Plants form a critical component of long-term space exploration, providing a number of services beyond a sustainable food source. For instance, they can capture carbon dioxide, release oxygen, revitalize the atmosphere, and enrich extraterrestrial regolith. An important challenge for space exploration is identifying genetic variants that make plants capable of performing these mission-critical functions under prevailing deep space conditions. Such plant genotypes could then be directly incorporated into human space missions while the genes governing their resilience can be introduced into target plant species. The most extreme terrestrial habitats are the only environments on Earth that feature some conditions analogous to those present on the Moon or Mars. For instance, ferns, gymnosperms, and flowering plants in the high Atacama Desert experience near Martian aridity, and moss inhabiting the volcano Licancabur in the tropical Andes receives ultraviolet B (UVB) irradiance approaching that on the Martian surface (Cabrol et al., 2014; Häder and Cabrol, 2020). Hundreds of species of bryophytes, algae, and two flowering plant species dwelling in the Antarctic cold desert experience dryness, temperature ranges, and, during the seasonal ozone hole, elevated UVB levels quite similar to those on Mars (Colesie et al., 2023). Plants from these extreme environments have evolved adaptations to sustain life under such harsh conditions, thus are likely to harbor genes that confer an advantage in human space exploration. These plants, however, differ considerably from model plants prevalent in space biology research and are likely to require alternative experimental techniques.

The waxy cuticle coating aerial organs forms an effective barrier between plant cells and the external environment, and its occurrence some 400 million years ago in bryophyte ancestors has been considered one of the greatest adaptations of plants that allowed them to establish and conquer dry land (Riederer and Schreiber, 2001; Domínguez, Heredia-Guerrero, and Heredia, 2015; Hõrak, 2017; Renault et al., 2017; Xue et al., 2017; Ziv et al., 2018). At present, waxy cuticles alongside tiny leaves, spines, taproots, or water-storage tissues continue to be instrumental to plant survival in the harshest places on Earth. Cuticular wax is typically a complex mixture of dozens of compounds with diverse long hydrocarbon chains (C20–C37) or ring structures that consist of fully saturated fatty acid derivatives (Yeats and Rose, 2013). Some waxes are epicuticular and form an outermost coating at the tissue/air interface, while others are superimposed and integrated within a three-dimensional network of cutin biopolymer (Buschhaus and Jetter, 2011). A high content of cuticular waxes that are non-polar result in a plant surface that is hydrophobic or super-hydrophobic. Surface hydrophobicity influences the wettability, self-cleaning behavior, and light reflection at the cuticle interface (Koch and Ensikat, 2008). Hydrophobic surfaces help plants conserve water by reducing the amount lost through evaporation and thus are instrumental to the drought-resistance of plants inhabiting arid climates (Tomasi and Abdel-Haleem, 2023). Hydrophobic surfaces also protect plants from water-related damage such as fungal growth or ice formation on leaves, giving an advantage to plants in extremely cold environments.

Molecular analysis of plant responses to the spaceflight environment typically occurs post-flight when scientific payload is returned to Earth. To adequately preserve molecular processes relevant to plant physiological adaptation to spaceflight, plant tissue needs to be fixed in-flight. To be effective, intracellular fixation must rapidly halt enzymatic reactions without disturbing or modifying cell physiology in the spaceflight-adapted state. Immersing plant tissue in a concentrated aqueous solution of ammonium and cesium sulfates known as RNAlater is currently the most popular method of in-flight plant fixation (Ferl et al., 2011; Kruse et al., 2017; Manzano et al., 2020). To achieve proper fixation, RNAlater needs to be absorbed to the plant surface and translocated into the cells. However, two phenomena that occur at the tissue/air surface and tissue/liquid interface affect RNAlater interactions with plant surfaces that are hydrophobic. First, there is a tendency for plants to trap air and retain thin air layers known as gas films when air makes initial contact with hydrophobic plant surfaces that are then submersed in any polar liquid (Figure 1). Air bubbles are hydrophobic due to the physical properties of nitrogen and oxygen molecules, which are non-polar and have no charge and therefore do not form hydrogen bonds with water molecules but have high affinity for hydrophobic cuticular waxes. The air bubbles present a physical obstacle to tissue–RNAlater contact. Second, the asymmetric sharing of electrons between oxygen and hydrogen atoms in water molecules results in high surface tension and droplet formation (Figure 1). Consequently, aqueous solutions such as RNAlater on hydrophobic surfaces coalesce instead of evenly spreading, which impedes RNAlater adherence (Nairn et al., 2016). It has been shown that hydrophobic plant surfaces have low wettability reflected in water contact angles above 90° and limited ability to retain fluid droplets (Brewer and Nuñez, 2007; Aryal and Neuner, 2010; Aryal and Neuner, 2016). In addition to contact interactions at the surface, RNAlater absorption through the hydrophobic surface can be further limited by low cuticle permeability (Figure 1). Permeability is governed by diffusivity associated with the size of RNAlater molecules in relation to the plant cuticle structure; for cuticles with rich and tightly arranged waxy compounds, diffusivity remains low. Finally, cuticles rich in waxes exhibit low solubility resulting from low affinity of RNAlater molecules for the waxy compounds (Figure 1) (Khayet and Fernández, 2012).

Freezing is another effective method that can be considered for in-flight tissue fixation. Since freezing-based fixation is a physical and not a chemical process, it is suitable for any plant irrespective of its structure or chemical composition and thus can be employed for plants with hydrophobic surfaces. To avoid triggering cold responses and changes to gene expression, plant tissue needs to be frozen very quickly (Kidokoro et al., 2022). Snap-freezing in liquid nitrogen is the fastest tissue freezing technique that has proven successful in numerous biological investigations; however, it cannot be performed on the International Space Station (ISS). Additionally, rapid freezing causes intracellular water molecules to transition from amorphous fluid to a rigid lattice, while salts in cell fluids become concentrated at toxic levels. Ice crystals can tear cellular structures, and disruption of lysosomes leads to release of RNases. Thus, tissues intended for molecular analysis need to remain deep-frozen until processed.

The moss Ceratodon purpureus has a long history as a model for the study of gravitropism, and the species has been successfully deployed in multiple spaceflight experiments to the ISS (Sack, 1993; Sievers et al., 1996; Kern and Sack, 2001; Kern et al., 2005; Cove and Quatrano, 2006). The moss isolates used in these studies originated from central Europe. However, the species has a very broad distribution, spanning all seven continents (McDaniel and Shaw, 2005) and occupies polar and high mountain habitats characterized by extreme cold, drought, low atmospheric pressure, and considerable UVB radiation (McDaniel and Shaw, 2005; Clarke and Robinson, 2008a). Moreover, Ceratodon is found in regions with substantial background ionizing radiation of geological origin and is among one of the first plant colonizers of exclusion zones post nuclear fallout containing toxic levels of radionuclides (Kovalchuk et al., 2004; Mousseau and Møller, 2020). The species is tolerant of both chemical and UV mutagenesis, and the annotated genome of C. purpureus provides a critical resource for transcriptomic analyses of responses to environmental challenges (Thornton et al., 2005). Moreover, C. purpureus undergoes efficient gene-targeting knock-outs and knock-ins using CRISPR-based approaches (Tavernier et al., 2023).

Here, we present the design of plant payload and fixation procedures developed for the spaceflight investigation ARTEMOSS (ANT1 Radiation Tolerance Experiment with Moss in Orbit on the Space Station). ARTEMOSS deploys the moss C. purpureus isolate ANT1, which was collected in Antarctica—the most extreme of terrestrial environments, characterized by severe aridity, cold, and high seasonal UVB fluxes. ARTEMOSS addresses growth, development, and physiology, as reflected by gene expression, of ANT1 in the combined environments of ground-based simulated deep-space ionizing radiation and spaceflight microgravity and requires moss tissue fixation in-flight. We thus present approaches to improve fixation in RNAlater of Antarctic moss with hydrophobic surfaces, along with extensive testing of deep-freeze fixation tailored for the microgravity environment.

We here demonstrate that total RNA obtained from Antarctic moss tissue fixed in RNAlater is highly degraded and inadequate for downstream transcriptomic analysis. Antarctic moss eludes efficient fixation in RNAlater, despite the integration of various surfactants that modify the adherence of RNAlater to the moss tissue and combinations of surfactants with various types of oils that facilitate RNAlater permeation. We demonstrated that deep-freezing moss tissue on Petri plates provides adequate tissue fixation and allows the extraction of high-quality RNA suitable for gene expression profiling. After evaluating availability and tissue fixation time for various deep-freezing conditions, we designed the ARTEMOSS payload with considerations of constrained space and crew time.

Dispensing RNAlater on the moss tissue on a Petri plate revealed the presence of air bubbles adhered to moss filaments. The non-spherical air bubbles forming airfilm is indicative of the hydrophobic or super-hydrophobic surface rich in cuticular waxes (Krasowska et al., 2009). Moss C. purpureus isolate ANT1 was collected at Casey Bay in Antarctica—the driest and coldest of all terrestrial environments. Although the chemical characterization of the external surface of ANT1 is not available, plants from very cold or arid environments exhibit particularly high cuticular wax content, which is presumably instrumental in averting water loss and desiccation and is unlike plants from tropical and subtropical regions with abundant rainfall (Busta et al., 2016; Goldsmith et al., 2017; Hõrak, 2017; Waterman et al., 2017; Xue et al., 2017; Waterman et al., 2018). Additionally, other traits adaptive to survival in Antarctica have been identified in ANT1. For instance, the alkali extraction of cell-wall-bound phenolics, combined with methanol extraction of soluble phenolics, determined that ANT1 had six times higher concentration of cell-wall-bound UV-screening compounds than the concentration of intracellular UV screens (Clarke and Robinson, 2008a; Waterman et al., 2018). ANT1 also showed an exceptionally high content of biflavonoids and unknown phenolic compounds that are strongly bound to cell walls as well as being located inside the cells; they play dual roles as both UV-screening and antioxidant compounds that give this species advantage under damaging UV conditions during seasonal stratospheric ozone depletion (Waterman et al., 2017).

Total RNA extracted from the moss tissue submersed in RNAlater and retaining airfilm was of very poor quality, indicating inefficient fixation of Antarctic moss tissue in RNAlater. The presence of encapsulating air bubbles may limit the surface interaction between tissue and RNAlater. The addition of surfactants of different chemical composition to RNAlater reduced air bubble retention on the moss tissue and exposed most of the tissue surface to contact with RNAlater. However, surfactants did not satisfactorily improve the quality of total RNA extracted from the fixed moss tissue. This was unexpected, since the performance of surfactants in dispersing, spreading, sticking, and wetting has been successfully deployed in agriculture to improve the absorption of various active agents (Castro et al., 2013). In molecular biology, surfactants are successfully used as enhancements for the “floral dip” transformation of plants (Huynh et al., 2022). This suggested that poor fixation in RNAlater did not result solely from the ineffective fixative-tissue surface interaction and that dislodging air bubbles improves RNAlater adherence to the moss tissue but was still not sufficient to achieve effective ANT1 tissue fixation in RNAlater. Although the addition of surfactants to RNAlater did not improve ANT1 fixation, this strategy may be effective with other plants, particularly as the variety of plant species used in spaceflight investigations expands. Even highly hydrophilic plant surfaces when exposed to air trap air bubbles; however, in the presence of the buoyancy force on the ground, the submersion of plant tissue in a liquid causes air bubbles to detach and travel upward to the liquid surface. In microgravity, however, the lack of gravity-driven buoyancy and sedimentation within fluids causes non-departing small bubbles to coalesce and form sizable bubbles or even dry patches on a surface of biological or solid materials, irrespective of their hydrophobicity. Thus, the inability of air bubbles to naturally dislodge or burst likely compromises any liquid chemical delivery to a plant. Hence, targeting air bubble surface tension by using surfactants, like those tested in this investigation, could destabilize air bubbles, prevent coalescence, and ultimately remove bubbles from the system, thus improving biological experimentations in microgravity.

Modifying the permeation of RNAlater through the Antarctic moss tissue by adding oils in combination with surfactants resulted in a slightly better quality of extracted RNA. The minor improvement was unexpected since it has been demonstrated that the cuticular waxes form a permeation barrier to polar solvents, and their solubilization opens pathways that enable transport across the cuticle (Staiger et al., 2019). Hence, the symptoms observed of a change in cuticle texture are referred to as “softening” or even “dewaxing,” which dramatically improve the movement of polar solvents through the cuticle (Rourke and Gibbs, 1999). In agriculture, the addition of various types of emulsifiable oils increases the permeability of plant cuticles to foliage-applied polar pesticides (Nicetic et al., 2011). In our study, RNAlater in the presence of the petroleum oil-based adjuvant preserved RNA better than when mixed with orange oil-based adjuvant, suggesting that paraffin oil but not orange oil improved RNAlater permeation through the cuticular waxes. Since oil efficacy on modifying plant waxes depends on wax composition, structure, and chemical properties, it can be speculated that paraffin oil had higher affinity for waxes present in Antarctic moss cuticle than orange oil and thus solubilized waxes more effectively. The possibility exists that a carefully selected combination of an appropriate oil with detergents added to RNAlater would render adequate Antarctic moss tissue fixation and preserve good quality RNA. We also cannot rule out a scenario in which the inability of RNAlater to permeate the moss cells results from the presence of compounds other than, or in addition to, cuticular waxes.

As an alternative to RNAlater, we evaluated the efficacy of ethanol for Antarctic moss tissue fixation (Supplementary Material). Fixation in 70% ethanol resulted in high-quality RNA extracted from the moss tissue, but not at lower 30% and 50% concentrations, even though air bubbles never formed on moss tissue submersed at any concentration. Although ethanol seemed to be an effective fixative for plants with hydrophobic surfaces, it is not suitable for use in spaceflight due to its high flammability.

We demonstrated that deep-freezing Antarctic moss tissue is an effective fixation method that results in the preservation of very high-quality total RNA. The time required for the moss tissue on a Petri plate to achieve −5°C differed by 100 s between −80°C and −150°C freezers; however, the time for the moss tissue to achieve a deep-frozen state was substantially longer, by nearly 1,000 s (16 min 40 s) in the −80°C freezer than in the −150°C freezer, establishing a strong preference for using the GLACIER freezer over MELFI to fix our plant payload in-flight. We recreated a freezing scenario in microgravity by separating the Petri plates from the metal freezer surface with a layer of Styrofoam. Although the moss tissue on Petri plates placed on Styrofoam froze later than the moss tissue on Petri plates in direct contact with metal surface, the difference depended on the Petri plate configuration from 110 s (1 min 50 s) to 280 s (4 min 40 s) at −80°C. We demonstrated, however, that the substantial difference in the moss tissue freezing time resulted from a Petri plate being directly exposed to chilling air or being insulated by other Petri plates in a stack. Petri plates on both edges of the five-plate stacks achieved a deep-frozen state much faster than those in the middle of the stack, insulated by two Petri plates on each side. That difference was particularly large when the five-plate stack was placed on Styrofoam and reached 510 s (8 min 30 s) between the moss tissue on the top and middle Petri plate. The moss tissue on the top Petri plate froze slightly faster than that on the bottom Petri plate, although the five-plate stack was placed in the freezer horizontally and not vertically. This suggested that the moss tissue froze faster when separated from the cold air only by a plastic Petri plate lid as on the top Petri plate than when the tissue was separated from the cold air by a plastic Petri plate bottom and a layer of the media, as on the bottom of the Petri plate (Figure 7B). Thus, the least insulation of plant tissue from the cold air would shorten the deep-freeze fixation time. Therefore, for ARTEMOSS, we decided to construct stacks from just three instead of five Petri plates. Although the time to deep-freeze moss tissue on the Petri plates varied depending on the plate’s location within a stack, the difference in time did not affect the quality of RNA obtained from the moss tissue assessed by RIN. RNA extracted from the moss tissue harvested from Petri plates at each location within the plate stacks had RIN above 7.0.

Neither the effects of the sudden temperature drop below freezing or the differences in brief exposures to temperature falling from 0°C to −150°C on gene expression have been investigated in plants. However, even if some genes were activated during the deep-freezing process, they will appear in spaceflight but also in ground control gene expression profiles, and therefore such genes will not be wrongly asserted as spaceflight unique. Furthermore, numerous previous spaceflight experiments on Arabidopsis used the freezing method, predominantly in the −80°C MELFI freezer, to successfully preserve protein or transcript profiles; none reported enrichment in the cold-activated genes in the spaceflight samples (Kiss et al., 2011; Mazars et al., 2014; Barbero Barcenilla et al., 2023; Land et al., 2023).

Terrestrial plants from the edges of the limits of life have evolved adaptive traits that are seemingly favorable to thriving in the harsh environments inherent in human space exploration. However, these very adaptations may present challenges to routine experimental procedures, and working with plants from extreme environments will frequently require employing unconventional technological solutions. Based on the findings of this study, we designed in-flight fixation operations for the ARTEMOSS payload consisting of Antarctic moss on Petri plates that would best secure the transcript profile expressed in orbit. We will substitute the standard method of fixation in RNAlater with deep-freezing in GLACIER at −150°C. We will arrange the Petri plates in three-plate stacks to preserve the physical space and crew time at the fixation step, yet limit variations in the moss tissue freezing time on respective Petri plates. Three biological replicas per treatment will occupy each Petri plate position, top, middle, or bottom across a total of five three-plate stacks to correct for minor discrepancies in the moss tissue freezing time. The three-plate stacks at ground control will be placed on Styrofoam in the −150°C freezer to recreate fixing conditions in microgravity more accurately, where no contact with metal surface is maintained.

As we are poised to return to the Moon and take our terrestrial plants with us, chemical on-site fixation of plant tissue designated for sample return may not be an ideal approach. To achieve proper chemical fixation on the lunar surface, plants would need to grow in hardware preloaded with a fixative brought from Earth which would considerably increase upmass. Moreover, with time plants on the Moon will likely be grown in on site regolith in open containers rather than on nutrient media in hardware confinement. Therefore, the deep-freezing method, as evaluated in this investigation, which does not require special hardware or additional upmass, can be a viable alternative to preserve plant samples on the lunar surface for return to Earth. The necessity of returning ultracold geological samples has been long advocated. Here, we further demonstrate the value of incorporating long-term cryogenic containment systems on return missions for preserving biological investigations.