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Comparison of Microgravity Analogs to Spaceflight in Studies of Plant Growth and Development

Comparison of Microgravity Analogs to Spaceflight in Studies of Plant Growth and Development RevIew published: 06 December 2019 doi: 10.3389/fpls.2019.01577 Comparison of Microgravity Analogs to Spaceflight in Studies of Plant Growth and Development 1 2 3 4 John Z. Kiss *, Chris Wolverton , Sarah E. Wyatt , Karl H. Hasenstein 5,6,7 and Jack J.W.A. van Loon 1 2 Department of Biology, University of North Carolina—Greensboro, Greensboro, NC, United States, Department of Botany & Microbiology, Ohio Wesleyan University, Delaware, OH, United States, Molecular and Cellular Biology Program, Department of Environmental & Plant Biology, Ohio University, Athens, OH, United States, Biology Department, University of Louisiana at Lafayette, Lafayette, LA, United States, DESC (Dutch Experiment Support Center), Department of Oral and Maxillofacial Surgery/Oral Pathology, Amsterdam University Medical Center, Amsterdam, Netherlands, Academic Centre for Dentistry Amsterdam (ACTA), VU-University, Amsterdam, Netherlands, European Space Agency (ESA) Technology Center (ESTEC), Life & Physical Science, Instrumentation and Life Support Laboratory, TEC-MMG, Noordwijk, Netherlands Life on Earth has evolved under the influence of gravity. This force has played an important role in shaping development and morphology from the molecular level to the whole organism. Although aquatic life experiences reduced gravity effects, land plants have evolved under a 1-g environment. Understanding gravitational effects requires changing the magnitude of this force. One method of eliminating gravity’s influence is to enter into a free-fall orbit around the planet, thereby achieving a balance between Edited by: Valérie Legué, centripetal force of gravity and the centrifugal force of the moving object. This balance Université Clermont Auvergne, is often mistakenly referred to as microgravity, but is best described as weightlessness. France In addition to actually compensating gravity, instruments such as clinostats, random- Reviewed by: Tomomichi Fujita, positioning machines (RPM), and magnetic levitation devices have been used to eliminate Hokkaido University, Japan effects of constant gravity on plant growth and development. However, these platforms Yutaka Miyazawa, do not reduce gravity but constantly change its direction. Despite these fundamental Yamagata University, Japan differences, there are few studies that have investigated the comparability between *Correspondence: John Z. Kiss these platforms and weightlessness. Here, we provide a review of the strengths and jzkiss@uncg.edu weaknesses of these analogs for the study of plant growth and development compared to spaceflight experiments. We also consider reduced or partial gravity effects via spaceflight Specialty section: This article was submitted to and analog methods. While these analogs are useful, the fidelity of the results relative to Plant Abiotic Stress, spaceflight depends on biological parameters and environmental conditions that cannot a section of the journal Frontiers in Plant Science be simulated in ground-based studies. Received: 19 July 2019 Keywords: Arabidopsis, clinostat, plant growth, simulated microgravity, random positioning machine, reduced Accepted: 12 November 2019 gravity, spaceflight experiments Published: 06 December 2019 Citation: Kiss JZ, Wolverton C, Wyatt SE, INTRODUCTION Hasenstein KH and van Loon JJWA (2019) Comparison of Microgravity Plants have evolved under the influence of Earth’s gravity, a force of “1 g.” This ubiquitous force ae ff cts Analogs to Spaceflight in Studies of plant growth, development, and morphology at all levels, from the molecular to the whole plant Plant Growth and Development. (Vandenbrink et al., 2014). In addition, gravity underlies other physical phenomena like buoyancy, Front. Plant Sci. 10:1577. doi: 10.3389/fpls.2019.01577 convection, and sedimentation, which ae ff ct many physical and chemical processes and therefore Frontiers in Plant Science | www.frontiersin.org 1 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs also shape plant growth and development. For example, buoyancy A clinostat is a device that rotates specimens around one or more ae ff cts gas exchange, cellular respiration, and photosynthesis, but axes. A number of different types of clinostats have been used to itself is a function of varying densities (Braun et al., 2018). study plant growth and development as well as to address basic Studying the direct and indirect effects of gravity on plant issues in fundamental biology. These clinostats can be divided growth, however, is complicated by the difficulty of changing gravity into several types: one-axis clinostats with slow (1–4 rpm) or fast on Earth. One means of reducing gravity’s influence is to establish (50–120 rpm) rotation and clinostats with two or three axes of free fall and eliminate the effect of gravity either for a few seconds in rotation. If the rate of rotation for the axes varies, such systems so-called drop towers and parabolic flights or for the long term by are distinguished as random positioning machines (RPMs). In using orbital free fall, which creates weightlessness. This condition addition to these instruments, magnetic levitation has been used is achieved by the balance between Earth’s gravity and the velocity to balance gravity (Kamal et al., 2016). required to maintain free fall (Kiss, 2015). Experiments focusing on plant growth and development have been carried out in this One-Axial Clinostats environment almost from the advent of human spaceflight in the e fir Th st experiments to expose plants to altered gravity 1960s (Wolverton and Kiss, 2009; Vandenbrink and Kiss, 2016). environments were performed nearly 160 years before humans Fascinating insights into plant biology have been provided reached low-Earth orbit. Early in the 19th century, T.A. Knight by spaceflight studies aboard orbiting spacecraft. For instance, used a water wheel as a centrifuge to expose oat seedlings to at the cell/molecular level, changes in the cell cycle (Manzano variable acceleration, demonstrating that plants were indeed et al., 2009; Matía et al., 2010) and the cell wall (Soga et al., 2002; sensing this physical force when carrying out “geotropic” growth Johnson et al., 2015) have been observed when plants develop in (Knight, 1806). Later in the same century, Sachs developed a microgravity. Recently, there have been a plethora of spaceflight device, which he named a “klinostat,” to alter the effects of gravity experiments on the effects of varying gravity levels on gene by constantly rotating its longitudinal axis horizontally, thereby expression in plants (Paul et al., 2012; Correll et al., 2013; Kwon averaging the presumed effect of the gravitational force over the et al., 2015; Johnson et al., 2017; Paul et al., 2017; Choi et al., rotated axis (Sachs, 1882). 2019). And facilitated by the absence of significant gravitational Clinostats have been employed as a control for the accelerations in spaceflight, novel mechanisms of phototropism gravitational force in numerous studies investigating plant (Molas and Kiss, 2009) have been discovered in flowering plants development and responses to directional stimuli (Figure 1). (Millar et al., 2010; Kiss et al., 2012; Vandenbrink et al., 2016). The clinostat has frequently been used for studies in which the On the applied side of plant space research, there has also been researcher wished to reorient the organ or cell in the gravitational progress on cultivating plants for use in bioregenerative life field for a period of time, then eliminate, as much as possible, support systems (Braun et al., 2018). the influence of constant gravity on the organ. Such was the use Because of the scarcity of access to spaceflight, researchers of clinostats in experiments investigating both the presentation have used other approaches to minimize or eliminate constant time, perception time, and lag time of the gravitropic response 1-g conditions (Kiss, 2015). These methods include drop towers of various species and organs (Pickard 1973; Johnsson and (samples are weightlessness for seconds), parabolic flights in Pickard, 1979; Kiss et al., 1989). The theoretical justification specialized airplanes (samples are weightlessness for approximately for the use of clinostats was elaborated by Dedolph and Dipert 10–20 s), and sounding rockets (minutes of weightlessness) as (1971); they demonstrated the importance of the rotation rate attractive alternatives (see also Beysens and van Loon, 2015). on the effectiveness of the clinostat due to its influence on the Sounding rockets are retrieved in the same general area aer t ft heir sedimentation path of the starch statoliths, thought to be the launch without entering into orbit. In the free-fall phase, these primary means of gravity susception in plants (Kiss, 2000). missions typically provide 3–8 min of microgravity (Böhmer They found that rotation rates of 2–4 rpm corresponded to a and Schleiff, 2019 ). In recent years, private companies such as more effective randomization because it minimized the path Blue Origin and Virgin Galactic are promising suborbital flight length of statolith sedimentation. with several minutes of microgravity (Pelton, 2019). However, for In addition to their use as a means of minimizing the most systems in plant biology, these suborbital methods provide a unidirectional effects of gravity, several studies have incorporated period of weightlessness that is too short to effectively assay growth modified versions of the clinostat that expose the axial organ to and development. A conceptual alternative to these methods of a fractional g treatment either through a programmed rotation creating brief free-fall conditions is to develop conditions in which pattern or through the incorporation of a centrifuge as the the direction of the gravity vector is constantly changing through innermost rotating axis of the clinostat. These instruments have the use of clinostats and similar devices. been key in estimating the threshold acceleration necessary to activate gravity perception and growth responses (Shen-Miller et al., 1968; Brown et al., 1995; Laurinavicius et al., 1998; Galland CLINOSTATS et  al., 2004; Duemmer et al., 2015; Bouchern-Dubuisson et al., Clinostats have been developed since gravity was identified as a 2016; Frolov et al., 2018), as well as identifying cellular-level major contributor of plant growth and development by Knight, responses of plants to microgravity (Murakami and Yamada, 1988; Sachs, and Ciesielski in the late 1800s (reviewed in Hoson et al., Kraft et al., 2000 ; Dauzart et al., 2016; Manzano et al., 2018) and the 1997; van Loon 2007; Hasenstein, 2009; Herranz et al., 2013). persistence of the gravity stimulus (John and Hasenstein, 2011). Frontiers in Plant Science | www.frontiersin.org 2 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs comparisons with space experiments and have been widely used by many researchers (e.g., Brown et al., 1995; Kraft et al., 2000 ). Fast-Rotating Clinostats Slow-rotating clinostats as described above simply consider the overall geometry and develop a scheme of rotation that fulfills −3 certain conditions (such as centrifugal accelerations less than 10 g). However, fast-rotating clinostats (typically 50–120 rpm) also utilize the path of sedimentation in a fluid, usually an aqueous growth medium for small (<1 mm) organisms (Aleshcheva et al., 2016; Warnke et al., 2016). In liquids, sedimentation and a relatively slow rotation result in potentially significant artifacts including spirally movements from centrifugation, sedimentation, and a viscosity-dependent FIGURe 1 | A standard two-dimensional clinostat used to grow Medicago Coriolis force. When the speed of rotation is increased as in a seedlings (green arrows) at 1 rpm. Blue arrow indicated the direction of rotation. Scale bar, 10.5 cm. fast-rotating clinostat, sedimentation of a particle will be less than the movement of the liquid, thereby resulting in a reduced radius that finally produces a smaller diameter than the size of the particle or a cell. u Th s, in the conditions as found in the Despite their usefulness for temporarily changing the fast-rotating clinostat, the rotation stabilizes the fluid around unidirectional force of gravity, there is also evidence that such the particle, which in turn eliminates the gravity effects for all treatments introduce their own sets of stimuli that may compete practical purposes. While the fast-rotating clinostat can provide with or confound interpretation of those pathways of most interest conditions that mimic weightlessness very well, it is limited to to the user (Hasenstein and van Loon, 2015). Centrifugal forces small organisms such as unicells or bacteria but, generally, not resulting from rotation about one or two axes varies as a function applicable for plant studies (Cogoli, 1992). of the position of the organ under study along the radius and speed of rotation, and the organ will experience variable g levels across its axis. Because the force of gravity itself is never altered, Non-Uniformly Rotating Clinostats the bending due to differential growth will cause the position of In addition to positioning one-axial clinostats at certain angles to the organ with respect to the radius of rotation to change over mimic fractional gravity levels (<1 g), it is possible to achieve a the course of an experiment. For example, the growth of an axial similar condition by changing the rate of horizontal rotation such organ over the course of a long-term experiment will result in that the rotation is stopped during the bottom time (Brungs et al., the organ experiencing a change in acceleration if the growth 2016). The bottom dwell time determines the effective residual direction is away from the center of the axis of rotation. This acceleration. When uniform rotation represents complete gravity factor is one source of complexity when interpreting the results of −1 compensation for a 1-rpm (~0.1 rad s ) clinostat, extending each clinostat experiments, as indicated by the observation that plants rotation by the amount of gravity that is supposed to be established, respond differently when rotated around one axis versus the other for example 0.1 g, would require a bottom dwell time of 6 s. The (John and Hasenstein, 2011; Hasenstein and van Loon, 2015). extra 6 s relative to the normal rotation of 60 s (=1 rpm) spent at Long-term experiments on clinostats are particularly the “bottom” position (Figure  2) creates 0.1 g net acceleration. challenging because as the organs increase in mass, the changing Because additional acceleration or deceleration needs to be weight distribution will cause bending stresses and other non- minimized, the movement requires precise algorithms and motor random mechanical stimulation that will vary as a function of control. The advantage of such designs is that fractional g-levels the specific load-bearing structure of each organ. Thus, growing can be established. Nonetheless, this principle also depends on plants on a rotating clinostat can result in mechanical stress (van rotation and therefore suffers from the same shortcomings as Loon, 2007; Manzano et al., 2009). The use of clinostats to study standard clinostats (Hasenstein and van Loon, 2015). developmental effects of gravity are also limited because of their inability to control for constantly changing loads and rotational Random Positioning Machines forces, thus restricting their usefulness with plants mainly to studies of directional growth responses. Thus, many factors, e limi Th ted ability to average gravity effects by horizontal rotation led to the evolution of RPMs in order not to generate constant such as weighting distribution and rotation velocity, need to be considered when designing clinostats for life science experiment accelerations in any particular direction (Kraft et al., 2000 ; van Loon, 2007; Herranz et al., 2013). The idea is to provide a more complex (Brown et al., 1996). Despite these disadvantages, the simplicity and availability of motion patterns than constant rotation around one or two axes such that no directional preference remains. Ideally rotation should occur clinostats are the main reasons that these devices are the most common approach to attempt to simulate altered gravity conditions. around all three spatial axes (x, y, and z, i.e., pitch, yaw, and roll) and would require a three gimbal or Cardan suspension. However, Although the artifacts associated with clinostats require caution of the assessment of gravitational effects, they can provide valuable most RPM systems are based on two axes or an “altazimuth mount” Frontiers in Plant Science | www.frontiersin.org 3 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs MAGNeTIC LevITATION In contrast to the various clinostats that attempt to randomize the effect of gravity by changing the direction of its vector, magnetic forces counteract the gravity force by a magnetic force that results from a magnetic gradient and the diamagnetic susceptibility of the object which together generate a force that can be equal to gravity (Geim et al., 1999; Kamal et al., 2016). Interestingly, based on the orientation of the magnetic core, this gradient exists in two opposing directions such that in a vertically oriented magnetic field the top gradient balances gravity effects on biological, i.e., diamagnetic material at the point where F  = F . e Th opposite mag g pole of the magnetic gradient also generates a 1-g force equivalent and therefore provides a 2-g equivalent (1 g attributed to the FIGURe 2 | Motion profile of a an object experiencing fractional gravity as a result of non-uniform rotation. The trace of a point rotating around an axis magnetic gradient in addition to the original gravity). While shows an extended resting position during phase T . The ratio between the 1 the effect of magnetic gradients and diamagnetic properties dwell time in the bottom position (T ) and complete rotation (T ) corresponds 1 0 of the levitated object (e.g., frogs, seeds, or seedlings) balances to the fractional gravity experienced by plants, provided that the dwell time the effect of gravity (i.e., stably suspend biological objects is does not exceed the gravity perception time. space), the very strong magnetic field (about 15 T) and gradient is likely to ae ff ct the movement of charged particles (ions) and therefore alters the physiological conditions which aeff ct gene such that the two axes are mounted perpendicular to each other expression (Paul et al., 2006). In addition, the small space in a (Figure 3). This arrangement is sufficient to position any object on magnet bore, the requirement to cool magnets while maintaining the experimental platform in any desirable direction (i.e., the vector “room temperature” for biological objects, and to provide light, normal to the experimental platform can point in any direction). contributes to the complexity of magnetic levitation. The required u Th s, seedlings that develop on an RPM appear to grow randomly strong magnetic field and gradient (about 1,400 T /m) also require as achieved in spaceflight ( Figure 4). specialized magnetic systems that are expensive to operate. Randomness is achieved when the rotational angle differs Additional research is needed to determine which systems between the two axes and changes over time. While these best mimic reduced gravity conditions, especially for plants that systems provide the best gravity compensation, they do so occupy a large volume and are therefore ae ff cted by any gradient despite apparently exceeding the maximum permissible angular of rotational, inertial, or magnetic conditions. Despite the above- −1 acceleration (approx. 30 deg s for a 10-cm radius). Apparently mentioned complications, the ability to produce partial or even better results are obtained when the sum of both axes movements excess gravity forces makes magnetic gradients an attractive −1 exceeds 60–80 deg s (Brungs et al., 2016). While this puzzling alternative to clinostat-based research. As indicated earlier, observation deserves future studies, it also highlights some the precise and narrow space that corresponds to the desired postulated gravisensing mechanisms, namely that the movement level makes studies on whole plants problematic because the of the suspected gravity sensors (starch-filled amyloplasts; Kiss, compensation point averages all forces acting on the levitated 2000) is sensitive to mechanostimulation, and thus describes object by susceptibility, density, and distance. u Th s, the most dynamic gravisensing (Hasenstein, 2009). valuable aspect of high-gradient magnetic fields is the ability to Nevertheless, in gravity-perceiving root columella cells, the precisely move (levitate) cellular organelles, such as statoliths in position of amyloplasts was similar in weightlessness in spaceflight roots (Kuznetsov and Hasenstein, 1996), hypocotyls (Kuznetsov and on the RPM, but was significantly different between and Hasenstein, 1997), rhizoids (Kuznetsov and Hasenstein, 2001), spaceflight and two-axial clinostats ( Figure 5). For in vitro systems and seedlings (Hasenstein and Kuznetsov, 1999). In addition, like Arabidopsis cell cultures, it is important to realize that in fluid- magnetic levitation has been shown to be a useful ground-based lfi led experimental containers, there is also a fluid shear applied proxy for microgravity in a number of other systems including to the cells (Leguy et al., 2017). This issue can be mitigated by osteoblast cells (Hammer et al., 2009), Drosophila melanogaster increasing the cell substrate viscosity (Kamal et al., 2019). (Herranz et al., 2012), and bacteria (Dijkstra et al., 2010). u Th s, the RPM can be a useful proxy for weightlessness for certain biological parameters, as shown in studies with plant cells, Drosophila, and mammalian cell cultures (Kraft et al., 2000 ; CeNTRIFUGeS Herranz et al., 2010; Wuest et al., 2015). In addition, due to the difficulty, availability, and cost of spaceflight experiments, the Although it sounds somewhat counterintuitive, we can also explore RPM may in fact be one of the best substitutes/analogs especially the effects of microgravity by the application of centrifuges. This when this instrument can potentially generate results comparable reduced gravity paradigm (RGP) is based on the premise that to those in true microgravity. This scenario is true especially when adaptations seen going from a hypergravity level to a lower gravity the changes in direction are faster than the response time of the level are similar to changes seen going from 1 g to microgravity object (e.g., plant body) to gravity (Borst and van Loon, 2009). (van Loon, 2016). Using such a paradigm, we are not focusing Frontiers in Plant Science | www.frontiersin.org 4 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs FIGURe 3 | Three random positioning machines (RPMs) each with two independently driven perpendicular frames. The discrete rotation axes allow the implementation of slip rings to provide power and exchange data with the experiment that can be mounted onto the inner frame. Both the full-sized RPM (A) and the two desktop models (B) are shown with 10-cm square Petri dishes (pd). The diameter of the disk (asterisk) on the full-sized RPM is 40 cm and provides the generation of partial gravity. on the absolute acceleration values but rather on the responses Kiss, 2016). In contrast, we know little about plant physiology in generated due to the change between the two accelerations levels. reduced gravity environments, which are less than the normal e p Th remise of such an experiment is that the plant sample has 1 g that characterizes Earth-based studies. Reduced gravity can to be adapted and stable to a higher gravity level such as 2 g. also be termed partial-g or fractional-g. The exploration of the en, a Th s the g-level is lowered to 1 g, the plant will respond to this Moon and Mars will be important in the future and will rely reduced gravity level. It is hypothesized that the processes in such upon optimized plant cultivation because plants will be essential adaptations are of the same type as one would see going from 1 g for life support systems (Kiss, 2014). Therefore, it is important into free fall, although the magnitude might be different. Thus, this to develop new knowledge about the biology of plants at the reduced gravity paradigm is best used for stable and steady systems lunar and Martian g-levels, 0.17 g and 0.38 g, respectively. Studies at a certain higher g level combined with measuring a relatively fast on plants in partial gravity environments also can provide new responding phenomenon when reducing the acceleration load. information on basic biological questions such as what is the threshold of gravisensing in plants (e.g., Kiss et al., 1989; Perbal, 2009; Duemmer et al., 2015). To establish partial gravity on-board sounding rockets or ReDUCeD OR PARTIAL GRA vITY STUDIeS orbiting laboratories, a centrifuge is needed to produce the Numerous studies on plant growth and development have been desired accelerations. Centrifuges can be used to generate any performed in space (Wolverton and Kiss, 2009; Vandenbrink and acceleration from near zero to 1 g. Especially 1-g experiments Frontiers in Plant Science | www.frontiersin.org 5 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs FIGURe 4 | Arabidopsis seedlings grown in spaceflight hardware for 3.5 days in the dark. Arrowheads indicate the hypocotyl apex. (A) Seedlings that germinated and developed on the random positioning machine (RPM) are disoriented. (B) Ground controls (GR) are oriented to the gravity vector which is toward the bottom of the photograph. Scale bar, 6 mm. Figure is from Kraft et al. (2000) and is used with permission from Springer Nature publishers. are valuable as in-flight controls, which provide context for the In experiments in which directional light and fractional gravity analyses of spaceflight experiments ( Vandenbrink and Kiss, were applied simultaneously, Kiss and colleagues reported strong 2016). Fortunately, there are several facilities on the International positive phototropism in response to unilateral red light in the Space Station (ISS) that are equipped with centrifuges, and the ISS stem-like hypocotyls and roots of plants grown in microgravity can be used to study partial gravity effects on plant development. (Millar et al., 2010). In time course studies, shoots had positive phototropism in response to red light in microgravity and at 0.1 g, and the curvature was not significantly different between two Plant Responses in Reduced or Partial gravity conditions (Figure 6A). However, the red-light-based Gravity in Spaceflight phototropism at 0.3 g was not significantly different from the A series of experiments have recently been performed on the ISS red-light phototropic response of the 1-g control, and there was with Arabidopsis thaliana and have focused on 1) the interaction significant reduction of red-light phototropism at 0.3 g and 1 g between gravitropism and phototropism in microgravity and (see also Kiss et al., 2012). fractional gravity (Kiss et al., 2012; Vandenbrink and Kiss, 2016; In experiments with seedlings, roots exhibited a strong positive Vandenbrink et al., 2016) and 2) identification of the threshold phototropism in response to unidirectional red illumination in for gravity perception in roots in the wild-type and starchless microgravity conditions (Figure 6B). In contrast to the experiments (pgm-1) mutants (Wolverton, in progress). These experiments with shoots, the red-light-based phototropic response in roots at 0.1 g utilized the European Modular Cultivation System (EMCS) was reduced and not significantly different from the red phototropic which had onboard centrifuges allowing for gravitational ranges curvature at 0.3 g and 1 g. Thus, our fractional gravity experiments from microgravity to small fractions of a g up to 1 g (Kiss et al., demonstrated a reduction of red-light-based phototropic curvature 2014). The EMCS was decommissioned in 2017, but international in the shoot-like hypocotyls at the level of 0.3 g, but the level of 0.1 space agencies have developed hardware such as Cell Biology g was enough to reduce the red-light-based phototropism in roots. Experiment Facility (CBEF) and Biolab support research at This range of fractional gravity is approximately the same as the fractional g (Brinckmann, 2012). g-levels found on the Moon and Mars, 0.17 g and 0.38 g, respectively. Frontiers in Plant Science | www.frontiersin.org 6 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs FIGURe 5 | Plastid position in central columella cells of root tips of Arabidopsis seedlings grown on the ground (GR), during spaceflight ( FL), on a random positioning machine (RPM), and on a clinostat (CL). These cells are involved in gravity perception (Kiss, 2000). There is no statistical difference (P > 0.05) between the FL and RPM samples as indicated by *, while the FL samples are significantly different ( P < 0.05) from the CL specimens as indicaated by ** and determined by an ANOVA followed by a Tukey post-test. FIGURe 6 | Time course studies of positive phototropic curvature Taken together, our results suggest that this range of reduced g in seedlings of Arabidopsis at indicated gravity levels in a spaceflight represents a significant sensory threshold. experiment. Different letters indicate significant differences among the plots. This hypothesis is being investigated further in a separate series (A) Response of the shoot-like hypocotyls of Arabidopsis seedlings to red of experiments designed to test the threshold force required to light. The response at 0.3 g was not significantly different from the value of the 1-g control, and there was attenuation of red-light phototropism at 0.3 g activate gravity sensing and response of Arabidopsis seedlings in and 1 g. (B) Response of the roots of Arabidopsis seedlings to red light. The the EMCS. Ground-based clinostat experiments have estimated responses at 0.1 g and 0.3 g were not significantly different from the value of the gravity perception threshold at or around 0.003 g (Shen- the 1-g control, and these values were attenuated compared to the robust Miller et al., 1968; Laurinavicius et al., 1998; Duemmer et al., response in microgravity. Figure is adapted from Kiss et al. (2012). 2015). This threshold was tested in these space experiments, and the analysis currently is in progress. Extending these results to include the starchless mutant in addition to wild-type seedlings 2018). In another recent study using Arabidopsis tissue culture will allow for the comparison of gravity perception threshold in cells, cell proliferation and growth were uncoupled under roots that lack sedimenting statoliths (Kiss et al., 1989), which we simulated reduced gravity also using an RPM (Kamal et al., 2018). predict will require greater accelerations to activate perception e r Th esults of these few studies are promising and encourage and response in these seedlings. future exploration of simulated partial gravity for other biological systems. Successful application of partial gravity simulation could develop into new avenue of research. For example, the simulated Plant Responses to Simulated Partial or Mars gravity of 0.38 g could be used in various biological studies Reduced Gravity Using Analogs to help prepare for a human mission to Mars. While the main focus of this paper has been on the simulation of microgravity, we also see that there is potential to use the analog devices to simulate partial or reduced gravity conditions that are CONCLUSIONS AND FUTURe DIReCTIONS found on the Moon and Mars (Kiss, 2014). This approach has been recently used in RPM studies of the effects of simulated Numerous studies have compared the biological effects of clinostats and other microgravity analogs to space experiments partial gravity on the balance between cell growth and cell proliferation during early plant development (Manzano et al., (e.g., Brown et al., 1996; Kraft et al., 2000 ; Herranz et al., 2013; Frontiers in Plant Science | www.frontiersin.org 7 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs Huang et al., 2018). The experiments to date suggest that while Nevertheless, in this era of the International Space Station, these devices may be useful tools in some cases, there are great we must take advantage of its unique facilities to compare effects differences observed between plants that grow and develop on observed in clinostats and other space simulators. We also should these devices and plants that are grown in weightlessness during use the centrifuges available on the ISS to systematically explore spaceflight. In fact, rotation on certain types of clinostats may the effects of partial gravity on plant growth and development. have deleterious effects in some biological systems ( Hensel and Understanding plant biology in space under different gravity Sievers, 1980; Kozeko et al., 2018; Ruden et al., 2018). levels will be useful as we develop technologies needed for human e Th conditions under which ground-based simulation can habitation of other worlds. provide useful information and compare various gravitational regimens need to be systematically determined through carefully controlled experiments in which ground analog studies are AUTHOR CONTRIBUTIONS compared with spaceflight experiments. A problem with past studies of microgravity simulators/analogs is that it can be JK wrote the first draft of the manuscript. CW, SW, KH, and JL difficult to compare results between spaceflight experiments also wrote and edited sections of the manuscript. All authors to those of simulation devices (Herranz et al., 2013). Thus, contributed to manuscript revision, read, and approved the ground-based experiments should be performed to maximize submitted version. comparability between spaceflight and experimental ground- based devices. For example, in plant studies, factors to consider include identical seed stock, growth substrate, nutrient media, FUNDING and light composition and intensity. In addition, containers should be identical for space and ground-based studies. This Financial support provided by grants from the National latter consideration can be made difficult by the reluctance of Aeronautics and Space Administration (NASA) to JK space agencies to provide access to their expensive spaceflight (#80NSSC17K0546 and #NNX12AO65G), CW (#NNX15 hardware (Kiss, 2015). AG55G), and KH (#80NSSC17K0344). facility programme. Microgravity Sci. 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Space Sci. 3, 21. doi: 10.3389/fspas.2016.00021 Copyright © 2019 Kiss, Wolverton, Wyatt, Hasenstein and van Loon. iTh s is Vandenbrink, J. P., and Kiss, J. Z. (2016). Space, the final frontier: A critical review an open-access article distributed under the terms of the Creative Commons of recent experiments performed in microgravity. Plant Sci. 243, 115–119. doi: Attribution License (CC BY). The use, distribution or reproduction in other 10.1016/j.plantsci.2015.11.004 forums is permitted, provided the original author(s) and the copyright owner(s) Vandenbrink, J. P., Kiss, J. Z., Herranz, R., and Medina, F. J. (2014). Light and are credited and that the original publication in this journal is cited, in accordance gravity signals synergize in modulating plant development. Front. Plant Sci. 5, with accepted academic practice. No use, distribution or reproduction is permitted 563. doi: 10.3389/fpls.2014.00563 which does not comply with these terms. 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Comparison of Microgravity Analogs to Spaceflight in Studies of Plant Growth and Development

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RevIew published: 06 December 2019 doi: 10.3389/fpls.2019.01577 Comparison of Microgravity Analogs to Spaceflight in Studies of Plant Growth and Development 1 2 3 4 John Z. Kiss *, Chris Wolverton , Sarah E. Wyatt , Karl H. Hasenstein 5,6,7 and Jack J.W.A. van Loon 1 2 Department of Biology, University of North Carolina—Greensboro, Greensboro, NC, United States, Department of Botany & Microbiology, Ohio Wesleyan University, Delaware, OH, United States, Molecular and Cellular Biology Program, Department of Environmental & Plant Biology, Ohio University, Athens, OH, United States, Biology Department, University of Louisiana at Lafayette, Lafayette, LA, United States, DESC (Dutch Experiment Support Center), Department of Oral and Maxillofacial Surgery/Oral Pathology, Amsterdam University Medical Center, Amsterdam, Netherlands, Academic Centre for Dentistry Amsterdam (ACTA), VU-University, Amsterdam, Netherlands, European Space Agency (ESA) Technology Center (ESTEC), Life & Physical Science, Instrumentation and Life Support Laboratory, TEC-MMG, Noordwijk, Netherlands Life on Earth has evolved under the influence of gravity. This force has played an important role in shaping development and morphology from the molecular level to the whole organism. Although aquatic life experiences reduced gravity effects, land plants have evolved under a 1-g environment. Understanding gravitational effects requires changing the magnitude of this force. One method of eliminating gravity’s influence is to enter into a free-fall orbit around the planet, thereby achieving a balance between Edited by: Valérie Legué, centripetal force of gravity and the centrifugal force of the moving object. This balance Université Clermont Auvergne, is often mistakenly referred to as microgravity, but is best described as weightlessness. France In addition to actually compensating gravity, instruments such as clinostats, random- Reviewed by: Tomomichi Fujita, positioning machines (RPM), and magnetic levitation devices have been used to eliminate Hokkaido University, Japan effects of constant gravity on plant growth and development. However, these platforms Yutaka Miyazawa, do not reduce gravity but constantly change its direction. Despite these fundamental Yamagata University, Japan differences, there are few studies that have investigated the comparability between *Correspondence: John Z. Kiss these platforms and weightlessness. Here, we provide a review of the strengths and jzkiss@uncg.edu weaknesses of these analogs for the study of plant growth and development compared to spaceflight experiments. We also consider reduced or partial gravity effects via spaceflight Specialty section: This article was submitted to and analog methods. While these analogs are useful, the fidelity of the results relative to Plant Abiotic Stress, spaceflight depends on biological parameters and environmental conditions that cannot a section of the journal Frontiers in Plant Science be simulated in ground-based studies. Received: 19 July 2019 Keywords: Arabidopsis, clinostat, plant growth, simulated microgravity, random positioning machine, reduced Accepted: 12 November 2019 gravity, spaceflight experiments Published: 06 December 2019 Citation: Kiss JZ, Wolverton C, Wyatt SE, INTRODUCTION Hasenstein KH and van Loon JJWA (2019) Comparison of Microgravity Plants have evolved under the influence of Earth’s gravity, a force of “1 g.” This ubiquitous force ae ff cts Analogs to Spaceflight in Studies of plant growth, development, and morphology at all levels, from the molecular to the whole plant Plant Growth and Development. (Vandenbrink et al., 2014). In addition, gravity underlies other physical phenomena like buoyancy, Front. Plant Sci. 10:1577. doi: 10.3389/fpls.2019.01577 convection, and sedimentation, which ae ff ct many physical and chemical processes and therefore Frontiers in Plant Science | www.frontiersin.org 1 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs also shape plant growth and development. For example, buoyancy A clinostat is a device that rotates specimens around one or more ae ff cts gas exchange, cellular respiration, and photosynthesis, but axes. A number of different types of clinostats have been used to itself is a function of varying densities (Braun et al., 2018). study plant growth and development as well as to address basic Studying the direct and indirect effects of gravity on plant issues in fundamental biology. These clinostats can be divided growth, however, is complicated by the difficulty of changing gravity into several types: one-axis clinostats with slow (1–4 rpm) or fast on Earth. One means of reducing gravity’s influence is to establish (50–120 rpm) rotation and clinostats with two or three axes of free fall and eliminate the effect of gravity either for a few seconds in rotation. If the rate of rotation for the axes varies, such systems so-called drop towers and parabolic flights or for the long term by are distinguished as random positioning machines (RPMs). In using orbital free fall, which creates weightlessness. This condition addition to these instruments, magnetic levitation has been used is achieved by the balance between Earth’s gravity and the velocity to balance gravity (Kamal et al., 2016). required to maintain free fall (Kiss, 2015). Experiments focusing on plant growth and development have been carried out in this One-Axial Clinostats environment almost from the advent of human spaceflight in the e fir Th st experiments to expose plants to altered gravity 1960s (Wolverton and Kiss, 2009; Vandenbrink and Kiss, 2016). environments were performed nearly 160 years before humans Fascinating insights into plant biology have been provided reached low-Earth orbit. Early in the 19th century, T.A. Knight by spaceflight studies aboard orbiting spacecraft. For instance, used a water wheel as a centrifuge to expose oat seedlings to at the cell/molecular level, changes in the cell cycle (Manzano variable acceleration, demonstrating that plants were indeed et al., 2009; Matía et al., 2010) and the cell wall (Soga et al., 2002; sensing this physical force when carrying out “geotropic” growth Johnson et al., 2015) have been observed when plants develop in (Knight, 1806). Later in the same century, Sachs developed a microgravity. Recently, there have been a plethora of spaceflight device, which he named a “klinostat,” to alter the effects of gravity experiments on the effects of varying gravity levels on gene by constantly rotating its longitudinal axis horizontally, thereby expression in plants (Paul et al., 2012; Correll et al., 2013; Kwon averaging the presumed effect of the gravitational force over the et al., 2015; Johnson et al., 2017; Paul et al., 2017; Choi et al., rotated axis (Sachs, 1882). 2019). And facilitated by the absence of significant gravitational Clinostats have been employed as a control for the accelerations in spaceflight, novel mechanisms of phototropism gravitational force in numerous studies investigating plant (Molas and Kiss, 2009) have been discovered in flowering plants development and responses to directional stimuli (Figure 1). (Millar et al., 2010; Kiss et al., 2012; Vandenbrink et al., 2016). The clinostat has frequently been used for studies in which the On the applied side of plant space research, there has also been researcher wished to reorient the organ or cell in the gravitational progress on cultivating plants for use in bioregenerative life field for a period of time, then eliminate, as much as possible, support systems (Braun et al., 2018). the influence of constant gravity on the organ. Such was the use Because of the scarcity of access to spaceflight, researchers of clinostats in experiments investigating both the presentation have used other approaches to minimize or eliminate constant time, perception time, and lag time of the gravitropic response 1-g conditions (Kiss, 2015). These methods include drop towers of various species and organs (Pickard 1973; Johnsson and (samples are weightlessness for seconds), parabolic flights in Pickard, 1979; Kiss et al., 1989). The theoretical justification specialized airplanes (samples are weightlessness for approximately for the use of clinostats was elaborated by Dedolph and Dipert 10–20 s), and sounding rockets (minutes of weightlessness) as (1971); they demonstrated the importance of the rotation rate attractive alternatives (see also Beysens and van Loon, 2015). on the effectiveness of the clinostat due to its influence on the Sounding rockets are retrieved in the same general area aer t ft heir sedimentation path of the starch statoliths, thought to be the launch without entering into orbit. In the free-fall phase, these primary means of gravity susception in plants (Kiss, 2000). missions typically provide 3–8 min of microgravity (Böhmer They found that rotation rates of 2–4 rpm corresponded to a and Schleiff, 2019 ). In recent years, private companies such as more effective randomization because it minimized the path Blue Origin and Virgin Galactic are promising suborbital flight length of statolith sedimentation. with several minutes of microgravity (Pelton, 2019). However, for In addition to their use as a means of minimizing the most systems in plant biology, these suborbital methods provide a unidirectional effects of gravity, several studies have incorporated period of weightlessness that is too short to effectively assay growth modified versions of the clinostat that expose the axial organ to and development. A conceptual alternative to these methods of a fractional g treatment either through a programmed rotation creating brief free-fall conditions is to develop conditions in which pattern or through the incorporation of a centrifuge as the the direction of the gravity vector is constantly changing through innermost rotating axis of the clinostat. These instruments have the use of clinostats and similar devices. been key in estimating the threshold acceleration necessary to activate gravity perception and growth responses (Shen-Miller et al., 1968; Brown et al., 1995; Laurinavicius et al., 1998; Galland CLINOSTATS et  al., 2004; Duemmer et al., 2015; Bouchern-Dubuisson et al., Clinostats have been developed since gravity was identified as a 2016; Frolov et al., 2018), as well as identifying cellular-level major contributor of plant growth and development by Knight, responses of plants to microgravity (Murakami and Yamada, 1988; Sachs, and Ciesielski in the late 1800s (reviewed in Hoson et al., Kraft et al., 2000 ; Dauzart et al., 2016; Manzano et al., 2018) and the 1997; van Loon 2007; Hasenstein, 2009; Herranz et al., 2013). persistence of the gravity stimulus (John and Hasenstein, 2011). Frontiers in Plant Science | www.frontiersin.org 2 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs comparisons with space experiments and have been widely used by many researchers (e.g., Brown et al., 1995; Kraft et al., 2000 ). Fast-Rotating Clinostats Slow-rotating clinostats as described above simply consider the overall geometry and develop a scheme of rotation that fulfills −3 certain conditions (such as centrifugal accelerations less than 10 g). However, fast-rotating clinostats (typically 50–120 rpm) also utilize the path of sedimentation in a fluid, usually an aqueous growth medium for small (<1 mm) organisms (Aleshcheva et al., 2016; Warnke et al., 2016). In liquids, sedimentation and a relatively slow rotation result in potentially significant artifacts including spirally movements from centrifugation, sedimentation, and a viscosity-dependent FIGURe 1 | A standard two-dimensional clinostat used to grow Medicago Coriolis force. When the speed of rotation is increased as in a seedlings (green arrows) at 1 rpm. Blue arrow indicated the direction of rotation. Scale bar, 10.5 cm. fast-rotating clinostat, sedimentation of a particle will be less than the movement of the liquid, thereby resulting in a reduced radius that finally produces a smaller diameter than the size of the particle or a cell. u Th s, in the conditions as found in the Despite their usefulness for temporarily changing the fast-rotating clinostat, the rotation stabilizes the fluid around unidirectional force of gravity, there is also evidence that such the particle, which in turn eliminates the gravity effects for all treatments introduce their own sets of stimuli that may compete practical purposes. While the fast-rotating clinostat can provide with or confound interpretation of those pathways of most interest conditions that mimic weightlessness very well, it is limited to to the user (Hasenstein and van Loon, 2015). Centrifugal forces small organisms such as unicells or bacteria but, generally, not resulting from rotation about one or two axes varies as a function applicable for plant studies (Cogoli, 1992). of the position of the organ under study along the radius and speed of rotation, and the organ will experience variable g levels across its axis. Because the force of gravity itself is never altered, Non-Uniformly Rotating Clinostats the bending due to differential growth will cause the position of In addition to positioning one-axial clinostats at certain angles to the organ with respect to the radius of rotation to change over mimic fractional gravity levels (<1 g), it is possible to achieve a the course of an experiment. For example, the growth of an axial similar condition by changing the rate of horizontal rotation such organ over the course of a long-term experiment will result in that the rotation is stopped during the bottom time (Brungs et al., the organ experiencing a change in acceleration if the growth 2016). The bottom dwell time determines the effective residual direction is away from the center of the axis of rotation. This acceleration. When uniform rotation represents complete gravity factor is one source of complexity when interpreting the results of −1 compensation for a 1-rpm (~0.1 rad s ) clinostat, extending each clinostat experiments, as indicated by the observation that plants rotation by the amount of gravity that is supposed to be established, respond differently when rotated around one axis versus the other for example 0.1 g, would require a bottom dwell time of 6 s. The (John and Hasenstein, 2011; Hasenstein and van Loon, 2015). extra 6 s relative to the normal rotation of 60 s (=1 rpm) spent at Long-term experiments on clinostats are particularly the “bottom” position (Figure  2) creates 0.1 g net acceleration. challenging because as the organs increase in mass, the changing Because additional acceleration or deceleration needs to be weight distribution will cause bending stresses and other non- minimized, the movement requires precise algorithms and motor random mechanical stimulation that will vary as a function of control. The advantage of such designs is that fractional g-levels the specific load-bearing structure of each organ. Thus, growing can be established. Nonetheless, this principle also depends on plants on a rotating clinostat can result in mechanical stress (van rotation and therefore suffers from the same shortcomings as Loon, 2007; Manzano et al., 2009). The use of clinostats to study standard clinostats (Hasenstein and van Loon, 2015). developmental effects of gravity are also limited because of their inability to control for constantly changing loads and rotational Random Positioning Machines forces, thus restricting their usefulness with plants mainly to studies of directional growth responses. Thus, many factors, e limi Th ted ability to average gravity effects by horizontal rotation led to the evolution of RPMs in order not to generate constant such as weighting distribution and rotation velocity, need to be considered when designing clinostats for life science experiment accelerations in any particular direction (Kraft et al., 2000 ; van Loon, 2007; Herranz et al., 2013). The idea is to provide a more complex (Brown et al., 1996). Despite these disadvantages, the simplicity and availability of motion patterns than constant rotation around one or two axes such that no directional preference remains. Ideally rotation should occur clinostats are the main reasons that these devices are the most common approach to attempt to simulate altered gravity conditions. around all three spatial axes (x, y, and z, i.e., pitch, yaw, and roll) and would require a three gimbal or Cardan suspension. However, Although the artifacts associated with clinostats require caution of the assessment of gravitational effects, they can provide valuable most RPM systems are based on two axes or an “altazimuth mount” Frontiers in Plant Science | www.frontiersin.org 3 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs MAGNeTIC LevITATION In contrast to the various clinostats that attempt to randomize the effect of gravity by changing the direction of its vector, magnetic forces counteract the gravity force by a magnetic force that results from a magnetic gradient and the diamagnetic susceptibility of the object which together generate a force that can be equal to gravity (Geim et al., 1999; Kamal et al., 2016). Interestingly, based on the orientation of the magnetic core, this gradient exists in two opposing directions such that in a vertically oriented magnetic field the top gradient balances gravity effects on biological, i.e., diamagnetic material at the point where F  = F . e Th opposite mag g pole of the magnetic gradient also generates a 1-g force equivalent and therefore provides a 2-g equivalent (1 g attributed to the FIGURe 2 | Motion profile of a an object experiencing fractional gravity as a result of non-uniform rotation. The trace of a point rotating around an axis magnetic gradient in addition to the original gravity). While shows an extended resting position during phase T . The ratio between the 1 the effect of magnetic gradients and diamagnetic properties dwell time in the bottom position (T ) and complete rotation (T ) corresponds 1 0 of the levitated object (e.g., frogs, seeds, or seedlings) balances to the fractional gravity experienced by plants, provided that the dwell time the effect of gravity (i.e., stably suspend biological objects is does not exceed the gravity perception time. space), the very strong magnetic field (about 15 T) and gradient is likely to ae ff ct the movement of charged particles (ions) and therefore alters the physiological conditions which aeff ct gene such that the two axes are mounted perpendicular to each other expression (Paul et al., 2006). In addition, the small space in a (Figure 3). This arrangement is sufficient to position any object on magnet bore, the requirement to cool magnets while maintaining the experimental platform in any desirable direction (i.e., the vector “room temperature” for biological objects, and to provide light, normal to the experimental platform can point in any direction). contributes to the complexity of magnetic levitation. The required u Th s, seedlings that develop on an RPM appear to grow randomly strong magnetic field and gradient (about 1,400 T /m) also require as achieved in spaceflight ( Figure 4). specialized magnetic systems that are expensive to operate. Randomness is achieved when the rotational angle differs Additional research is needed to determine which systems between the two axes and changes over time. While these best mimic reduced gravity conditions, especially for plants that systems provide the best gravity compensation, they do so occupy a large volume and are therefore ae ff cted by any gradient despite apparently exceeding the maximum permissible angular of rotational, inertial, or magnetic conditions. Despite the above- −1 acceleration (approx. 30 deg s for a 10-cm radius). Apparently mentioned complications, the ability to produce partial or even better results are obtained when the sum of both axes movements excess gravity forces makes magnetic gradients an attractive −1 exceeds 60–80 deg s (Brungs et al., 2016). While this puzzling alternative to clinostat-based research. As indicated earlier, observation deserves future studies, it also highlights some the precise and narrow space that corresponds to the desired postulated gravisensing mechanisms, namely that the movement level makes studies on whole plants problematic because the of the suspected gravity sensors (starch-filled amyloplasts; Kiss, compensation point averages all forces acting on the levitated 2000) is sensitive to mechanostimulation, and thus describes object by susceptibility, density, and distance. u Th s, the most dynamic gravisensing (Hasenstein, 2009). valuable aspect of high-gradient magnetic fields is the ability to Nevertheless, in gravity-perceiving root columella cells, the precisely move (levitate) cellular organelles, such as statoliths in position of amyloplasts was similar in weightlessness in spaceflight roots (Kuznetsov and Hasenstein, 1996), hypocotyls (Kuznetsov and on the RPM, but was significantly different between and Hasenstein, 1997), rhizoids (Kuznetsov and Hasenstein, 2001), spaceflight and two-axial clinostats ( Figure 5). For in vitro systems and seedlings (Hasenstein and Kuznetsov, 1999). In addition, like Arabidopsis cell cultures, it is important to realize that in fluid- magnetic levitation has been shown to be a useful ground-based lfi led experimental containers, there is also a fluid shear applied proxy for microgravity in a number of other systems including to the cells (Leguy et al., 2017). This issue can be mitigated by osteoblast cells (Hammer et al., 2009), Drosophila melanogaster increasing the cell substrate viscosity (Kamal et al., 2019). (Herranz et al., 2012), and bacteria (Dijkstra et al., 2010). u Th s, the RPM can be a useful proxy for weightlessness for certain biological parameters, as shown in studies with plant cells, Drosophila, and mammalian cell cultures (Kraft et al., 2000 ; CeNTRIFUGeS Herranz et al., 2010; Wuest et al., 2015). In addition, due to the difficulty, availability, and cost of spaceflight experiments, the Although it sounds somewhat counterintuitive, we can also explore RPM may in fact be one of the best substitutes/analogs especially the effects of microgravity by the application of centrifuges. This when this instrument can potentially generate results comparable reduced gravity paradigm (RGP) is based on the premise that to those in true microgravity. This scenario is true especially when adaptations seen going from a hypergravity level to a lower gravity the changes in direction are faster than the response time of the level are similar to changes seen going from 1 g to microgravity object (e.g., plant body) to gravity (Borst and van Loon, 2009). (van Loon, 2016). Using such a paradigm, we are not focusing Frontiers in Plant Science | www.frontiersin.org 4 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs FIGURe 3 | Three random positioning machines (RPMs) each with two independently driven perpendicular frames. The discrete rotation axes allow the implementation of slip rings to provide power and exchange data with the experiment that can be mounted onto the inner frame. Both the full-sized RPM (A) and the two desktop models (B) are shown with 10-cm square Petri dishes (pd). The diameter of the disk (asterisk) on the full-sized RPM is 40 cm and provides the generation of partial gravity. on the absolute acceleration values but rather on the responses Kiss, 2016). In contrast, we know little about plant physiology in generated due to the change between the two accelerations levels. reduced gravity environments, which are less than the normal e p Th remise of such an experiment is that the plant sample has 1 g that characterizes Earth-based studies. Reduced gravity can to be adapted and stable to a higher gravity level such as 2 g. also be termed partial-g or fractional-g. The exploration of the en, a Th s the g-level is lowered to 1 g, the plant will respond to this Moon and Mars will be important in the future and will rely reduced gravity level. It is hypothesized that the processes in such upon optimized plant cultivation because plants will be essential adaptations are of the same type as one would see going from 1 g for life support systems (Kiss, 2014). Therefore, it is important into free fall, although the magnitude might be different. Thus, this to develop new knowledge about the biology of plants at the reduced gravity paradigm is best used for stable and steady systems lunar and Martian g-levels, 0.17 g and 0.38 g, respectively. Studies at a certain higher g level combined with measuring a relatively fast on plants in partial gravity environments also can provide new responding phenomenon when reducing the acceleration load. information on basic biological questions such as what is the threshold of gravisensing in plants (e.g., Kiss et al., 1989; Perbal, 2009; Duemmer et al., 2015). To establish partial gravity on-board sounding rockets or ReDUCeD OR PARTIAL GRA vITY STUDIeS orbiting laboratories, a centrifuge is needed to produce the Numerous studies on plant growth and development have been desired accelerations. Centrifuges can be used to generate any performed in space (Wolverton and Kiss, 2009; Vandenbrink and acceleration from near zero to 1 g. Especially 1-g experiments Frontiers in Plant Science | www.frontiersin.org 5 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs FIGURe 4 | Arabidopsis seedlings grown in spaceflight hardware for 3.5 days in the dark. Arrowheads indicate the hypocotyl apex. (A) Seedlings that germinated and developed on the random positioning machine (RPM) are disoriented. (B) Ground controls (GR) are oriented to the gravity vector which is toward the bottom of the photograph. Scale bar, 6 mm. Figure is from Kraft et al. (2000) and is used with permission from Springer Nature publishers. are valuable as in-flight controls, which provide context for the In experiments in which directional light and fractional gravity analyses of spaceflight experiments ( Vandenbrink and Kiss, were applied simultaneously, Kiss and colleagues reported strong 2016). Fortunately, there are several facilities on the International positive phototropism in response to unilateral red light in the Space Station (ISS) that are equipped with centrifuges, and the ISS stem-like hypocotyls and roots of plants grown in microgravity can be used to study partial gravity effects on plant development. (Millar et al., 2010). In time course studies, shoots had positive phototropism in response to red light in microgravity and at 0.1 g, and the curvature was not significantly different between two Plant Responses in Reduced or Partial gravity conditions (Figure 6A). However, the red-light-based Gravity in Spaceflight phototropism at 0.3 g was not significantly different from the A series of experiments have recently been performed on the ISS red-light phototropic response of the 1-g control, and there was with Arabidopsis thaliana and have focused on 1) the interaction significant reduction of red-light phototropism at 0.3 g and 1 g between gravitropism and phototropism in microgravity and (see also Kiss et al., 2012). fractional gravity (Kiss et al., 2012; Vandenbrink and Kiss, 2016; In experiments with seedlings, roots exhibited a strong positive Vandenbrink et al., 2016) and 2) identification of the threshold phototropism in response to unidirectional red illumination in for gravity perception in roots in the wild-type and starchless microgravity conditions (Figure 6B). In contrast to the experiments (pgm-1) mutants (Wolverton, in progress). These experiments with shoots, the red-light-based phototropic response in roots at 0.1 g utilized the European Modular Cultivation System (EMCS) was reduced and not significantly different from the red phototropic which had onboard centrifuges allowing for gravitational ranges curvature at 0.3 g and 1 g. Thus, our fractional gravity experiments from microgravity to small fractions of a g up to 1 g (Kiss et al., demonstrated a reduction of red-light-based phototropic curvature 2014). The EMCS was decommissioned in 2017, but international in the shoot-like hypocotyls at the level of 0.3 g, but the level of 0.1 space agencies have developed hardware such as Cell Biology g was enough to reduce the red-light-based phototropism in roots. Experiment Facility (CBEF) and Biolab support research at This range of fractional gravity is approximately the same as the fractional g (Brinckmann, 2012). g-levels found on the Moon and Mars, 0.17 g and 0.38 g, respectively. Frontiers in Plant Science | www.frontiersin.org 6 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs FIGURe 5 | Plastid position in central columella cells of root tips of Arabidopsis seedlings grown on the ground (GR), during spaceflight ( FL), on a random positioning machine (RPM), and on a clinostat (CL). These cells are involved in gravity perception (Kiss, 2000). There is no statistical difference (P > 0.05) between the FL and RPM samples as indicated by *, while the FL samples are significantly different ( P < 0.05) from the CL specimens as indicaated by ** and determined by an ANOVA followed by a Tukey post-test. FIGURe 6 | Time course studies of positive phototropic curvature Taken together, our results suggest that this range of reduced g in seedlings of Arabidopsis at indicated gravity levels in a spaceflight represents a significant sensory threshold. experiment. Different letters indicate significant differences among the plots. This hypothesis is being investigated further in a separate series (A) Response of the shoot-like hypocotyls of Arabidopsis seedlings to red of experiments designed to test the threshold force required to light. The response at 0.3 g was not significantly different from the value of the 1-g control, and there was attenuation of red-light phototropism at 0.3 g activate gravity sensing and response of Arabidopsis seedlings in and 1 g. (B) Response of the roots of Arabidopsis seedlings to red light. The the EMCS. Ground-based clinostat experiments have estimated responses at 0.1 g and 0.3 g were not significantly different from the value of the gravity perception threshold at or around 0.003 g (Shen- the 1-g control, and these values were attenuated compared to the robust Miller et al., 1968; Laurinavicius et al., 1998; Duemmer et al., response in microgravity. Figure is adapted from Kiss et al. (2012). 2015). This threshold was tested in these space experiments, and the analysis currently is in progress. Extending these results to include the starchless mutant in addition to wild-type seedlings 2018). In another recent study using Arabidopsis tissue culture will allow for the comparison of gravity perception threshold in cells, cell proliferation and growth were uncoupled under roots that lack sedimenting statoliths (Kiss et al., 1989), which we simulated reduced gravity also using an RPM (Kamal et al., 2018). predict will require greater accelerations to activate perception e r Th esults of these few studies are promising and encourage and response in these seedlings. future exploration of simulated partial gravity for other biological systems. Successful application of partial gravity simulation could develop into new avenue of research. For example, the simulated Plant Responses to Simulated Partial or Mars gravity of 0.38 g could be used in various biological studies Reduced Gravity Using Analogs to help prepare for a human mission to Mars. While the main focus of this paper has been on the simulation of microgravity, we also see that there is potential to use the analog devices to simulate partial or reduced gravity conditions that are CONCLUSIONS AND FUTURe DIReCTIONS found on the Moon and Mars (Kiss, 2014). This approach has been recently used in RPM studies of the effects of simulated Numerous studies have compared the biological effects of clinostats and other microgravity analogs to space experiments partial gravity on the balance between cell growth and cell proliferation during early plant development (Manzano et al., (e.g., Brown et al., 1996; Kraft et al., 2000 ; Herranz et al., 2013; Frontiers in Plant Science | www.frontiersin.org 7 December 2019 | Volume 10 | Article 1577 Kiss et al. Microgravity Analogs Huang et al., 2018). The experiments to date suggest that while Nevertheless, in this era of the International Space Station, these devices may be useful tools in some cases, there are great we must take advantage of its unique facilities to compare effects differences observed between plants that grow and develop on observed in clinostats and other space simulators. We also should these devices and plants that are grown in weightlessness during use the centrifuges available on the ISS to systematically explore spaceflight. In fact, rotation on certain types of clinostats may the effects of partial gravity on plant growth and development. have deleterious effects in some biological systems ( Hensel and Understanding plant biology in space under different gravity Sievers, 1980; Kozeko et al., 2018; Ruden et al., 2018). levels will be useful as we develop technologies needed for human e Th conditions under which ground-based simulation can habitation of other worlds. provide useful information and compare various gravitational regimens need to be systematically determined through carefully controlled experiments in which ground analog studies are AUTHOR CONTRIBUTIONS compared with spaceflight experiments. A problem with past studies of microgravity simulators/analogs is that it can be JK wrote the first draft of the manuscript. CW, SW, KH, and JL difficult to compare results between spaceflight experiments also wrote and edited sections of the manuscript. All authors to those of simulation devices (Herranz et al., 2013). Thus, contributed to manuscript revision, read, and approved the ground-based experiments should be performed to maximize submitted version. comparability between spaceflight and experimental ground- based devices. For example, in plant studies, factors to consider include identical seed stock, growth substrate, nutrient media, FUNDING and light composition and intensity. In addition, containers should be identical for space and ground-based studies. This Financial support provided by grants from the National latter consideration can be made difficult by the reluctance of Aeronautics and Space Administration (NASA) to JK space agencies to provide access to their expensive spaceflight (#80NSSC17K0546 and #NNX12AO65G), CW (#NNX15 hardware (Kiss, 2015). AG55G), and KH (#80NSSC17K0344). facility programme. Microgravity Sci. 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Space Sci. 3, 21. doi: 10.3389/fspas.2016.00021 Copyright © 2019 Kiss, Wolverton, Wyatt, Hasenstein and van Loon. iTh s is Vandenbrink, J. P., and Kiss, J. Z. (2016). Space, the final frontier: A critical review an open-access article distributed under the terms of the Creative Commons of recent experiments performed in microgravity. Plant Sci. 243, 115–119. doi: Attribution License (CC BY). The use, distribution or reproduction in other 10.1016/j.plantsci.2015.11.004 forums is permitted, provided the original author(s) and the copyright owner(s) Vandenbrink, J. P., Kiss, J. Z., Herranz, R., and Medina, F. J. (2014). Light and are credited and that the original publication in this journal is cited, in accordance gravity signals synergize in modulating plant development. Front. Plant Sci. 5, with accepted academic practice. No use, distribution or reproduction is permitted 563. doi: 10.3389/fpls.2014.00563 which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 10 December 2019 | Volume 10 | Article 1577

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