Plant Science In Space!

Fundamental plant space biology

The Space Shuttle era, from the early days of sortie missions where biological science was conducted only as small payloads on the shuttle itself through to the days of extended missions with laboratory modules and visits to space stations, saw a large expansion of plant science (Halstead and Dutcher, 1987; Dutcher et al., 1994). Plant payloads were a large fraction of the science complements of flight activities from the late 1980s to the retirement of the Shuttle. These studies were enabled by increasingly advanced hardware that began to eliminate some of the difficulties of growing plants in space, e.g., the use of LED lighting, condensate recycling, and porous tube watering techniques (Morrow et al., 1994, 1995; Morrow and Crabb, 2000; Link et al., 2003), and injected crew interaction, which allowed manipulation of experiments on orbit and produced increased agency programmatic attention to plant biology. There were multiple opportunities to study metabolism, development, molecular gene expression, with increasing attention to plant tropisms.

The following sections highlight many of the insights gained in fundamental plant biology through research enabled by the Space Shuttle, and examples of experiments that played a role in these insights have been organized in the context of Space Shuttle mission numbers—either associated with a sortie flight experiment or experiments that transitioned from shuttle to the ISS. The contributions of plant space biology are of course not limited to research associated with the specific Space Shuttle missions highlighted here; plant research has been conducted on Skylab, Salyut, and Mir; on satellites and sounding rockets; and in a variety of analog environments on the Earth. Additional discussions of plant space biology outside the scope of this review can be found within a number of reviews and related articles, and a selection of articles reach beyond the present review

Metabolism

Overall plant metabolism and photosynthesis were studied with wheat on STS-51 and STS-64 where it was noted that the shoots of space-grown seedling were 25% smaller than ground controls, and the CO₂-saturated photosynthetic rate at saturating light intensities in space-grown plants were similarly reduced compared with ground control plants (Tripathy et al., 1996; Volovik et al., 1999). Brassica plants on STS-87 had altered chloroplast morphology and altered chlorophyll a/b ratios (Adamchuk et al., 1999; Jiao et al., 1999, 2004). Wheat on STS-110, however, did not differ from ground controls in photosynthetic activity at moderate light levels, yet did show reductions in whole chain electron transport in saturating light (Monje et al., 2005; Stutte et al., 2005). From these experiments, overall growth and photosynthesis appear to be influenced by spaceflight, although the specifics of the effects can be influenced by differences in hardware capabilities, experimental setup, and plant type. The relationship between plant nutrition, photosynthesis, and gas exchange in the spaceflight environment has been recently reviewed (Wolff et al., 2012).

Secondary metabolism also appears altered by spaceflight, affecting both the production of interesting secondary compounds and potentially interactions with plant pathogen (reviewed by Tuominen et al., 2009). For example, isoflavonoid biosynthesis is increased in spaceflight compared with matched ground controls (Levine 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, 1988). However, in brassica stems, the concentration of 3-butenyl glucosinolate was increased on orbit compared with ground controls. Nonetheless, plants do appear to be more susceptible to pathogens and microbial interactions in spaceflight (Musgrave et al., 2005). In spaceflight, soybean seedlings were more susceptible to infection by Phytophthora (Leach et al., 2001; Ryba-White et al., 2001) and wheat plants were more susceptible to Neotyphodium (Bishop et al., 1997). Recent work launched on STS-135 included an experiment designed to study the symbiotic relationship between the nitrogen-fixing bacterium Sinorhizobium meliloti and the legume Medicago truncatula (Stutte and Roberts, 2011).

Development and reproduction

Once plants were determined to be able to grow on orbit, studies were initiated to explore spaceflight-induced changes in growth and development. Early experiments with Hemerocalis sp. and Haplopappus gracilis showed that roots could initiate from plantlets on orbit, but that spaceflight compromised apical dominance by affecting the emergence of axillary branches (Levine et al., 1990; Krikorian et al., 1992; Levine and Krikorian, 1992). Additionally, some mitotic index abnormalities and chromosomal anomalies were observed, and cultured cells of orchard-grass (Dactylis glomerata L.) were impaired in embryogenesis, leading to some concern about spaceflight seed development (Conger et al., 1998). The development of subcellular features has also been explored. Protoplasts of Brassica napus were launched as part of the first International Microgravity Laboratory (IML-1) on STS-42, and the regeneration of the cell wall in these cells was observed with an on-orbit microscope. The cells maintained in microgravity showed delayed cell division, cell aggregation, and cell wall regeneration compared with comparable ground controls. These observations further contributed to the concern that microgravity may have a negative impact on plant cell development (Rasmussen et al., 1994). However, as spaceflight hardware and growth conditions improved, concern over potential development difficulties largely subsided (Krikorian, 1996a, 1996b) as protocols for germination and development of several species in space became well established (Hoehn et al., 1992; Guikema et al., 1994; Stutte et al., 2005, 2006).

The general growth and levels of the plant growth regulators indole acetic acid and abscisic acid were not seriously impacted in Zea mays after 5 d in orbit on mission STS-34 (Schulze et al., 1992). However, phenomena associated with the more subtle interactions of auxin and gravity are affected by spaceflight (Ueda et al., 2000). For example, peg formation at the transition zone between hypocotyl and root in cucurbits is bilateral on orbit, but unilateral on the ground (Takahashi et al., 1999; Kamada et al., 2000). And a general reduction in reproductive structures and development remained (Kuang et al., 1996a, 1998, 2000a; Musgrave et al., 1997, 2000; Musgrave and Kuang, 2001, 2003), so that concern continued about the development of seeds on orbit (Mashinsky et al., 1994; Jiang et al., 1998; Musgrave et al., 2000). That concern was relieved on STS-51, when Arabidopsis thaliana (Arabidopsis) was grown in more advanced spaceflight hardware and demonstrated that early reproductive development can occur normally provided there are no tangential impediments to pollination or fertilization, and especially to the gaseous environment presented to the plants (Kuang et al., 1995, 1996a; Musgrave et al., 1997). Arabidopsis grown on STS-54 and STS-58, showed changes in leaf ultrastructure, chlorophyll, and starch content that varied depending on air flow (Musgrave et al., 1998) in experiments that were again enabled by improvements in spaceflight plant growth hardware and conditions (Morrow et al., 1994; Porterfield et al., 2000). Later experiments on STS-68 specifically characterized Arabidopsis seed development on orbit and showed no significant difference from ground controls (Kuang et al., 1996b). The ability to successfully grow a plant from seed and produce viable seeds on orbit was a major milestone in plant space biology.

Genomic responses

Molecular genetic data joined spaceflight plant biology analyses as sample preservation and sample return capabilities allowed the recovery of high quality RNA. An early study employed RNase-protection assays to reveal a slight increase in the mRNA of the enzyme alcohol dehydrogenase (ADH) (Porterfield et al., 1997), and this hallmark of hypoxic stress was also reflected in the activity of the ADH enzyme in the spaceflight environment (Porterfield et al., 2000). The first set of published spaceflight array data indicated that there were a number of genes that showed moderate changes between flight and ground controls in Arabidopsis that was launched as seedlings on STS-93, with the most dramatic differential expressions in response pathways that may not necessarily confer an adaptive advantage, though the results did not suggest any particular environmental stress (Paul et al., 2001, 2005b). In wheat grown in different hardware and germinated on orbit, there were virtually no statistically significant differences between the gene expression patterns of space-grown and ground control plants (Stutte et al., 2006). However, in the fern Ceratopteris richardii, over 5% of the genes examined from germinating spores were differentially expressed. Many of these differentially expressed genes encoded proteins associated with plant stress responses, but a large contingent represented genes associated with calcium-mediated processes, not surprising since calcium signaling is central to spore orientation and germination (Salmi and Roux, 2008; Salmi et al., 2011a, 2011b). Work with Arabidopsis seedlings also indicated that features of calcium-mediated signal transduction were affected by the orbital environment. Changes in tissue-specific expression patterns of the Adh::GUS reporter gene between spaceflight and ground control plants suggested that an aspect of calcium signaling was disrupted in these plants (Paul et al., 2001). Although these results seem inconsistent, it must be recognized that these initial molecular studies were conducted in very different plant species, in different spaceflight hardware and with different analytical platforms.

Further advances in spaceflight operations and hardware and the increased use of model plant systems now allow increasingly well-designed and controlled whole transcriptome analyses (Paul et al., 2005a; Ferl et al., 2011). On STS-135, a BRIC (Biological Research in a Canister) carried Arabidopsis (Col-0) etiolated seedlings and undifferentiated tissue culture callus (Paul et al., 2012b; Zupanska et al., 2013). The etiolated seedlings displayed qualitatively moderate but highly significant changes in the expression of genes associated with drought stress, wounding, and calcium- and auxin-mediated signaling, and cell wall development. A number of genes were also highly induced (5× or greater) by spaceflight that encoded proteins typically involved with pathogen defense and environmental stress responses. Nondifferentiated cultured Arabidopsis cells displayed a dramatic response with many genes showing very high levels (greater than 10×) of differential expression in spaceflight. The most highly represented genes in this category were those encoding heat shock related proteins and transcription factors, a response that was not seen in seedlings (Paul et al., 2012b).

These data and other spaceflight transcriptome data currently under analysis strongly suggest that plant adaptation to spaceflight requires changes in gene expression, and at least part of the adaptive response appears to be associated with cell wall remodeling. Further, the ability to sense gravity’s influence is present even in single cells.

Tropisms

Explorations of the effect of the lack of gravity on plant tropisms were among the very first structured spaceflight experiments with terrestrial biology, and the ability to conduct tropism experiments without the overriding influence of gravity enabled by spaceflight has had lasting appeal in the plant research community (reviewed by Correll and Kiss, 2008; Chebli and Geitmann, 2011).

It was discovered early on that plants maintain inherent growth patterns that are independent of gravity. Sunflower seedlings flown on the Spacelab 1 mission that had initiated circumnutation before launch continued hypocotyl oscillations in microgravity (Brown and Chapman, 1984). Later orbital studies with wheat seedlings provided another demonstration that aerial tissue responds in certain preprogrammed manners independent of gravity. Dark-grown coleoptiles expand in microgravity with a curvature oriented away from the caryopsis, suggesting an inherent growth pattern that is not reliant on gravity for a directional cue (Heathcote et al., 1995). Yet, it does not take much in the way of gravitational force (as little as 0.10 g) to dramatically influence these growth patterns (Brown et al., 1995; Johnsson et al., 1995). The contribution of gravity to intrinsic growth patterns has been most recently demonstrated with Arabidopsis carried by the Space Shuttle to the International Space Station (ISS). In these experiments, the oscillations of the side stems of the inflorescence (the primary axillary and lateral inflorescence stems) clearly occurred in the absence of gravity. Rosette leaves also demonstrated self-sustained rhythmic movement within the daily cycle on orbit. The introduction of 0.80 g on an orbital centrifuge enhanced the oscillations, but it is not yet clear how gravity elicits this response (Johnsson et al., 2009; Solheim et al., 2009).

Nonvascular plants exhibit these inherent patterns of growth as well. The protonemata of the moss Ceratodon purpureus are highly phototropic, and typically the phototropism will overwhelm the effects of gravity. However, when grown in the absence of both light and gravity an inherent pattern of growth for the moss filaments emerges. Once they reached a threshold of developmental age, the protonemata tended to grow in an arc, and the overall patterns of the densely packed moss cultures took on the look of clock-wise spirals (Kern and Sack, 2001; Kern et al., 2001, 2005). These results further support the conclusion that plants maintain intrinsic patterns of growth that are founded in inherent cellular characteristics and independent of environmentally driven tropisms.

But what of the mechanisms that underlie well-established tropic responses? Gravitropism and phototropism are the two primary tropic influences in plant growth and development, and while the quality and quantity of light can be manipulated in terrestrial experiments, the force of gravity can never be completely removed. And for this reason, the spaceflight environment has been a powerful experimental platform for exploring the nature of tropisms—gravitropism as well as other tropisms that would otherwise be masked by the effects of gravity on Earth.

Mechanisms underlying the starch–statolith model of root gravitropism were examined in spaceflight in a number of studies. In an early experiment launched on STS-61A in Spacelab, lentil seeds were germinated on orbit and examined for patterns of growth. When the fixed material was examined for ultrastructural differences of the gravisensing root cells, it was discovered that, although there were no qualitative differences in these cells among plants grown in microgravity, in the orbital 1-g centrifuge or the ground control plants, the distribution of statoliths within these cells was different in microgravity. In the flight samples, the staoliths were larger and distributed in the proximal region of the cell (not randomly, as is seen with clinorotation) (Perbal et al., 1987; Perbal and Driss-Ecole, 1989). These results led to the discovery that statoliths are not entirely free to sediment within the cell volume, but rather are attached to the cytoskeletal actin filaments via myosin. As a motor protein, myosin could contribute to the displacement of statoliths from the distal pole of the cell toward the proximal pole, as is seen in the transition from the 1-g centrifuge to microgravity (Perbal et al., 1997; Volkmann et al., 1999; Driss-Ecole et al., 2000; Perbal and Driss-Ecole, 2002). It is important to note that this observation can only be made in an environment where the motive force exerted by myosin can be observed without the overwhelming background force normally exerted by gravity.

The starch–statolith model was further explored in Arabidopsis, primarily by taking advantage of the diversity of mutants and cultivars in Arabidopsis. In an experiment comparing Arabidopsis mutants deficient in starch biosynthesis to wild-type plants, the distribution of the amyloplasts in the gravity sensing columella cells was different between flight and ground controls (Hilaire et al., 1997). Three Arabidopsis mutants that differed in the degree of deficiency in starch metabolism were launched on STS-81. After a period of growth in microgravity, the mutants were given a 1-g stimulus in an orbital centrifuge to assess the ability of each of the plant lines to respond to the introduction of gravity. In each case, the amount of starch was directly correlated to the degree of the response to gravity, thereby supporting the starch–statolith model for gravity sensing (Kiss et al., 1998). Additional experiments with starch-deficient Arabidopsis mutants were conducted on STS-84. These experiments examined the distribution of the amyloplasts in the columella cells, patterns of growth, and also in various Arabidopsis ecotypes. It was concluded from this study that the Landsberg erecta (Ler) line and C24 ecotype were best suited to cultivation in a spaceflight environment (Kiss et al., 2000; Kraft et al., 2000).

Although we tend to look for commonality of the underlying mechanisms of tropic responses, there are a surprising number of variations among plants—even among cultivars of the same species—and again, the orbital environment can assist the discovery of subtle phenotypes.

Launched as part of the second International Microgravity Laboratory (IML-2) on STS-65, transgenic Brassica napus was used to make the direct comparison of normally gravitropic wild-type plants with otherwise identical agravitropic transgenic lines. Although the two lines have distinctly different growth patterns in unit gravity, there were no discernible differences between wild-type and the transgenic plants on orbit; growth patterns, root morphology, cellular ultrastructure, and amyloplast distribution were identical in both lines. The conclusion derived from these observations was that removing the plants from the influence of gravity also removed the phenotypic differences normally observed between these lines (Iversen et al., 1996).

As noted, Arabidopsis has been widely studied on orbit, and the results of each experiment contribute to a complex picture of the tropic response of this plant. In the dark, the roots of etiolated seedlings of ecotype Landsberg were seen to display a skew to the right (Millar et al., 2011), while the roots of etiolated Col-0 grew randomly (Paul et al., 2012b). Rice cultivars have also been shown to respond with differing levels of spontaneous curvature when grown in microgravity; cultivar Koshihikari coleoptiles show more prominent intrinsic curvature than that of the dwarf cultivar Tan-ginbozu (Hoson et al., 1999). In contrast, roots of light-grown Arabidopsis Col-0 on orbit show clear negative phototropism with a slight skew to the left, and ecotype Wassilewskija (Ws) roots skew strongly to the right while maintaining a negatively phototropic direction of growth (Paul et al., 2012a). Not all plant roots are negatively phototropic, even in unit gravity (Kutschera and Briggs, 2012). One of these light-indifferent plants is another member of the Brassicaceae family, Lepidium sativum, and on orbit L. sativum roots appear to grow randomly (Johnsson et al., 1996).

The manipulation of light in the absence of gravity can reveal nuances of phototropism that are normally masked on earth. The TROPI experiment spanned several Space Shuttle flights and increments on the ISS and focused on dissecting the influence of light quality through the use of orbital hardware specialized to deliver specific wavelengths on the background of variable gravity. These experiments in the absence of gravity revealed a heretofore unknown red-light phototropic response in Arabidopsis hypocotyls. It was also discovered that in the absence of gravity the photoreceptors attuned respectively to red and blue light were differentially sensitive to changes in gravitational force introduced by an orbital centrifuge (Kiss, 2009; Millar et al., 2010; Kiss et al., 2012).

Another subtle influence of tropism that can be overwhelmed by the influence of gravity on Earth is the presence of an ionic gradient in the growth medium. Gradients of H⁺, as well as other signaling ions, such as calcium, have been shown to influence cellular alignments and tropic responses in many plant systems (Blancaflor et al., 1998; Chatterjee et al., 2000; Fasano et al., 2001; Plieth and Trewavas, 2002; Monshausen et al., 2007). The germinating spores from the fern Ceratopteris richardii have been used as a model for examining the effects of calcium ion currents in cellular orientation in both terrestrial and orbital venues. It was hypothesized that C. richardii spores use gravity for polar axis alignment, and in the initial experiments on orbit it was shown that in the absence of gravity, although spores germinated and apparently developed normally, the polarity of that development was random (Roux et al., 2003). In short-term milligravity experiments on parabolic aircraft, calcium channel blockers successfully disrupted gravity-directed cell polarity, which further supported the premise that gravity perception in C. richardii germinating spores was mediated by mechanosensitive calcium channels (Salmi et al., 2011a, 2011b). Water is of course the carrier of ionic gradients, but water itself can affect tropic responses in plant roots when it is present as a gradient. There has been speculation that gravitropism interferes with hydrotropism (Takahashi et al., 2009) and a recent ISS payload, HydroTropi, was designed to dissect the mechanisms mediating the interactions of gravity, hydrotropism and auxin-mediated signaling.