A new era of lunar exploration has started under the umbrella of the Artemis Program at NASA (Chavers et al., 2019). In the first phase (by 2024), NASA plans to return to the Moon and accomplishes the following tasks: (1) to send robotic landers and payloads to the lunar surface; (2) to assemble the Gateway outpost in lunar orbit; and (3) to resume the first human landings on the surface of the Moon since 1972. NASA’s Artemis Program will use a coordinated approach utilizing the resources of the entire agency including NASA centers with a possible focus on landing near the South Pole of the Moon. In the second phase (by 2028), NASA plans to establish a sustained human presence on the Moon. The agency believes that the technologies and instruments developed during Artemis will be useful for future human missions to Mars.
In addition to human spaceflight that is part of Artemis, NASA has developed the Commercial Lunar Payload Services (CLPS) program to send relatively small (<500 kg) robotic landers to the Moon (Chavers et al., 2019). The goals of CLPS include determining whether there are lunar resources for future human missions and conducting science experiments related to Artemis. The first landers under CLPS to land on the Moon are scheduled to be built by Astrobotic (Pittsburgh, PA, USA) and Intuitive Machines (Houston, TX, USA). Based on our previous research on the International Space Station (ISS), we predicted that lunar gravity may be insufficient for normal plant growth and development while Martian gravity is adequate (Kiss et al., 2012; Vandenbrink et al., 2016). These results have profound implications for human exploration plans and the use of a Moon base as a stepping stone to Mars.
We have proposed a plant biology experiment on one of the early CLPS lunar landers that would be in a greenhouse housed in a CubeSat system. Thus, Arabidopsis thaliana (L.), a widespread worldwide species, would be grown in a previously designed 1U CubeSat habitat system for plant growth (Kitto et al., 2020). CubeSats are small devices developed for space research and are composed of multiples of 10′10′11 cm (1U) cubic units (Swartwout, 2013). Recently, an experiment with a prototype of this plant growth CubeSat was performed on the ISS (Kitto et al., 2020). Although there were several technical issues with this untended payload, seedlings of A. thaliana were successfully grown.
The first CLPS lunar landers have severe restrictions on power and mass, and mission resources available for biological experiments are very limited. In designing experiments with CubeSat on the Moon, a broad spectrum of temperature tolerance needs to be considered for growing Arabidopsis plants since strict temperature control requires major power resources. The surface temperature on the Moon can vary from −173 °C to +127 °C (Kim, 2020) depending on whether it is lunar day or night (each is 13.5 Earth days). Thus, it will be difficult to have multiple heaters and coolers for these experiments, so we need to develop strategies for the widest temperature range possible for growing plants.
This widest temperature range is in contrast to our previous experiments conducted with Arabidopsis seedlings on the Space Shuttle and ISS (Kiss, 2015). In these previous experiments, our planned temperature range was 20–25 °C with the optimum being 22–23 °C. Even with different types of plant growth hardware including Biorack (Katembe et al., 1998; Kiss et al., 1999), Biological Research in Canisters (BRIC) (Millar et al., 2011), and the European Modular Cultivation System (EMCS) (Kiss et al., 2011; Vandenbrink et al., 2016), the optimal temperature goal was easily attained. In the longer term, ground-based experiments (about 40 days), A. thaliana plants were grown at 10–26 °C (Schmuths et al., 2006). However, based on the mission profiles to date, our planned lunar experiment is limited to about 10 days, so we also want to ensure sufficient growth within this time frame.
Fortunately, since A. thaliana is a widespread worldwide species and is native to Europe, Asia, and Africa, it can grow in its natural environments under various temperature conditions. Thousands of populations of A. thaliana have been collected in the range of this plant, and these ecotypes vary considerably in genotype and phenotype since this species has successfully adapted to many different local environmental conditions (Alonso-Blanco et al., 2016).
Several studies have focused on the physiology and development of natural ecotypes of A. thaliana from different latitudinal distributions. For instance, differences in foliar vascular architecture, photosynthetic capacity, and transpiration rate were noted in ecotypes from Italy, Poland, and Sweden (Adams et al., 2016). In a related study, differences in freezing tolerance were found between the Italian and Swedish ecotypes (Park et al., 2018). Similarly, Zhen and Ungerer (2008) tested 71 Arabidopsis natural populations originated from 15°N to 60°N latitudinal range and found that northern ecotypes are more tolerant of freezing. In a more extensive series of experiments with over 100 ecotypes from throughout the European range of Arabidopsis, seed dormancy and flowering time patterns were correlated with the latitudinal gradient of the plant (Debieu et al., 2013). Interestingly, Li et al. (1998) noted that northern ecotypes tend to have a smaller plant size. In another study, Schmuths et al. (2006) tested for germination and emergence of the radicle at different temperatures of natural ecotypes of Arabidopsis from different latitudinal distributions.
Thus, for the reasons for our proposed study, we tested for temperature effects on germination and seedling growth from Arabidopsis ecotypes from different latitudes. The parameters used for germination and morphology are standard ones to assess the general health of plants in space missions (Kiss, 2015). We tested in the range of 10–35 °C because the possible temperatures within the contraints of using the robotic landers lie within these ranges.
Thus, our working hypothesis regarding ecotypes of A. thaliana is that the optimal temperatures for plant growth would be correlated to latitudinal distributions of this species. In other words, ecotypes from warmer climates like Africa would grow best at warmer temperatures and those from colder regions like Scandinavia would grow best at colder temperatures (Alonso-Blanco et al., 2016). Thus, if we were to use several ecotypes (in one lunar experiment) from wide latitudinal distributions, then we could extend the tolerable temperature range for the robust growth of Arabidopsis. This approach would potentially allow us to increase the temperature tolerance range of growing plants in the CubeSat that is part of a proposed CLPS lunar lander mission.
MATERIALS AND METHODS
We studied several natural ecotypes of the plant A. thaliana (L.) Heynh. Seeds of these ecotypes were obtained from two sources and are listed in Table 1. Seed stocks of most of the ecotypes used were from the ABRC Arabidopsis Biological Resource Center (https://abrc.osu.edu/). [Note that these seed stocks also are available from the Arabidopsis Information Resource (TAIR) at https://www.arabidopsis.org/]. Additional seed stocks were obtained from Dr. Michael F. Thomashow of Michigan State University, and these lines are described in the studies by Ågren and Schemske (2012), Gehan et al. (2015), and Oakley et al. (2017).
Key characteristics of the seed stocks of the nine ecotypes used in these studies.
|Name||Code||Latitude||Altitude (m)||Mean temperature (°C) Spring/Fall||Origin||Source||Stock #|
|Cape Verde Is||Cvi-0||N16°||1200||NA||Cape Verde||ABRC||CS8580|
Seeds of A. thaliana were surface-sterilized with 70% (v/v) of ethanol and a drop of Triton X-100 (200 ml) for 5 min. Seeds were first rinsed in 95% (v/v) of ethanol two times for 1 min each and then rinsed three times with sterilized water for 1 min each. Gridded square Petri dishes 100 ′ 100 ′ 15 mm (Cat. #60872, VWR International, LLC) were filled with 1.2% (w/v) of agar with growth medium, which consisted of one-half-strength Murashige and Skoog salts medium and 1% (w/v) of sucrose at pH 5.5 and is described in the study by Kiss et al. (1997). A layer of sterilized nitrocellulose film (Cat. # V7131, Promega Corp., Madison, WI, USA) was placed on top of the solidified nutrient agar.
Following surface sterilization, seeds were placed in two rows into each Petri dish. The agar dishes with seeds were then wrapped in Parafilm and left for 72 h at 4°C to stimulate germination. After seed stratification, the plates were placed vertically and continuously illuminated for 10 days with white light fluorescent tubes (120–140 mmol m−2s−1) in one growth chamber at 25, 30, or 35°C and in another growth chamber (approximately 240–260 mmol m−2s−1) at 11 °C or 15 °C, respectively. For germination tests, the number of seeds used for each ecotype and temperature is shown in Table 2. [However, we believe that it may be difficult to add a stratification step to the lunar experiment given our understanding of mission constraints.]
Number of seedlings grown from each ecotype and used for the temperature experiments.
|Seed code||Growth temperature|
|11 °C||15 °C||25 °C||30 °C||35 °C|
Image and Data Analyses
Seedlings in Petri dishes were imaged periodically with a Canon EOS Rebel T6 DSLR camera and with a Epson V600 Photo Scanner at termination on the 10th day. Growth and morphometricproperties were measured using the Fiji-win64 software. For each ecotype, the following growth parameters were measured: number of leaves, area of leaves, main root length, number of secondary roots, and the total root length. All analyses were performed with the RGui 64-bit R 3.5.1 for Windows with R Commander package (R Development Core Team, 2008).
Seed germination was assayed in the nine ecotypes of A. thaliana from different natural populations at 15, 25, and 35 °C and from four populations at 11 and 30 °C (Tables 1 and 2 and Figure 1), respectively. The highest germination occurred at 15 and 25 °C (Figure 1B,C). Minimal seed germination of 22% or less occurred at 35 °C. Across all temperature conditions studied, the highest seed germination occurred in the Col-0 ecotype. In addition, the SW and Mt-0 ecotypes had good seed germination in the range of 11–30 °C (Figure 1A,D).
We also examined the morphological features of the plants at different temperature treatments. In terms of leaf development, as with the data on germination, the best results across all ecotypes were at 15 and 25 °C (Figure 2). Thus, the greatest number of leaves (Figure 2A) and the largest leaf area (Figure 2B) occurred when seedlings were incubated at 15 and 25 °C. Similar to the seed germination studies, the best results were obtained with the Col-0 and the Mt-0 ecotypes. At the 30 °C incubation point, only the Col-0, SW, and Mt-0 (but not the IT ecotype) showed limited growth of leaves, while there was no growth for any of the ecotypes tested at 35 °C.
The results of the studies of root development (Figure 3) were generally similar to those obtained with investigations of the leaves. In terms of the main root (Figure 3A), the greatest length across all ecotypes was at 15 and 25 °C. The best performers on this parameter again were the Col-0 and the Mt-0 ecotypes. Across all ecotypes, the largest number of secondary roots (Figure 3B) occurred when the seedlings were incubated at 15 and 25 °C, with the Col-0 and the Mt-0 ecotypes exhibiting the greatest number of secondary roots. In terms of the final measurement considered, the length of the total root network (including both the primary and secondary roots; Figure 3C), the results are similar to the previous two parameters. For all three root criteria, there was little or no growth in any of the ecotypes at 30 and 35 °C (Figure 3).
Images of seedlings of the Col-0 (Figure 4) and the Mt-0 (Figure 5) ecotypes illustrate the differences in the development of the shoot and root systems at the different temperatures studied. Seedlings of the Col-0 show robust growth with a well-developed shoot system and an extensive primary and secondary root networks when incubated at 15 and 25 °C, but there was also good growth at 11 °C (Figure 4). Mt-0 seedlings grow well at 15 °C but have more robust growth at 25 °C (Figure 5). Taken together, when considering seed germination and seedling development, the Col-0 ecotype performed better than the other ecotypes studied at all temperatures tested in these experiments.
Studies with Different Ecotypes of A. thaliana
Hundreds of ecotypes of A. thaliana exist in nature (Alonso-Blanco et al., 2016), so our working plan was to send several ecotypes from varying latitudes on a mission to optimize the success of germination and growth of seedlings and plants in a lunar CubeSat greenhouse (Kitto et al., 2020). We predicted that a mixture of ecotypes from colder (higher latitude) and warmer (lower latitude) environments would allow us to have successful growth of seedlings in our proposed experiment. While other WT strains of A. thaliana have been used in previous space flight studies (Vandenbrink and Kiss, 2016), most notably Landsberg (Ler) and Wassilewskija (WS), we chose to focus on strains other than Ler and WS since there were many studies on the broad ecological distribution of these other strains.
However, our initial prediction was not supported by the results obtained in the present study. The Columbia ecotype (Col-0), which can be traced to origins in Poland at 53°N latitude (Fernandez et al., 2018), performed best at a wide range of temperatures from 11 to 30 °C. However, while there was seed germination at 30 °C and even at 35 °C, acceptable growth occurred in the range of 11–25 °C. The next best ecotype in terms of these germination and growth parameters was Mt-0, which is from a Libyan population 28°N latitude (Alonso-Blanco et al., 2016). However, based on the studies to date, Col-0 alone is our preferred choice in these proposed experiments as it performed much better than all of the other genotypes tested in these studies.
Importance of Optimization of Spaceflight Experiments
Spaceflight opportunities are relatively rare and expensive (Vandenbrink and Kiss, 2016), so it is important to do extensive ground-based testing of many parameters to optimize the flight experiments (Kiss, 2015). In our previous spaceflight experiments, we performed extensive testing to ensure success (Katembe et al., 1998). For example, in the initial EMCS project, we performed extensive ground-based studies as the hardware and the facility were both new. Thus, we tested the effects of storage of seeds on germination and growth as well as cold storage procedures following the termination of the experiment (Kiss et al., 2009). In some of our later EMCS experiments, we improved the lighting and imaging by using infrared illumination to provide high-quality images of the seedlings (Vandenbrink et al., 2019), and ground-based studies were important in identifying and optimizing these parameters (Kiss et al., 2014). Thus, in the present study, we continued using this general approach to determine the optimal temperature range for growing plants in a lunar greenhouse experiment.
In contrast to our initial working hypothesis of using several ecotypes of A. thaliana from populations from varying latitudes to have a wider range of optimal plant growth, we plan to use a single ecotype, Columbia (Col-0), in our proposed lunar studies in a 1U CubeSat greenhouse habitat. Based on the results presented in this study, Col-0 should produce good seed germination and robust plant growth at the range of 11–25 °C. This 14 °C range is broader than the 5 °C (20–25 °C) range used in our previous plant biology space experiments with Arabidopsis (Kiss et al., 2014), but it is within a temperature range used in previous ground-based experiments (e.g., Schmuths et al., 2006).
This wider temperature range will give engineers greater flexibility in designing a thermal system in the CubeSat that will be tethered to and acquire power from the robotic lunar lander (Kitto et al., 2020). Our future research will help to establish additional parameters of growing plants in the lunar environment, which will be important in the long-term for using plants as part of a bioregenerative life support system needed for human exploration of the Moon and perhaps Mars (Kiss, 2014).
We thank Ms. Alexandra Settle for her help with seedling growth measurements. We also thank our collaborators from NASA, Drs. Chris McKay and Robert Bowman, for their helpful advice and many thoughtful discussions on lunar biology. Financial support was provided by NASA grant 80NSSC17K0546.
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Adams WW, Stewart JJ, Cohu CM, Muller O, Demmig-Adams B (2016) Habitat temperature and precipitation of)| false Arabidopsis thalianaecotypes determine the response of foliar vasculature, photosynthesis, and transpiration to growth temperature. Frontiers in Plant Science 7, 1026. 27504111
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Park S, Gilmour SJ, Grumet R, Thomashow MF (2018) CBF-dependent and CBF-independent regulatory pathways contribute to the differences in freezing tolerance and cold-regulated gene expression of two)| false Arabidopsisecotypes locally adapted to sites in Sweden and Italy. PLoS One 13, e0207723. 30517145
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Vandenbrink JP, Herranz R, Poehlman WL, Feltus FA, Villacampa A, Ciska M, Medina FJ, Kiss JZ (2019) RNA-seq analyses of)| false Arabidopsis thalianaseedlings after exposure to blue-light phototropic stimuli in microgravity. American Journal of Botany 106, 1466–1476. 10.1002/ajb2.1384 31709515
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