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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.

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).

The few studies on the evolution of epiphytism in ferns have mostly focused on xerophytic and humus-collecting strategies, neglecting hygrophytes that are abundant in rainforests, such as the trichomanoids (Hymenophyllaceae). Using a phylogenetic approach, we studied the acquisition of epiphytism in this lineage, with the aim of identifying ecological anatomical adaptations and verifying the regressive epiphytic ‘bryophyte-like’ strategy previously suggested for the group. Inferred evolution of anatomy and morphology, regression and ecology (more particularly colonial epiphytism) were analysed and compared using a maximum likelihood approach. Regressive evolution of anatomy and morphology is revealed in the three clades of colonial epiphytes, probably linked to the selection of water acquisition by blades rather than by regressed roots. However, the ‘bryophyte-like’ strategy is restricted to some taxa (especially Didymoglossum). Furthermore, a relationship is revealed between large metaxylem and climbing habit. Diversification of colonial epiphytes (and some individual epiphytes) and hemi-epiphytism would have occurred in the upper Cretaceous and Tertiary, in accordance with the timing of diversification of modern ferns and the evolution of epiphytism in other fern families in the first angiosperm-dominated forests. This was here performed by selecting hygrophilous strategies that are unique in vascular plants. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 573–593.

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].

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

The dose-response curve of the gravitropic reaction is often used to evaluate the gravisensing of plant organs. It has been proposed (Larsen 1957) that the response (curvature) varies linearly as a function of the logarithm of the dose of gravistimulus. As this model fitted correctly most of the data obtained in the literature, the presentation time (tp, minimal duration of stimulation in the gravitational field to induce a response) or the presentation dose (dp, minimal quantity in g.s of stimulation to induce a response) were estimated by extrapolating down to zero curvature the straight line representing the response as a function of the logarithm of the stimulus. This method was preferred to a direct measurement of dp or tp with minute stimulations, since very slight gravitropic response cannot be distinguished from the background oscillations of the extremity of the organs. In the present review, it is shown that generally the logarithmic model (L) does not fit the experimental data published in the literature as well as the hyperbolic model (H). The H model in its simplest form is related to a response in which a ligand-receptor system is the limiting phase in the cascade of events leading to the response (Weyers et al. 1987). However, it is demonstrated that the differential growth, responsible for the curvature (and the angle of curvature), would vary as a hyperbolic function of the dose of stimulation, even if several steps involving ligand-receptor systems are responsible for the gravitropic curvature. In the H model, there is theoretically no presentation time (or presentation dose) since the curve passes through the origin. The value of the derivative of the H function equals a/b and represents the slope of the cune at the origin. It could be therefore used to estimate gravisensitivity. This provides a measurement of graviresponsiveness for threshold doses of stimulation. These results imply that the presentation time (or presentation dose) derived from the L model cannot be used anymore as an estimate of gravisensitivity. On the contrary, the perception time (minimal duration of a repeated stimulation which induces a response), which is less than 1 s, should be related to the perception of gravity. The consequences of these results on the mode of action and the nature of graviperception are discussed.

11 p.-8 fig.-3 tab., Space experiments provide a unique opportunity to advance our knowledge of how plants respond to the space environment, and specifically to the absence of gravity. The European Modular Cultivation System (EMCS) has been designed as a dedicated facility to improve and standardise plant growth in the International Space Station (ISS). The EMCS is equipped with two centrifuges to perform experiments in microgravity and with variable gravity levels up to 2.0 g. Seven experiments have been performed since the EMCS was operational on the ISS. The objectives of these experiments aimed to elucidate phototropic responses (experiments TROPI-1 and -2), root gravitropic sensing (GRAVI-1), circumnutation (MULTIGEN-1), cell wall dynamics and gravity resistance (Cell wall/Resist wall), proteomic identification of signalling players (GENARA-A) and mechanism of InsP3 signalling (Plant signalling). The role of light in cell proliferation and plant development in the absence of gravity is being analysed in an on-going experiment (Seedling growth). Based on the lessons learned from the acquired experience, three preselected ISS experiments have been merged and implemented as a single project (Plant development) to study early phases of seedling development. A Topical Team initiated by European Space Agency (ESA), involving experienced scientists on Arabidopsis space research experiments, aims at establishing a coordinated, long-term scientific strategy to understand the role of gravity in Arabidopsis growth and development using already existing or planned new hardware., Experimental work reported in this paper and performed in the authors’ laboratories was supported by the French Space Agency (CNES), the Norwegian Research Council, the Spanish National Plan for Research, Development and Innovation (different grants) and the ELIPS Programme of the European Space Agency (ESA). Specifically, the activities related to the ‘Arabidopsis Topical team’ were supported by an ESA grant.

18 p.-8 fig.-2 tab. Mazars, Christian et alt., The ‘‘GENARA A’’ experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in mg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in mg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected., Funding was supported by CNRS -CNES - Université Paul Sabatier.

Because symbionts are experiencing major morphological changes during ectomycorrhiza development, the expression of genes encoding cytoskeletal proteins is likely altered. To test this contention, we have cloned and characterized in a alpha-tubulin cDNA (EgTubA1) from Eucalyptus globulus. A poorly-aggressive isolate (No. 270) of the ectomycorrhizal basidiomycete Pisolithus tinctorius caused no changes in root transcript levels of EgTubA1, whereas a drastic up-regulation in its expression was observed between 3 to 4 days after contact with the aggressive isolate 441. This enhanced alpha-tubulin expression coincided with the increase lateral root formation induced by fungal colonisation. The changes in alpha-tubulin expression support a role for cytoskeleton components in ectomycorrhiza development.

5 páginas -- PAGS nros. 169-174, The study of the modifications induced by altered gravity in functions of plant cells is a valuable tool for the objective of the survival of terrestrial organisms in conditions different from those of the Earth. We have used the system “cell proliferation–ribosome biogenesis”, two inter-related essential cellular processes, with the purpose of studying these modifications. Arabidopsis seedlings belonging to a transformed line containing the reporter gene GUS under the control of the promoter of the cyclin gene CYCB1, a cell cycle regulator, were grown in a Random Positioning Machine, a device known to accurately simulate microgravity. Samples were taken at 2, 4 and 8 days after germination and subjected to biometrical analysis and cellular morphometrical, ultrastructural and immunocytochemical studies in order to know the rates of cell proliferation and ribosome biogenesis, plus the estimation of the expression of the cyclin gene, as an indication of the state of cell cycle regulation. Our results show that cells divide more in simulated microgravity in a Random Positioning Machine than in control gravity, but the cell cycle appears significantly altered as early as 2 days after germination. Furthermore, higher proliferation is not accompanied by an increase in ribosome synthesis, as is the rule on Earth, but the functional markers of this process appear depleted in simulated microgravity-grown samples. Therefore, the alteration of the gravitational environmental conditions results in a considerable stress for plant cells, including those not specialized in gravity perception

The GRAVI-1 experiment was brought on board the International Space Station by Discovery (December 2006) and carried out in January 2007 in the European Modular Cultivation System facility. For the first run of this experiment, lentil seedlings were hydrated and grown in microgravity for 15 h and then subjected for 13 h 40 min to centrifugal accelerations ranging from 0.29 x 10(-2) g to 0.99 x 10(-2) g. During the second run, seedlings were grown either for 30 h 30 min in microgravity (this sample was the control) or for 21 h 30 min and then subjected to centrifugal accelerations ranging from 1.2 x 10(-2) g to 2.0 x 10(-2) g for 9 h. In both cases, root orientation and root curvature were followed by time-lapse photography. Still images were downlinked in near real time to ground Norwegian User Support and Operations Center during the experiment. The position of the root tip and the root curvature were analyzed as a function of time. It has been shown that in microgravity, the embryonic root curved strongly away from the cotyledons (automorphogenesis) and then straightened out slowly from 17 to 30 h following hydration (autotropism). Because of the autotropic straightening of roots in microgravity, their tip was oriented at an angle close to the optimal angle of curvature (120 degrees -135 degrees ) for a period of 2 h during centrifugation. Moreover, it has been demonstrated that lentil roots grown in microgravity before stimulation were more sensitive than roots grown in 1 g. In these conditions, the threshold acceleration perceived by these organs was found to be between 0 and 2.0 x 10(-3) g and estimated punctually at 1.4 x 10(-5) g by using the hyperbolic model for fitting the experimental data and by assuming that autotropism had no or little impact on the gravitropic response. Gravisensing by statoliths should be possible at such a low level of acceleration because the actomyosin system could provide the necessary work to overcome the activation energy for gravisensing.

11p.-2 fig.-6 tab., Growing plants in space for using them in bioregenerative life support systems during long-term human spaceflights needs improvement of our knowledge in how plants can adapt to space growth conditions. In a previous study performed on board the International Space Station (GENARA A experiment STS-132) we evaluate the global changes that microgravity can exert on the membrane proteome of Arabidopsis seedlings. Here we report additional data from this space experiment, taking advantage of the availability in the EMCS of a centrifuge to evaluate the effects of cues other than microgravity on the relative distribution of membrane proteins. Among the 1484 membrane proteins quantified, 227 proteins displayed no abundance differences between µ g and 1 g in space, while their abundances significantly differed between 1 g in space and 1 g on ground. A majority of these proteins (176) were over-represented in space samples and mainly belong to families corresponding to protein synthesis, degradation, transport, lipid metabolism, or ribosomal proteins. In the remaining set of 51 proteins that were under-represented in membranes, aquaporins and chloroplastic proteins are majority. These sets of proteins clearly appear as indicators of plant physiological processes affected in space by stressful factors others than microgravity., The authors would like to thank the National Aeronautics and Space Administration (NASA) who successfully performed the spaceflight experiment; they also thank the astronauts for performing the required tasks on board the ISS. We acknowledge the Norwegian User Support and Operations Center team (NUSOC) for the ground and space preparation of the GENARA-A experiment and we thank the European Aeronautic Defense and Space Company (Astrium EADS) for the design and building of the hardware. We also thank the European Space Agency (ESA) and the Centre National d’Etudes Spatiales(CNES) for their scientific and financial support.

45 ref.; The auxin-binding protein 1 (ABP1) has been proposed to be involved in the perception of the phytohormone at the plasma membrane. Site-directed mutagenesis was performed on highly conserved residues at the C terminus of ABP1 to investigate their relative importance in protein folding and activation of a functional response at the plasma membrane. Detailed analysis of the dynamic interaction of the wild-type ABP1 and mutated proteins with three distinct monoclonal antibodies recognizing conformation-dependent epitopes was performed by surface plasmon resonance. The influence of auxin on these interactions was also investigated. The Cys(177) as well as Asp(175) and Glu(176) were identified as critical residues for ABP1 folding and action at the plasma membrane. On the contrary, the C-terminal KDEL sequence was demonstrated not to be essential for auxin binding, interaction with the plasma membrane, or activation of the transduction cascade although it does appear to be involved in the stability of ABP1. Taken together, the results confirmed that ABP1 conformational change is the critical step for initiating the signal from the plasma membrane.