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.

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.

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.

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.

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 x93Plant Signaling in Microgravity x94 (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 x931g x94 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 x931 x94g have uncovered regulatory mechanisms that are both conserved and altered between the wild type and transgenic seedlings.

Life in spaceflight demonstrates remarkable adaptive processes within the specialized environments of space vehicles which are subject to the myriad of attending and unique environmental issues associated with orbital trajectories. To examine the adaptive processes that occur in plants in space leaves and roots from Arabidopsis seedlings that were grown from seed for 12 days on the International Space Station and preserved on orbit in RNAlater were returned to earth and analyzed using iTRAQ broad scale proteomics procedures.

Life in spaceflight demonstrates remarkable adaptive processes within the specialized environments of space vehicles which are subject to the myriad of attending and unique environmental issues associated with orbital trajectories. To examine the adaptive processes that occur in plants in space leaves and roots from Arabidopsis seedlings that were grown from seed for 12 days on the International Space Station and preserved on orbit in RNAlater were returned to earth and analyzed using iTRAQ broad scale proteomics procedures.

The project focuses on understanding the transcriptional and post-transcriptional mechanisms that regulate early seedling development in spaceflight and microgravity. One of the goals of the PRR experiment was to study the role of small regulatory RNAs in plant response to the space environment using Arabidopsis thaliana.

We address a key baseline question of whether gene expression changes are induced by the orbital environment, and then we ask whether undifferentiated cells, cells presumably lacking the typical gravity response mechanisms, perceive spaceflight. Arabidopsis seedlings and undifferentiated cultured Arabidopsis cells were launched in April, 2010, as part of the BRIC-16 flight experiment on STS-131. Biologically replicated DNA microarray and averaged RNA digital transcript profiling revealed several hundred genes in seedlings and cell cultures that were significantly affected by launch and spaceflight. The response was moderate in seedlings; only a few genes were induced by more than 7-fold, and the overall intrinsic expression level for most differentially expressed genes was low. In contrast, cell cultures displayed a more dramatic response, with dozens of genes showing this level of differential expression, a list comprised primarily of heat shock-related and stress-related genes. This baseline transcriptome profiling of seedlings and cultured cells confirms the fundamental hypothesis that survival of the spaceflight environment requires adaptive changes that are both governed and displayed by alterations in gene expression. The comparison of intact plants with cultures of undifferentiated cells confirms a second hypothesis: undifferentiated cells can detect spaceflight in the absence of specialized tissue or organized developmental structures known to detect gravity.

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.