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 | Pectin21 |
915 | ν(C-O-C) In plane, symmetric | Cellulose,12 lignin22 |
1000 | Aromatics, in-plane CH3 rocking of polyene | Proteins,23 carotenoids24, 25 |
1048 | ν(C-O)+ν(C-C)+δ(C-O-H) | Cellulose,12 lignin22 |
1115 | -C = C- | Carotenoids24, 25 |
1155 | -C = C- | Carotenoids24, 25 |
1186 | -C = C- | Carotenoids24, 25 |
1215 | -C = C- | Carotenoids24, 25 |
1288 | δ(C-C-H) | Aliphatic, cellulose, lignin22 |
1326 | δCH2 bending | Aliphatic, cellulose, lignin22 |
1335 | δCH2 bending | Aliphatics20 |
1382 | δCH2 bending | Aliphatics20 |
1440 | δ(CH2)+δ(CH3) | Aliphatics20 |
1525 | -C = C- (in plane) | Carotenoids24, 25 |
1608 | ν(C-C) Aromatic ring + σ(CH) | Lignin26, 27 |
1678 | Amide I | Proteins23 |
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.