* PREMISE OF THE STUDY: In spaceflight experiments, tissues for morphologic study are fixed in 3% glutaraldehyde, while tissues for molecular study are fixed in RNAlater; thus, an experiment containing both study components requires multiple fixation strategies. The possibility of using RNAlater-fixed materials for standard SEM-based morphometric investigation was explored to expand the library of tissues available for analysis and maximize usage of samples returned from spaceflight, but these technologies have wide application to any situation where recovery of biological resources is limited. * METHODS AND RESULTS: RNAlater-fixed samples were desalinated in distilled water, dehydrated through graded methanol, plunged into liquid ethane, and transferred to cryovials for freeze-substitution. Sample tissues were critical point dried, mounted, sputter-coated, and imaged. * CONCLUSIONS: The protocol resulted in acceptable SEM images from RNAlater-fixed Arabidopsis thaliana tissue. The majority of the tissues remained intact, including general morphology and finer details such as root hairs and trichomes.
Research Containing: RNAlater
In plants, sensitive and selective mechanisms have evolved to perceive and respond to light and gravity. We investigated the effects of microgravity on the growth and development of Arabidopsis thaliana (ecotype Landsberg) in a spaceflight experiment. These studies were performed with the Biological Research in Canisters (BRIC) hardware system in the middeck region of the space shuttle during mission STS-131 in April 2010. Seedlings were grown on nutrient agar in Petri dishes in BRIC hardware under dark conditions and then fixed in flight with paraformaldehyde, glutaraldehyde, or RNAlater. Although the long-term objective was to study the role of the actin cytoskeleton in gravity perception, in this article we focus on the analysis of morphology of seedlings that developed in microgravity. While previous spaceflight studies noted deleterious morphological effects due to the accumulation of ethylene gas, no such effects were observed in seedlings grown with the BRIC system. Seed germination was 89% in the spaceflight experiment and 91% in the ground control, and seedlings grew equally well in both conditions. However, roots of space-grown seedlings exhibited a significant difference (compared to the ground controls) in overall growth patterns in that they skewed to one direction. In addition, a greater number of adventitious roots formed from the axis of the hypocotyls in the flight-grown plants. Our hypothesis is that an endogenous response in plants causes the roots to skew and that this default growth response is largely masked by the normal 1 g conditions on Earth.
Ground testing of Arabidopsis preservation protocol for the microarray analysis to be used in the ISS EMCS Multigen-2 experiment
Gene expression analysis using microarrays has proved to be an important method in life science. The opportunity to grow higher plants on the International Space Station (ISS) opens up the possibility for gene expression profiling of plants grown in microgravity. The work presented focuses on how to meet the scientific requirements of plant growth and the sample preservation, given the technical and operational constraints associated with space research. The growth chamber (Multigen-2 Science Testing Unit) and a protocol suggested to be used in the European Modular Cultivation System (EMCS) Multigen-2 experiment on the ISS to grow and later preserve Arabidopsis seedlings, were tested on ground. The results showed that most of the plants developed normally. In order to avoid high population stress the number of seedlings per growth area should be reduced. The RNAlater preservation method to be used in the space experiment was compared with a quick freeze in Liquid Nitrogen (LN2). The RNA from samples preserved in RNAlater at room temperature for 24 h was slightly more degraded than the RNA from the LN2 preserved samples (RNA integrity number, RIN: 7.7 and 8.6, respectively). However, the RNA quality and quantity was satisfactory for microarray analysis. Of the genes analysed, 74 genes (0.28%) were significantly differentially expressed, most of them showing moderate to low regulation. Among the genes induced in the RNAlater preserved samples, three salt inducible transcription factors (ZAT10, SZF1 and SZF2) were identified, suggesting that the high salt concentration in RNAlater causes salt stress before the transcription stopped. In conclusion, the Multigen-2 preservation protocol tested here will allow for the genes regulated by microgravity in the space experiment to be revealed. The results do indicate that not all the biological processes are stopped instantly by the RNAlater. The limited diffusion indirectly caused by the microgravity may potentially result in a different degree of salt stress in the test compared to the 1 × g control during the space experiment. This has to be accounted for during the evaluation of the results. Since slightly degraded RNA was observed, further optimalisation of the preservation protocol will be performed.
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.