Introduction

Plant microgravity science has been one of the most active fields in space biology since the first satellite was launched in 1957. Studies on higher plants in space have two main goals: bioregenerative life support systems for long-term missions and fundamental plant biology research. Long-term human space exploration missions require a life support system capable of regenerating all the essentials for astronauts’ survival. Plants are considered a key factor in such a bioregenerative system, because photosynthesis can provide food, such as carbohydrates, proteins, lipid oils, and vitamins, as well as the oxygen that is vital to maintaining a balance between the partial pressures of oxygen and carbon dioxide. Studies on plants in bioregenerative life support systems have been extensively reviewed (Salisbury and Bugbee 1988; Kliss et al. 1994; Tako et al. 2010; Wheeler 2010; Paul et al. 2013a). This review will focus on short-term and long-term space experiments using higher plants, with an emphasis on their perception of, responses to, and adaptation to microgravity under spaceflight conditions (Fig. 1).

Fig. 1
figure 1

Schematic diagram of short- and long-term responses of plant cells toward microgravity

Full size image

The ability of plant organs to use gravity to guide growth is called gravitropism; gravitropism maximizes the uptake of water and nutrients from the soil by roots and solar energy capture by leaves on the ground. Many studies have demonstrated that plant growth is severely impaired in space (Briarty and Maher 2004; De Micco et al. 2011; Paul et al. 2013b). Plants grown in microgravity (i.e., on spaceflights) or in simulated microgravity (i.e., on clinostats) exhibit spontaneous curvatures or changes in growth direction, called automorphosis (Hoson et al. 1995; Driss-Ecole et al. 2008) or automorphogenesis (Stanković et al. 1998). Recent advances in plant genomics and proteomics and microgravity experiments on spaceflights, which allow real changes in the orientation and/or intensity of the gravity vector, allow the mechanisms of perception, response, and adaptation of plants to microgravity signals to be addressed. The action of microgravity has been hypothesized to depend on the duration of exposure (Claassen and Spooner 1994; Matía et al. 2009). Upon exposure to short-term (seconds to hours) microgravity, plant cells initially exhibit abiotic stress responses, such as dramatically increasing the production of reactive oxygen species (ROS) and other radicals, while in long-term (days to months) microgravity, plants change metabolically and adapt to oxidative stress (Tables 1 and 2). However, key questions remain. How does microgravity affect plant growth and development? Do particular molecular signals exist? And, if so, are they specific to the distinct phases of microgravity response and adaptation?

Table 1 Current publications focusing on genomics and proteomics of plants under microgravity according to PubMed
Full size table

Drop towers, sounding rockets, and aircrafts during parabolic flight can be used to offer a short-term microgravity. Although the duration of these platforms is very short (seconds to minutes), it is enough to cause signaling changes in calcium, lipids, and cytosolic pH that alter ROS signals, the redistribution of auxin, and changes in metabolic activity leading to an adaptive plant response. The opportunities for long-term space experiments are very scarce. Therefore, ground-based facilities, such as clinostats, have been developed to simulate microgravity, but the influence of gravity will never fully be neutralized on the ground (Briegleb 1992).

Satellites, spacecraft, and space shuttles can offer relatively long-term days to months) microgravity. Many of the insights into fundamental plant biology in space were enabled by these types of space platforms. For example, the effects of microgravity on overall plant metabolism, growth, development, and reproduction were determined (Zheng et al. 2008; De Micco et al. 2014). The genomic and proteomic responses of plants to space flight were also analyzed (Table 1 and 2). The upmass and capsule volume are still limiting factors for the study of long-term plant responses to space environments.

Table 2 Current publications focusing on the response of genomics and proteomics of plants under simulated microgravity and/or hypergraivity conditions according to PubMed
Full size table

Reception and Response

Strong evidence now shows that plants can perceive gravity on the time scale of a second. For example, the sedimentation of statoliths (amyloplasts) in gravity-sensing columella cells of Arabidopsis roots occurred in less than 1 s (Hejnowicz et al. 1998; Perbal et al. 2002; Perbal and Driss-Ecole 2003), and the redistribution of auxin and root curvature growth could be observed within 10 s (Leitz et al. 2009). However, the underlying molecular mechanisms of these responses in plant cells remains unknown. Numerous signaling pathways, such as Ca 2+, lipid, and cytosolic pH, have been implicated in short-term microgravity-induced signal transduction (Table 1).

Calcium Signaling

Environmental changes are signaled through a transient increase in the intracellular Ca 2+ concentration, which is also thought to be an important second messenger for sensing and responding to gravity (reviewed in Plieth 2005, Toyota et al. 2008). The plasma membrane is considered the primary site for sensing microgravity, which causes an influx of Ca 2+. The movement of Ca 2+ in the wall might regulate extension growth and that free intracellular Ca 2+ might mediate signaling in statocytes in response to an altered gravity vector. However, the mechanistic understanding of the role of Ca 2+ in response to microgravity still lack (Leitz et al. 2009) Recently, the responses of cytosolic calcium, hydrogen peroxide H 2 O 2), and related gene and protein expressions in Arabidopsis cell cultures on parabolic flights has been studied (Hausmann et al. 2014). Ca 2+-dependent genes, such as members of the Ca 2+–binding family and Ca 2+-dependent protein kinases, exhibited increased expression at the end of the microgravity phase of the parabola (Hausmann et al. 2014). A close interrelationship between Ca 2+ and ROS signaling was also reported (Wong et al. 2007; Takeda et al. 2008). For example, seconds of exposure to microgravity could increase both cytosolic Ca 2+ and H 2 O 2 levels in parallel (Hausmann et al. 2014). Ca 2+ influx caused by microgravity can activate a Ca 2+-dependent protein kinase (CDPK), which phosphorylates the N-terminal region of the H 2 O 2-producing NADPH oxidase, thus increasing H 2 O 2 production. The latter led to the up-regulation of H 2 O 2 scavengers such as ascorbate peroxidases (APX4, APX6), glutathione peroxidase, catalases (CAT1), and superoxide dismutase. H 2 O 2 production and degradation will consequently reach a new balance in long-term microgravity.

Lipid Signaling

Membrane lipids can serve as signaling molecules in addition to their structural function because of their specific distribution in cellular compartments (Toker 2002). Most of these molecules, such as phosphatidylinositol (PtdIns) and its phosphorylated derivatives, the water-soluble inositol (1,4,5), trisphoshate (InsP3), sterols, and sphingolipids, are involved in vesicle trafficking, which is essential for the auxin transport facilitators PINs recirculation during gravitropic responses (Smith et al. 2013; Boutté and Grebe 2009). InsP3 is involved in releasing intracellular calcium after altered gravity stimuli, and its signal peaks appear at both the sensing site and elongation zone of roots (Krinke et al. 2007; Im et al. 2010; Gillaspy 2011; Perera et al. 1999; Perera et al. 2001). Although the downstream receptor of InsP3 is still unknown, it participates in regulating the redistribution of auxin in response to altered gravity. For example, when InsP3 was hydrolyzed or its synthesis blocked by chemical inhibitors like U73122, the establishment of an asymmetric auxin distribution in roots was delayed, and gravitropic response was also attenuated (Perera et al. 2001; Andreeva et al. 2010; Salinas-Mondragon et al. 2010).

Cytosolic pH Signaling

The pH in apoplasts of central columella cells of roots increased from 7.2 to 7.6 during statolith sedimentation (Fasano et al. 2001). This alkalinization may be an additional result of calcium ion channel activation, which usually activates a plasma membrane H +/OH −conductance (Boonsirichai et al. 2006; Monshausen et al. 2011; Sato et al. 2014). However, this alkalinization is absent in starchless pgm mutants (Fasano et al. 2001). Transcriptomic data obtained in microgravity showed that many genes are closely related to calcium processes and cytosolic pH signaling (Paul et al. 2011, 2012). The capacity to regulate cytoplasmic pH has been suggested to be a crucial factor in determining cell survival under microgravity.

Microgravity modifies the dynamics of fluids and small particles, thus altering the diffusion of gases (e.g., carbon dioxide and oxygen) and the uptake of water and nutrients (Wolff et al. 2014). Plant cells grown in such an environment might exhibit hypoxia. Oxygen-deficit stress induces cytoplasmic acidification. To prevent acidosis, plant cells grown under microgravity can enhance the activity of fermentative pathway (Paul et al. 2001) and probably accumulate amino acids, such as alanine, to regulate the pH within the anoxic cells. The lack of high-resolution instrumentation is a major obstacle to studying the effects of microgravity on intra- and extracellular pH (Ruyters and Braun 2014). The use of novel fluorescent pH reporter proteins, like Ptilosarcus gurneyi green fluorescent protein (Pt-GFP), and fluorescent biosensor technology enables more accurate measurement of intracellular pH (Monshausen et al. 2011; Gjetting et al. 2012; 2013; Geilfus et al. 2014) . Fluorescence microscopy and confocal live-cell imaging have begun to be used in space experiments (Ruyters and Braun 2014) and may provide more details about changes in intra- and extracellular pH in space.

Auxin Reflux and Signaling

Asymmetric application of H 2 O 2 promoted gravitropism in maize roots, indicating the involvement of the auxin signaling pathway (Joo et al. 2001). Recently, comparisons of global differential gene expression in Arabidopsis wild-type (WT) and mutant (i.e.,pin2 and pin3) seedlings grown under parabolic flight conditions were performed using DNA microarrays,the results demonstrated that the regulation of auxin responsive genes in transient microgravity is PIN3-dependent. PIN2-mediated auxin responses are located downstream of the primary transient microgravity response (Aubry-Hivet et al. 2014). These data partially corroborate previous proteomic analysis of Arabidopsis WT and pin2 roots under clinorotation and hypergravity conditions (Tan et al. 2011). In addition, three-dimensional clinorotation remarkably increased the expressions of PsPIN1 and PsAUX1 in pea epicotyls (Hoshino et al. 2004). Notably, these proteins are regulated by light as well as gravity (Ruyters and Braun 2014; Vandenbrink et al. 2014). Only a microgravity environment could distinguish the two stimuli (Vandenbrink et al. 2014). Studies have been carried out in microgravity recently on the International Space Station (ISS) (Vandenbrink et al. 2014). The impacts and cross-talk of light and gravity, nevertheless, in particular on indole-3-acetic acid transport, require further evaluation.

Metabolic Changes

Rapid metabolic responses in higher plants were also observed in short-term hypergravity and microgravity. For example, transient microgravity enhanced carbohydrate metabolism in WT and pin2 but not in pin3 roots (Aubry-Hivet et al. 2014), indicating an increased demand for energy during the response. Increased phosphorylation of triosephosphate isomerase (glycolysis), pyruvate dehydrogenase (glycolysis), and citrate synthase (citrate cycle) was detected in Arabidopsis callus cells upon exposure to microgravity for 20 s (Hausmann et al. 2014). Thus, short-term microgravity stimulation is sufficient to alter the transcript levels of many genes, mainly Ca 2+- and ROS-related ones.

Adaptation

The long-term and short-term effects of microgravity on plant growth and development appear to be different. For example, a number of metabolic products, ions, and signal molecules accumulated during short-term microgravity, but their levels did not continue to increase during long-term microgravity. Long-term adaptation to microgravity is essential to plant growth and proper development in spaceflight conditions. Plants show a strong capacity to withstand long-term spaceflight conditions via metabolic changes and adaptation to oxidative stress (Table 1). Primary and secondary metabolic changes in plants on U.S. space shuttles and the ISS have been recently reviewed (Paul et al. 2013a); three main strategies are usually used by plants: altered metabolism, adaptation to oxidative stress, and enhancement of other tropic responses.

Metabolic Adaptation

Characterizing metabolic changes has been generally considered among the most direct approaches for studying physiological responses of plant cells to microgravity (Tripathy et al. 1996; Hampp et al. 1997; Volovik et al. 1999). Genomic and proteomic changes have been analyzed in spaceflight experiments. For example, in Arabidopsis callus 14d grown on board the Chinese spacecraft SZ-8, many key enzymes of the carbohydrate metabolic pathway, such as GDH1 and GDH2, were greatly upregulated, ensuring an effective cellular energy state during the adaption to microgravity (Zhang et al. 2015). The impact of microgravity on cellular trafficking and energy state might alter plant cell construction and metabolism. A thinner cell wall and decrease in cell wall constituents (polysaccharides) were observed under spaceflight conditions (Hoson et al. 2001; Soga et al. 2002; Nedukha 1997). Concerted changes in transcriptional and protein expression patterns and physiological traits have been noticed under long-term spaceflight stress. The expressions of cell wall-associated CsExps genes in cucumber seedlings peg were reduced 4-fold in microgravity compared with those in the ground control (Link et al. 2001). Lignin production is often reduced in spaceflight along with the activities of phenylalanine ammonia lyase and peroxidase, as particularly shown in pine seedlings (Cowles et al. 1984; Cowles et al. 1988). In Brassica stems, the concentration of 3-butenyl glucosinolate increased in orbit compared with ground controls.

Adaptation to Oxidative Stress

Removal of ROS species produced under spaceflight stress in plant cells is essential to minimizing oxidative damage to biological macromolecules. Reconstruction of antioxidant defense mechanisms to scavenge the ROS is key to plant adaptation to microgravity. Using transgenic Arabidopsis plants harboring the alcohol dehydrogenase promoter linked to a β -glucuronidase (GUS) reporter gene, Paul et al. (2001) showed that spaceflight affects stress-signal perception and transduction. The adaptive response was observed in altered expression of heat shock proteins (HSPs) in Arabidopsis cell cultures,HSP genes were induced exclusively in response to prolonged microgravity on board the ISS and under sustained clinorotation, but transient microgravity intervals in parabolic flight and various hypergravity conditions failed to induce them (Paul et al. 2005; Zupanska et al. 2013; Kozeko and Kordyum 2009).

Increases in Other Tropic Responses

Many plants can complete their life cycles in microgravity and spaceflight experiments (reviewed by Correll and Kiss 2008; Correll and Kiss 2011). According to early space experiments, gravity was not necessary to maintain normal growth and proper development. However, more recent studies have indicated that those results could be attributed to the flexible adaptation mechanisms of plants. Analyses of Arabidopsis plants carried by the space shuttle to the ISS demonstrated how gravity contributed to intrinsic growth patterns (Johnsson et al. 2009; Solheim et al. 2009). To compensate for the lack of gravitropism, plants under microgravity strengthened other tropic responses. For example, Arabidopsis hypocotyls display increased blue-light phototropism (Millar et al. 2010). Recently JAXA’s HydroTropi experiments on the ISS showed that roots of cucumber seedlings became sensitive to moisture gradients, bending strongly toward moistened medium, and auxin-inducible genes such as CsIAA1 andCsPIN5 were expressed much more strongly in the responsive position of bending roots (Moriwaki et al. 2013).

Conclusions

Our knowledge of the short-term and long-term microgravity responses of plants in space has been greatly improved by recent advances in plant genomics and proteomics and in growth chambers allowing adequate environmental control for plant cultivation on space flights. Genome-wide DNA microarrays and global proteomic analyses have shown that, shortly after microgravity exposure, numerous genes are transiently up-regulated, many of which function in general stress responses, such as Ca 2+, lipid, and cytosolic pH signaling (Aubry-Hivet et al. 2014; Hausmann et al. 2014). However, after long-term acclimation, only a few genes remain at the elevated level and they are often functionally important for microgravity adaptation, probably via oxidative stress tolerance, metabolic changes, and tropic growth (Paul et al. 2005; Mazars et al. 2014; Zhang et al. 2015).

In the coming decade, plant research in space should consider the molecular regulatory network level, with emphasis on an advanced understanding of long-term microgravity effects and the molecular basis of plant adaptation to space. Plant growth facilities and sophisticated scientific equipment would be perfect for use in space. Genomic and proteomic studies will offer the promise of new data to better resolve the mechanism of plant responses and adaptation to short- and long-term microgravity. Long-term, uninterrupted, multigenerational, plant experiments will be possible on board the ISS and Chinese space station, which is expected to be fully operational by 2020.