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19 p.-8 fig.-1 tab., Long-duration space missions will need to rely on the use of plants in bio-regenerative life support systems (BLSSs) because these systems can produce fresh food and oxygen, reduce carbon dioxide levels, recycle metabolic waste, and purify water. In this scenario, the need for new experiments on the effects of altered gravity conditions on plant biological processes is increasing, and significant efforts should be devoted to new ideas aimed at increasing the scientific output and lowering the experimental costs. Here, we report the design of an easy-to-produce and inexpensive device conceived to analyze the effect of interaction between gravity and light on root tropisms. Each unit consisted of a polystyrene multi-slot rack with light-emitting diodes (LEDs), capable of holding Petri dishes and assembled with a particular filter-paper folding. The device was successfully used for the ROOTROPS (for root tropisms) experiment performed in the Large Diameter Centrifuge (LDC) and Random Positioning Machine (RPM) at ESA's European Space Research and Technology centre (ESTEC). During the experiments, four light treatments and six gravity conditions were factorially combined to study their effects on root orientation of Brassica oleracea seedlings. Light treatments (red, blue, and white) and a dark condition were tested under four hypergravity levels (20 g, 15 g, 10 g, 5 g), a 1 g control, and a simulated microgravity (RPM) condition. Results of validation tests showed that after 24 h, the assembled system remained unaltered, no slipping or displacement of seedlings occurred at any hypergravity treatment or on the RPM, and seedlings exhibited robust growth. Overall, the device was effective and reliable in achieving scientific goals, suggesting that it can be used for ground-based research on phototropism-gravitropism interactions. Moreover, the concepts developed can be further expanded for use in future spaceflight experiments with plants., This work was supported by the ESA-HRE CORA contract #4000127705/19/NL/PG/pt. Financial support was also provided by ESA-TEC contract #4000116223/16/NL/KML/hh to Jack J.W.A. van Loon, by Spanish Agencia Estatal de Investigación (grant RTI2018–099309-B-I00, co-funded by EU-ERDF) to F. Javier Medina, and by NASA (grant 80NSSC17K0546) to John Z. Kiss.

18 p.-9 fig., Premise: Light and gravity are fundamental cues for plant development. Our understanding of the effects of light stimuli on plants in space, without gravity, is key to providing conditions for plants to acclimate to the environment. Here we tested the hypothesis that the alterations caused by the absence of gravity in root meristematic cells can be counteracted by light., Methods: Seedlings of wild‐type Arabidopsis thaliana and two mutants of the essential nucleolar protein nucleolin (nuc1, nuc2) were grown in simulated microgravity,either under a white light photoperiod or under continuous darkness. Key variables of cell proliferation (cell cycle regulation), cell growth (ribosome biogenesis),and auxin transport were measured in the root meristem using in situ cellular markers and transcriptomic methods and compared with those of a 1 g control., Results: The incorporation of a photoperiod regime was sufficient to attenuate or suppress the effects caused by gravitational stress at the cellular level in the root meristem. In all cases, values for variables recorded from samples receiving light stimuli in simulated microgravity were closer to values from the controls than values from samples grown in darkness. Differential sensitivities were obtained for the two nucleolin mutants., Conclusions: Light signals may totally or partially replace gravity signals, significantly improving plant growth and development in microgravity. Despite that, molecular alterations are still compatible with the expected acclimation mechanisms, which need to be better understood. The differential sensitivity of nuc1 and nuc2 mutants to gravitational stress points to new strategies to produce more resilient plants to travel with humans in new extraterrestrial endeavors., This work was funded by the Agencia Estatal de Investigación of the Spanish Ministry of Science an Innovation, Grants#ESP2015‐64323‐R and #RTI2018‐099309‐B‐I00 (co‐funded by EU‐ERDF) to F.J.M., and Bonus Recherche from the UPVD to J.S.V. The use of the facilities of microgravity simulation was provided by the ESA‐CORA‐Ground Based Facilities Program, contract Ref. #4000105761 to F.J.M. and R.H. A.M. was recipient of a contract of the Program for Young Researchers Training of the Agencia Estatal de Investigación of the Spanish Ministry of Science an Innovation Ref. #BES‐2013‐063933.

35 p.-5 tab.-4 tab.supl., We performed a series of experiments to study the interaction between phototropism and gravitropism in Arabidopsis thaliana as part of the Seedling Growth Project on the International Space Station. Red-light-based and blue-light-based phototropism were examined in microgravity and at 1g, a control that was produced by an on-board centrifuge. At the end of the experiments, seedlings were frozen and brought back to Earth for gene profiling studies via RNASeq methods. In this paper, we focus on five genes identified in these space studies by their differential expression in space: one involved in auxin transport and four others encoding genes for: a methyltransferase subunit, a transmembrane protein, a transcription factor for endodermis formation, and a cytoskeletal element (an intermediate filament protein). Time course studies using mutant strains of these five genes were performed for blue-light and red-light phototropism studies as well as for gravitropism assays on ground. Interestingly, all five of the genes had some effects on all the tropisms under the conditions studied. In addition, RT-PCR analyses examined expression of the five genes in wild-type seedlings during blue-light-based phototropism. Previous studies have supported a role of both microfilaments and microtubules in tropism pathways. However, the most interesting finding of the present space studies is that NFL, a gene encoding an intermediate filament protein, plays a role in phototropism and gravitropism, which opens the possibility that this cytoskeletal element modulates signal transduction in plants., This work was supported by the National Aeronautics and Space Administration [grant 80NSSC17K0546 to John Z. Kiss] and by the Spanish Plan Estatal de Investigación Científica y Desarrollo Tecnológico [Grant RTI2018-099309-B-I00, co-funded by EU-ERDF, to F. Javier Medina].

15 p.-6 fig., In a microgravity environment, without any gravitropic signal, plants are not able to define and establish a longitudinal growth axis. Consequently, absorption of water and nutrients by the root and exposure of leaves to sunlight for efficient photosynthesis is hindered. In these conditions, other external cues can be explored to guide the direction of organ growth. Providing a unilateral light source can guide the shoot growth, but prolonged root exposure to light causes a stress response, affecting growth and development, and also affecting the response to other environmental factors. Here, we have investigated how the protection of the root from light exposure, while the shoot is illuminated, influences the direction of root growth in microgravity. We report that the light avoidance mechanism existing in roots guides their growth towards diminishing light and helps establish the proper longitudinal seedling axis in simulated microgravity conditions. This process is regulated by flavonols, as shown in the flavonoid-accumulating mutant transparent testa 3, which shows an increased correction of the root growth direction in microgravity, when the seedling is grown with the root protected from light. This finding may improve the efficiency of water and nutrient sourcing and photosynthesis under microgravity conditions, as they exist in space, contributing to better plant fitness and biomass production in space farming enterprises, necessary for space exploration by humans., The funds for this research were provided by Agencia Estatal de Investigación of the Spanish Ministry of Science and Innovation, Grants #ESP2015-64323-R and #RTI2018-099309-B-I00 (co-funded by EU-ERDF), awarded to FJM. The clinostat was granted by the United Nations Zero-Gravity Instrument Project (ZGIP). AV was a recipient of a contract of the Spanish National Program for Young Researchers Training (BES-2016-077976).

24 p.-9 fig., Gravitropism and phototropism play a primary role in orienting root growth. Tropistic responses of roots mediated by gravity and light have been extensively investigated, and a complex mutual interaction occurs between these two tropisms. To date, most studies have been conducted in 1 g, microgravity, or simulated microgravity, whereas no studies investigated root phototropism in hypergravity. Therefore, we studied the effects of several gravity treatments with those of different light wavelengths on root growth orientation. Here, we report growth and curvature of Brassica oleracea roots under different g levels, from simulated microgravity up to 20 g, and unilateral illumination with different spectral treatments provided by light emitting diodes. Microgravity was simulated with a random positioning machine whereas hypergravity conditions were obtained using the Large Diameter Centrifuge at the laboratories of the European Space Agency in the Netherlands. Four light treatments (white light, blue light, red light, and dark) were used in this study. Overall, roots of seedlings grown in the dark were longer than those developed under unilateral light treatments, regardless of the gravity level. Unilateral blue light or white light stimulated a negative phototropism of roots under all g levels, and root curvature was not affected by either hypergravity or simulated microgravity compared to 1 g. Results also confirmed previous findings on the effect of light intensity on root curvature and highlighted the relevance of blue-light photon flux density in root phototropism. Roots illuminated with red light showed a weak curvature in simulated microgravity but not in hypergravity. Moreover, root curvature under red light was similar to dark-grown roots in all g levels, suggesting a possible involvement of surface-dependent phenomena in root skewing under either red light or dark conditions. Further studies can confirm phototropic responses of B. oleracea in the weightless environment of orbiting spacecraft. Nevertheless, according to our findings, directional lighting represents an effective stimulus to guide root growth in a wide range of gravity conditions., This work was supported by the ESA-HRE CORA contract #4000127705/19/NL/PG/pt. Financial support was also provided by ESA-TEC contract #4000116223/16/NL/KML/hh to Jack J.W.A. van Loon, by Spanish Agencia Estatal de Investigación (Grant RTI2018-099309-B-I00, co-funded by EU- ERDF) to F. Javier Medina, and by NASA (Grant 80NSSC17K0546) to John Z. Kiss.

Simulated microgravity and partial gravity research on Earth is a necessary complement to space research in real microgravity due to limitations of access to spaceflight. However, the use of ground-based facilities for reduced gravity simulation is far from simple. Microgravity simulation usually results in the need to consider secondary effects that appear in the generation of altered gravity. These secondary effects may interfere with gravity alteration in the changes observed in the biological processes under study. In addition to microgravity simulation, ground-based facilities are also capable of generating hypergravity or fractional gravity conditions whose effects on biological systems are worth being tested and compared with the results of microgravity exposure. Multiple technologies (2D clinorotation, random positioning machines, magnetic levitators, or centrifuges) and experimental hardware (different containers and substrates for seedlings or cell cultures) are available for these studies. Experimental requirements should be collectively and carefully considered in defining the optimal experimental design, taking into account that some environmental parameters, or life-support conditions, could be difficult to be provided in certain facilities. Using simulation facilities will allow us to anticipate, modify, or redefine the findings provided by the scarce available spaceflight opportunities.

31 p.-5 fig.-2 tab.-2 fig. supl., Plant cell proliferation is affected by microgravity during spaceflight, but involved molecular mechanisms, key for space agronomy goals, remain unclear. To investigate transcriptomic changes in cell cycle phases caused by simulated microgravity, an Arabidopsis immobilized synchronous suspension culture was incubated in a Random Positioning Machine. After simulation, a transcriptomic analysis was performed with two subpopulations of cells (G2/M and G1 phases enriched) and an asynchronous culture sample. Differential expression was found at cell proliferation, energy/redox and stress responses, plus unknown biological processes gene ontology groups. Overall expression inhibition was a common response to simulated microgravity, but differences peak at the G2/M phase and stress response components change dramatically from G2/M to the G1 subpopulation suggesting a differential adaptation response to simulated microgravity through the cell cycle. Cell cycle adaptation using both known stress mechanisms and unknown function genes may cope with reduced gravity as an evolutionary novel environment., This work was supported by the Spanish “Plan Estatal de Investigación Científica y Técnica y de Innovación” of the Ministry of Economy, Industry and Competitiveness [to F.J.M., Grant numbers AYA2012-33982 and ESP2015-64323-R, co-funded by ERDF], by a pre-doctoral fellowship from CSIC, Spain [to K.Y.K., JAE-PreDoc Program, Ref. JAEPre_2010_01894], the ESA-ELIPS Program [to R.H., ESA GIA Project, contract number 4000105761], and ESA support [to J.v.L., via contract TEC-MMG/2012/263 and 4000107455/12/NL/PA].

The response of plants to the spaceflight environment and microgravity is still not well understood, although research has increased in this area. Even less is known about plants’ response to partial or reduced gravity levels. In the absence of the directional cues provided by the gravity vector, the plant is especially perceptive to other cues such as light. Here, we investigate the response of Arabidopsis thaliana 6-day-old seedlings to microgravity and the Mars partial gravity level during spaceflight, as well as the effects of red-light photostimulation by determining meristematic cell growth and proliferation. These experiments involve microscopic techniques together with transcriptomic studies. We demonstrate that microgravity and partial gravity trigger differential responses. The microgravity environment activates hormonal routes responsible for proliferation/growth and upregulates plastid/mitochondrial-encoded transcripts, even in the dark. In contrast, the Mars gravity level inhibits these routes and activates responses to stress factors to restore cell growth parameters only when red photostimulation is provided. This response is accompanied by upregulation of numerous transcription factors such as the environmental acclimation-related WRKY-domain family. In the long term, these discoveries can be applied in the design of bioregenerative life support systems and space farming., This research was funded by the Agencia Estatal de Investigación of the Spanish Ministry of Science an Innovation, Grants #ESP2015-64323-R and #RTI2018-099309-B-I00 (co-funded by EU-ERDF) to F.J.M., by pre-doctoral fellowships to A.M. and A.V. from the Spanish National Program for Young Researchers Training (MINECO, Ref. #BES-2013-063933, #BES-2016-077976) and the Seedling Growth Project for ISS experimentation #LSRA2009-0932/ 1177, a shared project of ESA-ELIPS Program and NASA, to F.J.M. and J.Z.K. In addition, J.Z.K. is funded by Grants NNX12A065G and 80NSSC17K0546. This research is related to the Space Omics TT funded by the ESA contract 4000131202/20/NL/PG to R.H.

25 p.-4 fig., Any space exploration initiative, such as the human presence in the Moon and Mars, must incorporate plants for life support. To enable space plant culture we need to understand how plants respond to extraterrestrial conditions, adapt to them, and compensate their deleterious effects at multiple levels. Gravity is a major difference between the terrestrial and the extraterrestrial environment. Gravitropism is the process of establishing a growth direction for plant organs according to the gravity vector. Gravity signals are sensed at specialized tissues by the motion of amyloplasts called statoliths and transduced to produce a cellular polarization capable of influencing the transport of auxin. Gravity alterations eventually result in changes in the lateral balance of auxin in the root, producing deviations of the growth direction. Under microgravity, auxin changes affect the root meristem causing increased cell proliferation and decreased cell growth. The nucleolus, the nuclear site of production of ribosomes, is a marker of this unbalance, which could alter plant development. At the molecular level, microgravity induces a reprogramming of gene expression that mostly affects plant defense systems against abiotic stresses, indicating that these categories of genes are involved in the adaptation to extraterrestrial habitats. Nevertheless, no specific genes for plant response to gravitational stress have been identified. Despite this stress, plants survive, developing until the adult stage and reproducing under microgravity conditions. A major research challenge is to identify environmental factors, such as light, which could interact, modulate, or balance the impact of gravity, contributing to the tolerance and survival of plants under spaceflight conditions. Understanding the crosstalk between light and gravity sensing will contribute to the success of the next generation agriculture in human settlements outside the Earth., Work performed in the authors’ laboratory was supported by different grants of the Spanish National Agency for Research of theSpanish Government, e.g. Grants # ESP2015-64323-R and #RTI2018-099309-B-I00 (co-funded by EU-ERDF).

6 p., The authors belong to the ESA Space Omics Topical Team funded by the ESA grant/contract 4000131202/20/NL/PG/pt “Space Omics: Towards an integrated ESA/NASA –omics database for spaceflight and ground facilities experiments” to RH.

30 p.-10 fig.-1 tab., Plant biology is an important area for the future of space exploration, but biological spaceflight experiments have been always constrained by the hardware capabilities. The European Modular Cultivation System (EMCS) unit was an incubator for small organisms, such as Arabidopsis thaliana, built by the European Space Agency (ESA) and was decommissioned in 2018. Here, we describe the FixBox system as add-on hardware to provide fixation capabilities to the plant growth cassettes, which, initially, were not designed to be used for that purpose. Tests were performed to ensure the successful use of this device in the EMCS facility. We also evaluate the required adaptations to the hardware, e.g., to guarantee that the reduced fluid motion in microgravity does not cause any bubbles that could impair the quality of fixation. Arabidopsis thaliana seedlings grown during spaceflight were fixed in the FixBox either in glutaraldehyde or formaldehyde. Electron microscopical images and confocal microscopy immunofluorescent localizations showed an excellent preservation of both cell ultrastructure and antigen conformation. Thus, it is possible to modify existing hardware to comply with the scientific requirements to augment the existing capabilities on board the ISS. In addition, it is also possible to reuse culture chambers from predesigned experimental containers into new modular subunits as FixBox. Similarly, we can design new hardware compatible with a novel cultivation chamber on board, such as is available in BIOLAB, to be used later with FixBox. Lessons learned for future space plant biology researchers include how to manage the number of hardware requirements and constraints on how to preserve the biological samples., This work was supported by the Agencia Estatal de Investigación of the Spanish Ministry of Science an Innovation, Grants #ESP2015–64323-R and #RTI2018–099309-B-I00 (co-funded by EU-ERDF), awarded to FJM, and by pre-doctoral fellowships to (AM), (MV) and (AV) from the Spanish National Program for Young Researchers Training (MINECO, Ref. BES-2013-063933, BES-2016-077976). This research was supported also by grants (NNX12A0656 and 80NSSC17K0546) from NASA to JZK. The Seedling Growth Project spaceflights and activities to the ISS were funded by ILSRA2009–0932/1177 of ESA-ELIPS Program. The FixBox has been built under a contract to SENER Aeroespacial SA by ESA (4000105697/12/NL/FC) to support the SG project.

Background Light and gravity are fundamental cues for plant development on Earth. In space, understanding the effects of changing conditions affecting to these two stimuli is key for optimizing the life support systems to come with space exploration. Simulated microgravity is useful as a complement to real spaceflight experiments into refining our knowledge of early plant development adaptation to extraterrestrial environments. Results In wild type Arabidopsis and in mutants of the two genes of the essential nucleolar protein nucleolin (nuc1 and nuc2), the use of an extended toolbox of cell proliferation, cell growth and ribosome biogenesis markers, has allowed us to show that the incorporation of an illumination regime, in this case photoperiod, has been sufficient to attenuate or suppress the effects caused by gravitational stress at the cellular level in the root meristem. These results are consistent with other experiments carried out at real and simulated microgravity in which early plant development is nominal when the environmental conditions are optimal (nutrients, light, temperature, humidity). Conclusions Light signals may total or partially replace gravity signals significantly improving plant growth and development in microgravity. Despite that, molecular alterations are still compatible with the expected adaptation mechanisms that should be better understood. The differential sensitivity of nuc1 and nuc2 mutants to gravitational stress points to new strategies to produce more resilient plants to travel with humans in new extraterrestrial endeavors.

Zero gravity is an environmental challenge unknown to organisms throughout evolution on Earth. Nevertheless, plants are sensitive to altered gravity, as exemplified by changes in meristematic cell proliferation and growth. We found that synchronized Arabidopsis-cultured cells exposed to simulated microgravity showed a shortened cell cycle, caused by a shorter G2/M phase and a slightly longer G1 phase. The analysis of selected marker genes and proteins by quantitative polymerase chain reaction and flow cytometry in synchronic G1 and G2 subpopulations indicated changes in gene expression of core cell cycle regulators and chromatin-modifying factors, confirming that microgravity induced misregulation of G2/M and G1/S checkpoints and chromatin remodelling. Changes in chromatin-based regulation included higher DNA methylation and lower histone acetylation, increased chromatin condensation, and overall depletion of nuclear transcription. Estimation of ribosome biogenesis rate using nucleolar parameters and selected nucleolar genes and proteins indicated reduced nucleolar activity under simulated microgravity, especially at G2/M. These results expand our knowledge of how meristematic cells are affected by real and simulated microgravity. Counteracting this cellular stress is necessary for plant culture in space exploration.

Understanding plant adaptive responses to the space environment is a requisite for enabling space farming. Spaceflight produces deleterious effects on plant cells, particularly affecting ribosome biogenesis, a complex stress-sensitive process coordinated with cell division and differentiation, known to be activated by red light. Here, we have used mutants from the two nucleolin genes in Arabidopsis (NUC1 and NUC2), encoding the main regulator of the ribosome biogenesis in the nucleolus, in order to better understand their role in adaptive response mechanisms to stress. Thus, we show that nucleolin stress-related gene NUC2 can compensate the environmental stress provided by darkness in nuc1 plants, while nuc2 plants are not able to provide a complete response to red light. These ground control findings, as part of the ESA/NASA Seedling Growth spaceflight experiments will determine the basis for the identification of a genetic background enabling an adaptive advantage for plants in future space experiments.

Summary Understanding plant adaptive responses to the space environment is a requisite for enabling space farming. Spaceflight produces deleterious effects on plant cells, particularly affecting ribosome biogenesis, a complex stress-sensitive process coordinated with cell division and differentiation, known to be activated by red light. Here, in a series of ground studies, we have used mutants from the two Arabidopsis nucleolin genes (NUC1 and NUC2, nucleolar regulators of ribosome biogenesis) to better understand their role in adaptive response mechanisms to stress on Earth. Thus, we show that nucleolin stress-related gene NUC2 can compensate for the environmental stress provided by darkness in nuc1 plants, whereas nuc2 plants are not able to provide a complete response to red light. These ground control findings, as part of the ESA/NASA Seedling Growth spaceflight experiments, will determine the basis for the identification of genetic backgrounds enabling an adaptive advantage for plants in future space experiments., Graphical Abstract, Highlights • Ribosome synthesis is a target of spaceflight stressor effects on plant development • Nucleolin mutants promote a differential response to light/darkness stress • Red light and NUC2 may counteract the spaceflight alterations in gene expression • Ground controls are important for the interpretation of spaceflight -omics experiments, Plant Biology; Omics; Space Sciences

36 p.-11 fig.-1 tab., Red light is able to compensate for deleterious effects of microgravity on root cell growth and proliferation. Partial gravity combined with red light produces differential signals during the early plant development. Light and gravity are environmental cues used by plants throughout evolution to guide their development. We have investigated the cross-talk between phototropism and gravitropism under altered gravity in space. The focus was on the effects on the meristematic balance between cell growth and proliferation, which is disrupted under microgravity in the dark. In our spaceflight experiments, seedlings of three Arabidopsis thaliana genotypes, namely the wild type and mutants of phytochrome A and B, were grown for 6 days, including red-light photoactivation for the last 2 days. Apart from the microgravity and the 1g on-board control conditions, fractional gravity (nominally 0.1g, 0.3g, and 0.5g) was created with on-board centrifuges. In addition, a simulated microgravity (random positioning machine, RPM) experiment was performed on ground, including both dark-grown and photostimulated samples. Photoactivated samples in spaceflight and RPM experiments showed an increase in the root length consistent with phototropic response to red light, but, as gravity increased, a gradual decrease in this response was observed. Uncoupling of cell growth and proliferation was detected under microgravity in darkness by transcriptomic and microscopic methods, but red-light photoactivation produced a significant reversion. In contrast, the combination of red light and partial gravity produced small but consistent variations in the molecular markers of cell growth and proliferation, suggesting an antagonistic effect between light and gravity signals at the early plant development. Understanding these parameters of plant growth and development in microgravity will be important as bioregenerative life support systems for the colonization of the Moon and Mars., Funding for this study was provided mainly by the Spanish National Plan for Research and Development (MINECO-ERDF co-funding) Grant ESP2015-64323-R to FJM. The access to ISS and RPM facilities was granted by ESA-ELIPS ILSRA-2009-0932 to FJM and GBF Program GIA Project (contract# 4000105761) to RH. This research was supported also by grants (NNX12A0656 and 80NSSC17K0546) from NASA to JZK and the French Space Agency—CNES to ECD and VPL. MAV and AM were recipients of grants of the Spanish National Program for Young Researchers Training (Refs. BES-2010-035741 and BES-2013-063933, respectively).

Gravity is the only component of Earth environment that remained constant throughout the entire process of biological evolution. However, it is still unclear how gravity affects plant growth and development. In this study, an in vitro cell culture of Arabidopsis thaliana was exposed to different altered gravity conditions, namely simulated reduced gravity (simulated microgravity, simulated Mars gravity) and hypergravity (2g), to study changes in cell proliferation, cell growth, and epigenetics. The effects after 3, 14, and 24-hours of exposure were evaluated. The most relevant alterations were found in the 24-hour treatment, being more significant for simulated reduced gravity than hypergravity. Cell proliferation and growth were uncoupled under simulated reduced gravity, similarly, as found in meristematic cells from seedlings grown in real or simulated microgravity. The distribution of cell cycle phases was changed, as well as the levels and gene transcription of the tested cell cycle regulators. Ribosome biogenesis was decreased, according to levels and gene transcription of nucleolar proteins and the number of inactive nucleoli. Furthermore, we found alterations in the epigenetic modifications of chromatin. These results show that altered gravity effects include a serious disturbance of cell proliferation and growth, which are cellular functions essential for normal plant development., This work was supported by the Spanish “Plan Estatal de Investigación Científica y Ténica y de Innovación” of the Ministry of Economy, Industry and Competitiveness [Grant numbers AYA2012–33982 and ESP2015–64323-R, co-funded by ERDF], by a pre-doctoral fellowship to [Kh.Y.K.] from CSIC, Spain [JAE-PreDoc Program, Ref. JAEPre_2010_01894] the ESA-ELIPS Program [ESA SEGMGSPE_Ph1 Project, contract number 4200022650], and ESA support via contract TEC-MMG/2012/263.

11 p.-8 fig.-1 tab., Clinostats and Random Positioning Machine (RPM) are used to simulate microgravity, but, for space exploration, we need to know the response of living systems to fractional levels of gravity (partial gravity) as they exist on Moon and Mars. We have developed and compared two different paradigms to simulate partial gravity using the RPM, one by implementing a centrifuge on the RPM(RPMHW), the other by applying specific software protocols to driving the RPM motors (RPMSW). The effects of the simulated partialgravity were tested in plant root meristematic cells, a system with known response to real and simulated microgravity. Seeds of Arabidopsis thaliana were germinated under simulated Moon (0.17 g) and Mars (0.38 g) gravity. In parallel, seeds germinated under simulated microgravity (RPM), or at 1 g control conditions. Fixed root meristematic cells from 4-day grown seedlings were analyzed for cell proliferation rate and rate of ribosome biogenesis using morphometrical methods and molecular markers of the regulation of cell cycle and nucleolar activity. Cell proliferation appeared increased and cell growth was depleted under Moon gravity,compared with the 1g control. The effects were even higher at the Moon level than at simulated microgravity, indicating that meristematic competence (balance between cell growth and proliferation) is also affected at this gravity level. However, the results at the simulated Mars level were close to the 1g static control. This suggests that the threshold for sensing and responding to gravity alteration in the root would be at a level intermediate between Moon and Mars gravity. Both partial g simulation strategies seem valid and show similar results at Moon g levels, but further research is needed, in space flight and simulation facilities,especially around and beyond Mars g levels to better understand more precisely the differences and constrains in the use of these facilities for the space biology community., Funding: for [JvL]: Grant ALW-GO-MG/10-07 from the Netherlands Organization for Scientific (NWO) Research Earth and Life Sciences (ALW)via the Netherlands Space Office (NSO) and the ESA contract 4000107455/12/NL/PA.For [FJM]: Grant ESP2015-64323-R from the Spanish National Plan for Research and Development (MINECO-ERDF co-funding). For [RH]: ESA-ELIPS Program ESA GIA Project, contract number 4000105761. [AM] was recipient of a fellowship of the Spanish National Program for Young Researchers Training (MINECO, Ref. BES-2013- 063933).

Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

Zero gravity is an environmental challenge unknown to organisms throughout evolution on Earth. Nevertheless, plants are sensitive to altered gravity, as exemplified by changes in meristematic cell proliferation and growth. We found that synchronized Arabidopsis-cultured cells exposed to simulated microgravity showed a shortened cell cycle, caused by a shorter G2/M phase and a slightly longer G1 phase. The analysis of selected marker genes and proteins by quantitative polymerase chain reaction and flow cytometry in synchronic G1 and G2 subpopulations indicated changes in gene expression of core cell cycle regulators and chromatin-modifying factors, confirming that microgravity induced misregulation of G2/M and G1/S checkpoints and chromatin remodelling. Changes in chromatin-based regulation included higher DNA methylation and lower histone acetylation, increased chromatin condensation, and overall depletion of nuclear transcription. Estimation of ribosome biogenesis rate using nucleolar parameters and selected nucleolar genes and proteins indicated reduced nucleolar activity under simulated microgravity, especially at G2/M. These results expand our knowledge of how meristematic cells are affected by real and simulated microgravity. Counteracting this cellular stress is necessary for plant culture in space exploration.

13 p.-2 fig., Gravity plays a role in modulating plant growth and development and its alteration induces changes in these processes. Microgravity research has recently been extended to the use of in vitro plant cell cultures which are considered as an ideal model system to study cell proliferation and growth. In general, among the ground-based facilities available for microgravity simulation, the 2D pipette clinostat had been previously considered a suitable facility to be used for unicellular biological models although studies using single plant cell cultures raised some concerns. The incompatibility comes from the standard requirement of shaking a suspension culture for assuring its viability and active proliferation status in the control samples. Moreover, a related issue applies to the use of the random positioning machine (RPM) for cell suspension experiments. Here, we demonstrate an alternative culture method based on the immobilization of the culture before the altered gravity treatment occurs, such that it behaves as a solid object. Our immobilization procedure preserved plant cell culture viability without compromising basic cell properties as viability, morphology, cell cycle phases distribution, or chromatin organization, when compared with a standard cell suspension under shaking as a control. This approach should allow the space biology community to improve the quantity and quality of plant cell results in future simulated microgravity experiments or spaceflight opportunities., This work was supported by grants of the Spanish National Plan for Research and Development, Ref. Nos.AYA2012-33982 and ESP2015-64323-R, the Dutch Space Research Organization (NWOALW-SRON) [Grant number MG-057] and the ESA-ELIPS Program[ESA SEGMGSPE Ph1 Project, contract number 4200022650]. KYK was supported by the Spanish CSIC JAE-PreDoc Program (Ref.JAEPre 2010 01894).

13 p.-3 fig.- 6 fig. supl., Motility and aging in Drosophila have proven to be highly modified under altered gravity conditions (both in space and ground simulation facilities). In order to find out how closely connected they are, five strains with altered geotactic response or survival rates were selected and exposed to an altered gravity environment of 2g. By analysing the different motile and behavioural patterns and the median survival rates, we show that altered gravity leads to changes in motility, which will have a negative impact on the flies' survival. Previous results show a differential gene expression between sessile samples and adults and confirm that environmentally-conditioned behavioural patterns constrain flies' gene expression and life span. Therefore, hypergravity is considered an environmental stress factor and strains that do not respond to this new environment experience an increment in motility, which is the major cause for the observed increased mortality also under microgravity conditions. The neutral-geotaxis selected strain (strain M) showed the most severe phenotype, unable to respond to variations in the gravitational field. Alternatively, the opposite phenotype was observed in positive-geotaxis and long-life selected flies (strains B and L, respectively), suggesting that these populations are less sensitive to alterations in the gravitational load. We conclude that the behavioural response has a greater contribution to aging than the modified energy consumption in altered gravity environments. © 2012 Springer Science+Business Media Dordrecht., This work was supported by Grants from the Spanish “Plan Nacional de Investigación Científica y Desarrollo Tecnológico” Ref. AYA2009-07792-E/ESP to RH, from the Dutch Organisation of Scientific research (NWO) #MG-057 to JvL and by the access to the Ground Base Facilities: ESA SEGMGSPE_Ph1 Project to JvL, FJM and RH.

33 p.-7 fig., Environmental gravity modulates plant growth and development, and these processes are influenced by the balance between cell proliferation and differentiation in meristems. Meristematic cells are characterized by the coordination between cell proliferation and cell growth, that is, by the accurate regulation of cell cycle progression and the optimal production of biomass for the viability of daughter cells after division. Thus, cell growth is correlated with the rate of ribosome biogenesis and protein synthesis. We investigated the effects of simulated microgravity on cellular functions of the root meristem in a sequential study. Seedlings were grown in a clinostat, a device producing simulated microgravity, for periods between 3 and 10days. In a complementary study, seedlings were grown in a Random Positioning Machine (RPM) and sampled sequentially after similar periods of growth. Under these conditions, the cell proliferation rate and the regulation of cell cycle progression showed significant alterations, accompanied by a reduction of cell growth. However, the overall size of the root meristem did not change. Analysis of cell cycle phases by flow cytometry showed changes in their proportion and duration, and the expression of the cyclin B1 gene, a marker of entry in mitosis, was decreased, indicating altered cell cycle regulation. With respect to cell growth, the rate of ribosome biogenesis was reduced under simulated microgravity, as shown by morphological and morphometric nucleolar changes and variations in the levels of the nucleolar protein nucleolin. Furthermore, in a nucleolin mutant characterized by disorganized nucleolar structure, the microgravity treatment intensified disorganization. These results show that, regardless of the simulated microgravity device used, a great disruption of meristematic competence was the first response to the environmental alteration detected at early developmental stages. However, longer periods of exposure to simulated microgravity do not produce an intensification of the cellular damages or a detectable developmental alteration in seedlings analyzed at further stages of their growth. This suggests that the secondary response to the gravity alteration is a process of adaptation, whose mechanism is still unknown, which eventually results in viable adult plants., This work was supported by the Spanish “Plan Estatal de Investigación Científica y Ténica y de Innovación”of the Ministry of Economy and Competitiveness [Grant numbers AYA2012-33982 and ESP2015-64323-R, co-funded by ERDF, and pre-doctoral fellowships to [A.I.M.] and [I.M.](Program FPI)], the French “Centre National d’Etudes Spatiales” (CNES) (G. GauquelinKoch) and the Dutch Space Research Organization (NWO-ALW-SRON) [Grant number MG057].

Changes in the cell growth rate of an in vitro cellular system in Arabidopsis thaliana induced by short exposure to an altered gravity environment have been estimated by a novel approach. The method consisted of defining three structural nucleolar types which are easy and reliable indicators of the ribosome biogenesis activity and, consequently, of protein biosynthesis, a parameter strictly correlated to cell growth in this cellular system. The relative abundance of each nucleolar type was statistically assessed in different conditions of gravity. Samples exposed to simulated microgravity for 200 min showed a significant decrease in nucleolar activity compared to 1g controls, whereas samples exposed to hypergravity (2g) for the same period showed nucleolar activity slightly increased. These effects could be considered as an early cellular response to the environmental alteration, given the short duration of the treatment. The functional significance of the structural data was validated by a combination of several different well-known parameters, using microscopical, flow cytometry, qPCR, and proteomic approaches, which showed that the decreased cell growth rate was decoupled from an increased cell proliferation rate under simulated microgravity, and the opposite trend was observed under hypergravity. Actually, not all parameters tested showed the same quantitative changes, indicating that the response to the environmental alteration is time-dependent. These results are in agreement with previous observations in root meristematic cells and they show the ability of plant cells to produce a response to gravity changes, independently of their integration into plant organs., AIM and AM were supported by the Spanish FPI fellowship program. RH was supported by a Spanish Science and Technology (Especialización en Organismos Internacionales)/CDTI scholarship (ESA Spanish trainee program) and also supported by a CSIC JAE-Doc contract. This work was supported by Grants from the Spanish “Plan Nacional de Investigación Científica y Desarrollo Tecnológico” (Ministerio de Economía y Competitividad, Spain), Refs. AYA2010-11834-E and AYA2012-33982 to FJM and AYA2009-07792-E to RH and by the access to the Ground Based Facilities: ESA SEGMGSPE_Ph1 Project (contract number 4200022650) to JVL, FJM, and RH.

Understanding the effects of gravity on biological organisms is vital to the success of future space missions. Previous studies in Earth orbit have shown that the common fruitfly ( Drosophila melanogaster ) walks more quickly and more frequently in microgravity, compared with its motion on Earth. However, flight preparation procedures and forces endured on launch made it difficult to implement on the Earth's surface a control that exposed flies to the same sequence of major physical and environmental changes. To address the uncertainties concerning these behavioural anomalies, we have studied the walking paths of D. melanogaster in a pseudo-weightless environment (0 g *) in our Earth-based laboratory. We used a strong magnetic field, produced by a superconducting solenoid, to induce a diamagnetic force on the flies that balanced the force of gravity. Simultaneously, two other groups of flies were exposed to a pseudo-hypergravity environment (2 g *) and a normal gravity environment (1 g *) within the spatially varying field. The flies had a larger mean speed in 0 g * than in 1 g *, and smaller in 2 g *. The mean square distance travelled by the flies grew more rapidly with time in 0 g * than in 1 g *, and slower in 2 g *. We observed no other clear effects of the magnetic field, up to 16.5 T, on the walks of the flies. We compare the effect of diamagnetically simulated weightlessness with that of weightlessness in an orbiting spacecraft, and identify the cause of the anomalous behaviour as the altered effective gravity.

6 páginas, 2 figuras, 1 tabla -- PAGS nros. 299-304, In the GENE experiment performed during an 11-day Soyuz Mission to the International Space Station (ISS), we intended to determine if microgravity affects Drosophila metamorphosis processes. Control experiments were performed including a 1g ground control parallel to the ISS flight samples and a Random Position Machine microgravity simulated control. A preliminary analysis of the results indicates that five hundred to one thousand genes change their expression profiles depending on the cut-off levels selected. Especially affected among them are the mitochondrial ones (an example with the respiratory chain is presented). We show here that there is a synergic effect of the constraints introduced to meet the requirements of the space experiment (mainly, a cold step and the use of hermetically closed Type-I containers). The cold transport step to the launch site was introduced to slow down the pupal development. The hermetically closed Type I containers were required to ensure the containment of the fixative (acetone) in the experiment. As shown here, the oxygen concentration inside the container was not optimal but fully compatible with pupal development. It is highly likely that such combined environmental effects will become a common finding in these types of studies as they become more complicated and extensive. They could open the way to understand how the gene expression patterns and the actual phenotypes can adjust to the environment. These findings indicate the importance of a vigorous ground based program in support of real microgravity experiments. Only then we can utilize the ISS in order to understand the consequences of the modified environment in outer space on living organisms, This work was supported by Grants from The Spanish "Plan Nacional de Investigación Científica y Desarrollo Tecnológico" Ref. nos. ESP2001-4522-PE, and ESP2003-09475-C02-01 and from The Netherlands Institute for Space Researchs, NW0-SROM, MG-057

5 páginas -- PAGS nros. 293-297, Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of cell cycle progression and the rate of ribosome biogenesis (this latter taken to estimate cell growth) have been studied using morphometric markers or parameters evaluated by light and electron microscopy in real microgravity on the International Space Station (ISS) and in ground-based simulated microgravity, using the Random Positioning Machine and the Magnetic Levitation Instrument. Results showed enhanced cell proliferation but depleted cell growth in both real and simulated microgravity, indicating that the two processes are uncoupled, unlike the situation under normal gravity on Earth in which they are strictly co-ordinated events. It is concluded that microgravity is an important stress condition for plant cells compared to normal ground gravity conditions

22 p.-6 fig., Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended., This work was supported by grants of the Spanish National Plan for Research and Development, Ref. Nos. AYA2010-11834-E, and AYA2012-33982, access to Magnet facilities by the European Union (EUROMAGNET II) Project 2010.17 (NSO06-209) to FJM, the GBF project #4200022650 and #4000105761 to RH and ESA grant contract 4000107455112/NL/PA to JvL. KYK was supported by the Spanish CSIC JAE-PreDoc Program (Ref.JAEPre_2010_01894).

Simulated microgravity and partial gravity research on Earth is highly convenient for every space biology researcher due to limitations of access to spaceflight. However, the use of ground-based facilities for microgravity simulation is far from simple. Microgravity simulation usually results in the need to consider additional environmental parameters which appear as secondary effects in the generation of altered gravity. These secondary effects may interfere with gravity alteration in the changes observed in the biological processes under study. Furthermore, ground-based facilities are also capable of generating hypergravity or fractional gravity conditions, which are worth being tested and compared with the results of microgravity exposure. Multiple technologies (2D clinorotation, random positioning machines, magnetic levitators or centrifuges), experimental hardware (proper use of containers and substrates for the seedlings or cell cultures), and experimental requirements (some life support/environmental parameters are more difficult to provide in certain facilities) should be collectively considered in defining the optimal experimental design that will allow us to anticipate, modify, or redefine the findings provided by the scarce spaceflight opportunities that have been (and will be) available.

Cell proliferation is a crucial cellular process which influences development. In plants, meristems are formed by actively proliferating cells, in which the main expression of proliferation is the existence of a cell division cycle. Many cell activities are influenced by the cell proliferation status and cell cycle progression, among them ribosome biogenesis, which is morphologically expressed as the nucleolus. The connection is established through nucleolar proteins, which regulate the synthesis and processing of preribosomal precursors and, at the same time, are targets of various cell cycle regulators, such as certain kinases. Nucleolin is one of these nucleolar proteins, whose level increases with cell proliferation and depends on the cell cycle stages. Not only the levels, but also other important features of the protein, such as its distribution in situ in the nucleolus, its phosphorylation and its physiological degradation, depend on these parameters. Furthermore, since the nucleolar structure is highly sensitive to functional variations, distinct nucleolar structures, regarding the nucleolar size and the distribution of nucleolar subcomponents, have been defined for each period of the cell cycle, using synchronized cells. In addition to increasing our knowledge of cellular physiology, these relationships can be used to mark the proliferative state of the cell and the periods of cell cycle.

16 p.-1 fig.

16 p.-4 fig.-2 tab., Simulated microgravity and partial gravity research on Earth is highly convenient for every space biology researcher due to limitations of access to spacefl ight. However, the use of ground-based facilities for microgravity simulation is far from simple. Microgravity simulation usually results in the need to consider additional environmental parameters which appear as secondary effects in the generation of altered gravity. These secondary effects may interfere with gravity alteration in the changes observed in the biological processes under study. Furthermore, ground-based facilities are also capable of generating hypergravity or fractional gravity conditions, which are worth being tested and compared with the results of microgravity exposure. Multiple technologies (2D clinorotation, random positioning machines, magnetic levitators or centrifuges), experimental hardware (proper use of containers and substrates for the seedlings or cell cultures), and experimental requirements (some life support/environmental parameters are more diffi cult to provide in certain facilities) should be collectively considered in defi ning the optimal experimental design that will allow us to anticipate, modify, or redefi ne the fi ndings provided by the scarce spacefl ight opportunities that have been (and will be) available., Most of the results and comments included in this book chapter have been the consequence of the authors’ participation in “ESA Access to GBF” Project Nos. 4200022650 and 4000105761 in close collaboration with GBF managers Dr. van Loon (DESC), Dr. Hemmersbach(DLR), Dr. Pereda-Loth (Toulouse University), Dr. Hill (Nottingham University), and Dr. Christianen (Nijmegen University). Work performed in the authors’ laboratory was financially supported by the Spanish Plan Nacional de Investigación Científica y Desarrollo Tecnológico, Grant Ref. No. AYA2012-33982.

Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

18 p.-3 fig., Tropisms are growth-mediated plant movements that help plants to respond to changes in environmental stimuli. The availability of water and light, as well as the presence of a constant gravity vector, are all environmental stimuli that plants sense and respond to via directed growth movements (tropisms). The plant response to gravity (gravitropism) and the response to unidirectional light (phototropism) have long been shown to be interconnected growth phenomena. Here, we discuss the similarities in these two processes, as well as the known molecular mechanisms behind the tropistic responses. We also highlight research done in a microgravity environment in order to decouple two tropisms through experiments carried out in the absence of a significant unilateral gravity vector. In addition, alteration of gravity, especially the microgravity environment, and light irradiation produce important effects on meristematic cells, the undifferentiated, highly proliferating, totipotent cells which sustain plant development. Microgravity produces the disruption of meristematic competence, i.e., the decoupling of cell proliferation and cell growth, affecting the regulation of the cell cycle and ribosome biogenesis. Light irradiation, especially red light, mediated by phytochromes, has an activating effect on these processes. Phytohormones, particularly auxin, also are key mediators in these alterations. Upcoming experiments on the International Space Station will clarify some of the mechanisms and molecular players of the plant responses to these environmental signals involved in tropisms and the cell cycle., Financial support was provided by the National Aeronautics and Space Administration through grants NNX10AM86G and NNX12AO65G to John Z. Kiss. Financial support to F. Javier Medina and Raul Herranz was provided by the Spanish “Plan Nacional de Investigación Científica, Desarrollo Tecnológico e Innovación” Grant Ref. No. AYA2012-33982, and by European Space Agency, Program “Access to Ground Based Facilities,” Grants Ref. Nos. 4200022650 and 4000105761.

9 p.-3 tab.-1 fig., Earth-based microgravity simulation techniques are required due to space research constraints. Using diamagnetic levitation, we exposed Arabidopsis thaliana in vitro callus cultures to environments with different levels of effective gravity and magnetic field strengths (B) simultaneously. The environments included simulated 0g* at B=10.1 T, an internal 1g* control (B=16.5 T), and hypergravity (2g* at B=10.1 T). Furthermore, samples were also exposed to altered gravity environments that were created with mechanical devices, such as the Random Positioning Machine (simulated μg) and the Large Diameter Centrifuge (2g). We have determined the proteomic signature of cell cultures exposed to these altered-gravity environments by means of the difference gel electrophoresis (DiGE) technique, and we have compared the results with microarray-based transcriptomes from the same samples. The magnetic field itself produced a low number of proteomic alterations, but the combination of gravitational alteration and magnetic field exposure produced synergistic effects on the proteome of plants (the number of significant changes is 3-7 times greater). Tandem mass spectrometry identification of 19 overlapping spots in the different conditions corroborates a major role of abiotic stress and secondary metabolism proteins in the molecular adaptation of plants to unusual environments, including microgravity. Copyright © 2013, Mary Ann Liebert, Inc. 2013., The authors acknowledge funding from the Spanish Space Program in the ‘‘Plan Nacional de Investigacion Cientifica y Desarrollo Tecnologico’’ AYA2009-07952, AYA2010-11834-E to F.J.M. and AYA2009-07792-E to R.H. RPM/LDC access was possible due to the Dutch Space Research Organization NWO-ALW-SRON grant MG-057 to J.vL. and the SEGMGSPE_Ph1 access to ESA GBF project contract 4200022650 to R.H., F.J.M., and J.vL. Magnetic levitation at the High Field Magnet Laboratory in Nijmegen was granted by Euro-MagNET II under the EU contract n 228043 and by the Stichting voor Fundamenteel Onderzoek der Materie (FOM),financially supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). PRIDE files have been generated in the CSIC CNB computational proteomics unit from CSIC & ProteoRed: Carlos III Networked Proteomics Platform.

17 p.-1 tab.-5 fig. Herranz et alt., Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms.However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities.Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature. Key Words: 2-D clinostat—3-D clinostat—Gravity—Magnetic levitation—Random positioning machine—Simulated microgravity—Space biology., The joint effort to compare our results has been funded by the ESA Access to Ground Based Facilities SEGMGSPE_Ph1 Project ‘‘Systematic Evaluation of the ground based (micro-) gravity simulation paradigms available in Europe.First Phase: Similarities and Differences between the different approaches (ESA contract 4200022650).’’ Nevertheless, primary research reviewed in this manuscript has been possible by grants from the Spanish Space Program in the ‘‘Plan Nacional de Investigacion Cientifica y Desarrollo Tecnologico’’AYA2009-07792-E to R.H., AYA2009-07952 and AYA2010-11834-E to F.J.M., German Space Administration grants on behalf of Bundesministerium fu¨ r Wirtschaft und Technologie 50WB0527 to R.H., 50WB0815 to M.B., 50WB0828 to M.L., 50WB0921 to O.U., and Dutch Space Research Organization NWO-ALW-SRON grant MG-057 to J.J.W.A.vL. Magnetic levitation at the High Field Magnet Laboratory in Nijmegen was granted by EuroMagNET II under the EU contract n 228043 and by the Stichting voor Fundamenteel Onderzoek der Materie (FOM), financially supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO). Research using the University of Nottingham’s superconducting magnet was supported by grants from the UK Engineering and Physical Sciences Research Council (EPSRC), Nos. GR/S83005/01 and EP/G037647/1. R.J.A.H acknowledges EPSRC for support under a Research Fellowship EP/I004599/1 and C-DIP grant EP/J005452/1.

Capítulo 41.-- Open Access: License CC BY 3.0, This work was supported by grants from the Spanish National Plan for Research, Development and Innovation (BFU 2008-00666/BMC, BFU 2009-08977 and AYA2009- 07952).