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28 p.-7 fig.-3 tab., 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., Work performed in the authors’ laboratory was financially supported by the Spanish Plan Estatal de Investigación Científica y Desarrollo Tecnológico, Grants #ESP2015-64323-R and #RTI2018-099309-B-I00 (co-funded by EU-ERDF) to F.J.M. and a grant from European Space Agency contract# 4000107455/12/NL/PA awarded to J.J.W.A.v.L.

50 p.-8 fig.-8 fig. supl.-2 tab.supl., 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., 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 [Grant numbers AYA2012-33982 and ESP2015-64323-R, co-funded by ERDF], by a predoctoral 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.

6 p.-6 fig., 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 1g controls with respect to suspension cultures. Standard 1g 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 1g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1g controls., This work was supported by grants of the Spanish National Plan for Research and Development, Ref. Nos. AYA2010-11834-E, and AYA2012-33982 and by European Space Agency (ESA) Access to GBFs contract numbers 4200022650 & 4000105761. KYK was sup-ported by the Spanish Consejo Superior de Investigaciones Científicas (CSIC) JAE-PreDoc Program (Ref. JAEPre_2010_01894)

292 p.-92 fig.-21 tab., [EN] Gravity is a key environmental cue for life on Earth, the only one that has remained constant throughout evolution. Environmental gravity is a particular challenge for the growth of terrestrial plants and alteration of absence of gravity is a novel and phylogenetically unknown environmental change for them. The effects of a change in the environmental gravity on Arabidopsis root meristematic cells have been approached up to now in our laboratory in a relatively small number of microgravity experiments performed in space and in ground based facilities (GBFs). A disruption of the coordination of cell growth and proliferation was shown to occur in response to altered gravity in these undifferentiated, highly proliferating cells. This response may be specifically triggered in the meristem by general mechanisms of graviresponse, such as graviresistance and gravitropism, including altered regulation of auxin polar transport, although the specific mechanisms of gravity sensing and response which operating on meristematic cells are unknown. In this work we aim to define whether these gravity responses are purely cellular, or depend on the tissue level. To achieve this objective we have used a biological model system consisting of Arabidopsis thaliana cell cultures in vitro, in which neither specialized structures for gravity sensing, such as statoliths, nor extracellular signal transduction pathways, like auxin polar transport, are known to be present. Novel cellular and molecular methods, available for the in vitro cell culture model, have been used to get a deeper understanding on the mechanisms operating at individual cells to alter cell growth and proliferation under gravitational stress. To disclose the impact of gravity on the plant biological processes, we require suitable ground based facilities (GBFs) for microgravity simulation. GBFs are valuable tools for preparing spaceflight experiments, and they also serve as stand-alone platforms for gravitational research. First of all, we performed a systematic and comparative study of the suitability of several available GBFs to provide a reliable microgravity simulation for plant cell cultures. The 2D pipette clinostat and magnetic levitation facilities were found to do not comply with our requirements. The method of choice consisted of a first embedding of cells in agarose followed by incubation, either in the Random Positioning Machine (RPM; microgravity), or in the Large Diameter Centrifuge (LDC; hypergravity). This immobilization approach, adapted and developed by us, has proved successful to reproduce several altered gravity environments while preserving plant cell culture viability. Plus, novel modes of operation of the RPM (RPMHW and RPMSW) were assayed for partial g simulation. This approach, directed towards the simulation of the Moon and Mars gravity conditions, yielded interesting data and revealed as highly promising., We exposed in vitro cell cultures to different levels of altered gravity to study cell growth, cell proliferation and whole genome effects, including transcriptomic changes and chromatin remodeling. Asynchronous cell cultures were exposed to the simulated microgravity (RPM), the Moon (RPMHW), Mars (RPMSW), and 2g hypergravity (LDC), for 3h, 14h and 24h. Cell growth and cell proliferation were similarly uncoupled by altered gravity as in meristematic cells from seedlings. These alterations were stronger under hypogravity conditions, while the hypergravity effect was weaker. Distribution of cell cycle phases was gradually disrupted through the exposure time, in addition to cell cycle regulators; Cyclin B1 expression was altered, while the antigen “Prolifera” was increased. Ribosome biogenesis was decreased as inferred by decreased nucleolin and fibrillarin levels, and the increased number of inactive nucleoli. In addition, an effect on the epigenetic regulation of gene expression was noted, including increased DNA methylation and depleted histone acetylation. The use of aphidicolin synchronization allowed a deep study of each cell cycle phase and of the cell proliferation rate through a period of 72h. Under 1g control we linked the morphofunctional features of the nucleolus to cell cycle phases. Compact nucleoli appeared at G1 phase and S phase (double sized). The G2 phase was characterized by large nucleoli, some of them vacuolated, and by the highest nucleolar protein levels, reflecting a high rate of ribosome synthesis. Under simulated microgravity nucleolar activity was reduced versus 1g, especially during G2/M. Furthermore, in these conditions, the extra-nucleolar transcription by RNA polymerase II was depleted, while condensed chromatin increased. Cell cycle acceleration was demonstrated, being particularly observed at the G2/M phase. The length of this phase showed a considerable variation in the different gravity conditions studies, whose arrangement, from shortest to longest was: the Moon (simulated), microgravity (simulated), Mars (simulated), Earth, and hypergravity. Consequently, G2/M checkpoint disruption was considered a key cell cycle target for altered gravity effects., Finally, transcriptomic analyses were performed. Global transcriptome response to simulated microgravity was obtained in both G1 and G2/M subpopulations of synchronous cultures and in asynchronous cultures after 14h of incubation, being generally repressed. Differential GO groups affected were abiotic stress, cell cycle regulation and mitochondrial (unknown function) genes. In fact, G2/M checkpoint genes were clearly downregulated, causing an accelerated cell cycle, while the G1 checkpoint genes were slightly upregulated, allowing the cell to partially compensate the acceleration. Plant response to microgravity alteration works through a unique and complex mechanism; differential activation of several environmental stress pathways suggests synergistic effects at different cell cycle subpopulations. A new pathway, including mitochondrial genes, has been involved in altered gravity responses, maybe connected to additional mitochondrial activity and ROS production as a rapid response to microgravity. An additional methodological contribution of this work was the production of adapted protocols and materials for future spaceflight research. The definition of morphofunctional nucleolar models, together with the development of transgenic fluorescent Arabidopsis cultures will allow the use of “in vivo” observation of the samples by microscopic techniques. They can be implemented in ISS experiments to enhance the scientific outcomes of the space biology programs., The conclusions we have reached from this Doctoral Thesis Work are as follows: 1. We have found that the best method to expose a plant cell culture system to altered gravity environments, using ground based facilities, is the immobilization by agarose embedding. This method preserves cell viability, allows cell synchronization and avoids unwanted mechanical stimuli. 2. Using the biological system of in vitro plant cell culture, the best instruments to reproduce several altered gravity environments were the Random Positioning Machine (RPM), for simulated microgravity, the Large Diameter Centrifuge (LDC) for hypergravity, and the modified RPM, based on Hardware or Software modes of operation, for simulated partial g, i.e. levels of gravity between 0 and 1, comprising the Moon and Mars gravity conditions. In this latter case, further research is required to confirm their interchangeability. Other instruments tested have not given satisfactory results. The 2D Pipette Clinostat, often used for animal cellular systems, is not suitable, especially for long term experiments, due to technical problems in the 1g control samples (including cell viability issues). The magnetic levitation approach, while quite versatile in terms of partial g simulation, raises important concerns due to the high energy magnetic fields, and also to technical problems in the 0g* alternative experiences. 3. Other important technical achievements have also resulted from the work with plant cell cultures under altered gravity conditions. These achievements include: a. The adaptation of powerful cell biology techniques to be used in our Arabidopsis in vitro model system for the first time: morphofunctional nucleolar models, EdU labelling assay, cell synchronization and quantitative colocalization techniques. b. The establishment of new transgenic cell cultures, which have been successfully derived from previously established mutant/marker lines., 4. Similarly to the effects observed in root meristematic cells of seedlings exposed to microgravity, either real (“Root” experiment performed in the ISS) or simulated, the coordination of fundamental plant cell developmental processes is disrupted under reduced gravity conditions (simulated microgravity and partial gravity - the Moon and Mars) in plant cell cultures, while hypergravity (2g) produces an opposite and weaker misbalance on the plant cell growth and proliferation equilibrium. 5. Cell cycle is accelerated under simulated microgravity and the Moon conditions, due to relaxation of the cell cycle checkpoints, particularly at the G2/M transition, resulting in a higher cell proliferation rate. This proliferation increase is accompanied by a reduced cell size, corroborated by a depleted nucleolar activity, taken as an estimation of cell growth. 6. Cell cycle is decelerated under hypergravity conditions, producing a significantly longer cell cycle. While cell size and some cell growth parameters are not significantly affected, an increase in the nucleolar activity is inferred from the analysis of nucleolar models. 7. Mars gravity conditions produce an intermediate effect: while cell proliferation is initially increased, due to a shorter G2/M phase, and cell growth is decreased (as in other reduced gravity conditions), G1 phase is particularly extended to produce a longer cell cycle, thus resembling the effect of hypergravity. 8. The effects of altered gravity on the plant cell culture in vitro should be explained in the context of a system without known professional gravisensitive cells, such as seedling statoliths. A likely interpretation should take into account the unspecific graviresistance mechanism, which involves gravity sensing by non-differentiated cells, but a universal, still unknown mechanism of gravity perception would also play a prominent role. The existence of this mechanism is supported by the plethora of known physiological processes which involve cell polarity or spatial organization of cells. The overlapping of different systems of graviperception could lead to “confusing” the system with contradictory signals, which could explain the results of the partial gravity simulation, somehow striking. 9. An extensive effect of simulated microgravity at the overall genome level has been differentially detected through the cell cycle phases. Transcriptomic responses have been confirmed by proteomic analyses and this is consistent with epigenetic modifications.10. Epigenetic modifications, both hypermethylation of DNA and histone deacetylation, are a key component in the regulation of gene expression that allows the plant cells to cope with altered gravity environments. Chromatin modifications and remodeling effects are probably influencing the altered progression rates of Arabidopsis cell cycle, since variable condensed/decondensed chromatin states have been observed through the cell cycle.11. Plant response to altered gravity, rather than being based in a small group of genes or transduction pathways, relies in a complex mechanism, characterized by a unique response against a novel environmental stress, suggesting a synergistic effect which combines elements of multiple abiotic stress pathways. The implication of these results for sustainable agriculture on Earth and in Life Support Systems in space is certain., [ES] La gravedad es el único factor ambiental, esencial para el desarrollo de la vida en la tierra, que ha permanecido constante durante la evolución. La gravedad es un desafío constante para el crecimiento de las plantas terrestres y la alteración o la ausencia de la gravedad es un cambio ambiental nuevo y filogenéticamente desconocido para estos seres vivos. Los efectos del cambio de la gravedad ambiental sobre las células meristemáticas de Arabidopsis se han abordado en nuestro laboratorio en un número relativamente pequeño de experimentos en microgravedad en el Espacio y en instalaciones de simulación en Tierra, en los que hemos descubierto una importante alteración de la coordinación entre la proliferación y el crecimiento celular que es característica de estas células indiferenciadas, altamente proliferantes. En la base de estas alteraciones se encuentran mecanismos específicos de respuesta de la planta a la señal gravitatoria, como la graviresistencia o el gravitropismo, en los que está implicada la regulación del transporte polar de auxinas, aunque el mecanismo específico que opera en las células meristemáticas es desconocido. El objetivo principal de esta Tesis Doctoral es definir si estas respuestas gravitatorias son puramente celulares o dependen de la organización tisular, y para ello hemos utilizado un sistema modelo de cultivo celular in vitro de plantas de Arabidopsis thaliana, en el que no se conoce la presencia de estructuras especializadas para la gravisensibilidad, como los estatolitos, ni la de cascadas de señalización tisular, como el transporte de auxinas. Mediante nuevos abordajes experimentales, disponibles en este modelo en cultivo in vitro, se pretende investigar la respuesta de las células individuales al estrés gravitatorio, concretada en los procesos de proliferación y crecimiento celular y en los mecanismos implicados., Para desvelar el impacto de la gravedad en los procesos biológicos de las plantas se precisan instalaciones de microgravedad simulada en tierra (Ground Based Facilities - GBF) adecuadas. Las GBF son valiosas para la preparación de experimentos espaciales, y también como instalaciones independientes en investigación gravitacional. En primer lugar hemos realizado un estudio comparativo y sistemático de la idoneidad de varias GBFs para proporcionar una simulación de microgravedad fiable para los cultivos de células vegetales en suspensión. Los estudios realizados en el clinostato de pipetas 2D y en las instalaciones de levitación magnética mostraron que estos dispositivos no daban una respuesta totalmente satisfactoria a los requerimientos exigidos. El método finalmente seleccionado consistió en la inclusión de las células en agarosa previa a su incubación en la Máquina de Posicionamiento Aleatorizado (Random Positioning Machine - RPM) para experimentos en microgravedad simulada, o en la Centrifuga de Gran Diámetro (Large Diameter Centrifuge - LDC), para experimentos en hipergravedad. El abordaje experimental de inmovilización del cultivo en suspensión, adaptado y desarrollado en este trabajo, ha sido un éxito, tanto por su reproducibilidad de los ambientes de gravedad alterada como por la preservación de la viabilidad del cultivo celular de plantas. Además, se han investigado nuevos modos de operación de la RPM (RPMHW y RPMSW) para obtener gravedad parcial (entre 0g y 1g). Esta nueva capacidad del dispositivo, enfocada a la simulación de las condiciones de la Luna y Marte, ha dado resultados de gran interés y promete nuevos avances de la investigación en esta línea. Los cultivos celulares in vitro de Arabidopsis se expusieron a diferentes niveles de gravedad alterada para estudiar el crecimiento y la proliferación celular así como los efectos a nivel de genoma completo, incluidos los cambios transcriptómicos y el remodelado de la cromatina. Los cultivos celulares asincrónicos se expusieron a las condiciones simuladas de microgravedad (RPM), la gravedad de la Luna (RPMHW), la de Marte (RPMSW), así como a la hipergravedad 2g (LDC) durante 3h, 14h y 24h. El crecimiento y la proliferación celular se desacoplaron de forma similar a lo observado en células meristemáticas de plántulas. Estas alteraciones fueron mayores en condiciones de gravedad reducida, pero más leves en hipergravedad. La distribución de células en las fases del ciclo celular se fue alterando gradualmente con el tiempo de exposición; además, se observaron cambios en la expresión de genes reguladores del ciclo celular, como Ciclina B1 o el antígeno “Prolifera”. La biogénesis de ribosomas disminuyó, según mostró la disminución en los niveles de nucleolina y fibrilarina, y el aumento en el número de nucleolos inactivos. Además, se detectó un efecto sobre la regulación epigenética de la expresión génica, comprendiendo un aumento en la metilación del DNA y una disminución en la acetilación de histonas., El uso de sincronización por afidicolina permitió un estudio en profundidad de cada una de las fases del ciclo celular y de la tasa de proliferación celular, a lo largo de un período de 72 h. En condiciones de gravedad 1g control se definió un patrón ultraestructural concreto del nucleolo y de sus subcomponentes para cada fase del ciclo celular. Los nucleolos del tipo morfológico compacto aparecieron en las fases G1 y S, en esta última con un tamaño incrementado al doble. La fase G2 se caracterizó por nucleolos de gran tamaño, algunos de ellos del tipo vacuolado, y por los más altos niveles de las proteínas nucleolares nucleolina y fibrilarina, como reflejo de una elevada tasa de síntesis de ribosomas. En condiciones de microgravedad simulada la actividad nucleolar descendió respecto al control 1g, sobre todo en la subpoblación G2/M. Además, la trascripción extra-nucleolar por la RNA polimerasa II se redujo y se encontró un incremento en la proporción de cromatina condensada. Se demostró la aceleración del ciclo celular, debida esencialmente a un acortamiento de la fase G2/M. Este periodo muestra una duración variable, dependiendo de los niveles de gravedad, cuya ordenación, de menor a mayor es: la Luna (simulada), microgravedad (simulada), Marte (simulada), La Tierra hasta el periodo más largo en el caso de la hipergravedad. Por tanto, el punto de control G2/M aparece como una diana clave para los efectos de la gravedad alterada sobre el ciclo celular y su perturbación parece ser la causa principal de los cambios en la duración del ciclo inducidos por los cambios gravitatorios. Por último se realizaron estudios sobre los cambios en el transcriptoma que aparecían tras la exposición a la gravedad alterada de los cultivos celulares in vitro. Comparamos la respuesta global del genoma a la microgravedad simulada, tanto en las subpoblaciones G1 y G2/M de cultivos sincrónicos, como en cultivos asincrónicos expuestos durante 14 h, apareciendo la transcripción global generalmente reprimida en todos ellos. Los principales grupos GO que aparecieron afectados incluyeron genes de estrés abiótico, regulación del ciclo celular y genes mitocondriales de función desconocida. De hecho, los genes que regulan el punto de control G2/M se mostraron claramente reprimidos, provocando un ciclo celular acelerado, mientras que la regulación del punto de control G1 estaba levemente potenciada, permitiendo una recuperación parcial de la aceleración del ciclo originada en la transición G2/M. En consecuencia, la respuesta de las plantas a la microgravedad opera a través de un mecanismo único y complejo; la activación diferencial de varias rutas del estrés ambiental sugiere un efecto sinérgico en diferentes subpoblaciones del ciclo celular. Una nueva ruta de señalización, que incluye genes mitocondriales, se ha relacionado con la respuesta a la gravedad alterada, posiblemente en conexión con la actividad mitocondrial y producción adicional de radicales libres (ROS) como respuesta rápida a la microgravedad., Una contribución adicional de este trabajo a los procedimientos metodológicos de nuestro laboratorio es la producción de protocolos y materiales adaptados para próximos experimentos espaciales. La definición de modelos nucleolares morfofuncionales, junto al desarrollo de cultivos transgénicos fluorescentes de Arabidopsis permitirá el uso de muestras in vivo para observación microscópica. Su implementación en experimentos de vuelo a la ISS potenciará los retornos científicos futuros de los programas de biología espacial. Las conclusiones obtenidas de este trabajo de Tesis Doctoral son las siguientes: 1. Hemos comprobado que el mejor método para exponer un cultivo celular de plantas a un ambiente de gravedad alterada, en instalaciones de simulación en tierra, es la inmovilización por inclusión en agarosa. Este método mantiene la viabilidad celular, permite la sincronización celular y evita estímulos mecánicos no deseados. 2. Mediante el sistema biológico de cultivo celular vegetal in vitro, las instalaciones que mejor reproducen varios ambientes de gravedad alterada fueron la Máquina de Posicionamiento Aleatorizado (RPM), para microgravedad simulada, la Centrífuga de Gran Diámetro (LDC) para hipergravedad, y una versión de la RPM que usa modos de funcionamiento nuevos basados en Hardware o Software, para simular gravedad parcial, es decir, niveles de gravedad entre 0 y 1g, que incluyen las condiciones gravitatorias de La Luna y de Marte. En este último caso, se requiere investigación adicional para confirmar la equivalencia de los equipamientos. Otros instrumentos utilizados no han proporcionado resultados satisfactorios. El clásico clinostato 2D de pipetas para sistemas celulares no es adecuado, especialmente para experimentos a largo plazo, debido a problemas técnicos de los controles 1g (incluida una viabilidad celular comprometida). El abordaje de levitación magnética, aunque muy versátil para simulación de gravedad parcial, despierta importantes preocupaciones tanto por la presencia de campos magnéticos muy intensos como por los problemas técnicos de varias simulaciones alternativas para 0g*., 3. Otros avances técnicos conseguidos durante la realización de esta tesis con cultivos celulares de plantas en condiciones de gravedad alterada son: a. La adaptación de potentes técnicas de biología celular a nuestro sistema modelo in vitro de Arabidopsis por primera vez: los modelos morfofuncionales del nucléolo, el ensayo de tinción con EdU, la sincronización celular y técnicas cuantitativas de colocalización. b. La obtención de nuevos cultivos transgénicos, que han sido derivados con éxito desde líneas mutantes o con genes marcadores prestablecidas en nuestro grupo. 4. Tal y como se observó en las células meristemáticas de la raíz de plántulas expuestas a microgravedad, tanto real (Experimento “Root” en la ISS) o simulada, la coordinación de los procesos celulares fundamentales en el desarrollo de las plantas se pierde en condiciones de gravedad reducida (microgravedad simulada y gravedad parcial; la Luna y Marte) en cultivos celulares de planta, mientras que la hipergravedad (2g) produce un desequilibrio menor y en sentido opuesto entre el crecimiento y la proliferación celular. 5. El ciclo celular se acelera en las condiciones de microgravedad simulada y la Luna, debido a una relajación en los puntos de control del ciclo celular, concretamente en la transición G2/M, causando una mayor tasa de proliferación celular. Este aumento en la proliferación se acompaña de una reducción en el tamaño celular, corroborado por una actividad nucleolar disminuida, considerada como una estimación del crecimiento celular. 6. El ciclo celular se retrasa en las condiciones de hipergravedad, causando un ciclo celular significativamente más largo. Aunque el tamaño celular y algunos parámetros del crecimiento celular no están afectados significativamente, se puede inferir un aumento en la actividad nucleolar cuando se analiza la distribución de los modelos nucleolares a 2 g. 7. Las condiciones gravitatorias de Marte producen un efecto intermedio; aunque la proliferación celular aumenta inicialmente, debido a un periodo G2/M acortado, y el crecimiento celular disminuye (como en otras condiciones de gravedad reducida), la fase G1 es particularmente extendida hasta el punto de promover un ciclo celular más largo, por lo tanto recordando el efecto de la hipergravedad., 8. Los efectos de la gravedad alterada en cultivos celulares vegetales in vitro se debería explicar en el contexto de un sistema en el que las células gravisensibles profesionales, como los estatolitos de las plántulas, están ausentes o se desconocen. Una explicación probable sería considerar que el mecanismo inespecífico de la graviresistencia implica la gravisensibilidad en las células no diferenciadas. Por otro lado, un mecanismo universal, aun no descrito para la percepción de la gravedad, que sería responsable de la plétora de procesos fisiológicos que afectan a la polaridad celular y la organización espacial de las células, también podría estar jugando un papel fundamental. El solapamiento de diferentes sistemas de gravipercepción podría provocar la “confusión” del sistema con señales contradictorias, lo que explicaría los resultados de la gravedad parcial simulada. 9. La microgravedad simulada provoca un efecto extensivo y diferencial durante las fases del ciclo celular a nivel de genoma completo. Las respuestas transcriptómicas han sido confirmadas por análisis proteómicos y concuerdan con las modificaciones epigenéticas. 10. Las modificaciones epigenéticas, tanto la hipermetilación del DNA como la deacetilación de histonas, son componente clave de la regulación de la expresión génica que permite a las células vegetales lidiar con ambientes de gravedad alterada. Las modificaciones y la remodelación de la cromatina probablemente están relacionados con la alteración de las tasas de proliferación del ciclo celular de Arabidopsis, dado que el estado de condensación/decondensación de la cromatina varía con la progresión del ciclo celular. 11. La respuesta de las plantas a la gravedad alterada, más que basarse en un pequeño grupo de genes o cascadas de transducción especificas descansa en un mecanismo complejo, caracterizado por una respuesta única ante un estrés ambiental novedoso, que sugiere un efecto sinérgico que combina elementos de muchas vías de respuesta a estrés abiótico. La implicación de estos resultados a la agricultura sostenible en la Tierra y los sistemas de soporte vital en el Espacio es evidente., Becas JAE-preDoc 2010. Ministerio de Economía y Competitividad (AYA2009-07952 & AYA2012-33982). Proyectos de la Agencia Espacial Europea (GBF proyectos # 4000105761 & 4200022650) y la Unión Europea (EUROMAGNET II Project 2010.17 (NSO06-209)

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).

39 p.-7 fig.-2 tab.-9 tab. supl., Microgreens are rich functional crops with valuable nutritional elements that have health benefits when used as food supplements. Growth characterization,nutritional composition profile of 21 varieties representing five species of the Brassica genus asmicrogreens were assessed under light-emitting diodes(LEDs) conditions. Microgreens were grown under four different LEDs ratios(%); red:blue 80:20 and 20:80 (R80:B20 and R20:B80), or red:green:blue 70:10:20 and 20:10:70 (R70:G10:B20 and R20:G10:B70). Results indicated that supplemental lighting with green LEDs (R70:G10:B20) enhanced vegetative growth and morphology, while blue LEDs (R20:B80) increased the mineral and vitamin contents. Interestingly, by linking the nutritional content with the growth yield to define the optimal LEDs setup, we found that the best lighting to promote the microgreen growth was the green LEDs combination (R70:G10:B20). Remarkably, under the green LEDs combination (R70:G10:B20) conditions,the microgreens of Kohlrabi purple, Cabbage red, Broccoli, Kale Tucsan, Komatsuna red, Tatsoi and Cabbage green, which can benefit human health in conditions with limited food, had the highest growth and nutritional content., This research work is a part of a project received seed funding from the Dubai Future Foundation through the Guaana.com open research platform(grant no. MBR026). Dr. Mortaza is supported from ERDF project “Plants as a tool from sustainable global development” No. CZ.02.1.01/0.0/0.0/16_019/0000827.

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.