Raman Spectroscopy as a Tool for Assessing Plant Growth in Space and on Lunar Regolith Simulants

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The feasibility of such endeavors depends on both innovative engineering concepts and successful adaptation of life forms that exist on Earth to inhospitable environs. In such missions plants will play a vital role as life support systems, supplying astronauts with food, oxygen, carbon dioxide recycling, and psychological well-being benefits. Therefore, understanding the adaptability of plants to harsher environments, including different gravitational forces and growth on extraterrestrial soils will be required. In this study, we investigate the potential of Raman spectroscopy (RS), a modern analytical technique, in a non-invasive and non-destructive assessment of changes in the biochemistry of plants exposed to zero gravity on the International Space Station and during growth on lunar regolith simulants on Earth. We report that RS can sense changes in plant carotenoids, pectin, cellulose, and phenolics, which in turn, could be used to gauge the degree of plant stress to the new environments. Our findings also demonstrate that RS can monitor the efficiency of soil supplements that can be used to mitigate nutrient-free regolith media. We conclude that RS can serve as a highly efficient, fast, and label-free approach for monitoring plant health in exotic environments. Biological sciences/Biochemistry Biological sciences/Plant sciences Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Plants occupy nearly all regions on our planet, including deserts, oceans, and high-altitude regions. They help form ecological niches, produce oxygen and food for animals and humans. They will also be essential components for successful colonization of the Moon and Mars. During the past decade, substantial effort was made to understand how plant biochemistry changes in such exotic environments 1 , 2 , We previously showed that Arabidopsis thaliana seedlings cultivated aboard the International Space Station (ISS) exhibited elevated oxidative stress and genome oxidation, yet did not demonstrate a noticeable reduction in telomere length or physiological size 1 . We further showed that plants can be successfully cultivated for at least two generations on lunar regolith simulant if the regolith is treated with chemical antioxidants or if plants are genetically modified to express enhanced antioxidant activity (cite the ref 2 here). Nevertheless, plants grown on this substrate do not prosper and exhibit reduced biomass and fertility in the next generation, which is accompanied by telomere shortening, decreased telomerase enzyme activity, and increased genome oxidation. These findings indicate that further mitigation efforts will be required to maintain genome integrity of plants grown in such conditions to enable long-term sustainability 2 . Confirmatory and quantitative analysis of the biochemical changes associated with growth in these unusual conditions requires sophisticated equipment that cannot be possessed by space shuttles, including the ISS. This problem catalyzed the search for robust and reliable sensors for non-invasive, non-destructive, and label-free assessment of changes in plant biochemistry induced by many environmental factors. Our group hypothesized that Raman spectroscopy (RS), could be used to meet these strict requirements. 3 RS is based on the phenomenon of inelastic light scattering 4 , 5 . In this case, photons that hit the sample of interest excite the molecules to high vibrational state and scatter back both higher and lower than the incident light. Importantly, the change in the photon energy, which is also known as Raman Shift, directly depends on the chemical structure of molecules present in the sample 6 – 8 . Consequently, RS can probe the structure and composition of the sample of interest. Furthermore, because RS is non-invasive and non-destructive, it is ideal for analysis of life systems 9 . During the past decade, several companies have developed hand-held Raman spectrometers that can be used directly in the field or a greenhouse 9 . Expanding upon these advantages of RS, Lee and co-workers investigated the potential of this technique in non-invasive diagnostics of citrus greening disease 10 , 11 . It was found that RS could be used to accurately identify citrus greening in both oranges and grapefruits. Furthermore, RS enables specificity by accurate differentiation between the infected plants and nutritional deficient plants 10 , 11 . Recently reported studies employed high-performance liquid chromatography (HPLC) to identify biochemical changes in plants that were sensed by RS 12 . It was found that RS primarily detected changes in lutein and other carotenoids. It was also shown that RS sensed changes in the concentration of small phenolic compounds, such as coumaric acid, that occurred as a result of plant infection with bacterial, fungal, or viral pathogens 12 . In the current study, we examine biochemical changes in A. thaliana that were grown aboard the ISS. We reasoned that the hand-held nature of RS would be advantageous for on-site measurements compared to classical analytical techniques such as HPLC and HPLC-MS. These traditional methods are laborious and invasive. Furthermore, they generate hazardous waste that would be dangerous in the constrained environment of ISS. A direct comparison of RS to HPLC demonstrated that RS effectively probes changes in the concentration of carotenoids and low molecular weight phenylpropanoids, such as caffeic acid, in plants. 13 , 14 Recent results by Juarez and co-workers confirm that calibration of RS using HPLC overcomes the need of chromatographic techniques for detection and quantification of metal-induced toxicities in plants. 13 Therefore, RS holds promise for assessing stress-related plant responses on the ISS. We were also motivated by NASA’s beyond Low Earth Orbit colonization plans, and employed RS to examine changes in the biochemistry of plants grown on lunar regolith simulants. Finally, we investigated the extent to which RS could be used to monitor the improvement in the vegetation of regolith-grown plants exposed to antioxidant bio stimulants such as glutathione, proline, and ascorbic acid. Results and Discussion Raman spectra acquired from 12-day-old seedlings of Earth-grown A. thaliana exhibited vibrational bands that can be assigned to carotenoids, pectin, cellulose and phenylpropanoids, depicted in Fig. 1 and Table 1 . We also observed aliphatic vibrations (1326, 1335, and 1440 cm - 1 ), which could not be assigned to the specific class of compounds due to the presence of these chemical groups in nearly all classes of biological molecules. Raman spectra were also acquired from 12-day-old seedlings of ISS-grown A. thaliana and they had similar vibrational fingerprints. However, we found that intensity of the phenylpropanoid peak at 1608 cm - 1 for ISS-grown plants was substantially greater in comparison to the spectra acquired from the leaves of Earth-grown A. thaliana . An increase in the intensity of this vibrational band indicates an increase in the concentration of low molecular weight phenylpropanoids. These molecular species are involved in the plant response to biotic and abiotic stresses 15 – 17 , arguing that ISS-grown plants experience spaceflight-induced stress that is not observed in Earth-grown plants. This conclusion is further supported by the observed decrease in the intensity of vibrational bands that can be assigned to carotenoids (1115, 1155, 1186, 1215 and 1525 cm - 1 ). In the case of biotic or abiotic stresses, plants activate enzymatic degradation of carotenoids producing abscisic acid 18 , 19 . Plant carotenoids can be also oxidized and fragmented by reactive oxygen species (ROS), which results in the formation of β-lonone, β-cyclocitrals that activate plant defense mechanisms. 18 – 20 These results are in good agreement with the previously reported by Shippen and co-workers of higher ROS activity in ISS-grown plants compared to A. thaliana grown on Earth 1 . In the spectra acquired from ISS-grown A. thaliana seedlings, we observed an increase in the intensity of the vibrational band centered at 747 cm - 1 , which can be assigned to pectin. Additionally, we identified small statistically insignificant changes in the intensity of the 1048 cm - 1 peak, which originates from cellulose. Based on these results, we can conclude that spaceflight alters the structure and composition of pectin, with minimal changes to cellulose, in A. thaliana . Additional studies are required to fully understand the chemical nature of changes in these highly important biomolecules in plants. Table 1 Vibrational band assignments for leaf spectra of A. thaliana . Band Vibrational mode Assignment 742 γ(C–O-H) of COOH Pectin 21 915 ν(C-O-C) In plane, symmetric Cellulose, 12 lignin 22 1000 Aromatics, in-plane CH 3 rocking of polyene Proteins, 23 carotenoids 24 , 25 1048 ν(C-O)+ν(C-C)+δ(C-O-H) Cellulose, 12 lignin 22 1115 -C = C- Carotenoids 24 , 25 1155 -C = C- Carotenoids 24 , 25 1186 -C = C- Carotenoids 24 , 25 1215 -C = C- Carotenoids 24 , 25 1288 δ(C-C-H) Aliphatic, cellulose, lignin 22 1326 δCH 2 bending Aliphatic, cellulose, lignin 22 1335 δCH 2 bending Aliphatics 20 1382 δCH 2 bending Aliphatics 20 1440 δ(CH 2 )+δ(CH 3 ) Aliphatics 20 1525 -C = C- (in plane) Carotenoids 24 , 25 1608 ν(C-C) Aromatic ring + σ(CH) Lignin 26 , 27 1678 Amide I Proteins 23 We also performed a partial-least squared discriminant analysis (PLS-DA) to investigate the accuracy of RS-based differentiation between ISS- and Earth-grown A. thaliana , Table 2 . Previously reported results by our and other research groups demonstrate that PLS-DA performs as well or even better compared to other supervised chemometric algorithms such as support vector machine (SMV), linear discriminant analysis (LDA) or soft independent modelling by class analogy (SIMCA). 5 , 7 , 28 – 30 Therefore, we utilized this chemometric method for the analysis of our spectroscopic data. Our results showed that PLS-DA enabled 90% accurate identification of ISS- and 96.5% accurate identification of Earth-grown A. thaliana . Table 2 Confusion matrix of PLS-DA model that indicates accuracy of identification of Raman spectra acquired from ISS- and Earth-grown plants. ISS-grown Earth-grown Accuracy, % Predicted as ISS-grown 27 1 90 Predicted as Earth-grown 3 28 96.5 These results demonstrate that RS could be used as a robust and reliable tool to identify gravity-induced stresses in plants. Next, we investigated the extent to which RS could be used to probe the response of plants grown for 20 days on the lunar regolith simulant LMS-1, media that can be used to mimic the outer crust of the Moon 2 . RS revealed a substantial increase in the intensity of phenylpropanoids and a decrease in the intensity of carotenoids in the Raman spectra acquired from plants grown on lunar regolith simulant compared to plants grown on Earth soil, Fig. 2 . Again, these RS findings were consistent with evidence of a stress response for plants grown in lunar regolith simulant 2 Although no significant changes in the concentration of pectin were detected, we observed statistically significant differences in the intensities of the vibrational band that can be assigned to cellulose (1048 cm - 1 ). Specifically, the intensity of this band was stronger in the spectra acquired from plants grown in the lunar regolith simulant compared to plants grown in conventional Earth soil. These results indicate that regolith-induced stress caused a substantial increase in the synthesis of cellulose which may help to facilitate the adaptation of A. thaliana to this exotic substrate. We previously showed that the poor or toxic nutritional environment of regolith could be partially overcome by addition of an antioxidant cocktail composed of 0.75mM gluthathione, 0.75mM ascorbic acid and 0.75mM proline, or by its individual components 2 . Expanding upon this, we investigated whether RS could be used to monitor bio stimulant-induced improvement in the plant health, Fig. 3 . Based on the changes of the intensities of carotenoids and cellulose vibrations, we found that antioxidant treatment with all three components of the cocktail was the most efficient of all bio stimulants, as depicted on Fig. 3 . Specifically, plants grown on the regolith simulant soil treated with antioxidants exhibited comparable intensities of carotenoids and cellulose vibrations to the control plants that were grown in Earth soil. These results demonstrate that antioxidants minimize enzymatic degradation of plant carotenoids into abscisic acid. High concentration of carotenoids in these plants also suggest that low levels of ROS that are also known to fragment and degrade carotenoids. Thus, antioxidants minimize regolith-induced stress of plants. Our results further showed that the individual components of the antioxidant cocktail were able to significantly alter the carotenoid and cellulose RS profiles, Fig. 3 . Consistent with these observations, the antioxidant cocktail, as well as individual proline and gluthathione treatments substantially improved plant growth and reduced genome oxidation with respect to untreated lunar regolith 2 . In addition to LMS-1 Lunar Mare Simulant, other lunar regolith simulants are commercially available that replicate specific lunar environments with high fidelity (Long-Fox et al., 2023). 31 These include LHS-1 Lunar Highlands Simulant, and LSP-2 Lunar South Pole simulant. We also investigated the extent to which treatment of three different types of lunar regolith with antioxidant cocktail composed of glutathione, ascorbic acid and proline could change the concentration of phenylpropanoids, carotenoids, pectin and cellulose. For these experiments we examined more mature plants that were 47-days-old. We found that treatment of lunar highlands and lunar mare simulants with the cocktail composed of glutathione, ascorbic acid and proline increased the concentration of phenylpropanoids, carotenoids, and pectin. Interestingly, no substantial changes in the concentration of cellulose were observed, Fig. 4 . Such treatment-induced improvements were minor in plants grown on lunar south pole simulants. These findings indicate that the antioxidant-induced increase in the concentration of phenylpropanoids, carotenoids, and pectin in A. thaliana depends on type of simulant that plants were grown on. Furthermore, compared to 20-day-old LMS-1 grown plants, Fig. 2 , 47-day-old LMS-1 plants exhibited reduced levels of carotenoids and pectin relative to the control. The older plants also showed decreased phenylpropanoids and cellulose. These results indicate that secondary metabolite production is dynamic and evolves throughout plant development in lunar regolith simulant. Taken together, our results indicate that RS is a useful tool for non-invasive and non-destructive assessment of the effect of bio-stimulants on the health of plants grown in regolith. Moreover, our RS results are consistent with the results from other biochemical assays and highlight the potential challenges in preserving plant wellbeing in lunar regolith simulant despite efforts at bio-stimulation 2 . Consequently, these results underscore the feasibility of utilizing Raman spectroscopy for non-invasive and non-destructive evaluation of the overall health of plants cultivated in regolith. Materials and methods Plant Materials: Plant growth : For seedlings grown on the ISS Arabidopsis thaliana seeds (35S::HF-RPL18 in the Columbia-0 background; ABRC stock # CS66056) were affixed to a polyethersulfone (PES) membrane using guar gum 2 . Each membrane accommodated fifteen seeds and was subsequently placed onto square 10 cm 0.5x Murashige and Skoog plates with 1% agar. Following a 19-day period of darkness at 4°C, the plates were integrated into the Veggie growth chambers on the ISS and at Kennedy Space Center, where the plants were cultivated for 12 days under 16 h/8 h long day conditions. The growth conditions included 100 mol/m2/s of red (630 nm), blue (455 nm), and green (530 nm) light, simulating Veggie units. Ground control plates underwent seeding, growth, and harvesting with a 48-hour delay from the flight samples. Environmental conditions such as temperature, CO 2 , and relative humidity were monitored continuously on the ISS and replicated with a 48-hour delay in the ground control environmental chamber. The average temperature maintained was 23°C with 44% relative humidity, and CO 2 levels were typically around 1750 ppm. Following removal from the Veggie, astronauts or ground control staff collected the PES membranes and seedlings using forceps, which were then protected in the lid of the Petri dish, wrapped with foil, and swiftly transferred to a − 130°C cold bag for rapid freezing. Subsequently, the seedlings were stored at − 80°C, shipped on dry ice, and kept at − 80°C until processing. For plant grown on lunar regolith simulant seeds from Col-0 were procured from The Arabidopsis Biological Resource Center (ABRC) at Ohio State University, stock CS28994 2 . The seeds underwent surface sterilization in 2.7% sodium hypochlorite for 7 min, followed by stratification at 4°C for three days. Subsequently, they were directly transferred to their designated treatment of either regolith simulant (LMS-1, LSP-2 and LHS, Exolith Lab, Oviedo, FL, USA) or Earth soil (Sunshine, Mix 5, Sun Gro Horticulture, Agawam, MA, USA). Germination took place under a 12-h photoperiod with an average illuminance of 5000 (+/- 250) lux achieved using lights with a 3000:6500 Kelvin ratio, and maintained at a constant temperature of 22°C. The plants were allowed to grow under these conditions for five weeks or until growth ceased, upon which the entire plants were harvested for analysis. Raman Spectroscopy : Raman spectra were collected from the surface of plant leaves with a hand-held Resolve Agilent spectrometer equipped with an 830-nm laser source. The following experimental parameters were used for all collected spectra: 1 s integration time, 495 mW power, and baseline spectral subtraction by device software. Thirty spectra were collected from each group of plants. Spectra shown in the manuscript are processed through MATLAB, normalizing at the 1440cm - 1 peak (include explanation if necessary) and applied with Savitzky-Golay smoothing using a polynomial order of 0. Machine learning model In this work, we utilized partial least-squared discriminant analysis (PLS-DA). This supervised version of principal component analysis achieves dimensionality reduction with the information of target variables together with the good insight into the causes of discrimination through weights and loadings that assists in conducting exploratory data analysis 32 . Conclusions Our findings demonstrate that RS is a useful tool for the confirmatory sensing of changes in plant biochemistry caused by zero gravity and growth in Lunar regolith simulants. Notably, there is an established correlation between carotenoids and phenylpropanoids with stress response and oxidative damage; both plant materials subjected to spaceflight conditions and those cultivated in lunar environments exhibited alterations in the levels of these molecules. The observed changes in carotenoids and phenylpropanoids in space-flown plants and plants grown in simulants of lunar soil suggest a response to stress factors and oxidative challenges experienced in these unique settings. Our results also show that RS could be used to monitor the efficiency of soil supplements that were capable of mitigating the negative effects of nutrient-free regolith media and, consequently, improve the plant health. Thus, RS represents a highly efficient, quick, and label-free approach for monitoring plant health in exotic environments. Notes: The authors declare no competing financial interests. Declarations The authors declare no competing financial interests. ACKNOWLEDGMENTS This study was supported by funds from Texas A&M AgriLife Research, Texas A&M University Governor’s University Research Initiative (GURI) grant program of (12-2016/M1700437), by the National Institutes of Health (R01 GM065383 to D.E.S), the National Aeronautics and Space Administration (80NSSC19K1481 to S.E.W; 80NSSC23K0302 to D.E.S and S.E.W). and the NASA Postdoctoral Program at Kennedy Space Center administered by Oak Ridge Associated Universities (to A.M.). We extend our gratitude to the APEx-07 team, with special acknowledgment to Gerard Newsham, Anne Marie Campbell, Erica Bugardner, and Susan Manning-Roach, based at Kennedy Space Center, for their dedication to experiment validation, flight preparation, and return. We also express our appreciation to astronauts Thomas Pesquet, Mark Vande Hei, and Megan McArthur for their assistance with experiment takedown and harvest on the ISS. References Barcenilla, B. B.; Meyers, A. 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Additional Declarations (Not answered) Cite Share Download PDF Status: Published Journal Publication published 27 May, 2025 Read the published version in npj Microgravity → Version 1 posted Editorial decision: revise 23 Jan, 2025 Review # 2 received at journal 12 Jan, 2025 Reviewer # 2 agreed at journal 21 Dec, 2024 Review # 1 received at journal 24 Sep, 2024 Reviewer # 1 agreed at journal 02 Sep, 2024 Reviewers invited by journal 01 Aug, 2024 Editor assigned by journal 26 Jul, 2024 Submission checks completed at journal 26 Jul, 2024 First submitted to journal 25 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4801715","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":334788722,"identity":"ec1c3659-d2ee-4148-9e51-c1c6a09dc414","order_by":0,"name":"Dmitry Kurouski","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYDCCMxDKgI29ASpygGgtPIdJ1cIgkUykFr4zZ8w+fGyrM+aTfH/w0802Bjm+Gwn4tUie7TGeObPtsBmbdDKzdG4bg7EkIS0G53mMmXnbDtgAtTCAtCRuIFJLnQ2b5GHm30At9YS1AB0G1MJsxibBzAayJcGAoF/OHCtmnHHusDEbT7KZdc45CcOZZx7g18J3Jnkzw4eyOsP57Qcf384ps5HnO07AFnQgQZryUTAKRsEoGAXYAQAwv0BZPy+leAAAAABJRU5ErkJggg==","orcid":"","institution":"Texas A\u0026M","correspondingAuthor":true,"prefix":"","firstName":"Dmitry","middleName":"","lastName":"Kurouski","suffix":""},{"id":334788723,"identity":"ce85cf36-d593-429b-9fab-fbea0047a1a5","order_by":1,"name":"Axell Rodriguez","email":"","orcid":"","institution":"Texas A\u0026M","correspondingAuthor":false,"prefix":"","firstName":"Axell","middleName":"","lastName":"Rodriguez","suffix":""},{"id":334788724,"identity":"9fc651b3-b826-4a3a-a9a5-47aad6b5f80e","order_by":2,"name":"Borja Barbero Barcenilla","email":"","orcid":"https://orcid.org/0000-0002-5529-5147","institution":"Texas A\u0026M","correspondingAuthor":false,"prefix":"","firstName":"Borja","middleName":"Barbero","lastName":"Barcenilla","suffix":""},{"id":334788725,"identity":"80ee14b2-6dab-4573-bd5d-7759ab0d79b3","order_by":3,"name":"Emily Hall","email":"","orcid":"","institution":"Texas A\u0026M","correspondingAuthor":false,"prefix":"","firstName":"Emily","middleName":"","lastName":"Hall","suffix":""},{"id":334788726,"identity":"99be3c2a-a089-4526-a8cd-94883af7f6f5","order_by":4,"name":"Ishan Kundel","email":"","orcid":"","institution":"Texas A\u0026M","correspondingAuthor":false,"prefix":"","firstName":"Ishan","middleName":"","lastName":"Kundel","suffix":""},{"id":334788727,"identity":"57c6d5b8-aec2-4424-9449-185c5c0d6b62","order_by":5,"name":"Alexander Meyers","email":"","orcid":"","institution":"NASA Postdoctoral Program","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Meyers","suffix":""},{"id":334788728,"identity":"36cd39a5-02a3-4c65-9609-3f2c062b61cd","order_by":6,"name":"Sarah Wyatt","email":"","orcid":"https://orcid.org/0000-0001-7874-0509","institution":"Ohio University","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Wyatt","suffix":""},{"id":334788729,"identity":"90c90df7-8357-4700-a5bc-adff3536304f","order_by":7,"name":"Dorothy Shippen","email":"","orcid":"","institution":"Texas A\u0026M","correspondingAuthor":false,"prefix":"","firstName":"Dorothy","middleName":"","lastName":"Shippen","suffix":""}],"badges":[],"createdAt":"2024-07-25 12:07:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4801715/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4801715/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41526-025-00479-8","type":"published","date":"2025-05-27T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63470732,"identity":"4a912faf-4fa6-4dcb-ab71-30f52ced2508","added_by":"auto","created_at":"2024-08-28 13:11:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":288679,"visible":true,"origin":"","legend":"\u003cp\u003eAveraged Raman spectra (A) acquired from ISS- and Earth-grown \u003cem\u003eA. thaliana\u003c/em\u003e. Changes in the intensities of vibrational bands that can be assigned to phenylpropanoids (1608 cm\u003csup\u003e-1\u003c/sup\u003e), carotenoids, (1525 cm\u003csup\u003e-1\u003c/sup\u003e), pectin (747 cm\u003csup\u003e-1\u003c/sup\u003e) and cellulose (1048 cm\u003csup\u003e-1\u003c/sup\u003e) are reported by ANOVA graphs (B). Means of the intensities of vibrational bands are shown by dots; standard deviations by horizontal lines.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4801715/v1/712e61e0636c6f108a9d89aa.jpeg"},{"id":63470730,"identity":"4bcbea2e-a850-455c-9a63-5769de8233b6","added_by":"auto","created_at":"2024-08-28 13:11:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27125,"visible":true,"origin":"","legend":"\u003cp\u003eAveraged Raman spectra (A) acquired from Earth- and LMS-1 lunar regolith-grown \u003cem\u003eA. thaliana\u003c/em\u003e. Changes in the intensities of vibrational bands that can be assigned to phenylpropanoids (1608 cm\u003csup\u003e-1\u003c/sup\u003e), carotenoids, (1525 cm\u003csup\u003e-1\u003c/sup\u003e), pectin (747 cm\u003csup\u003e-1\u003c/sup\u003e) and cellulose (1048 cm\u003csup\u003e-1\u003c/sup\u003e) are reported by ANOVA graphs (B). Means of the intensities of vibrational bands are shown by dots; standard deviations by horizontal lines.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4801715/v1/5045111924f56fe63a43143c.png"},{"id":63470733,"identity":"ecfc7048-9a85-4fe9-acc9-322e29f53db6","added_by":"auto","created_at":"2024-08-28 13:11:28","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":474289,"visible":true,"origin":"","legend":"\u003cp\u003eAveraged Raman spectra (A-B) acquired from LMS-1 lunar regolith-grown \u003cem\u003eA. thaliana\u003c/em\u003e, and regolith washed with proline, ascorbic acid, glutathione, and antioxidants. Changes in the intensities of vibrational bands that can be assigned to carotenoids, (1525 cm\u003csup\u003e-1\u003c/sup\u003e) and cellulose (1048 cm\u003csup\u003e-1\u003c/sup\u003e) are reported by ANOVA graphs (C). Means of the intensities of vibrational bands are shown by dots; standard deviations by horizontal lines.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4801715/v1/00b8f774c064bd84c921344c.jpeg"},{"id":63470734,"identity":"1a8e6cf3-02e0-4811-a9c6-d9fd85c42886","added_by":"auto","created_at":"2024-08-28 13:11:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":788793,"visible":true,"origin":"","legend":"\u003cp\u003eAveraged Raman spectra (A) acquired from \u003cem\u003eA. thaliana\u003c/em\u003e grown on treated and untreated lunar regolith at 47 days old. Changes in the intensities of vibrational bands that can be assigned to phenylpropanoids (1610 cm\u003csup\u003e-1\u003c/sup\u003e), carotenoids, (1525 cm\u003csup\u003e-1\u003c/sup\u003e), pectin (747 cm\u003csup\u003e-1\u003c/sup\u003e) and cellulose (1050 cm\u003csup\u003e-1\u003c/sup\u003e) are reported by ANOVA graphs (B). Means of the intensities of vibrational bands are shown by dots; standard deviations by horizontal lines.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4801715/v1/29bd50e1bfdd59a40e8f2013.png"},{"id":83538128,"identity":"cc93ebde-84aa-43e0-8bb8-bd8d06b4be1e","added_by":"auto","created_at":"2025-05-28 07:09:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2670200,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4801715/v1/0c548388-3b73-4442-be80-282a295f1eb4.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Raman Spectroscopy as a Tool for Assessing Plant Growth in Space and on Lunar Regolith Simulants","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants occupy nearly all regions on our planet, including deserts, oceans, and high-altitude regions. They help form ecological niches, produce oxygen and food for animals and humans. They will also be essential components for successful colonization of the Moon and Mars. During the past decade, substantial effort was made to understand how plant biochemistry changes in such exotic environments \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, We previously showed that \u003cem\u003eArabidopsis thaliana\u003c/em\u003e seedlings cultivated aboard the International Space Station (ISS) exhibited elevated oxidative stress and genome oxidation, yet did not demonstrate a noticeable reduction in telomere length or physiological size \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. We further showed that plants can be successfully cultivated for at least two generations on lunar regolith simulant if the regolith is treated with chemical antioxidants or if plants are genetically modified to express enhanced antioxidant activity (cite the ref 2 here). Nevertheless, plants grown on this substrate do not prosper and exhibit reduced biomass and fertility in the next generation, which is accompanied by telomere shortening, decreased telomerase enzyme activity, and increased genome oxidation. These findings indicate that further mitigation efforts will be required to maintain genome integrity of plants grown in such conditions to enable long-term sustainability \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConfirmatory and quantitative analysis of the biochemical changes associated with growth in these unusual conditions requires sophisticated equipment that cannot be possessed by space shuttles, including the ISS. This problem catalyzed the search for robust and reliable sensors for non-invasive, non-destructive, and label-free assessment of changes in plant biochemistry induced by many environmental factors. Our group hypothesized that Raman spectroscopy (RS), could be used to meet these strict requirements.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e RS is based on the phenomenon of inelastic light scattering \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In this case, photons that hit the sample of interest excite the molecules to high vibrational state and scatter back both higher and lower than the incident light. Importantly, the change in the photon energy, which is also known as Raman Shift, directly depends on the chemical structure of molecules present in the sample \u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Consequently, RS can probe the structure and composition of the sample of interest. Furthermore, because RS is non-invasive and non-destructive, it is ideal for analysis of life systems \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. During the past decade, several companies have developed hand-held Raman spectrometers that can be used directly in the field or a greenhouse \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eExpanding upon these advantages of RS, Lee and co-workers investigated the potential of this technique in non-invasive diagnostics of citrus greening disease \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. It was found that RS could be used to accurately identify citrus greening in both oranges and grapefruits. Furthermore, RS enables specificity by accurate differentiation between the infected plants and nutritional deficient plants \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Recently reported studies employed high-performance liquid chromatography (HPLC) to identify biochemical changes in plants that were sensed by RS \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. It was found that RS primarily detected changes in lutein and other carotenoids. It was also shown that RS sensed changes in the concentration of small phenolic compounds, such as coumaric acid, that occurred as a result of plant infection with bacterial, fungal, or viral pathogens \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the current study, we examine biochemical changes in \u003cem\u003eA. thaliana\u003c/em\u003e that were grown aboard the ISS. We reasoned that the hand-held nature of RS would be advantageous for on-site measurements compared to classical analytical techniques such as HPLC and HPLC-MS. These traditional methods are laborious and invasive. Furthermore, they generate hazardous waste that would be dangerous in the constrained environment of ISS. A direct comparison of RS to HPLC demonstrated that RS effectively probes changes in the concentration of carotenoids and low molecular weight phenylpropanoids, such as caffeic acid, in plants.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Recent results by Juarez and co-workers confirm that calibration of RS using HPLC overcomes the need of chromatographic techniques for detection and quantification of metal-induced toxicities in plants.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Therefore, RS holds promise for assessing stress-related plant responses on the ISS. We were also motivated by NASA\u0026rsquo;s beyond Low Earth Orbit colonization plans, and employed RS to examine changes in the biochemistry of plants grown on lunar regolith simulants. Finally, we investigated the extent to which RS could be used to monitor the improvement in the vegetation of regolith-grown plants exposed to antioxidant bio stimulants such as glutathione, proline, and ascorbic acid.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eRaman spectra acquired from 12-day-old seedlings of Earth-grown \u003cem\u003eA. thaliana\u003c/em\u003e exhibited vibrational bands that can be assigned to carotenoids, pectin, cellulose and phenylpropanoids, depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. We also observed aliphatic vibrations (1326, 1335, and 1440 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e), which could not be assigned to the specific class of compounds due to the presence of these chemical groups in nearly all classes of biological molecules. Raman spectra were also acquired from 12-day-old seedlings of ISS-grown \u003cem\u003eA. thaliana\u003c/em\u003e and they had similar vibrational fingerprints. However, we found that intensity of the phenylpropanoid peak at 1608 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e for ISS-grown plants was substantially greater in comparison to the spectra acquired from the leaves of Earth-grown \u003cem\u003eA. thaliana\u003c/em\u003e. An increase in the intensity of this vibrational band indicates an increase in the concentration of low molecular weight phenylpropanoids. These molecular species are involved in the plant response to biotic and abiotic stresses \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, arguing that ISS-grown plants experience spaceflight-induced stress that is not observed in Earth-grown plants. This conclusion is further supported by the observed decrease in the intensity of vibrational bands that can be assigned to carotenoids (1115, 1155, 1186, 1215 and 1525 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e). In the case of biotic or abiotic stresses, plants activate enzymatic degradation of carotenoids producing abscisic acid \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Plant carotenoids can be also oxidized and fragmented by reactive oxygen species (ROS), which results in the formation of β-lonone, β-cyclocitrals that activate plant defense mechanisms.\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e These results are in good agreement with the previously reported by Shippen and co-workers of higher ROS activity in ISS-grown plants compared to \u003cem\u003eA. thaliana\u003c/em\u003e grown on Earth \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the spectra acquired from ISS-grown \u003cem\u003eA. thaliana\u003c/em\u003e seedlings, we observed an increase in the intensity of the vibrational band centered at 747 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, which can be assigned to pectin. Additionally, we identified small statistically insignificant changes in the intensity of the 1048 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e peak, which originates from cellulose. Based on these results, we can conclude that spaceflight alters the structure and composition of pectin, with minimal changes to cellulose, in \u003cem\u003eA. thaliana\u003c/em\u003e. Additional studies are required to fully understand the chemical nature of changes in these highly important biomolecules in plants.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVibrational band assignments for leaf spectra of \u003cem\u003eA. thaliana\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVibrational mode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAssignment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e742\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eγ(C\u0026ndash;O-H) of COOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePectin\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e915\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eν(C-O-C) In plane, symmetric\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCellulose,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e lignin\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAromatics, in-plane CH\u003csub\u003e3\u003c/sub\u003e rocking of polyene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProteins,\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e carotenoids\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eν(C-O)+ν(C-C)+δ(C-O-H)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCellulose,\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e lignin\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-C\u0026thinsp;=\u0026thinsp;C-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarotenoids\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-C\u0026thinsp;=\u0026thinsp;C-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarotenoids\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-C\u0026thinsp;=\u0026thinsp;C-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarotenoids\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-C\u0026thinsp;=\u0026thinsp;C-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarotenoids\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eδ(C-C-H)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAliphatic, cellulose, lignin\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1326\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eδCH\u003csub\u003e2\u003c/sub\u003e bending\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAliphatic, cellulose, lignin\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eδCH\u003csub\u003e2\u003c/sub\u003e bending\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAliphatics\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eδCH\u003csub\u003e2\u003c/sub\u003e bending\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAliphatics\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eδ(CH\u003csub\u003e2\u003c/sub\u003e)+δ(CH\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAliphatics\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-C\u0026thinsp;=\u0026thinsp;C- (in plane)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCarotenoids\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eν(C-C) Aromatic ring\u0026thinsp;+\u0026thinsp;σ(CH)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLignin\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1678\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmide I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProteins\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also performed a partial-least squared discriminant analysis (PLS-DA) to investigate the accuracy of RS-based differentiation between ISS- and Earth-grown \u003cem\u003eA. thaliana\u003c/em\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Previously reported results by our and other research groups demonstrate that PLS-DA performs as well or even better compared to other supervised chemometric algorithms such as support vector machine (SMV), linear discriminant analysis (LDA) or soft independent modelling by class analogy (SIMCA).\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Therefore, we utilized this chemometric method for the analysis of our spectroscopic data.\u003c/p\u003e \u003cp\u003eOur results showed that PLS-DA enabled 90% accurate identification of ISS- and 96.5% accurate identification of Earth-grown \u003cem\u003eA. thaliana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eConfusion matrix of PLS-DA model that indicates accuracy of identification of Raman spectra acquired from ISS- and Earth-grown plants.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eISS-grown\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEarth-grown\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccuracy, %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePredicted as ISS-grown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePredicted as Earth-grown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese results demonstrate that RS could be used as a robust and reliable tool to identify gravity-induced stresses in plants.\u003c/p\u003e \u003cp\u003eNext, we investigated the extent to which RS could be used to probe the response of plants grown for 20 days on the lunar regolith simulant LMS-1, media that can be used to mimic the outer crust of the Moon \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. RS revealed a substantial increase in the intensity of phenylpropanoids and a decrease in the intensity of carotenoids in the Raman spectra acquired from plants grown on lunar regolith simulant compared to plants grown on Earth soil, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Again, these RS findings were consistent with evidence of a stress response for plants grown in lunar regolith simulant \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough no significant changes in the concentration of pectin were detected, we observed statistically significant differences in the intensities of the vibrational band that can be assigned to cellulose (1048 cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e). Specifically, the intensity of this band was stronger in the spectra acquired from plants grown in the lunar regolith simulant compared to plants grown in conventional Earth soil. These results indicate that regolith-induced stress caused a substantial increase in the synthesis of cellulose which may help to facilitate the adaptation of \u003cem\u003eA. thaliana\u003c/em\u003e to this exotic substrate.\u003c/p\u003e \u003cp\u003eWe previously showed that the poor or toxic nutritional environment of regolith could be partially overcome by addition of an antioxidant cocktail composed of 0.75mM gluthathione, 0.75mM ascorbic acid and 0.75mM proline, or by its individual components \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Expanding upon this, we investigated whether RS could be used to monitor bio stimulant-induced improvement in the plant health, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on the changes of the intensities of carotenoids and cellulose vibrations, we found that antioxidant treatment with all three components of the cocktail was the most efficient of all bio stimulants, as depicted on Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Specifically, plants grown on the regolith simulant soil treated with antioxidants exhibited comparable intensities of carotenoids and cellulose vibrations to the control plants that were grown in Earth soil. These results demonstrate that antioxidants minimize enzymatic degradation of plant carotenoids into abscisic acid. High concentration of carotenoids in these plants also suggest that low levels of ROS that are also known to fragment and degrade carotenoids. Thus, antioxidants minimize regolith-induced stress of plants.\u003c/p\u003e \u003cp\u003eOur results further showed that the individual components of the antioxidant cocktail were able to significantly alter the carotenoid and cellulose RS profiles, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Consistent with these observations, the antioxidant cocktail, as well as individual proline and gluthathione treatments substantially improved plant growth and reduced genome oxidation with respect to untreated lunar regolith \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to LMS-1 Lunar Mare Simulant, other lunar regolith simulants are commercially available that replicate specific lunar environments with high fidelity (Long-Fox et al., 2023).\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e These include LHS-1 Lunar Highlands Simulant, and LSP-2 Lunar South Pole simulant. We also investigated the extent to which treatment of three different types of lunar regolith with antioxidant cocktail composed of glutathione, ascorbic acid and proline could change the concentration of phenylpropanoids, carotenoids, pectin and cellulose. For these experiments we examined more mature plants that were 47-days-old. We found that treatment of lunar highlands and lunar mare simulants with the cocktail composed of glutathione, ascorbic acid and proline increased the concentration of phenylpropanoids, carotenoids, and pectin. Interestingly, no substantial changes in the concentration of cellulose were observed, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Such treatment-induced improvements were minor in plants grown on lunar south pole simulants. These findings indicate that the antioxidant-induced increase in the concentration of phenylpropanoids, carotenoids, and pectin in \u003cem\u003eA. thaliana\u003c/em\u003e depends on type of simulant that plants were grown on. Furthermore, compared to 20-day-old LMS-1 grown plants, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, 47-day-old LMS-1 plants exhibited reduced levels of carotenoids and pectin relative to the control. The older plants also showed decreased phenylpropanoids and cellulose. These results indicate that secondary metabolite production is dynamic and evolves throughout plant development in lunar regolith simulant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, our results indicate that RS is a useful tool for non-invasive and non-destructive assessment of the effect of bio-stimulants on the health of plants grown in regolith. Moreover, our RS results are consistent with the results from other biochemical assays and highlight the potential challenges in preserving plant wellbeing in lunar regolith simulant despite efforts at bio-stimulation \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Consequently, these results underscore the feasibility of utilizing Raman spectroscopy for non-invasive and non-destructive evaluation of the overall health of plants cultivated in regolith.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials:\u003c/h2\u003e \u003cp\u003e \u003cb\u003ePlant growth\u003c/b\u003e: For seedlings grown on the ISS \u003cem\u003eArabidopsis thaliana\u003c/em\u003e seeds (35S::HF-RPL18 in the Columbia-0 background; ABRC stock # CS66056) were affixed to a polyethersulfone (PES) membrane using guar gum \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Each membrane accommodated fifteen seeds and was subsequently placed onto square 10 cm 0.5x Murashige and Skoog plates with 1% agar. Following a 19-day period of darkness at 4\u0026deg;C, the plates were integrated into the Veggie growth chambers on the ISS and at Kennedy Space Center, where the plants were cultivated for 12 days under 16 h/8 h long day conditions. The growth conditions included 100 mol/m2/s of red (630 nm), blue (455 nm), and green (530 nm) light, simulating Veggie units. Ground control plates underwent seeding, growth, and harvesting with a 48-hour delay from the flight samples. Environmental conditions such as temperature, CO\u003csub\u003e2\u003c/sub\u003e, and relative humidity were monitored continuously on the ISS and replicated with a 48-hour delay in the ground control environmental chamber. The average temperature maintained was 23\u0026deg;C with 44% relative humidity, and CO\u003csub\u003e2\u003c/sub\u003e levels were typically around 1750 ppm. Following removal from the Veggie, astronauts or ground control staff collected the PES membranes and seedlings using forceps, which were then protected in the lid of the Petri dish, wrapped with foil, and swiftly transferred to a \u0026minus;\u0026thinsp;130\u0026deg;C cold bag for rapid freezing. Subsequently, the seedlings were stored at \u0026minus;\u0026thinsp;80\u0026deg;C, shipped on dry ice, and kept at \u0026minus;\u0026thinsp;80\u0026deg;C until processing.\u003c/p\u003e \u003cp\u003eFor plant grown on lunar regolith simulant seeds from Col-0 were procured from The Arabidopsis Biological Resource Center (ABRC) at Ohio State University, stock CS28994 \u003csup\u003e2\u003c/sup\u003e. The seeds underwent surface sterilization in 2.7% sodium hypochlorite for 7 min, followed by stratification at 4\u0026deg;C for three days. Subsequently, they were directly transferred to their designated treatment of either regolith simulant (LMS-1, LSP-2 and LHS, Exolith Lab, Oviedo, FL, USA) or Earth soil (Sunshine, Mix 5, Sun Gro Horticulture, Agawam, MA, USA). Germination took place under a 12-h photoperiod with an average illuminance of 5000 (+/- 250) lux achieved using lights with a 3000:6500 Kelvin ratio, and maintained at a constant temperature of 22\u0026deg;C. The plants were allowed to grow under these conditions for five weeks or until growth ceased, upon which the entire plants were harvested for analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRaman Spectroscopy\u003c/b\u003e: Raman spectra were collected from the surface of plant leaves with a hand-held Resolve Agilent spectrometer equipped with an 830-nm laser source. The following experimental parameters were used for all collected spectra: 1 s integration time, 495 mW power, and baseline spectral subtraction by device software. Thirty spectra were collected from each group of plants. Spectra shown in the manuscript are processed through MATLAB, normalizing at the 1440cm\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e peak (include explanation if necessary) and applied with Savitzky-Golay smoothing using a polynomial order of 0.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMachine learning model\u003c/strong\u003e \u003cp\u003eIn this work, we utilized partial least-squared discriminant analysis (PLS-DA). This supervised version of principal component analysis achieves dimensionality reduction with the information of target variables together with the good insight into the causes of discrimination through weights and loadings that assists in conducting exploratory data analysis \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings demonstrate that RS is a useful tool for the confirmatory sensing of changes in plant biochemistry caused by zero gravity and growth in Lunar regolith simulants. Notably, there is an established correlation between carotenoids and phenylpropanoids with stress response and oxidative damage; both plant materials subjected to spaceflight conditions and those cultivated in lunar environments exhibited alterations in the levels of these molecules. The observed changes in carotenoids and phenylpropanoids in space-flown plants and plants grown in simulants of lunar soil suggest a response to stress factors and oxidative challenges experienced in these unique settings. Our results also show that RS could be used to monitor the efficiency of soil supplements that were capable of mitigating the negative effects of nutrient-free regolith media and, consequently, improve the plant health. Thus, RS represents a highly efficient, quick, and label-free approach for monitoring plant health in exotic environments.\u003c/p\u003e \u003cp\u003eNotes:\u003c/p\u003e \u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eThis study was supported by funds from Texas A\u0026amp;M AgriLife Research, Texas A\u0026amp;M University Governor\u0026rsquo;s University Research\u003c/p\u003e\n\u003cp\u003eInitiative (GURI) grant program of (12-2016/M1700437), by the National Institutes of Health (R01 GM065383 to D.E.S), the National Aeronautics and Space Administration (80NSSC19K1481 to S.E.W; 80NSSC23K0302 to D.E.S and S.E.W). and the NASA Postdoctoral Program at Kennedy Space Center administered by Oak Ridge Associated Universities (to A.M.). We extend our gratitude to the APEx-07 team, with special acknowledgment to Gerard Newsham, Anne Marie Campbell, Erica Bugardner, and Susan Manning-Roach, based at Kennedy Space Center, for their dedication to experiment validation, flight preparation, and return. We also express our appreciation to astronauts Thomas Pesquet, Mark Vande Hei, and Megan McArthur for their assistance with experiment takedown and harvest on the ISS.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarcenilla, B. B.; Meyers, A. D.; Castillo-Gonzalez, C.; Young, P.; Min, J. H.; Song, J.; Phadke, C.; Land, E.; Canaday, E.; Perera, I. Y.; et al. Arabidopsis telomerase takes off by uncoupling enzyme activity from telomere length maintenance in space. Nat Commun 2023, \u003cem\u003e14\u003c/em\u003e (1), 7854. 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Sensors and Actuators B: Chemical 2020, \u003cem\u003e303\u003c/em\u003e, 127243.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4801715/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4801715/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColonization of the Moon and other planets is an aspiration of NASA and may yield important benefits for our civilization. The feasibility of such endeavors depends on both innovative engineering concepts and successful adaptation of life forms that exist on Earth to inhospitable environs. In such missions plants will play a vital role as life support systems, supplying astronauts with food, oxygen, carbon dioxide recycling, and psychological well-being benefits. Therefore, understanding the adaptability of plants to harsher environments, including different gravitational forces and growth on extraterrestrial soils will be required. In this study, we investigate the potential of Raman spectroscopy (RS), a modern analytical technique, in a non-invasive and non-destructive assessment of changes in the biochemistry of plants exposed to zero gravity on the International Space Station and during growth on lunar regolith simulants on Earth. We report that RS can sense changes in plant carotenoids, pectin, cellulose, and phenolics, which in turn, could be used to gauge the degree of plant stress to the new environments. Our findings also demonstrate that RS can monitor the efficiency of soil supplements that can be used to mitigate nutrient-free regolith media. We conclude that RS can serve as a highly efficient, fast, and label-free approach for monitoring plant health in exotic environments.\u003c/p\u003e","manuscriptTitle":"Raman Spectroscopy as a Tool for Assessing Plant Growth in Space and on Lunar Regolith Simulants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-28 13:11:24","doi":"10.21203/rs.3.rs-4801715/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-01-23T08:17:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-01-13T01:59:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-12-22T01:40:09+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-09-24T13:45:38+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-09-02T14:26:51+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-08-01T13:49:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-26T10:25:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-26T10:25:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Microgravity","date":"2024-07-25T12:04:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-microgravity","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjmgrav","sideBox":"Learn more about [npj Microgravity](http://www.nature.com/npjmgrav/)","snPcode":"41526","submissionUrl":"https://submission.springernature.com/new-submission/41526/3","title":"npj Microgravity","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4bc0f505-d179-4fb2-99a3-01d9f8479a20","owner":[],"postedDate":"August 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35454540,"name":"Biological sciences/Biochemistry"},{"id":35454541,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-05-28T07:09:10+00:00","versionOfRecord":{"articleIdentity":"rs-4801715","link":"https://doi.org/10.1038/s41526-025-00479-8","journal":{"identity":"npj-microgravity","isVorOnly":false,"title":"npj Microgravity"},"publishedOn":"2025-05-27 04:00:00","publishedOnDateReadable":"May 27th, 2025"},"versionCreatedAt":"2024-08-28 13:11:24","video":"","vorDoi":"10.1038/s41526-025-00479-8","vorDoiUrl":"https://doi.org/10.1038/s41526-025-00479-8","workflowStages":[]},"version":"v1","identity":"rs-4801715","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4801715","identity":"rs-4801715","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}