The first biological experiments on the lunar show that low gravity conditions will acclimate to super-freezing

doi:10.21203/rs.3.rs-2372108/v1

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The first biological experiments on the lunar show that low gravity conditions will acclimate to super-freezing

https://doi.org/10.21203/rs.3.rs-2372108/v1

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For humanity to complete its ambitious solar system exploration, it is crucial to comprehend how terrestrial life reacts to differing planet gravity. We followed the life trajectory of an earth cotton seed's germination, development, and ultimate fate after prolonged exposure to extremely low temperatures using the life-regeneration ecosystem carried by Chang'e 4 probe, which landed on the Moon on January 3rd, 2019, for the first time in human history. In a controlled environment with similar characteristics, such as temperature, humidity, air pressure, and nutrition, we compared this life trajectory on the moon to that on Earth, except for the differences in gravity, light, and radiation. We discovered that the 1/6 g moon gravity speeds up seed germination. Surprisingly, Moon seedlings demonstrated rapid acclimatization to super-freezing (below minus 52 degrees Celsius) under 1/6 g lunar gravity, maintaining upright and green despite exposure to long-term extremely cold temperatures for 18–24 hours. Based on cellular and molecular reactions caused by moon-low gravity, we suggest probable mechanisms for cold resilience. These unique findings will enhance our understanding of how plants adapt to suboptimal environmental conditions in space.

Biological sciences/Biophysics

Biological sciences/Cell biology

Earth and environmental sciences/Environmental sciences

Health sciences/Medical research

In the past half-century, human beings have made great progress in exploring space and the solar system. The Perseverance mission of NASA in the United States, Tianwen 1 in China, and Hope in the United Arab Emirates, three bold spacecraft launched in the summer of 2020, signaled a new phase in the human exploration of Mars. A fundamental question is how terrestrial life reacts to the extraterrestrial environment as humanity prepares to achieve the ambitious goal of building bases on the moon and Mars. Due to a number of financial and technological challenges, this question remains largely unanswered. Organisms on earth exhibit strong adaptability to numerous environmental variables, such as temperature, humidity, nutrition, CO2 concentration, etc. There is, however, a paucity of information on how life on Earth reacts to different gravitational conditions in space. Exploring this is important since gravity is a crucial element in the development of terrestrial life and is extremely important to evolution¹.

Over the past 20 years, our understanding of the effects of microgravity on biological physiology has increased. By replicating brief periods of changed gravity, plant-based life regeneration ecosystems have aided scientists in their research of plant growth in a ground-controlled environment, and research researchers have been able to study the mechanisms of life adaption to a condition that is close to zero gravity due to the enhanced design of plant-growing chambers on the International Space Station (ISS)^2-6. Plant culture in space continues to be a top priority strategy for all space exploration plans since plants could be a crucial component of a long-term bio-regenerative life support system. Life-support systems have been continuously developed to give optimal growth conditions for plants in space⁷^,⁸, i.e., stably controlled light, temperature, air humidity, CO₂ concentration, and so on. A number of plants have been reported to be able to germinate and grow in ISS, including Arabidopsis thaliana⁴, Brassica rapa⁹, Cucumber¹⁰, Rice³, Triticum aestivum¹¹, and Pisum sativum¹². However, no previous experiment was carried out on low-gravity planets (e.g. Moon or Mars) - the potential destinations of planetary migration, because it is extremely difficult to conduct such experiments. It is still unknown how plants or other earth organisms respond to real low gravity and extreme environments on these planets.

To address the above questions, in 2019 we conducted the first experiment by humans to grow plants in the 1/6 g lunar gravity, which was reported by Science and Nature on January 15, 2019. The experiment was carried out by Chang’e 4 lander in a sealed, one atmospheric pressure, and climate-controlled ecosystem equipment which forms a mini biosphere under the condition of low gravity, intense radiation, and wide temperature variation (Fig. 1A-D). In this small container, the weight is 2.608 kg, the size is only Φ173mm×198 mm, and the inner space is only 0.82L. A biological warehouse fixed on the bottom contains rapeseeds, cotton seeds, potato seeds¹³, and Arabidopsis seeds, as well as yeast and fruit-fly eggs (Fig. 1B-D). Two radiators and multilayer heat insulation are designed to accurately control the container's internal temperature. All biological samples were covered by water-soluble PVA fiber cotton, and a pipe can load water from the storage tank to the biological warehouse. A light pipe on the container’s top surface was designed to import natural sunlight for plant photosynthesis. Other interfaces for telemetry and power, as well as CMOD, are mounted for accurate control. The biological growth can be recorded in real-time by two cameras in the upper part of the container.

Figure 1E illustrates the biological experiments performed on the Moon. The power of the ecosystem was switched on exactly 12.87 hours after the craft landed at 10:26 on Jan 3, 2019. Half an hour later, a water pipe successfully loaded water into the biological warehouse. The cameras and sensors recorded and transmitted the real-time data back to earth in the period that we named “the first lunar day after landing” until the power was switched off at 20:03 on Jan 12, 2019. After that, the Moon entered a so-called “first lunar night after landing”, which lasted for about 18 earth-days, and was dark and cold. The temperature of the container in such an environment cooled down quickly to about -52℃ in 23 hours after the power was switched off, according to the heat balance test performed on earth by China Academy of Space Technology (Test report number: KPZH-DLPG-05). Finally, the power re-switched on at 01:33 on Jan 31, 2019, marking the beginning of “the second lunar day after landing”. With temperature variation, humidity, air pressure, nutrition, photography time, and other parameters and growth conditions accurately matching those on the moon, we conducted the same experiments in parallel on the earth in a growth container. However, artificial light was used on the ground and there was no simulating radiation dose on the moon.

We recorded the life trajectory of plant germination, development, and final fate after the lethal strike from exposure to the 18 days of super-frozen lander lunar night in real-time. All physical parameters for the life-regeneration ecosystems carried by the Chang'e 4 probe or the parallel control on Earth were the same, except for space radiation and gravity (Fig. 2A), the latter was 1g on Earth and 1/6g on the Moon. As recorded by the cameras, both the Moon and the earth’s ecosystem grew one seedling. The moon seedling appeared ~ 22 hours after water injection and was slight, while the earth seedling grew up ~ 82 hours after water injection and was fleshy. This result indicates that 1/6 g low gravity accelerates the germination of seeds. Meanwhile, compared with the 1 g condition plant growth, the Moon seedling grew slower and developed an obviously shorter hypocotyl and thinner cotyledon (Fig. 2B-C). This is likely because there was insufficient light and intense radiation from the moon entering the vessel.

Generally, the absence of gravity is a stress condition for plants, specifically for the functions of meristematic cells¹⁴. Plants are supposed to sense the direction of gravity and re-orientate themselves according to the gravitational pressure model or starch-statolith hypothesis¹^,^15-17. It was reported that for plants grown on ISS, microgravity can modify the plant body shape, increase the number of cells with transverse microtubules, and suppress lateral expansion in shoot organs including hypocotyls and epicotyls¹⁸. Polar auxin transport was significantly disrupted in a PsPIN1 membrane localization-dependent manner after three days of Alaska pea seedlings being exposed to ISS microgravity¹⁹. Furthermore, RNA-seq analysis of Arabidopsis thaliana seedlings grown on ISS revealed that light-associated pathways (e.g., photosynthesis-antenna proteins and chlorophyll metabolism, which were involved in plants grown) were significantly downregulated in microgravity²⁰. Additionally, it was reported that growing Arabidopsis thaliana seedlings in the ISS for 4 days in complete darkness caused an increase in the rate of cell proliferation and a decrease in the rate of cell growth²¹. Meanwhile, A. thaliana seedlings in spaceflight had their mRNA sequenced, and the result showed that the expression of the genes associated with an increase in cell volume was strongly suppressed in the hypocotyl²². The above experiments suggest that the cotton seedling of the moon should adapt to 1/6 g gravity by mechano-transduction of cellular substances like microtubules, amyloplasts, or hormone auxins, which together with gene expression changes induced by microgravity, contribute to shaping up the short height and thin leaf features. It is also worth noting that, probably influenced by the temperature higher than 30℃ (Fig 2A), only cotton seeds germinated on the earth and the moon. Because the temperature exceeded 30 degrees Celsius when our container landed on the moon and started the growth experiment, and the high temperature lasted for nearly 5 days. Under such high-temperature conditions, only cotton seeds can germinate, other model plants including rapeseeds, potatoes, and Arabidopsis seeds, whose appropriate germination temperatures are lower than 30℃, didn’t germinate in either the Moon or the earth container. This was consistent with the results of our experiments on the ground many times.

A major challenge for life during space exploration is the extreme environment, especially the effects of low gravity because the gravitational field cannot be realistically modeled. Understanding how plants respond to extreme conditions under different gravity is essential for constructing the life support system for the future space base and understanding the survival of living systems in general. To gain some insight, we followed the cotton seedling's survival trajectory, which experienced extremely cold temperatures for an extended period during the lunar night under 1/6 g gravity. The seedling first grew for 190 hours after germination, then was exposed to dark and ultra-low temperatures (as low as -52℃ in the containers, according to a heat-balance-experiment performed in a simulated environment on earth) of the lunar night for ~18 earth days. During lunar night, the power for life-regeneration ecosystems was switched off to conserve energy. Surprisingly, on Jan 31, 2019, when the container power was re-switched on after being kept in frozen condition for ~18 days and rewarmed to room temperature, the moon seedling was still green and erect (Fig. 3A). In comparison, the seedlings on earth had already died, showing a fragmental and black-yellow color appearance (Fig. 3A). During the following 10 days of the second lunar day, the moon

seedling kept standing with green leaves, and finally crinkled (Fig. 3A). Although it seemed that the slim seedling didn’t continue to grow in the second lunar day, we suspect that this was due to oxygen exhaustion in the container (Fig. 2A). These observations mentioned above point to an extraordinary phenomenon: a slim seedling could survive after being attacked by sustained, super-cold under low gravity on the moon!

Studying the mechanism of this unusual phenomenon is difficult because the experiment cannot be repeated on Earth. Here, we suggest some potential mechanisms by considering possible answers to the following questions. First, what are the mechanisms for cold resistance in plants? Secondly, how does microgravity influence those cold adaptation mechanisms?

Previously, it was found that plants in Earth's boreal area possess very high freezing resistance. Under the seasonal programmed control of photoreceptors and the circadian clock system, plants acclimate to severe subzero temperatures in winter²³. Different tissues have different adaption mechanisms to subfreezing temperatures. Xylem parenchyma cells avoid intracellular freezing by deep super-cooling, while extracellular freezing is the most common mechanism of adaptation to freezing for many plant cells, e.g., cambium and leaf cells^23-25. Plant cells can tolerate the temperature of liquid nitrogen by extracellularly freezing²⁶. Overwintering vegetation buds of trees have been reported to survive freezing by various survival strategies like super-cooling or extra-organ freezing²⁷^,²⁸.

Although the mechanisms involved in the above-freezing resistance strategies are complicated and still unclear, it is thought that the following cellular physiologic processes are crucial for cold protection: 1)Membrane stability is a critical factor in cold tolerance, which is probably achieved both by changes in membrane lipid composition and accumulation of solutes in the surrounding cytosol, including soluble sugars, carbohydrates, proline and betaines^29-32; 2) Plant auxins are also involved in cold tolerance, among them, abscisic acid is a protective factor^32-34, while ethylene is a negative regulator³⁵^,³⁶; 3) Transient influx of calcium into the cytosol should be a key step in response to freezing³⁷^,³⁸; 4) A number of cold-responsive genes are activated during cold-acclimation, including heat shock, antioxidant, and metabolism associated genes involved in lipid or carbohydrate metabolism²⁴^,³⁷^,³⁹^,⁴⁰; 5) The probable involvement of mitochondria mediated energy metabolism⁴¹; and, 6) Cell cycle arrest or dormancy.

We speculate that some of the mechanisms above may be involved in the moon seedling's resistance to extremely cold temperatures. It was suggested that plants on earth develop freezing resistance through adaptation to long-term seasonal cold²³. How could the moon seedling develop expeditious acclimation to super-freezing in such a short time? The likely possibility is that the moon’s low gravity contributes to this process.

The impact of actual and simulated microgravity on certain cell components has been provided in an exciting review published in 2017. Briefly, the properties and functions of cytoplasmic membrane can be viewed as the most sensitive indicators of the effect of microgravity on the cell⁴². Chlorella cells' cytoplasmic membranes were observed to have higher degrees of sinuosity and complex folding configurations under microgravity, which were related to enhanced metabolism and active transport⁴³. Chlorella cells' cytoplasmic membranes were observed to have higher degrees of sinuosity and complex folding configurations under microgravity, which were related to enhanced metabolism and active transport⁴⁴. Membrane isolated from pea seedling roots grown in low gravity showed increased phosphatidylcholines and phosphatidylethanolamines⁴⁵, as well as enhanced unsaturated fatty acids and decreased saturated fatty acids content. Sterne, a crucial part of "lipid rafts" thought to be involved in microgravity-induced changes to membrane permeability and stress resistance, was significantly increased in the membrane from pea seedlings under clinostat circumstances⁴¹^,⁴⁵. A study about the seedling transcriptome and proteome of A.thaliana reported microgravity-induced membrane-bound proteins participating in the metabolism and cell defense⁶. These observations prompt us to suggest the following as a possible mechanism for the moon seedling's fast cold acclimation: 1/6 g gravity alters the components of the cell membrane, which in turn promotes the folding of the cytoplasmic membrane, and alters membrane permeability, ion transport, distribution of protective membrane proteins, and consequently contributes to freezing resistance.

How do plants translate such gravitational forces into initial cell membrane biochemical signaling cascades when exposed to sustained low gravity⁴⁶? As reviewed by Masatsugu and Simon⁴⁷, plants' gravitropic responses include both rapid phasic and extended tonic components. Numerous available data about plants’ rapid signaling events to mechanical/gravitropic stimulation indicate that cells sense and carry out signal transduction by sedimentation of amyloplasts, as well as the cytoplasmic increase of Ca²⁺ through mechanosensitive ion channels, reflecting auxin-triggered signaling^48-50. While the duration of mechanosensitive ion channel activity is too short of exhibiting a long-term gravitropism response, Masatsugu and Simon suggested that the cytoskeleton should be a candidate in this long-term perception system. A plant’s microtubule cytoskeleton plays a vital role in regulating cell expansion, and gravistimulation results in changes in the orientation of cortical microtubule directions in plants’ hypocotyls⁵¹. The cortical actin cytoskeleton underlying the cell membrane also appears to play a role in initial graviperception. The cortical actin cytoskeleton underlying the cell membrane also seems responsible for initial graviperception. As an essential component of the biochemical mechanosensory network⁵², actin is involved in clathrin-mediated endocytosis, which is influenced by cells’ membrane tension⁵³. Additionally, in vitro experiments showed that actin shells can control biomembrane buckling and wrinkle formation in reconstituted actin-and membrane systems⁵⁴. Most interestingly, Michele and his associates found that mechanical stretch can deform the nucleus, resulting in chromatin rheology and architecture change⁵⁵; the latter is generally believed to influence gene expression. These discoveries might provide one model for how plant cytoskeleton proteins operate to transduce the mechanical signal from long-term low gravity to membrane biochemical activity.

We think that several low gravity-induced physiological changes may also be beneficial to frozen protection in addition to gravity-associated membrane alteration: 1) Concentrations of soluble sugars, glucose, sucrose, and total starch were substantially increased in Space Shuttle Flight sweet potato stem⁵⁶, and soluble sugars enhanced in tobacco callus cells under rotating simulated microgravity⁵⁷;2) Low gravity in space had no discernible impact on rice shoot auxins, but it reduced the expression of ACC synthase³, which is a critical enzyme in the biosynthesis of ethylene also a negative regulator of cold resistance;3) Numerous experiments indicated that calcium in cells increased under actual or simulated low gravity conditions⁵^,⁵⁸^,⁵⁹. 4) Genes expression changed under real low gravity in space and simulated ground experiments. Functions of “major space genes” include Ca²⁺ and lipid signaling, cell membrane biosynthesis, total metabolism, carbohydrates and lipid response to stress, auxin-signaling, and heat shock protein (HSP) thought to support the organization of cytoskeleton in adaptive response to microgravity^60-63. In food-borne pathogen Listeria, low-shear modeled low gravity elevated cold resistance in a cold-stress-regulated gene dependent manner⁶⁴.

In conclusion, for the first time, we found that plant seeds can sprout on a planet under its true gravity, and the moon’s 1/6 g gravitational field accelerates the plant’s development. Most notably, it seemed that seedlings on the moon developed expeditious acclimation to super-freezing, keeping green and erect for several days after the exposure to the long-term super cold lunar night. We propose a hypothetical scenario for such fast acclimation of the moon seedlings to super-freezing stress. As summarized in figure3B, through moon-low gravity-induced adaption, plants can fold the cell’s cytoplasmic membrane, alter components and permeability of the membrane, enhance cytoplasmic soluble sugars as well as other carbohydrates, reduce ethylene production, provide an increased flow of calcium ions into cells, and alter “major space genes” expression, all of which help plants become more resistant to extreme cold.

This pioneering study of lunar plant growth explores how seedlings react to truly low gravity and extend our insight into how organisms can survive in extreme space environments. In future space missions, it will be exciting to test whether plants can withstand extremely low temperatures under microgravity environments. It will also be essential to find if such a phenomenon generalizes to other multicellular organisms such as animals.

ACKNOWLEDGMENTS

The authors wish to express their appreciation to the Shandong Institute of Space Electronic Technology, Institute of Optics and Electronics (CAS). Special thanks to Lunar Exploration and Space Engineering Center, Beijing Institute of Spacecraft System Engineering, Beijing Aerospace Control Center, National Astronomical Observatories (CAS) for program management and useful discussions. We also gratefully acknowledge the financial support of the third pre-research projects of the civil space project from China National Space Administration (CNSA), and the Key Technology for the Construction of Micro-Ecospheres Adapted to the Lunar Environment for this project.

AUTHOR CONTRIBUTIONS

GengXin Xie, general manager, proposed research; Jing Yang, main manuscript writing; YuXuan Xu participated in manuscript writing and research; YuanXun Zhang, Dan Qiu, JingHang Ding participated in research.

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No competing interests reported.

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The first biological experiments on the lunar show that low gravity conditions will acclimate to super-freezing

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