Plants grown in Apollo lunar regolith present stress-associated transcriptomes that inform prospects for lunar exploration

The Artemis program has renewed interest in lunar exploration and its implications for terrestrial biology. Plants are considered crucial for long-term lunar habitats, offering food, oxygen, water recycling, and carbon dioxide scrubbing. However, prior to this study, the interaction between lunar materials and terrestrial plants at the molecular level remained unexplored. Existing extraterrestrial plant growth systems largely rely on hydroponics, lacking information on in-situ resource utilization, like lunar regolith. This study aims to address this gap by investigating plant growth and gene expression in genuine lunar regolith, providing fundamental biological insights and informing the use of plants in lunar life support.

Extensive research exists on plant responses to spaceflight, documenting adaptations to gravity, radiation, and other space-related phenomena. Plants are valuable model organisms for understanding the physiological adaptation of terrestrial biology to space. However, most extraterrestrial plant growth systems rely on hydroponics due to the lack of knowledge on using in-situ resources like regolith. Previous research involving lunar samples and biology mainly focused on pathogen detection, with limited experiments exposing plants to lunar materials, but never using lunar regolith as the primary growth medium. This study addresses this significant gap in knowledge, providing critical data for future lunar exploration.

This study used *Arabidopsis thaliana* (Col-0) seeds sown directly onto samples from Apollo 11 (10084), 12 (12070), and 17 (70051), representing diverse regolith types (mature, submature, and immature). JSC-1A lunar simulant served as a terrestrial control. A 48-well plate system with a subsurface irrigation system using Rockwool and a 0.45-micron filter was developed. 900 mg aliquots of each regolith and simulant were used, with 4 replicate plates for each. Plants were grown in ventilated terrarium boxes under growth lights. Germination, growth rates, and rosette diameter were monitored daily. After 20 days, the aerial portions of the plants were harvested for transcriptome analysis using RNA-Seq. Differential gene expression was analyzed using edgeR, comparing lunar samples to the JSC-1A control. Data was parsed based on Apollo site and plant morphology (Severe, Small, Large) to identify stress responses.

Germination rates were near 100% across all samples, including the lunar regoliths. However, growth in lunar regolith was significantly slower and less uniform than in the JSC-1A control. Plants grown in Apollo 11 regolith showed the most severe growth retardation. Transcriptome analysis revealed that plants in all lunar regolith samples displayed significant differential gene expression, indicating a strong stress response. The majority (71%) of differentially expressed genes (DEGs) were associated with salt, metal, and reactive oxygen species (ROS) stresses. A subset of DEGs (29%) were related to nutrient metabolism. The number of DEGs varied by Apollo site: Apollo 11 (465), Apollo 12 (265), and Apollo 17 (113). Analysis of DEGs based on plant morphology (Severe, Small, Large) showed that even larger, seemingly healthy plants displayed stress responses. Severe plants showed over 1000 DEGs, primarily stress-related. Small plants showed fewer DEGs, mostly related to ROS. Large plants displayed DEGs associated with salt and drought stress. This suggests that even successful plants encounter significant metabolic challenges in lunar regolith.

This study demonstrates the potential of using lunar regolith as a plant growth substrate, fulfilling a long-standing gap in lunar plant biology research. However, the results also highlight significant challenges. The observed stress responses, particularly the prevalence of ionic and ROS stress, indicate that lunar regolith is far from a benign substrate. The differences in stress responses across Apollo sites and plant morphologies suggest that regolith properties (maturity, composition) play a crucial role in plant success. The observation that even large plants show significant stress highlights the metabolic burden of adaptation. Future research must focus on mitigating these stresses through soil amendments, genetic modification, or other strategies to improve plant growth and productivity in lunar environments.

Terrestrial plants can grow in lunar regolith, but the substrate presents significant challenges. The observed stress responses associated with ionic and ROS stress necessitate further research into soil amendments or genetic modifications for optimized plant growth. Future studies should investigate specific gene targets identified in this study to enhance plant tolerance to lunar regolith conditions, and also explore the use of different plant species with varying tolerances. This research is crucial for achieving sustainable lunar habitats.

The small scale of the experiment, using a limited number of regolith samples and plant replicates, may limit the generalizability of the findings. The use of a single plant species, *Arabidopsis thaliana*, prevents extrapolation to other plants. The study focused on short-term growth, and long-term effects of lunar regolith on plant health remain unknown. The potential influence of other lunar environmental factors (radiation, reduced gravity) was not addressed in this study.