Light signals counteract alterations caused by simulated microgravity in proliferating plant cells.

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.

From Spaceflight to Mars g-Levels: Adaptive Response of A. Thaliana Seedlings in a Reduced Gravity Environment Is Enhanced by Red-Light Photostimulation.

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.

Mammalian and Invertebrate Models as Complementary Tools for Gaining Mechanistic Insight on Muscle Responses to Spaceflight.

Bioinformatics approaches have proven useful in understanding biological responses to spaceflight. Spaceflight experiments remain resource intensive and rare. One outstanding issue is how to maximize scientific output from a limited number of omics datasets from traditional animal models including nematodes, fruitfly, and rodents. The utility of omics data from invertebrate models in anticipating mammalian responses to spaceflight has not been fully explored. Hence, we performed comparative analyses of transcriptomes of soleus and extensor digitorum longus (EDL) in mice that underwent 37 days of spaceflight. Results indicate shared stress responses and altered circadian rhythm. EDL showed more robust growth signals and Pde2a downregulation, possibly underlying its resistance to atrophy versus soleus. Spaceflight and hindlimb unloading mice shared differential regulation of proliferation, circadian, and neuronal signaling. Shared gene regulation in muscles of humans on bedrest and space flown rodents suggest targets for mitigating muscle atrophy in space and on Earth. Spaceflight responses of C. elegans were more similar to EDL. Discrete life stages of D. melanogaster have distinct utility in anticipating EDL and soleus responses. In summary, spaceflight leads to shared and discrete molecular responses between muscle types and invertebrate models may augment mechanistic knowledge gained from rodent spaceflight and ground-based studies.

Evaluation of growth and nutritional value of Brassica microgreens grown under red, blue and green LEDs combinations.

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 as microgreens 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.

Differential transcriptional profile through cell cycle progression in Arabidopsis cultures under simulated microgravity.

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.

Cell cycle acceleration and changes in essential nuclear functions induced by simulated microgravity in a synchronized Arabidopsis cell culture.

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.

RNA-seq analyses of Arabidopsis thaliana seedlings after exposure to blue-light phototropic stimuli in microgravity.

PREMISE: Plants synthesize information from multiple environmental stimuli when determining their direction of growth. Gravity, being ubiquitous on Earth, plays a major role in determining the direction of growth and overall architecture of the plant. Here, we utilized the microgravity environment on board the International Space Station (ISS) to identify genes involved influencing growth and development of phototropically stimulated seedlings of Arabidopsis thaliana. METHODS: Seedlings were grown on the ISS, and RNA was extracted from 7 samples (pools of 10-15 plants) grown in microgravity (µg) or Earth gravity conditions (1-g). Transcriptomic analyses via RNA sequencing (RNA-seq) of differential gene expression was performed using the HISAT2-Stringtie-DESeq2 RNASeq pipeline. Differentially expressed genes were further characterized by using Pathway Analysis and enrichment for Gene Ontology classifications. RESULTS: For 296 genes that were found significantly differentially expressed between plants in microgravity compared to 1-g controls, Pathway Analysis identified eight molecular pathways that were significantly affected by reduced gravity conditions. Specifically, light-associated pathways (e.g., photosynthesis-antenna proteins, photosynthesis, porphyrin, and chlorophyll metabolism) were significantly downregulated in microgravity. CONCLUSIONS: Gene expression in A. thaliana seedlings grown in microgravity was significantly altered compared to that of the 1-g control. Understanding how plants grow in conditions of microgravity not only aids in our understanding of how plants grow and respond to the environment but will also help to efficiently grow plants during long-range space missions.

The combined effects of real or simulated microgravity and red-light photoactivation on plant root meristematic cells.

MAIN CONCLUSION: 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.

A novel blue-light phototropic response is revealed in roots of Arabidopsis thaliana in microgravity.

MAIN CONCLUSION: Blue-light positive phototropism in roots is masked by gravity and revealed in conditions of microgravity. In addition, the magnitude of red-light positive phototropic curvature is correlated to the magnitude of gravity. Due to their sessile nature, plants utilize environmental cues to grow and respond to their surroundings. Two of these cues, light and gravity, play a substantial role in plant orientation and directed growth movements (tropisms). However, very little is currently known about the interaction between light- (phototropic) and gravity (gravitropic)-mediated growth responses. Utilizing the European Modular Cultivation System on board the International Space Station, we investigated the interaction between phototropic and gravitropic responses in three Arabidopsis thaliana genotypes, Landsberg wild type, as well as mutants of phytochrome A and phytochrome B. Onboard centrifuges were used to create a fractional gravity gradient ranging from reduced gravity up to 1g. A novel positive blue-light phototropic response of roots was observed during conditions of microgravity, and this response was attenuated at 0.1g. In addition, a red-light pretreatment of plants enhanced the magnitude of positive phototropic curvature of roots in response to blue illumination. In addition, a positive phototropic response of roots was observed when exposed to red light, and a decrease in response was gradual and correlated with the increase in gravity. The positive red-light phototropic curvature of hypocotyls when exposed to red light was also confirmed. Both red-light and blue-light phototropic responses were also shown to be affected by directional light intensity. To our knowledge, this is the first characterization of a positive blue-light phototropic response in Arabidopsis roots, as well as the first description of the relationship between these phototropic responses in fractional or reduced gravities.

Strain-specific variation of the decorin-binding adhesin DbpA influences the tissue tropism of the lyme disease spirochete.

Lyme disease spirochetes demonstrate strain- and species-specific differences in tissue tropism. For example, the three major Lyme disease spirochete species, Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii, are each most commonly associated with overlapping but distinct spectra of clinical manifestations. Borrelia burgdorferi sensu stricto, the most common Lyme spirochete in the U.S., is closely associated with arthritis. The attachment of microbial pathogens to cells or to the extracellular matrix of target tissues may promote colonization and disease, and the Lyme disease spirochete encodes several surface proteins, including the decorin- and dermatan sulfate-binding adhesin DbpA, which vary among strains and have been postulated to contribute to strain-specific differences in tissue tropism. DbpA variants differ in their ability to bind to its host ligands and to cultured mammalian cells. To directly test whether variation in dbpA influences tissue tropism, we analyzed murine infection by isogenic B. burgdorferi strains that encode different dbpA alleles. Compared to dbpA alleles of B. afzelii strain VS461 or B. burgdorferi strain N40-D10/E9, dbpA of B. garinii strain PBr conferred the greatest decorin- and dermatan sulfate-binding activity, promoted the greatest colonization at the inoculation site and heart, and caused the most severe carditis. The dbpA of strain N40-D10/E9 conferred the weakest decorin- and GAG-binding activity, but the most robust joint colonization and was the only dbpA allele capable of conferring significant joint disease. Thus, dbpA mediates colonization and disease by the Lyme disease spirochete in an allele-dependent manner and may contribute to the etiology of distinct clinical manifestations associated with different Lyme disease strains. This study provides important support for the long-postulated model that strain-specific variations of Borrelia surface proteins influence tissue tropism.

Astronaut omics and the impact of space on the human body at scale.

Future multi-year crewed planetary missions will motivate advances in aerospace nutrition and telehealth. On Earth, the Human Cell Atlas project aims to spatially map all cell types in the human body. Here, we propose that a parallel Human Cell Space Atlas could serve as an openly available, global resource for space life science research. As humanity becomes increasingly spacefaring, high-resolution omics on orbit could permit an advent of precision spaceflight healthcare. Alongside the scientific potential, we consider the complex ethical, cultural, and legal challenges intrinsic to the human space omics discipline, and how philosophical frameworks may benefit from international perspectives.

How are cell and tissue structure and function influenced by gravity and what are the gravity perception mechanisms?

Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap "Biology in Space and Analogue Environments" focusing on "How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?" The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.

How to obtain an integrated picture of the molecular networks involved in adaptation to microgravity in different biological systems?

Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.

Suboptimal evolutionary novel environments promote singular altered gravity responses of transcriptome during Drosophila metamorphosis.

BACKGROUND: Previous experiments have shown that the reduced gravity aboard the International Space Station (ISS) causes important alterations in Drosophila gene expression. These changes were shown to be intimately linked to environmental space-flight related constraints. RESULTS: Here, we use an array of different techniques for ground-based simulation of microgravity effects to assess the effect of suboptimal environmental conditions on the gene expression of Drosophila in reduced gravity. A global and integrative analysis, using "gene expression dynamics inspector" (GEDI) self-organizing maps, reveals different degrees in the responses of the transcriptome when using different environmental conditions or microgravity/hypergravity simulation devices. Although the genes that are affected are different in each simulation technique, we find that the same gene ontology groups, including at least one large multigene family related with behavior, stress response or organogenesis, are over represented in each case. CONCLUSIONS: These results suggest that the transcriptome as a whole can be finely tuned to gravity force. In optimum environmental conditions, the alteration of gravity has only mild effects on gene expression but when environmental conditions are far from optimal, the gene expression must be tuned greatly and effects become more robust, probably linked to the lack of experience of organisms exposed to evolutionary novel environments such as a gravitational free one.

Meristematic cell proliferation and ribosome biogenesis are decoupled in diamagnetically levitated Arabidopsis seedlings.

BACKGROUND: Cell growth and cell proliferation are intimately linked in the presence of Earth's gravity, but are decoupled under the microgravity conditions present in orbiting spacecraft. New technologies to simulate microgravity conditions for long-duration experiments, with stable environmental conditions, in Earth-based laboratories are required to further our understanding of the effect of extraterrestrial conditions on the growth, development and health of living matter. RESULTS: We studied the response of transgenic seedlings of Arabidopsis thaliana, containing either the CycB1-GUS proliferation marker or the DR5-GUS auxin-mediated growth marker, to diamagnetic levitation in the bore of a superconducting solenoid magnet. As a control, a second set of seedlings were exposed to a strong magnetic field, but not to levitation forces. A third set was exposed to a strong field and simulated hypergravity (2 g). Cell proliferation and cell growth cytological parameters were measured for each set of seedlings. Nucleolin immunodetection was used as a marker of cell growth. Collectively, the data indicate that these two fundamental cellular processes are decoupled in root meristems, as in microgravity: cell proliferation was enhanced whereas cell growth markers were depleted. These results also demonstrated delocalisation of auxin signalling in the root tip despite the fact that levitation of the seedling as a whole does not prevent the sedimentation of statoliths in the root cells. CONCLUSIONS: In our model system, we found that diamagnetic levitation led to changes that are very similar to those caused by real- [e.g. on board the International Space Station (ISS)] or mechanically-simulated microgravity [e.g. using a Random Positioning Machine (RPM)]. These changes decoupled meristematic cell proliferation from ribosome biogenesis, and altered auxin polar transport.

A Delphi study to identify and assess professional competencies in the education of optometrists.

PURPOSE: The aim of this study was to identify, assess, and reach a consensus on the professional competencies that optometrists must acquire during their undergraduate training at the University of Valladolid. The results obtained may be useful in the revision of the current curriculum. METHODS: A three round Delphi study was carried out with the participation of 116 participants grouped in 7 panels. In the first round, competencies were identified through an open-ended question and their frequency calculated. In the successive 2nd and 3rd rounds, the consensus and stability of each competency was established. RESULTS: A total of 56 professional competencies were identified, with 43 of them (77%) achieving a consensus among participants, of which the highest scores were obtained by the competencies that correspond to health functions of primary visual care. Out of the 13 competencies without consensus, for 11 of them this was due to the significant differences in assessment among the consulted participants; for 8 competencies (14%) response stability was found, while neither consensus nor stability were reached in 5 (9%) of the identified competencies. CONCLUSIONS: The results obtained with this Delphi study provide a set of relevant competencies for updating the curriculum of the university Degree in Optics and Optometry at the University of Valladolid, improving its suitability to current and future professional reality.

Perspectives for plant biology in space and analogue environments.

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.

Meta-analysis of the space flight and microgravity response of the Arabidopsis plant transcriptome.

Spaceflight presents a multifaceted environment for plants, combining the effects on growth of many stressors and factors including altered gravity, the influence of experiment hardware, and increased radiation exposure. To help understand the plant response to this complex suite of factors this study compared transcriptomic analysis of 15 Arabidopsis thaliana spaceflight experiments deposited in the National Aeronautics and Space Administration's GeneLab data repository. These data were reanalyzed for genes showing significant differential expression in spaceflight versus ground controls using a single common computational pipeline for either the microarray or the RNA-seq datasets. Such a standardized approach to analysis should greatly increase the robustness of comparisons made between datasets. This analysis was coupled with extensive cross-referencing to a curated matrix of metadata associated with these experiments. Our study reveals that factors such as analysis type (i.e., microarray versus RNA-seq) or environmental and hardware conditions have important confounding effects on comparisons seeking to define plant reactions to spaceflight. The metadata matrix allows selection of studies with high similarity scores, i.e., that share multiple elements of experimental design, such as plant age or flight hardware. Comparisons between these studies then helps reduce the complexity in drawing conclusions arising from comparisons made between experiments with very different designs.

Enhancing European capabilities for application of multi-omics studies in biology and biomedicine space research.

Following on from the NASA twins' study, there has been a tremendous interest in the use of omics techniques in spaceflight. Individual space agencies, NASA's GeneLab, JAXA's ibSLS, and the ESA-funded Space Omics Topical Team and the International Standards for Space Omics Processing (ISSOP) groups 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 coordination with international omics endeavors, 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.

How do gravity alterations affect animal and human systems at a cellular/tissue level?

The present white paper concerns the indications and recommendations of the SciSpacE Science Community to make progress in filling the gaps of knowledge that prevent us from answering the question: "How Do Gravity Alterations Affect Animal and Human Systems at a Cellular/Tissue Level?" This is one of the five major scientific issues of the ESA roadmap "Biology in Space and Analogue Environments". Despite the many studies conducted so far on spaceflight adaptation mechanisms and related pathophysiological alterations observed in astronauts, we are not yet able to elaborate a synthetic integrated model of the many changes occurring at different system and functional levels. Consequently, it is difficult to develop credible models for predicting long-term consequences of human adaptation to the space environment, as well as to implement medical support plans for long-term missions and a strategy for preventing the possible health risks due to prolonged exposure to spaceflight beyond the low Earth orbit (LEO). The research activities suggested by the scientific community have the aim to overcome these problems by striving to connect biological and physiological aspects in a more holistic view of space adaptation effects.