Author for correspondence: John Z. Kiss Tel: +1 513 529 4200 Email: email@example.com
•The aim of this study was to investigate phototropism in plants grown in microgravity conditions without the complications of a 1-g environment. Experiments performed on the International Space Station (ISS) were used to explore the mechanisms of both blue-light- and red-light-induced phototropism in plants.
•This project utilized the European Modular Cultivation System (EMCS), which has environmental controls for plant growth as well as centrifuges for gravity treatments used as a 1-g control. Images captured from video tapes were used to analyze the growth, development, and curvature of Arabidopsis thaliana plants that developed from seed in space.
•A novel positive phototropic response to red light was observed in hypocotyls of seedlings that developed in microgravity. This response was not apparent in seedlings grown on Earth or in the 1-g control during the space flight. In addition, blue-light-based phototropism had a greater response in microgravity compared with the 1-g control.
•Although flowering plants are generally thought to lack red light phototropism, our data suggest that at least some flowering plants may have retained a red light sensory system for phototropism. Thus, this discovery may have important implications for understanding the evolution of light sensory systems in plants.
Light and gravity interact in many ways throughout the growth and development of plants. Until recently, it has been difficult for plant biologists to study the distinct effects of these environmental stimuli without the complications of the other sensory inputs, particularly when considering phototropism or gravitropism. The British plant physiologist Malcolm B. Wilkins suggested that one of the most promising aspects regarding the era of spaceflight research was the new ability to study ‘pure’ phototropism. He wrote that without an effective gravity vector, such as in space laboratories in low Earth orbit, the ‘true nature of phototropism will finally be revealed’ (Wilkins, 1988).
It is important to note that, in the 1-g conditions found on Earth, once phototropic curvature is initiated in shoot or roots, induction of gravitropism is an inevitable consequence of the change in orientation of the curving organ (Hubert & Funke, 1937; Okada & Shimura, 1992; Mullen et al., 2000). In this situation, phototropism causes a competing gravitropic response, limiting the extent of curvature. Indeed, in studies of phototropism and gravitropism in seedlings grown on rotating clinostats, it was shown that decreased gravitropism could lead to increased phototropic curvatures of coleoptiles (Nick & Schäfer, 1988). Moreover, mutants with an impaired gravity response can display a more robust phototropic curvature (Vitha et al., 2000; Ruppel et al., 2001).
Phytochromes play key roles throughout the entire life cycle of flowering plants and mediate diverse functions from seed germination to seedling growth to senescence (Han et al., 2007). In Arabidopsis thaliana, there are five genes for phytochrome (PHYA, PHYB, PHYC, PHYD and PHYE; Franklin et al., 2005). All five phytochromes have been shown to be involved in both gravitropism and phototropism (e.g. Correll et al., 2003; Kiss et al., 2003; Lariguet & Fankhauser, 2004; Kumar & Kiss, 2006; Kumar et al., 2008). Members of the phytochrome family are structurally similar, and studies with specific phytochrome mutants have shown that, for some responses, the functions of different phytochromes are redundant (Schepens et al., 2004). However, for many responses, the different phytochromes display functionally different behaviors.
In contrast to most flowering plants, groups from older plant lineages such as mosses and ferns use red light as a cue in phototropism. For example, protonemata of mosses exhibit positive phototropism in response to red light (Kern & Sack, 1999; Mittmann et al., 2009). In addition, protonemata of most fern species also show red-light-induced positive phototropism (Suetsugu & Wada, 2007), while in a few species there is negative phototropism induced by red light (Kiss, 1994).
Previously, we reported a red-light-induced positive phototropism in roots of A. thaliana that was discovered using a feedback system to counter the effects of gravity and showed that this response is controlled by phytochromes (Kiss et al., 2003). In the present report, we used microgravity as a unique research tool to study phototropism in hypocotyls of seedlings grown in a laboratory on the International Space Station (ISS). Our results revealed that, in the absence of a gravitropic response, red light induced a phytochrome-dependent phototropic response in hypocotyls.
Materials and Methods
In these experiments, we studied wild-type (WT) seedlings of Arabidopsis thaliana [(L.) Heynh, Landsberg erecta (Ler) strain] and several phytochrome mutants. The mutant strains were phyA-201, phyB-1, and phyA-201.phyB-1 and are described by Hennig et al. (2002) and Kiss et al. (2003).
Details of the spaceflight and hardware
All of the experiments were performed in the European Modular Cultivation System (EMCS) lab facility on the ISS from October to December 2006 during Increment 14. The Experimental Containers (ECs) were launched on two space shuttle missions, STS-121 and STS-115, in 2006 and returned on three space shuttle missions, STS-116 (December 2006), STS-117 (June 2007), and STS-120 (November 2007). The project was termed ‘TROPI’, for the study of tropisms (Kiss et al., 2009).
The EMCS facility, which was in the Destiny module of the ISS, is an incubator with two centrifuges that is suitable for the growth of A. thaliana (Brinckmann, 1999, 2005). The hardware consisted of five cassettes per EC as described in Correll et al. (2005), and in each cassette we placed 14 seeds. The key features of this hardware are a lighting system with light-emitting diodes (LEDs), a water delivery system, and a substrate consisting of filter papers with embedded nutrient solution for plant growth. The fluence rate from the red LED array (660 nm) was 10 μmol m−2 s−1, and the fluence rate from the blue LED array (450 nm) was 40 μmol m−2 s−1. For the growth of the seedlings, a white LED array produced a fluence rate of 70 μmol m−2 s−1. All LEDs were manufactured by Purdy Electronics (Sunnyvale, CA, USA).
Three experimental runs were performed, with eight ECs per run. Surface-sterilized seeds were affixed with 1% (w/v) gum guar to a black gridded membrane (no. 66585; Pall Corp., Ann Arbor, MI, USA). The black membrane with seeds was affixed to the top of one layer of dry Whatman no. 17 filter paper, which had been preloaded with plant nutrients so that, when rehydrated, the growth medium would consist of one-half strength Murashige and Skoog salts with 1% (w/v) sucrose buffered with 1 mM MES (pH 5.5). The assembled cassettes were placed into ECs and stored until launch on the space shuttle to the ISS. Seeds were stored in hardware for 6–8 months before initiation of the experiment. Operational details of the space experiments were discussed in a recent methods paper (Kiss et al., 2009).
On the ISS, astronauts loaded the ECs into the EMCS unit and activated the experiment by hydrating the seeds, which was part of an automated series outlined in the time line (Fig. 1) and included recording of images captured on high-8 video tape. Several near-real-time video downlinks during experimental operations on the ISS provided input on the status of the experiment. Crew actions included changing the video tape and freezing the cassettes at the end of the experiment in an on-board −80°C freezer.
Post-flight data analyses
Frozen plant samples and video tapes were returned on space shuttle missions. To measure plant growth and curvature, the analog tapes were converted to digital video. Still images consisting of half-cassette fields of view, used to provide a high magnification view of the seedlings, were extracted from the digital tapes. Images from each half of the cassette were combined into a view of a single cassette. These images were then arranged in relative order according to the time line. Additional details are provided in our recent operations paper (Kiss et al., 2009).
Seedling growth and curvature were measured on these images using Image Pro Plus (Media Cybernetics, Bethesda, MD, USA). Seedlings that exhibited a shoot angle in excess of 30° in either direction from the vertical at the beginning of the stimulation phase (T = 0) were excluded from statistical analysis (as their photostimulus differed substantially from that of more oriented seedlings). To determine trends through time, simple linear regression analysis was conducted using the proc reg procedure in sas (SAS Institute, 2004). To determine if differences between treatments were significant, regression coefficients were compared using the proc glm procedure followed by contrast comparison in sas (SAS Institute, 2004). A t-test was used to compare growth rates under different light and gravity conditions using proc ttest in sas (SAS Institute, 2004). Growth was calculated as increments over starting values.
Because of operational constraints, seeds were stored in spaceflight hardware for 6, 7 and 8 months for runs 1, 2 and 3, respectively. An image of the three cassettes during run 1 is shown in Fig. 2. White LED arrays along the long axis of the cassette provided illumination for growth and initial orientation (Fig. 2a), and red or blue LED arrays along the shorter axis (oriented 90° from the white LEDs) provided the light for phototropic stimulation (Fig. 2b).
Seed germination on the ISS was reduced compared with the ground controls (Table 1; Fig. 2a). The average germination for all strains was 57.5%, while it was 92.6% in a ground control (Table 1). The percentage germination dropped precipitously in runs 2 and 3 relative to run 1. These decreases in germination were probably attributable to a relatively long storage time (i.e. 6–8 months) in spaceflight hardware (Kiss et al., 2009). However, seeds that did germinate exhibited robust growth and tropistic curvature (Fig. 2).
Table 1. Seed germination (%) of Arabidopsis thaliana in a ground control (n =478) and in three runs of the space experiments (n =560)
phy, phytochrome; WT, wild type.
One of the most interesting results from these experiments was that, in microgravity, hypocotyls of WT seedlings exhibited positive phototropic curvature in response to unidirectional red light (Fig. 2b). The curvature in microgravity (Fig. 3a) was statistically significant (P <0.0001, R2 = 0.0303) through time, whereas curvature of the 1-g control (from an on-board centrifuge) was not significant (P =0.24, R2 = 0.0018). In addition, the positive red-light phototropic response in microgravity was significantly different from that of the 1-g control (P <0.0001, F = 26.98).
The positive curvature response of WT hypocotyls to red illumination in microgravity was not significantly different from the response of hypocotyls of mutant phyB seedlings in microgravity (P =0.4972, R2 = 0.0020; Fig. 3b). However, hypocotyls of mutant phyA seedlings had a strong negative response to red light (P <0.0001, R2 = 0.1005; Fig. 3b). Comparing the responses of hypocotyls of phyA seedlings, there was a significantly greater (P <0.0001, F = 76.03) negative phototropic curvature in microgravity conditions relative to the 1-g control (Fig. 3c).
The effect of a 1-h red-light pretreatment on blue-light-induced phototropism was studied in 1-g and in microgravity (Fig. 4). In these experiments, seedlings were given a 1-h pretreatment with red light followed by continuous blue illumination (R-B) or continuous blue light alone (B). In the 1-g control, the positive phototropism of WT hypocotyls was enhanced by the red treatment, and this difference was statistically significant (P <0.0001, R2 = 0.2088). Comparing the blue-light phototropic response with the red-light pretreatment conditions, we noted that there was a significantly greater response in microgravity conditions relative to the 1-g control (P <0.0001, F = 124.76). Also, the blue-light phototropic response was greater than the response seen with red light only (Fig. 3a). Unfortunately, we could not obtain data from the blue-light-only treatment in microgravity to use in these comparisons because of lack of germination in those ECs (Kiss et al., 2009).
The growth rates of the WT, phyA, and phyB seedlings exhibited variability but were not significantly different (P >0.05) in microgravity compared with the 1-g controls (Fig. 5a,b). There were also no significant differences (P >0.05) in growth rates within a gravity treatment among the three genotypes. Thus, the differences in phototropism reported here were not a result of differences in growth rates of the different mutants or in the different treatments.
A novel red-light-induced phototropism in hypocotyls is revealed in microgravity conditions
The most significant result from our spaceflight experiments is the discovery of a novel positive phototropic response to red light in hypocotyls of A. thaliana. The red-light-based phototropism was observed in the microgravity conditions found in a spacecraft orbiting the Earth but was apparently suppressed by normal 1-g conditions. Specifically, the red-light-induced positive phototropism was statistically significant in hypocotyls of WT seedlings in microgravity (Figs 2, 3a) but was not significant in hypocotyls of WT seedlings in the 1-g control (Fig. 3a).
One possible explanation for the positive red-light phototropism we observed in spaceflight samples is found in the paper by Iino (1990), who suggested that a gradient of Pfr/Pr (far-red/red light-absorbing) forms of phytochrome is generated by unilateral red light through the tissues of the plant. As long-term red irradiation can depress cell elongation (Blaauw-Jansen & Blaauw, 1966), the gradient may cause a greater reduction in cell elongation on the side with greater fluence rates of red light. This theory was proposed by Blaauw (1919), but others have reported both reduced elongation on the lighted side of hypocotyls and enhanced elongation on the shaded side during phototropic responses (e.g. Baskin et al., 1985; Rich et al., 1987; Orbović & Poff, 1993). It is likely that, in microgravity, the differential elongation on either side of the organ is enhanced because the competition between negative gravitropism and the weaker positive red-light-induced phototropism of hypocotyls is eliminated (for a review, see Correll & Kiss, 2002).
The other report of possible phototropism directly induced by red light in flowering plants was in the study by Iino et al. (1984). These researchers described a positive phototropic curvature in response to red illumination in mesocotyls of corn (Zea mays) seedlings. This red-light-based response was much weaker than the blue-light-based positive phototropic curvature in mesocotyls and was absent in coleoptiles of the corn seedlings.
Before our recent spaceflight study, the only other demonstration of red-light-induced phototropism in flowering plants that was attributed to phytochromes A and B was a positive phototropism in roots of A. thaliana (Kiss et al., 2003). However, in a more recent study of root phototropism (Molas & Kiss, 2008), we showed that, while phytochromes A and B were both essential for a positive response to red light and acted in a complementary fashion, either photoreceptor acting without the other resulted in negative curvature in response to red light. Moreover, the signal transduction molecule PHYTOCHROME KINASE SUBSTRATE 1 (PKS1) is one of the first molecules interacting with either phytochrome A or B in the cytoplasm, and PKS1 is involved in regulating red-light-induced phototropism in roots (Molas & Kiss, 2008).
Plants such as mosses and ferns have versatile phototropic responses to both blue and red light (Mittmann et al., 2009). These plants have evolved a sophisticated light-sensing system to use red as well as blue light for regulation of these responses. One possible explanation is that mosses and ferns evolved in low-light aquatic niches that can filter red and blue light differently, so they had a need to utilize all parts of the spectrum (Suetsugu & Wada, 2007). Although flowering plants are generally thought to lack red-light phototropism, our data suggest that at least some flowering plants may have retained a red-light sensory system for phototropism. Thus, this discovery may have important implications for understanding the evolution of light sensory systems in plants.
However, the red-light phototropic response is weak and is not apparent in normal 1-g conditions on Earth, and further studies with mutants impaired in gravitropism may allow a better characterization of this novel response (Vitha et al., 2000; Ruppel et al., 2001). At this point it is not known why flowering plants evolved to use their blue-light photoreceptors for phototropism instead of the phytochromes.
Enhancement of blue-light-mediated phototropism in space
In addition to the red-light response we observed, blue-light-induced phototropism was enhanced in microgravity compared with the 1-g control in seedlings that had been pretreated with red light (Fig. 4). In a previous spaceflight experiment, Heathcote et al. (1995) found only minor increases in phototropic curvature of wheat (Triticum aestivum) coleoptiles under microgravity conditions. In those experiments, first positive responses in microgravity were slightly enhanced, both in magnitude and in duration, compared with the 1-g controls, but not to the extent predicted by clinostat studies. However, the results of Heathcote et al. (1995) were obtained from seedlings grown in nonventilated containers with limited diffusion paths to the spacecraft atmosphere, and this may have affected the space-grown plants. Indeed, accumulated cabin gases, particularly ethylene, may affect the results of spaceflight experiments with plants (Guisinger & Kiss, 1999; Kiss et al., 1999). However, in our present study, the atmospheric conditions, especially the concentration of ethylene, were precisely controlled by the EMCS system (Correll et al., 2005).
Does red light affect blue-light-based phototropism directly?
Phytochromes modulate blue-light-induced phototropism in shoots in such a way that brief exposure to red light before unilateral blue light results in an enhanced phototropic curvature (Liu & Iino, 1996). Both phytochromes A and B are involved in this red-light-induced enhancement of hypocotyl phototropism (Parks et al., 1996; Janoudi & Poff, 1997) as well as in red-light-dependent root phototropism (Kiss et al., 2003). Moreover, phytochrome activation is important for phototropism induced by continuous exposure to unilateral blue light in A. thaliana as phototropic curvature is strongly attenuated in the absence of phytochromes A and B (Hangarter, 1997).
An important question is whether red light indirectly affects phototropism by modulating gravitropism or if it affects phototropism more directly (Hangarter, 1997; Molas & Kiss, 2009). Because red-light exposure enhances the development of blue-light-dependent phototropic curvature in hypocotyls and red light can attenuate gravitropism, it has been suggested that red-light-induced enhancement of shoot phototropism may be a result of attenuation of gravitropism by red light (Parks et al., 1996). Thus, by reducing the antagonistic action of gravitropism, a more robust phototropic curvature could develop. Alternatively, phytochromes may affect the pathways of phototropic signaling more directly.
One of the goals of the present spaceflight project was to conduct an experiment to specifically test the hypothesis that red-light-dependent enhancement of phototropism is attributable to red-light effects on gravitropism. If the enhanced phototropic response caused by red light were mediated by red light attenuating gravitropism, under microgravity conditions, blue-light-induced phototropism in hypocotyls should be the same with and without previous red-light treatment, whereas, under 1-g, phototropism should be reduced in blue light alone.
We attempted to conduct this experiment in the microgravity conditions on the ISS. Four treatments were needed to fully address this issue, but, unfortunately, we were not able to obtain data from seedlings in microgravity exposed to blue illumination alone (Fig. 4). The main reasons for this loss of data were technical difficulties, especially low seed germination as a consequence of extended storage in spaceflight hardware (Kiss et al., 2009). However, the reduced responses of the phytochrome mutants directly to red light in microgravity (Fig. 3b,c) suggest that red light, in addition to attenuating gravitropism, may induce a weak phototropic response.
The promise and problems of microgravity research
Spaceflight research offers investigators the unique microgravity environment as a laboratory in which to answer novel questions in fundamental biology that may otherwise be difficult to resolve. A recent example of the utility of space experiments is provided by the studies of Johnsson et al. (2009) that deal with the mechanisms of circumnutation, which is the oscillatory or helical growth pattern around an axis in plants. In a spaceflight experiment on the ISS, these authors found that endogenous nutations occurred in stems of plants that developed completely in microgravity but also that gravitational accelerations amplified these circumnutations. The authors support a ‘combined’ model which incorporates two hypotheses – the endogenous model (Kiss, 2009) and the idea that circumnutation is related to and dependent upon gravity.
However, as our experiences have shown, spaceflight research poses many challenges both from technical and organizational perspectives (Kiss et al., 2009). Seed germination in the present study was lower compared with previous space experiments (e.g. Kiss et al., 1999; Driss-Ecole et al., 2008), which was probably a result of unexpected extended storage in hardware of up to 8 months. Also, because of the lack of chambers with adequate atmospheric control (Porterfield, 2002), effects potentially attributed to microgravity may be related to artifacts caused by accumulating gases such as ethylene (Kiss et al., 2000). Fortunately, our recent experiment used the excellent gas control system in the EMCS facility (Brinckmann, 2005) and we did not observe any ethylene-induced effects in plants grown in this space experiment.
An on-board centrifuge provided an essential 1-g control (Perbal & Driss-Ecole, 2002; Correll & Kiss, 2008) to help distinguish between true microgravity effects and indirect effects of spaceflight (e.g. ethylene gas, magnetic fields, vibrations and lack of convection; Iversen & Johnsson, 1996; Ferl et al., 2002). Many spaceflight experiments to date have lacked this important control (Krikorian, 1996). Fortunately, utilizing the EMCS unit, we were able to use centrifuges to provide a g-force control (Correll et al., 2005). This allowed us to determine that red-light-induced curvature in hypocotyls occurred in microgravity but was not detected in 1-g conditions (Fig. 3a).
Despite a number of technical problems, this spaceflight experiment demonstrated a red-light-induced phototropic response in hypocotyls that is normally masked by the 1-g conditions present during ground experiments. This discovery demonstrates that the unique microgravity conditions on the ISS make it possible to study a ‘pure’ phototropic response. Also, the red-light-enhanced, blue-light phototropism was of a greater magnitude in microgravity compared with the 1-g control, but additional studies to identify whether red light affects blue-light phototropism directly or indirectly by the attenuation of gravitropism are still needed. Laboratories in the newly completed ISS have the potential to allow the scientific community to carefully test these and other important questions in fundamental biology.
The authors thank the staff of Ames Research Center (especially Drs Mike Eodice, Marianne Steele, and Bob Bowman) of the National Aeronautics and Space Administration (NASA) for their efforts on behalf of this project. We also thank the Norwegian User Support and Operations Centre (N-USOC) in Trondheim, Norway for its technical support during operations on the ISS. Special thanks to Astronaut Thomas Reiter and other crew members for performing our experiments in space. Financial support was provided by NASA through grant NCC2-1200.