Plants will be an important component in bioregenerative systems for long-term missions to the Moon and Mars. Since gravity is reduced both on the Moon and Mars, studies that identify the basic mechanisms of plant growth and development in altered gravity are required to ensure successful plant production on these space colonization missions. To address these issues, we have developed a project on the International Space Station (ISS) to study the interaction between gravitropism and phototropism in Arabidopsis thaliana. These experiments were termed TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS) in 2006. In this paper, we provide an operational summary of TROPI and preliminary results on studies of tropistic curvature of seedlings grown in space. Seed germination in TROPI was lower compared to previous space experiments, and this was likely due to extended storage in hardware for up to 8 months. Video downlinks provided an important quality check on the automated experimental time line that also was monitored with telemetry. Good quality images of seedlings were obtained, but the use of analog video tapes resulted in delays in image processing and analysis procedures. Seedlings that germinated exhibited robust phototropic curvature. Frozen plant samples were returned on three space shuttle missions, and improvements in cold stowage and handing procedures in the second and third missions resulted in quality RNA extracted from the seedlings that was used in subsequent microarray analyses. While the TROPI experiment had technical and logistical difficulties, most of the procedures worked well due to refinement during the project.
Research Containing: Arabidopsis thaliana
In order to effectively study phototropism, the directed growth in response to light, we performed a series of experiments in microgravity to better understand light response without the “complications” of a 1-g stimulus. These experiments were named TROPI (for tropisms) and were performed on the European Modular Cultivation System (EMCS), a laboratory facility on the International Space Station (ISS). TROPI-1 was performed in 2006, and while it was a successful experiment, there were a number of technical difficulties. We had the opportunity to perform TROPI-2 in 2010 and were able to optimize experimental conditions as well as to extend the studies of phototropism to fractional gravity created by the EMCS centrifuge. This paper focuses on how the technical improvements in TROPI-2 allowed for a better experiment with increased scientific return. Major modifications in TROPI-2 compared to TROPI-1 included the use of spaceflight hardware that was off-gassed for a longer period and reduced seed storage (less than 2 months) in hardware. These changes resulted in increased seed germination and more vigorous growth of seedlings. While phototropism in response to red illumination was observed in hypocotyls of seedlings grown in microgravity during TROPI-1, there was a greater magnitude of red-light-based phototropic curvature in TROPI-2. Direct downlinking of digital images from the ISS in TROPI-2, rather than the use of analog tapes in TROPI-1, resulted in better quality images and simplified data analyses. In TROPI-2, improved cryo-procedures and the use of the GLACIER freezer during transport of samples back to Earth maintained the low temperature necessary to obtain good-quality RNA required for use in gene profiling studies.
The European Modular Cultivation System (EMCS) installed within the US laboratory module, Destiny, and/or the European experiment module, Columbus, onboard the International Space Station (ISS), is an ESA facility available for plant research and biological experiments. The EMCS facility uses standard experiment containers (ECs) mounted on centrifuges and provides life support such as water and gas supply systems as well as observation systems. The experiment-specific hardware such as the plant cultivation chamber, root phototropism observation chamber, and plant root gravitropism observation chamber is integrated into the EC. JAXA has five themes concerning space plant research, of which two-Cell Wall and Resist Wall-will include conducting space experiments using the EMCS facility; according to the present shuttle flight schedule, they are due to be launched in mid February 2007. The objectives of the Cell Wall / Resist Wall experiment include in-orbit growth of 10-cm-long inflorescence stems of Arabidopsis and subsequent, post-flight morphology, biological, gene expression, and cell-wall properties analyses on the ground. In this article, we describe the EMCS facility, the plant cultivation and onboard chemical fixation system. Furthermore, we also discuss the verification experiments conducted by JAXA.
Growth and Cell Wall Properties in Hypocotyls of Arabidopsis tua6 Mutant under Microgravity Conditions in Space
Seedlings of Arabidopsis α-tubulin 6 mutant (tua6) were cultivated under microgravity conditions in the European Modular Cultivation System on the International Space Station, and growth and cell wall properties of their hypocotyls were analyzed (the Resist Wall experiment). Seeds of tua6 mutant were shown to germinate and grow normally until the seedling stage under microgravity conditions, as at 1 G on the ground. The seedlings were naturally air-dried in orbit, which were then recovered and transported to earth. When the mechanical properties of the cell wall of rehydrated hypocotyls were examined with a tensile tester, the hypocotyls showed typical stress-strain and stress-relaxation curves, as normally fixed or frozen materials. Also, no prominent differences were detected in the extensibility or the stress-relaxation parameters of the cell wall between space-grown hypocotyls and ground controls, suggesting that tua6 hypocotyls formed the regular cell wall architecture under microgravity conditions. The results and lessons learned from the Resist Wall experiment are expected to provide the basis for the following space experiments to clarify the mechanism of gravity resistance in plants.
Life in spaceflight demonstrates remarkable acclimation processes within the specialized habitats of vehicles subjected to the myriad of unique environmental issues associated with orbital trajectories. To examine the response processes that occur in plants in space, leaves and roots from Arabidopsis (Arabidopsis thaliana) seedlings from three GFP reporter lines 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 by using iTRAQ broad-scale proteomics procedures. Using stringent criteria, we identified over 1500 proteins, which included 1167 leaf proteins and 1150 root proteins we were able to accurately quantify. Quantification revealed 256 leaf proteins and 358 root proteins that showed statistically significant differential abundance in the spaceflight samples compared to ground controls, with few proteins differentially regulated in common between leaves and roots. This indicates that there are measurable proteomics responses to spaceflight and that the responses are organ-specific. These proteomics data were compared with transcriptome data from similar spaceflight samples, showing that there is a positive but limited relationship between transcriptome and proteome regulation of the overall spaceflight responses of plants. These results are discussed in terms of emergence understanding of plant responses to spaceflight particularly with regard to cell wall remodeling, as well as in the context of deriving multiple omics data sets from a single on-orbit preservation and operations approach.
MAIN CONCLUSION: Cyclic leaf ascent and descent occur in synchrony and phase congruence with the lunisolar tidal force under a broad range of conditions. Digitized records of the vertical leaf movements of Arabidopsis thaliana were collected under space flight conditions in the International Space Station (ISS). Oscillations of leaf movements with periods of 45 and 90 min were found under light-adapted conditions, whereas in darkness, the periods were 45, 90, and 135 min. To demonstrate the close relationship between these oscillations and cyclical variations of the lunisolar gravitational force, we estimated the oscillations of the in-orbit lunisolar tide as they apply to the ISS, with the aid of the Etide software application. In general, in-orbit lunisolar gravitational profiles exhibited a periodicity of 45 min. Alignment of these in-orbit oscillations with the oscillations of Arabidopsis leaf movement revealed high degrees of synchrony and a congruence of phase. These data corroborate previous results which suggested a correlative relationship and a possible causal link between leaf movement rhythms obtained on ground and the rhythmic variation of the lunisolar tidal force.
The present suite of advanced space plant cultivation facilities require a significant level of resources to launch and maintain in flight. The facilities are designed to accommodate a broad size range of plant species and are, therefore, not configured to support the specific growth requirements of small plant species such as Arabidopsis thaliana at maximum efficiency with respect to mass and power. The facilities are equally not configured to support automated plant harvesting or tissue processing procedures, but rely on crew intervention and time. The recent reorganization of both spaceflight opportunities and allocation of limited in-flight resources demand that experiments be conducted with optimal efficiency. The emergence of A. thaliana as a dominant space flight model organism utilized in research on vegetative and reproductive phase biology provides strong justification for the establishment of a dedicated cultivation system for this species. This paper presents work on the design of a small plant cultivation facility directed at supporting research on the vegetative growth phase of A. thaliana . The design of the facility is based on the use of existing space flight hardware, and configured to support the fully automated germination of seed, cultivation of plants, and final termination of plant growth by chemical fixation and preservation of plant tissue.
Constraints in both launch opportunities and the availability of in-flight resources for Shuttle and Space Station life science habitat facilities has presented a compelling impetus to improve the operational flexibility, efficiency and miniaturization of many of these systems. Such advances would not only invigorate the level of research being conducted in low Earth orbit but also present the opportunity to expand life science studies to outer space and planetary bodies. Work has been directed towards the development of a miniature plant cultivation module (PCM) capable of supporting the automated and controlled growth and spectral monitoring of small plant species such as Arabidopsis thaliana. This paper will present data on the operational performance and efficiency of the cultivation module, and the extent to which such a system may be used to support plant growth studies in low Earth orbit and beyond.
Gravity control of growth form in Brassica rapa and Arabidopsis thaliana (Brassicaceae): Consequences for secondary metabolism
How gravity influences the growth form and flavor components of plants is of interest to the space program because plants could be used for food and life support during prolonged missions away from the planet, where that constant feature of Earth's environment does not prevail. We used plant growth hardware from prior experiments on the space shuttle to grow Brassica rapa and Arabidopsis thaliana plants during 16-d or 11-d hypergravity treatments on large-diameter centrifuge rotors. Both species showed radical changes in growth form, becoming more prostrate with increasing g-loads (2-g and 4-g). In Brassica, height decreased and stems thickened in a linear relationship with increasing g-load. Glucosinolates, secondary compounds that contribute flavor to Brassica, decreased by 140% over the range of micro to 4-g, while the structural secondary compound, lignin, remained constant at approximately 15% (w/w) cell wall dry mass. Stem thickening at 4-g was associated with substantial increases in cell size (47%, 226%, and 33% for pith, cortex, and vascular tissue), rather than any change in cell number. The results, which demonstrate the profound effect of gravity on plant growth form and secondary metabolism, are discussed in the context of similar thigmostresses such as touch and wind.