• The space exploration goals are pointing towards long-term manned missions to the Moon and Mars. To achieve such goals, the negative effects of ionizing radiation on organisms must be kept under a threshold. High levels of ionizing radiation can influence the functioning of Bioregenerative Life Support Systems (BLSS) due to their effects on both biotic components (i.e. producers, consumers, and decomposers) and non-living elements, which are integrated to achieve self-sustenance.
• The higher plants, key elements of BLSS, show better radio-resistance than animals but a standard behaviour has not been identified also due to the multiplicity of studies using different endpoints, plant materials (e.g. species/cultivar, phenological stage, nutritional status), and radiations (e.g. type, dose, protocols). Most available information derives from ground-based experiments with exposure to photon-type radiation rather than charged particles (i.e., protons and heavy ions), as well as to acute doses rather than chronic exposure, leaving the poor space fidelity of the ‘radiation analogues’ accelerators still an open issue.
• This presentation will summarize the main results we obtained, in the last 15 years, within ESA (European Space Agency) and ASI (Italian Space Agency) projects also in collaboration with the GSI Helmholtzzentrum für Schwerionenforschung (GmbH). The research has been aimed at understanding the effects of low and high LET (Linear Energy Transfer) radiation on morpho-anatomical, eco-physiological, biochemical, and nutritional aspects of different plant systems (e.g. seedlings, microgreens, adult plants) of various species including Asteraceae, Brassicaceae, Fabaceae, and Solanaceae.
• Such studies are useful to understand: a) the main mechanisms of radio-resistance, b) the impact on plants’ “regeneration” efficiency, and c) the effects on the nutritional quality of fresh food produced onboard. The results obtained also provide useful information to define the shielding requirements during the cultivation cycle of plants in different space contexts. They can also be applied to design nutritional countermeasures to intensify the physiological defences of astronauts against radiation exposure, through the integration of their diet with plant-derived fresh food, produced onboard, rich in antioxidants whose content is primed by the exposure to radiation itself. Finally, the presentation highlights the need for the scientific community to converge towards common standardization schemes and protocols as well as to adopt a multidisciplinary and transdisciplinary approach bringing together plant biology, medicine, microbiology, physics, and biotechnology, since the integration of skills and knowledge is a promising way to achieve the common goal of countermeasures development for radiation protection of astronauts.

Introduction/Background: Understanding how plants adapt to the unique pressures of spaceflight is critical for growing food in deep space. Microbes, including plant pathogens, were recently identified on the International Space Station (ISS) (Checinska Sielaff et al. 2016; Urbaniak et al. 2018; Khodadad et al. 2020), and disease loss threatens crop production in space (Urbaniak et al. 2018). To sustain long duration human space exploration, we need to grow and harvest edible crops and minimize crop loss from disease. This requires knowledge of plant immune responses during spaceflight, and how pathogen colonization and virulence are impacted by spaceflight conditions. However, our current knowledge of plant-microbe interactions in space is very limited (Foster et al. 2014).
Method/Experiment: The Advanced Plant Habitat (APH) is a plant growth system that provides improved growing conditions during spaceflight (Massa et al. 2016; John et al. 2021). We are investigating how the tomato immune system adapts to spaceflight when grown in the APH aboard the ISS. In early 2024, we grew both wild type and immune-deficient tomatoes in the APH and elicited defense responses with a chemical elicitor. Upon return to Earth, we will use genome-wide transcriptional profiling to compare the immune responses of space-grown tomatoes to ground controls. We are also investigating how tomato colonization by a fungal pathogen, Fusarium oxysporum, is altered by simulated microgravity on Earth using a custom-made 2D clinostat.
Results: Tomato plants grew well in the APH on the ISS and the experiment was successful. Tomato leaf samples arrived back to Earth in February 2024 and will be analyzed for gene expression in Spring 2024. Results will be shared at the conference.
Conclusion: We anticipate that our results will reveal fundamental insights into how the plant immune system responds to spaceflight conditions. Results will contribute to the development of sustainable crop production strategies in space and will enhance human exploration activities in space.

The growth of plants under weightlessness and other space conditions such as lack of buoyancy, elevated carbon dioxide (CO2) and substrate restrictions is commonly assumed to induce stress responses in plants. This ‘space syndrome’ has been defined based on experiments that compared ground controls with space-grown plants, but a comparison of subsequent space grown growth cycles has not been accomplished until the Advanced Plant Habitat (APH) 2 experiment, where radish plants were grown from seeds in two successive experiments on the International Space Station (ISS) for 27 days each. Leaf and bulb tissue from the space experiments (SEs) were compared with ground controls (GC) grown at Kennedy Space Center (KSC) under the same conditions as on the ISS, notably elevated CO2 (about 2500 ppm), and from lab controls (LC) grown under atmospheric CO2 but light and temperature conditions similar to the GC. Based on high-stringency RNAseq data (greater than 4-fold differences in up and downregulation and P values < 0.001) resulted in 4547 differentially transcribed genes for leaves and 1157 genes for bulbs, indicating that leaves are more sensitive to environmental conditions than bulbs.
Comparing leaf data from the first and second SE showed differential transcription of 227 genes, a number comparable to differences between the first SE and GC control (295 genes) and LC and SE 1 (183 genes). Comparing LC with SE 2 resulted in 820 differentially transcribed genes. The large difference between the two SE and the variability between the LC and GC material (1947 altered genes) suggests that space conditions per se do not cause consistent changes in gene transcription.
A similar conclusion can be reached from bulb data. SE 1 and 2 showed 175 differentially transcribed genes (61 up-, 114 downregulated). This number was higher than a comparison between GC and SE 1 (39 up- and 15 downregulated) but less than a comparison between LC and SE 1 (93 up- 125 downregulated) and SE2 (164 up- and 119 downregulated).
These comparisons and the inconsistent identity of affected genes lack of common pathways based on enrichment analyses and little overlap between the SEs but large discrepancies between KSC and laboratory controls suggest that the CO2 conditions may have a greater impact on plant cultivation than weightlessness. Therefore, the notion that plant cultivation in space leads to stress responses needs to be reexamined.

Introduction/Background:
NASA aims to establish sustainable colonies on the moon and Mars by 2050, with plants playing crucial roles in these ventures. Understanding how space flight and growth in extraterrestrial soil simulants affect the telomeres and telomerase of Arabidopsis thaliana is essential for successful plant cultivation in space environments. Measuring telomeres and telomerase serves as crucial indicators of prosperity and longevity in organisms. Telomeres, protective caps at the end of chromosomes, shield genetic material from degradation and maintain chromosomal stability. Understanding the dynamics of telomeres and telomerase activity provides insights into cellular aging, disease susceptibility, and overall organismal health. Thus, monitoring these markers offers valuable clues about an organism's potential for long-term survival and well-being.

Method/Experiment:
In this study, we investigated the impact of space flight and growth on extraterrestrial soil simulants on the telomeres and telomerase of Arabidopsis thaliana. Plants were grown aboard the ISS and in lunar regolith simulant to simulate extraterrestrial conditions. We utilized Southern blot and PCR-based assays to quantify telomere length and measure telomerase activity in our study. Levels of genome oxidation and oxidative stress were determined using biochemical assays. Organellar stability was evaluated through PCR-based amplification of specific genes.

Results:
Telomere length remained stable in plants grown on the ISS, despite a significant induction of telomerase enzyme activity, particularly in roots, where it increased by up to 150-fold. Ground-based studies confirmed that telomerase activity in Arabidopsis is elevated by various environmental stressors, independent of telomere length changes. A strong inverse correlation between genome oxidation and telomerase activity levels was observed, suggesting a potential redox protective role for plant telomerase.

In lunar regolith simulant, Arabidopsis exhibited arrest at a terminal vegetative state and activated multiple stress responses. Pre-washing the simulant with an antioxidant cocktail facilitated seed setting and viable second-generation plants. However, plants grown in lunar regolith simulant displayed increased genome oxidation and reduced biomass compared to Earth soil cultivation. Additionally, progressive telomere shortening and reduced telomerase enzyme activity were observed across various Arabidopsis accessions and regolith simulants.

Conclusion:
These findings underscore both the promise and challenges of ensuring genome integrity for successful plant growth in extraterrestrial environments. Telomere dynamics and telomerase activity are crucial factors to consider in the development of sustainable agricultural systems for space colonization efforts. Further research is needed to address these challenges and optimize plant growth in extraterrestrial habitats.