Operations of a spaceflight experiment to investigate plant tropisms

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.     

Changes in operational procedures to improve spaceflight experiments in plant biology in the European Modular Cultivation System

The microgravity environment aboard orbiting spacecraft has provided a unique laboratory to explore topics in basic plant biology as well as applied research on the use of plants in bioregenerative life support systems. Our group has utilized the European Modular Cultivation System (EMCS) aboard the International Space Station (ISS) to study plant growth, development, tropisms, and gene expression in a series of spaceflight experiments. The most current project performed on the ISS was termed Seedling Growth-1 (SG-1) which builds on the previous TROPI (for tropisms) experiments performed in 2006 and 2010. Major technical and operational changes in SG-1 (launched in March 2013) compared to the TROPI experiments include: (1) improvements in lighting conditions within the EMCS to optimize the environment for phototropism studies, (2) the use of infrared illumination to provide high-quality images of the seedlings, (3) modifications in procedures used in flight to improve the focus and overall quality of the images, and (4) changes in the atmospheric conditions in the EMCS incubator. In SG-1, a novel red-light-based phototropism in roots and hypocotyls of seedlings that was noted in TROPI was confirmed and now can be more precisely characterized based on the improvements in procedures. The lessons learned from sequential experiments in the TROPI hardware provide insights to other researchers developing space experiments in plant biology.     

Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function.     

Changes in plant medium composition after a spaceflight experiment: Potassium levels are of special interest

We present results on the analysis of 100 mL medium samples extracted from sterilized foam (Smithers-Oasis, Kent OH) used to support the growth of a representative dicotyledon (Haplopappus gracilis) and a representative monocotyledon (Hemerocallis cv Autumn Blaze) in NASA’s Plant Growth Unit (PGU) during a 5-day Space Shuttle flight and ground experiments. At recovery, the media remaining within replicate (n = 5) foam blocks (for both the spaceflight and ground experiments) were extracted under vacuum, filtered and subjected to elemental analyses. A unique aspect of this experiment was that all plants were either aseptically-generated tissue culture propagated plantlets or aseptic seedling clones. The design of the PGU facilitated the maintenance of asepsis throughout the mission (confirmed by post-flight microbial sampling) and thus any possible impact of microorganisms on medium composition was eliminated. Concentration levels of some elements remained the same, while some decreased and others increased. There was a significant two-fold difference between the final concentrations of potassium when the Earth-based and microgravity experiments were contrasted.     

3D information from 2D images recorded in the European Modular Cultivation System on the ISS

The European Modular Cultivation System (EMCS) on the ISS allows long-term biological experiments, e.g. on plants. Video cameras provide near real-time 2D images from these experiments. A method to obtain 3D coordinates and stereoscopic images from these 2D images has been developed and is described in this paper. The procedure was developed to enhance the data output of the MULTIGEN-1 experiment in 2007. One of the main objectives of the experiment was to study growth movements of the Arabidopsis plants and the effect of gravity on these. 3D data were important during parts of the experiment and the paper presents the method developed to acquire 3D data, the accuracy of the data, limitations to the technique and ways to improve the accuracy. Sequences of 3D data obtained from the MULTIGEN-1 experiment are used to illustrate the potential of this newfound capability of the EMCS. In the experiment setup, a positional depth accuracy of about ±0.4 mm for relative object distances and an absolute depth accuracy of about ±1.4 mm for time dependent phenomena was reached. The ability to both view biological specimens in 3D as well as obtaining quantitative 3D data added greatly to the scientific output of the MULTIGEN-1 experiment. The uses of the technique to other researchers and their experiments are discussed.     

Potential of salt-accumulating and salt-secreting halophytic plants for recycling sodium chloride in human urine in bioregenerative life support systems

This study addresses the possibility of growing different halophytic plants on mineralized human urine as a way to recycle NaCl from human wastes in a bioregenerative life support system (BLSS). Two halophytic plant species were studied: the salt-accumulating Salicornia europaea and the salt-secreting Limonium gmelinii. During the first two weeks, plants were grown on Knop’s solution, then an average daily amount of urine produced by one human, which had been preliminarily mineralized, was gradually added to the experimental solutions. Nutrient solutions simulating urine mineral composition were gradually added to control solutions. NaCl concentrations in the stock solutions added to the experimental and control solutions were 9 g/L in the first treatment and 20 g/L in the second treatment. The mineralized human urine showed some inhibitory effects on S. europaea and L. gmelinii. The biomass yield of experimental plants was lower than that of control ones. If calculated for the same time period (120 d) and area (1 m²), the amount of sodium chloride taken up by S. europaea plants would be 11.7 times larger than the amount taken up by L. gmelinii plants (486 g/m² vs. 41 g/m²). Thus, S. europaea is the better choice of halophyte for recycling sodium chloride from human wastes in BLSS.     

Effects of spaceflight (STS-87) on tropisms and plastid positioning in protonemata of the moss Ceratodon purpureus.

Apical cells of moss protonemata represent a single-celled system that perceives and reacts to light (positive and negative phototropism) and to gravity (negative gravitropism). Phototropism completely overrides gravitropism when apical cells are laterally irradiated with relatively high red light intensities, but below a defined light intensity threshold gravitropism competes with the phototropic reaction. A 16 day-long exposure to microgravity conditions demonstrated that gravitropism is allowed when protonemata are laterally illuminated with light intensities below 140 nmol m-2s-1. Protonemata that were grown in darkness in microgravity expressed an endogenous tendency to grow in arcs so that the overall culture morphology resembled a clockwise spiral. However this phenomenon only was observed in cultures that had reached a critical age and/or size. Organelle positioning in dark-grown apical cells was significantly altered in microgravity. Gravisensing most likely involves the sedimentation of starch-filled amyloplasts in a well-defined area of the tip cell. Amyloplasts that at 1-g are sedimented were clustered at the apical part of the sedimentation zone in microgravity. Clustering observed in microgravity or during clino-rotation significantly differs from sedimentation-induced plastid aggregations after inversion of tip cells at 1-g.     

Blue light-dependent phosphorylations of cryptochromes are affected by magnetic fields in Arabidopsis

The blue light receptor cryptochrome that could form radical pairs after exposure to blue light was suggested to be a magnetoreceptor based on the proposition that radical pairs were involved in the magnetoreception. But the effects of magnetic fields on the function of cryptochrome are poorly understood. Phosphorylation of cryptochrome in Arabidopsis was closely associated with the function of this photoreceptor. Here, we grew Arabidopsis seedlings in a 500 μT magnetic field and a near-null magnetic field and found that the 500 μT magnetic field enhanced the blue light-dependent phosphorylations of CRY1 and CRY2, and the near-null magnetic field weakened the blue light-dependent phosphorylation of CRY2 but not CRY1. Dephosphorylations of CRY1 and CRY2 in the darkness were slowed down in the 500 μT magnetic field, whereas dephosphorylations of CRY1 and CRY2 were accelerated in the near-null magnetic field. These results suggest that magnetic field with strength higher or weaker than the local geomagnetic field affects the activated states of cryptochromes, which thus modifies the functions of cryptochromes.     

Comparing IRI and a regional model with ionosonde measurements in Pakistan

We have used the technique of expansion in Empirical Orthogonal Functions (EOFs) to develop regional models of the critical frequencies of E and F₂ layers (f_oE, f_oF₂), peak height (h_mF₂), and semi-thickness of F₂ layer (Y_mF₂) over Pakistan. In the present study levels of solar activity specified by Smoothed Sunspot Number (R) from 10 to 200 are taken into account. The magnetic dip angle for the model ranges from 30° to 60°. We have compared the regional model and the International Reference Ionosphere (IRI) with measurements of three ionosondes in Pakistan. The model parameters f_oE and f_oF₂ are found overall comparable to the observed hourly median values during daytime at Karachi (geographic latitude = 24.95°N, longitude = 67.13°E, magnetic inclination = 37°), Multan (30.18°N, 71.48°E, 45°) and Islamabad (33.75°N, 73.13°E, 51.5°) during the years 1988, 1996 and 2000. For h_mF₂ the computed values by regional and IRI model for the year 1995 are found close to each other. However, for Y_mF₂the results are better during daytime as compared to nighttime.     

Survival and death of the haloarchaeon Natronorubrum strain HG-1 in a simulated martian environment

Halophilic archaea are of interest to astrobiology due to their survival capabilities in desiccated and high salt environments. The detection of remnants of salty pools on Mars stimulated investigations into the response of haloarchaea to martian conditions. Natronorubrum sp. strain HG-1 is an extremely halophilic archaeon with unusual metabolic pathways, growing on acetate and stimulated by tetrathionate. We exposed Natronorubrum strain HG-1 to ultraviolet (UV) radiation, similar to levels currently prevalent on Mars. In addition, the effects of low temperature (4, −20, and −80 °C), desiccation, and exposure to a Mars soil analogue from the Atacama desert on the viability of Natronorubrum strain HG-1 cultures were investigated. The results show that Natronorubrum strain HG-1 cannot survive for more than several hours when exposed to UV radiation equivalent to that at the martian equator. Even when protected from UV radiation, viability is impaired by a combination of desiccation and low temperature. Desiccating Natronorubrum strain HG-1 cells when mixed with a Mars soil analogue impaired growth of the culture to below the detection limit. Overall, we conclude that Natronorubrum strain HG-1 cannot survive the environment currently present on Mars. Since other halophilic microorganisms were reported to survive simulated martian conditions, our results imply that survival capabilities are not necessarily shared between phylogenetically related species.     

Molecular analysis of the graviperception signal transduction in the flagellate Euglena gracilis: Involvement of a transient receptor potential-like channel and a calmodulin

Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavior responses. The organism shows pronounced negative gravitaxis. This movement is based on physiological mechanisms, which in the past had been only indirectly assessed. It was shown that mechano-sensitive calcium channels are involved in the gravitaxis response. Recent studies have demonstrated that members of the transient receptor potential (TRP) family function as mechano-sensitive channels in several different cell types. We have sequenced part of a TRP gene in Euglena and applied RNA interference (RNAi) to confirm that these channels are involved in graviperception. It was found that RNAi against the putative TRP channel abolished gravitaxis. The genes of three calmodulins were sequences in Euglena, one of which was previously known in its protein structure (cal 1). The other two were unknown (cal 2 and cal 3). Cal 2 has been analyzed in detail. The biosynthesis of the corresponding proteins of cal 1 and cal 2 was inhibited by means of RNA interference to see whether this blockage impairs gravitaxis. RNAi of cal 1 leads to a long-term loss of free swimming in the cells (while euglenoid movement persists). It induced pronounced cell form aberrations and the division of cells was hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Thus cal 1 does not seem to be involved in gravitaxis. In contrast, the blockage of cal 2 has no pronounced influence on motility and cell form but leads to a complete loss of gravitactic orientation for more than 30 days showing that this calmodulin is an element in the signal transduction chain. The data are discussed in the context of the current model of the gravitaxis signal transduction chain in Euglena gracilis.     

The effect of geocenter motion on Jason-2 orbits and the mean sea level

We compute a series of Jason-2 GPS and SLR/DORIS-based orbits using ITRF2005 and the std0905 standards ( Lemoine et al., 2010). Our GPS and SLR/DORIS orbit data sets span a period of 2 years from cycle 3 (July 2008) to cycle 74 (July 2010). We extract the Jason-2 orbit frame translational parameters per cycle by the means of a Helmert transformation between a set of reference orbits and a set of test orbits. We compare the annual terms of these time-series to the annual terms of two different geocenter motion models where biases and trends have been removed. Subsequently, we include the annual terms of the modeled geocenter motion as a degree-1 loading displacement correction to the GPS and SLR/DORIS tracking network of the POD process. Although the annual geocenter motion correction would reflect a stationary signal in time, under ideal conditions, the whole geocenter motion is a non-stationary process that includes secular trends. Our results suggest that our GSFC Jason-2 GPS-based orbits are closely tied to the center of mass (CM) of the Earth consistent with our current force modeling, whereas GSFC’s SLR/DORIS-based orbits are tied to the origin of ITRF2005, which is the center of figure (CF) for sub-secular scales. We quantify the GPS and SLR/DORIS orbit centering and how this impacts the orbit radial error over the globe, which is assimilated into mean sea level (MSL) error, from the omission of the annual term of the geocenter correction. We find that for the SLR/DORIS std0905 orbits, currently used by the oceanographic community, only the negligence of the annual term of the geocenter motion correction results in a – 4.67 ± 3.40 mm error in the Z-component of the orbit frame which creates 1.06 ± 2.66 mm of systematic error in the MSL estimates, mainly due to the uneven distribution of the oceans between the North and South hemisphere.