiased manner. Mass spectrometry based proteomics can be rendered even more informative by addition of a labeling component to understand the dynamics of the changing protein content. In this study, we utilized a combination of proteomics techniques, namely label-free quantification and dynamic stable-isotope labeling by amino acids in cell culture (Dynamic SILAC) to characterize the microgravity stress response in primary cardiomyocytes.

These investigations studied the fundamentals of how plants perceive gravity and develop in microgravity. It informs how gene regulation is altered by spaceflight conditions. We noted expression changes in genes involved in hypoxia and heat shock responses, DNA repair, and cell wall structure between spaceflight samples compared to the ground controls. In addition, glycome profiling supported our expression analyses in that there was a difference in cell wall components between ground control and spaceflight-grown plants. Comparing our studies to those of the other BRIC-16 experiments demonstrated that, even with the same hardware and similar biological materials, differences in results in gene expression were found among these spaceflight experiments.