Adaptation of a 2-D Clinostat for Simulated Microgravity Experiments with Adherent Cells

The fast-rotating 2-D clinostat, a ground-based facility for investigations in simulated microgravity, is mainly used for experiments with cell suspensions. Here, we describe the adaptation of a 2-D clinostat for adherent cell investigations using commercially available slide flasks. As a gradient of residual accelerations is present in the slide flasks during clinorotation, the range of maximal g-values has to be adjusted to the investigated cells and type of analysis. For gene expression analysis, a harvesting slide chamber was constructed, allowing collection of cells exposed to defined g-values. Using this slide chamber, human 1F6 melanoma cell line, exposed in the ranges of ≤0.012 g, ≤0.024 g, or ≤0.036 g for 24 h, was harvested and the respective mRNA levels of guanylyl cyclase A (GC-A), an enzyme catalyzing cyclic GMP synthesis, were determined by real-time quantitative PCR analysis. Our results show that the down-regulation of GC-A mRNA levels in 1F6 melanoma cells depends on the residual acceleration values with a maximal reduction at ≤0.012 g. We further used the slide flasks by the clinorotation of murine RAW 264.7 macrophage cell line for f-actin analysis. The laser scanning microscopy images of cells exposed to g-values of ≤0.006 g for 1 h show an increase in the cell size of clinorotated cells, but no rearrangement in the f-actin filament system compared to static 1-g controls. Thus, 2-D clinostats equipped with slide flasks can be used for adherent cell experiments, however, the maximal g-values have to be carefully considered.     

Effect of Hypergravity on Endothelial Cell Function and Gene Expression

It is well known that endothelial cells (ECs), which play a major role in cardiovascular system functioning, are very sensitive to mechanical stimuli. It has been demonstrated that changes in inertial conditions (i.e. microgravity and hypergravity) can affect both phenotypic and genotypic expression in ECs. In this report we describe the effects of hypergravity on ECs isolated from bovine aorta (BAECs). ECs were repeatedly exposed to discontinuous hypergravity conditions (5 × 10 min at 10×g with 10 min at 1×g between sets), simulated in a hyperfuge. Then, cell morphology and metabolism were analyzed by autofluorescence techniques. The phenotypic expression of cytoskeleton constituents (β-actin, vimentin, tubulin), adhesion and survival signals (integrins), mediators of inflammation and angiogenesis was evaluated by immunocytofluorescence. Quantitative PCR (Q-PCR) with Low Density Arrays (LDAs) was used to evaluate modifications in gene expression. After hypergravity exposure, no significant changes were observed in cell morphology and energy metabolism. Cells remained adherent to the substratum, but integrin distribution was modified. Accordingly, the cytoskeletal network reorganized, documenting cell activation. There was a reduction in expression of genes controlling vasoconstriction and inflammation. Proapoptotic signals were downregulated. On the whole, the results documented that hypergravity exposure maintained EC survival and function by activation of adaptive mechanisms.     

The Impact of Hypergravity and Vibration on Gene and Protein Expression of Thyroid Cells

Experiments in space either on orbital missions on-board the ISS, or in suborbital missions using sounding rockets, like TEXUS as well as parabolic flight campaigns are still the gold standard to achieve real microgravity conditions in the field of gravitational biology and medicine. However, during launch, and in flight, hypergravity and vibrations occur which might interfere with the effects of microgravity. It is therefore important to know these effects and discriminate them from the microgravity effects. This can be achieved by ground-based facilities like centrifuges or vibration platforms. Recently, we have conducted several experiments with different thyroid cancer cell lines. This study, as part of the ESA-CORA-GBF 2010-203 project, focused on the influence of vibration and hypergravity on benign human thyroid follicular epithelial cells (Nthy-ori 3-1 cell line). Gene and in part protein expression regulation under both conditions were analyzed for VCAN, ITGA10, ITGB1, OPN, ADAM19, ANXA1, TNFA, ABL2, ACTB, PFN2, TLN1, EZR, RDX, MSN, CTGF, PRKCA, and PRKAA1 using quantitative real-time PCR and Western Blot. We found that hypergravity and vibration affected genes and proteins involved in the extracellular matrix, the cytoskeleton, apoptosis, cell growth and signaling. Vibration always led to a down-regulation, whereas hypergravity resulted in a more heterogeneous expression pattern. Overall we conclude that both conditions can influence gene regulation and production of various genes and proteins. As a consequence, it is important to perform control experiments on hypergravity and vibration facilities in parallel to flight experiments.     

From hypergravity to microgravity: Choosing the suitable simulator

Cardiovascular system functions are impaired in altered gravity conditions. In particular endothelial cells play a major role being responsible for the integrity of the vascular wall. Due to obvious difficulties in performing continuous and exhaustive experiments in space, most of the available data have been obtained so far using various simulators of hypergravity and microgravity (µg) conditions. The consistency of the data resides on the reliability of the simulator, being a critical point in the development of the research. We exposed the cell cultures to 1) hypergravity (launch condition) using MidiCAR at Dutch Experiment Support Center (DESC, NL); 2) simulated µg using the Rotating Wall Vessel (RWV) and the Random Positioning Machine (RPM). We used two different cellular models: human umbilical vein endothelial cells (HUVEC) and human leukocytes (U937). Only few experiments on cells using RPM have been reported. To assess the RPM best operative parameters we considered data from experiments in space on U937 as reference standard. Differently, cultures in modelled µg using RWV have been extensively reported. Our data on HUVEC indicate that the two µg simulators provide analogous results in terms of proliferation and cytoskeletal organization. Finally, to investigate the effects of spaceflight on different human cells, we developed a spaceflight-like protocol consisting of an initial hypergravity phase (launch), followed by a µg simulation (orbital flight). Using this protocol, results show that hypergravity limits in our models the effects on proliferation induced by modelled µg.     

Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) Hemocytes

Microgravity induces alterations in the functioning of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux-B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reactive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1g operation mode of the clinostat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this purpose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simulated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation) exposed to simulated microgravity (μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels measured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity during parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the centrifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground-based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.     

CECILIA, a versatile research tool for cellular responses to gravity

We describe a centrifuge designed and constructed according to current demands for a versatile instrument in cellular gravitational research, in particular protists (ciliates, flagellates). The instrument (called CECILIA,centrifuge forciliates) is suited for videomonitoring, videorecording, and quantitative evaluation of data from large numbers of swimming cells in a ground-based laboratory or in a drop tower/drop shaft under microgravity conditions. The horizontal rotating platform holds up to six 8mm-camcorders and six chambers holding the experimental cells. Under hypergravity conditions (up to 15 g) chambers can be rotated about 2 axes to adjust the swimming space at right angles or parallel to the resulting gravity vector. Evaluations of cellular responses to central acceleration — in the presence of gravitational 1 g — are used for extrapolation of cellular behaviour under hypogravity conditions. CECILIA is operated and monitored by computer using a custom-made soft-ware. Times and slopes of rising and decreasing acceleration, values and quality of steady acceleration are supervised online. CECILIA can serve as an on-ground research instrument for precursor investigations of the behaviour of ciliates and flagellates under microgravity conditions such as long-term experiments in the International Space Station.     

CECILIA,a versatile research tool for cellular responses to gravity

We describe a centrifuge designed and constructed according to current demands for a versatile instrument in cellular gravitational research, in particular protists (ciliates, flagellates). The instrument (called CECILIA,centrifuge forciliates) is suited for videomonitoring, videorecording, and quantitative evaluation of data from large numbers of swimming cells in a ground-based laboratory or in a drop tower/drop shaft under microgravity conditions. The horizontal rotating platform holds up to six 8mm-camcorders and six chambers holding the experimental cells. Under hypergravity conditions (up to 15 g) chambers can be rotated about 2 axes to adjust the swimming space at right angles or parallel to the resulting gravity vector. Evaluations of cellular responses to central acceleration — in the presence of gravitational 1 g — are used for extrapolation of cellular behaviour under hypogravity conditions. CECILIA is operated and monitored by computer using a custom-made soft-ware. Times and slopes of rising and decreasing acceleration, values and quality of steady acceleration are supervised online. CECILIA can serve as an on-ground research instrument for precursor investigations of the behaviour of ciliates and flagellates under microgravity conditions such as long-term experiments in the International Space Station.     

Simulating Parabolic Flight like <Emphasis Type="Italic">g</Emphasis>-Profiles on Ground - A Combination of Centrifuge and Clinostat

Clinostats and centrifuges are widely used to create simulated microgravity or hypergravity, respectively, in order to study the impact of gravity on biosystems. Here, we used a clinostat and a centrifuge in alternating modes of operation in order to create a simulated parabolic flight like g-profile. To our knowledge, it is the first time that both devices were run in connection. In order to test the method, we investigated the production of reactive oxygen species of immune cells (macrophages) during oxidative burst in an on-line kinetic approach, which has been extensively studied under real (parabolic flight) and simulated microgravity (clinostat) as well as under hypergravity conditions (centrifuge). Our results indicate that clinostat and centrifuge can be operated in an alternating way to simulate the repetitive changes of gravity during parabolic flight. Although the switch from one gravity level to the other could not be carried out as quickly as it takes place during actual parabolic flight due to technical and operational reasons, it can be concluded that running experiments in a clinostat aboard a centrifuge on ground are suitable for studying gravity-related phenomena.     

A Hypergravity Environment Induced by Centrifugation Alters Plant Cell Proliferation and Growth in an Opposite Way to Microgravity

Seeds of Arabidopsis thaliana were exposed to hypergravity environments (2g and 6g) and germinated during centrifugation. Seedlings grew for 2 and 4 days before fixation. In all cases, comparisons were performed against an internal (subjected to rotational vibrations and other factors of the machine) and an external control at 1g. On seedlings grown in hypergravity the total length and the root length were measured. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation, which were quantified by counting the number of cells per millimeter in the specific cell files, and cell growth, which were appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus. The expression of cyclin B1, a key regulator of entry in mitosis, was assessed by the use of a CYCB1:GUS genetic construction. The results showed significant differences in some of these parameters when comparing the 1g internal rotational control with the 1g external control, indicating that the machine by itself was a source of alterations. When the effect of hypergravity was isolated from other environmental factors, by comparing the experimental conditions with the rotational control, cell proliferation appeared depleted, cell growth was increased and there was an enhanced expression of cyclin B1. The functional meaning of these effects is that cell proliferation and cell growth, which are strictly associated functions under normal 1g ground conditions, are uncoupled under hypergravity. This uncoupling was also described by us in previous experiments as an effect of microgravity, but in an opposite way. Furthermore, root meristems appear thicker in hypergravity-treated than in control samples, which can be related to changes in the cell wall induced by altered gravity.     

Effect of Hypergravity on the Level of Heat Shock Proteins 70 and 90 in Pea Seedlings

Exposure to hypergravity induces significant changes in gene expression of plants which are indicative of stress conditions. A substantial part of the general stress response is up-regulation of heat shock proteins (Hsp) which function as molecular chaperones. The objective of this research was to test the possible changes in the Hsp70 and Hsp90 level in response to short-term hypergravity exposure. In this study 5-day-old etiolated pea seedlings were exposed to centrifuge-induced hypergravity (3–14 g) for 15 min and 1 h and a part of the seedlings was sampled at 1.5 and 24 h after the exposures. Western blot analysis showed time-dependent changes in Hsp70 and Hsp90 levels: an increase under hypergravity and a tendency towards recovery of the normal content during re-adaptation. The quantity and time of their expression was correlated with the g-force level. These data suggest that short-term hypergravity acts as a stress which could increase the risk of protein denaturation and aggregation. Molecular chaperons induced during the stress may have an essential role in counteracting this risk.     

Effects of hypergravity on the cell shape and on the organization of cytoskeleton and extracelluar matrix molecules of in vitro human dermal fibroblasts

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity ranging from 2 to 20 g for 8 days. Changes only appeared above 15 g. The majority of 20 g-subjected cells showed fine filipods in the shape of a star whereas most control cells had rounded shapes and spread by forming lamellipodia. Indirect immunofluorescence staining of vinculin, alpha-actinin and actin stress fibers showed changes of the arrangement anchoring points of stress fibers under hypergravity. Tubulin staining showed that the centrosomal material generally located above the nucleus in control cells had migrated to the nucleus side in 20 g-exposed cells. After 8 d of culture under 20 g hypergravity the thickness of fibronectin network seemed to be increased and bundles of fibrils appeared linking ordered arrays of fibers. The fibrils of collagen I formed better delimited and thicker bundles of fibers. We may assume that 20 g hypergravity can induce changes in fibroblast cell shape, migration way, and anchorage leading to a reorganization of extracellular matrix without concomitant change of cell proliferation.     

Effect of gravity on pool boiling heat transfer on thermal spray coating

This study deals with heat transfer enhancement surface manufactured by thermal spraying. Two thermal spraying methods using copper as a coating material, wire flame spraying (WFS) and vacuum plasma spraying (VPS), were applied to the outside of copper cylinder with 20 mm OD. The surface structure by WFS was denser than that by VPS. The effect of gravity on boiling heat transfer coeffcient and wall superheat at the onset of boiling were experimentally evaluated under micro- and hyper-gravity condition during a parabolic trajectory flight of an airplane. Pool boiling experiments in saturated liquid of HCFC123 were carried out for heat fluxes between 1.0 and 160 kW/m² and saturated temperature of 30 °C. As a result, the surface by VPS produced higher heat transfer coefficient and lower superheat at the onset of boiling under microgravity. For the smooth surface, the effect of gravity on boiling heat transfer coefficient was a little. For the coating, a large difference in heat transfer coefficient to gravity was observed in the moderate heat flux range. The heat transfer coefficinet decreased as gravity changed from the normal to hypergravity, and was improved as gravity changed from the hyperto microgravity. The difference in heat transfer coefficient between the normal and microgravity was a little. Heat transfer enhancement factor was kept over the experimental range of heat flux. It can be said that boiling behavior on thermal spray coating might be influenced by flow convection velocity.     

Hypergravity affects morphology and function in microvascular endothelial cells

Cardiovascular diseases are major health problems in astronauts and pilots. The basic problem in cardiovascular diseases is the loss of function by vascular endothelium. It has been demonstrated that changes in inertial conditions (i.e. hypo- and hypergravity) can affect both phenotypic and genotypic expression in endothelial cells. This report describes the effects observed in endothelial cells from coronary post-capillary venules after repeated exposures to hypergravity conditions, alternating with recovery periods. The results showed changes in gene expression, cell energy metabolism, morphology and cytoskeleton organization.     

Effects of Altered Gravity on the Cytoskeleton of Neonatal Rat Cardiocytes

As an intracellular load-bearing structure, the cytoskeleton is hypothesized to play a crucial role in gravity perception of the cell. Recent data show that the cytoskeleton, which includes actin microfilaments and microtubules, is involved in modulating both the electrical and the mechanical activities of the myocardium. The present study employed observation and quantified analyses of fluorescent images of cardiocytes under different gravity conditions. In acute gravitational change (micro- and hypergravity) induced by parabolic flight, we found disassembly of microtubules but enhanced polymerization of microfilaments, with rearrangement from G-actin to F-actin. In ground-based experiments, exposure of cardiocytes to 2×g hypergravity (centrifugation) led to increased width and number of actin fibers from 2 to 48 h, while microtubules showed no significant changes except polarization at 24 and 48 h. In contrast, exposure of cardiocytes to clinorotation led to disassembly of microtubules from 1 to 48 h, while microfilaments showed no significant changes except redistribution, which was accompanied by rounding of the cells (48 h). We assume that the sensitivity of microfilaments to hypergravity and that of microtubules to microgravity might contribute to the specific cytoskeletal changes observed in parabolic flight. These findings indicate different sensitivity and responses of microfilaments and microtubules to different gravitational changes, which might be part of functional adaptations of the cardiocytes to altered gravitational environments.     

Signaling through rho gtpases in microgravity (rho signaling) on iss (soyuz tma-1) belgian soyuz mission “odissea”

Rho GTPases, RhoA, Rac1 and Cdc42, are molecular switches in the intracellular signaling pathways, that relay the information collected by receptors to soluble mediators and insoluble extracellular matrix environment. The objective of this experiment was to investigate the impact of microgravity on cellular processes depending on Rho GTPases activity, i.e.cytoskeleton and focal adhesions organization, GTPases translocation to the membrane, nuclear translocation of signaling molecules and gene expression. WI26 fibroblasts were stably transfected by the constitutively active form of each of the Rho GTPase. Selected clone of the engineered cells and the wild-type cells were used during the belgian ODISSEA Soyuz mission to investigate the alterations of the mechanical and phenotypic expression of the cells induced by microgravity and their rescue by the engineered Rho GTPases. A failure in the time schedule, a disconnection of the experiment containers before the automatic activation of the fixation procedure, was responsible for the loss of the biological samples.     

Hypergravity to Explore the Role of Buoyancy in Boiling in Porous Media

Boiling in porous media is an active topic of research since it is associated with various applications, e.g. microelectronics cooling, wetted porous media as thermal barriers, food frying. Theoretical expressions customary scale boiling heat and mass transfer rates with the value of gravitational acceleration. Information obtained at low gravity conditions show a deviation from the above scaling law but refers exclusively to non-porous substrates. In addition, the role of buoyancy in boiling at varying gravitational levels (i.e. from microgravity—important to satellites and future Lunar and Martial missions, to high-g body forces—associated with fast aerial maneuvers) is still unknown since most experiments were conducted over a limited range of g-value. The present work aims at providing evidence regarding boiling in porous media over a broad range of hypergravity values. For this, a special device has been constructed for studying boiling inside porous media in the Large Diameter Centrifuge (LDC at ESA/ESTEC). LDC offers the unique opportunity to cancel the shear stresses and study only the effect of increased normal forces on boiling in porous media. The device permits measurement of the temperature field beneath the surface of the porous material and video recordings of bubble activity over the free surface of the porous material. The preliminary results presented from experiments conducted at terrestrial and hypergravity conditions, reveal for the first time the influence of increased levels of gravity on boiling in porous media.     

Changes in expression of genes involved in apoptosis in activated human T-cells in response to modeled microgravity

Space flights result in remarkable effects on various physiological systems, including a decline in cellular immune functions. Previous studies have shown that exposure to microgravity, both true and modeled, can cause significant changes in numerous lymphocyte functions. The purpose of this study was to search for microgravity-sensitive genes, and specifically for apoptotic genes influenced by the microgravity environment and other genes related to immune response. The experiments were performed on anti-CD3 and IL-2 activated human T cells. To model microgravity conditions we have utilized the NASA rotating wall vessel bioreactor. Control lymphocytes were cultured in static 1g conditions. To assess gene expression we used DNA microarray chip technology. We had shown that multiple genes (approximately 3–8% of tested genes) respond to microgravity conditions by 1.5 and more fold change in expression. There is a significant variability in the response. However, a certain reproducible pattern in gene response could be identified. Among the genes showing reproducible changes in expression in modeled microgravity, several genes involved in apoptosis as well as in immune response were identified. These are IL-7 receptor, Granzyme B, Beta-3-endonexin, Apo2 ligand and STAT1. Possible functional consequences of these changes are discussed.     

Synthesis and characterization of magnetic bioactive glass-ceramics containing Mg ferrite for hyperthermia

Novel magnetic bioactive glass-ceramics (M GC) were synthesized by doping Mg ferrite to wollastonite–fluorapatite-containing glass-ceramics. The phase composition was investigated by XRD. The magnetic property was measured by VSM. The in vitro bioactivity was investigated by simulated body fluid (SBF) soaking experiment. Cell growth on the surface of the material was evaluated by co-culturing osteoblast-like ROS17/2.8 cells with M GC. The results showed that CaSiO₃, Ca₂MgSi₂O₇, Ca₅(PO₄)₃F and Fe₂MgO₄ were the main phases of M GC. Under a magnetic field of 10,000 Oe, the saturation magnetization and coercive force of M GC were 7.2 emu/g and 175 Oe, respectively. After soaking in SBF for 14 days, a lot of hydroxyapatite containing CO₃^2? was observed on the surface of M GC. The experiment of co-culturing cells with M GC showed that osteoblast-like ROS17/2.8 cells could attach well on the surface of M GC. The material has the potential to be used as thermoseeds for hyperthermia.     

Optical Photoacoustic Detection of Circulating Melanoma Cells In Vitro

The purpose of this research was to investigate the sensitivity of a system for the detection of circulating melanoma cells based on the thermoelastic properties of melanoma. The method employs photoacoustic (PA) excitation coupled with an optical transducer capable of determining the presence of cells within the circulating system in vitro. The transducer is based on stress wave-induced changes of the optical reflectance of a glass–water interface, probed with a continuous laser beam that is incident at an angle close to the critical angle of total internal reflection. A frequency tripled Nd:YAG laser pumping an optical parametric oscillator was employed to provide 532nm and 620nm laser light with a pulse duration of 10ns. A custom-made flow chamber was used as an excitation and acoustic wave collection device. The targets were a human melanoma cell line HS 936 with an average diameter of about 15μm. Melanoma cells were suspended in 10mL of two types of media. The first one was Tyrode’s buffer in concentrations ranging from 10 to 50 cells per μL, and the second one included 10⁶ healthy white blood cells per mL of Tyrode’s buffer. PA pressure waves were detected by an optical stress transducer. Detection trials resulted in a detection threshold of the order of one individual cell, indicating the effectiveness of the proposed mechanism. Results imply the potential to assay simple blood samples, from healthy and metastatic patients, to test the presence of cancerous melanoma providing an unprecedented method for screening metastatic disease.     

Hypergravity of 10<Emphasis Type="Italic">g</Emphasis> Changes Plant Growth,Anatomy, Chloroplast Size,and Photosynthesis in the Moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

The photosynthetic and anatomical responses of bryophytes to changes in gravity will provide crucial information for estimating how these plant traits evolved to adapt to changes in gravity in land plant history. We performed long-term hypergravity experiments at 10g for 4 and 8 weeks using the moss Physcomitrella patens with two centrifuges equipped with lighting systems that enable long-term plant growth under hypergravity with irradiance. The aims of this study are (1) to quantify changes in the anatomy and morphology of P. patens, and (2) to analyze the post-effects of hypergravity on photosynthesis by P. patens in relation to these changes. We measured photosynthesis by P. patens for a population of gametophores (e.g., canopy) in Petri dishes and plant culture boxes. Gametophore numbers increased by 9% for a canopy of P. patens, with 24–27% increases in chloroplast sizes (diameter and thickness) in leaf cells. In a canopy of P. patens, the area-based photosynthesis rate (A _canopy) was increased by 57% at 10g. The increase observed in A _canopy was associated with greater plant numbers and chloroplast sizes, both of which involved enhanced CO₂ diffusion from the atmosphere to chloroplasts in the canopies of P. patens. These results suggest that changes in gravity are important environmental stimuli to induce changes in plant growth and photosynthesis by P. patens, in which an alteration in chloroplast size is one of the key traits. We are now planning an ISS experiment to investigate the responses of P. patens to microgravity.