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Advancements in plant space biology are required for the realization of human space exploration missions, where the re-supply of resources from Earth is not feasible. Until a few decades ago, space life science was focused on the impact of the space environment on the human body. More recently, the interest in plant space biology has increased because plants are key organisms in Bioregenerative Life Support Systems (BLSS) for the regeneration of resources and fresh food production. Moreover, plants play an important role in psychological support for astronauts. The definition of cultivation requirements for the design, realization, and successful operation of BLSS must consider the effects of space factors on plants. Altered gravitational fields and radiation exposure are the main space factors inducing changes in gene expression, cell proliferation and differentiation, signalling and physiological processes with possible consequences on tissue organization and organogenesis, thus on the whole plant functioning. Interestingly, the changes at the cellular and molecular levels do not always result in organismic or developmental changes. This apparent paradox is a current research challenge. In this paper, the main findings of gravity- and radiation-related research on higher plants are summarized, highlighting the knowledge gaps that are still necessary to fill. Existing experimental facilities to simulate the effect of space factors, as well as requirements for future facilities for possible experiments to achieve fundamental biology goals are considered. Finally, the need for making synergies among disciplines and for establishing global standard operating procedures for analyses and data collection in space experiments is highlighted.

Following on from the NASA twins’ study there has been a tremendous interest in the use of omics techniques in spaceflight. The individual space agency’s, NASA’s GeneLab, JAXA´s ibSLS and ESA-funded Space Omics Topical Team and the International Standards for Space Omics Processing (ISSOP) group have established several initiatives to support this growth. Here, we present recommendations from the Space Omics Topical Team to promote standard application of space omics in Europe. We focus on four main themes: i) continued participation in and co-ordination with international omics endeavours; ii) strengthening of the European space omics infrastructure including workforce and facilities; iii) capitalizing on the emerging opportunities in the commercial space sector, and iv) capitalizing on the emerging opportunities in human subjects research.

Discovering the adaptation mechanisms of plants to the space environment is essential for supporting human space exploration. Transcriptomic analyses allow the identification of adaptation response pathways by detecting changes in gene expression at the global genome level caused by the main factors of the space environment, namely altered gravity and cosmic radiation. This article reviews transcriptomic studies carried out from plants grown in spaceflights and in different ground-based microgravity simulators. Despite differences in plant growth conditions, these studies have shown that cell wall remodeling, oxidative stress, defense response, and photosynthesis are common altered processes in plants grown under spaceflight conditions. European scientists have significantly contributed to the acquisition of this knowledge, e.g., by showing the role of red light in the adaptation response of plants (EMCS experiments) and the mechanisms of cellular response and adaptation mostly affecting cell cycle regulation, using cell cultures in microgravity simulators.

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 European research community, via European Space Agency (ESA) spaceflight opportunities, has significantly contributed towards our current understanding of spaceflight biology. Recent molecular biology experiments include “omic” analysis, which provides a holistic and systems level understanding of the mechanisms underlying phenotypic adaptation. Despite vast interest in, and the immense quantity of biological information gained from space omics research, the knowledge of ESA-related space omics works as a collective remains poorly defined due to the recent exponential application of omics approaches in space and the limited search capabilities of pre-existing records. Thus, a review of such contributions is necessary to clarify and promote the development of space omics among ESA and ESA state members. To address this gap, in this review we: i) identified and summarised omics works led by European researchers, ii) geographically described these omics works, and iii) highlighted potential caveats in complex funding scenarios among ESA member states.

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.

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 en

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.

Clinorotation was the first method designed to simulate microgravity on ground and it remains the most common and accessible simulation procedure. However, different experimental settings, namely angular velocity, sample orientation, and distance to the rotation center produce different responses in seedlings. Here, we compare A. thaliana root responses to the two most commonly used velocities, as examples of slow and fast clinorotation, and to vertical and horizontal clinorotation. We investigate their impact on the three stages of gravitropism: statolith sedimentation, asymmetrical auxin distribution, and differential elongation. We also investigate the statocyte ultrastructure by electron microscopy. Horizontal slow clinorotation induces changes in the statocyte ultrastructure related to a stress response and internalization of the PIN-FORMED 2 (PIN2) auxin transporter in the lower endodermis, probably due to enhanced mechano-stimulation. Additionally, fast clinorotation, as predicted, is only suitable within a very limited radius from the clinorotation center and triggers directional root growth according to the direction of the centrifugal force. Our study provides a full morphological picture of the stages of graviresponse in the root tip, and it is a valuable contribution to the field of microgravity simulation by clarifying the limitations of 2D-clinostats and proposing a proper use.

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.

Plants are a necessary component of any system of bioregenerative life-support for human space exploration. For this purpose, plants must be capable of surviving and adapting to gravity levels different from the Earth gravity, namely microgravity, as it exists on board of spacecrafts orbiting the Earth, and partial-g, as it exists on the surface of the Moon or Mars. Gravity is a fundamental environmental factor for driving plant growth and development through gravitropism. Exposure to real or simulated microgravity produces a stress response in plants, which show cellular alterations and gene expression reprogramming. Partial-g studies have been performed in the ISS using centrifuges and in ground based facilities, by implementing adaptations in them. Seedlings and cell cultures were used in these studies. The Mars gravity level is capable of stimulating the gravitropic response of the roots and preserving the auxin polar transport. Furthermore, whereas Moon gravity produces alterations comparable, or even stronger than microgravity, the intensity of the alterations found at Mars gravity was milder. An adaptive response has been found in these experiments, showing upregulation of WRKY transcription factors involved in acclimation. This knowledge must be improved by incorporating plants to the coming projects of Moon exploration.

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 micro- gravity 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 simula- tion facilities will allow us to anticipate, modify, or redefine the findings provided by the scarce available spaceflight opportunities.

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.

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.

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.

The coming enterprises of space exploration by humans, e.g. the future colonization of the Moon and Mars, will require the utilization of plants as key components of Bioregenerative Life Support Systems. The space environment is very different from the Earth environment in many factors. Many of these adverse factors can be counteracted in spaceships, or in Martian or Lunar settlements, but the living beings must adapt to grow and survive in microgravity. The Earth gravity has remained constant in magnitude and direction throughout the entire history of our planet, including biological evolution. Gravity establishes the direction of plant growth through the process called gravitropism and this orientation is essential for the normal function of roots, stems and leaves, assuring the adequate nutrition of the plant. The phytohormone auxin is known to play a main role in gravitropism. Auxin is also a major regulator of plant development, since this hormone is ultimately responsible of the maintenance of meristematic cells, which are totipotent cells, continuously engaged in the cell cycle, and are the suppliers of differentiated cells for plant development. Meristematic competence is the balance between cell growth and cell proliferation occurring in meristematic cells. A major effect of the microgravity environment is the disruption of meristematic competence, comprising the increase of the proliferation rate and the decrease of the growth rate,estimated through the rate of production of ribosomes, the cellular factories of proteins. Microgravity also induces a noticeable reprogramming of gene expression. In meristematic cells, genes driving the cell cycle regulation are affected. In general, the systems responsible of the plant defense against abiotic stresses and the energy/redox systems are major targets of the gene reprogramming. Noticeably, no specific genes related to gravity alteration have been identified, although a significant proportion of altered genes enco

[ENG]Space exploration will require life support systems, in which plants can provide nutrients, oxygen, moisture, and psychological well-being and eliminate wastes. In alien environments, plants must adapt to a different gravity force, even the zero gravity of spaceflight. Under these conditions, essential cellular and molecular features related to plant development are altered and changes in gene expression occur. In lunar gravity, the effects are comparable to microgravity, while the gravity of Mars produces milder alterations. Nevertheless, it has been possible to develop and reproduce plants in space. Current research seeks to identify signals replacing gravity for driving plant growth, such as light. Counteracting gravitational stress will help in enabling agriculture in extraterrestrial habitats., [ES]La exploración espacial requerirá sistemas de soporte vital que incluyan plantas para proporcionar nutrientes, oxígeno, humedad y bienestar psicológico, y que sirvan además para eliminar desechos. En entornos extraterrestres, las plantas se han de adaptar a una gravedad diferente e incluso a la gravedad cero de los vuelos espaciales. En estas condiciones se alteran las características celulares y moleculares relacionadas con el desarrollo de las plantas y se producen cambios en la expresión génica. En la gravedad lunar, los efectos son comparables con la microgravedad, mientras que la gravedad de Marte provoca alteraciones más leves. Sin embargo, ya ha sido posible desarrollar y reproducir plantas en el espacio. Las investigaciones actuales tratan de identificar señales, como la luz, que reemplacen a la gravedad como impulsora del crecimiento vegetal. Contrarrestar el estrés gravitatorio ayudará a hacer posible la agricultura en hábitats extraterrestres., [CAT] L’exploració espacial requerirà sistemes de suport vital que incloguen plantes per a proporcionar nutrients, oxigen, humitat i benestar psicològic, i que servisquen a més per a eliminar deixalles. En entorns extraterrestres, les plantes s’han d’adaptar a una gravetat diferent i fins i tot a la gravetat zero dels vols espacials. En aquestes condicions s’alteren les característiques cellulars i moleculars relacionades amb el desenvolupament de les plantes i es produeixen canvis en l’expressió gènica.En la gravetat lunar, els efectes són comparables amb la microgravetat, mentre que la gravetat de Mart provoca alteracions més lleus. No obstant això, ja ha estat possible desenvolupar i reproduir plantes en l’espai. Les investigacions actuals tracten d’identificar senyals, com la llum, que reemplacen la gravetat com a impulsora del creixement vegetal. Contrarestar l’estrès gravitatori ajudarà a fer possible l’agricultura en hàbitats extraterrestres.

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.

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 qPCR 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 remodeling. 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.

On 20th July 1969, the Fresnedillas Control Station, near Madrid, received the first words of a human from the surface of the Moon. “That’s one small step for [a] man, one giant leap for mankind”, was the historical sentence recorded from Neil A. Armstrong, commander of the “Apollo XI” mission. Nowadays, fifty years after Armstrong’s epic achievement, space exploration by humans is commonly recognized as a highly exciting and attractive challenge and a powerful booster for scientific and technological progress in order to improve the human life on Earth (NASA et al. 2018). This is true despite some criticisms (minority, but significant) that question the high costs that it entails (Rinaldi 2016). The establishment of permanent settlements in the Moon and Mars is becoming a realistic venture day by day. After a decade of successful rover explorations to the surface of Mars (Voosen 2018), both ESA and NASA, and more recently the agencies from growing economies in Asian countries, are working to promote a manned mission, first to the Moon, and then to Mars. The European Space Agency (ESA), of which Spain is an active member, adheres to these objectives and is strongly committed in supporting and participating in these programs. The main aim of space life science is to understand how the space environment, and specifically altered gravity and radiation, affects the morphology, physiology and behaviour of living organisms, and to design countermeasures to enable terrestrial life, and particularly human life, to develop outside Earth. That is, how they perceive and respond to gravity and radiation and adapt to the space environment. There is a variety of disciplines, such as genetics, molecular, anatomical or physiological fields, which use a range of technologies to address these issues. In order to understand adaptations at the functional level it is necessary to comprehend adaptations at cellular and tissue levels. Also, basic research analysing biomolecules, cells and