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Biocompatibility studies in preparation for a spaceflight experiment on plant tropisms (TROPI)
Advances in Space Research 39 (2007) 1154–1160 www.elsevier.com/locate/asr
Biocompatibility studies in preparation for a spaceflight experiment on plant tropisms (TROPI) John Z. Kiss a,*, Prem Kumar a, Robert N. Bowman b, Marianne K. Steele b, Michael T. Eodice b, Melanie J. Correll c, Richard E. Edelmann a a
c
Department of Botany, Miami University, Oxford, OH 45056, USA b NASA Ames Research Center, Moffett Field, CA 94035, USA Department of Agricultural and Biological Engineering, University of Florida, Gainesville, FL 32611, USA Received 31 August 2006; received in revised form 8 December 2006; accepted 12 December 2006
Abstract The interaction among tropisms is important in determining the growth form of a plant. Thus, we have developed a project to study the interaction between two key tropistic responses (i.e., gravitropism and phototropism) to be performed in microgravity on the International Space Station (ISS). Specifically, we are interested in the role of red-light-absorbing phytochrome pigments in modulating tropisms in seedlings of Arabidopsis thaliana. This project, termed TROPI for tropisms, is to be performed on the European Modular Cultivation System (EMCS), which provides an incubator with atmospheric control, lighting, and high-resolution video. The EMCS has two rotating centrifuge platforms so that our experiments can be performed at microgravity, 1g (control), and fractional g-levels. In order to optimize these spaceflight experiments, we have continued ground-based technical tests as well as basic science experiments. Since the seeds will have to be stored for several months in hardware prior to use on the ISS, we tested the effects of long-term storage of seeds in the TROPI EUE (experimental unique equipment) on germination rates and plant growth. The EUE consists of five seedling cassettes with LED lighting and a water delivery system in an Experimental Container (EC). Preliminary studies showed that there were reduced seed germination and plant growth after several months of storage in the EUE. We determined that the likely source of this biocompatibility problem was the conformal coating of electrical components of the EUE, which was required by NASA for safety reasons. In order to alleviate this problem, carbon filters were added to both the seedling cassettes and to the base of the EC. We expect that these improvements to the hardware will result in healthy plants capable of robust tropistic responses in our spaceflight experiments. 2006 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: EMCS; Gravitropism; ISS; Plant space biology; Phototropism; Spaceflight hardware
1. Introduction On Earth it is very difficult to study the interacting effects of gravity and other stimuli such as light on plant growth. However, a project was launched on the space shuttle (missions STS-121 in July 2006 and STS-115 in September 2006) to examine these interactions in seedlings of Arabidopsis thaliana on the International Space Station (ISS). A major goal of these experiments to be performed *
Corresponding author. Tel.: +1 513 529 5428; fax: +1 513 529 4243. E-mail address: [email protected] (J.Z. Kiss).
on the ISS is to gain insights into the cellular and molecular mechanisms of phototropism, the directed growth of plants in response to light. Phototropism in stems has been more extensively studied compared to root phototropism, which when observed in most species is typically away from the light stimulus (i.e., negative phototropism; Whippo and Hangarter, 2006). However, since the phototropic response in roots is weak relative to gravitropism, it remained largely unstudied for decades (Sakai et al., 2000). Several reports have demonstrated that, in stems, interactions between positive phototropism and negative
0273-1177/$30 2006 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2006.12.017
J.Z. Kiss et al. / Advances in Space Research 39 (2007) 1154–1160
gravitropism determine the direction of growth in young seedlings (e.g., Hangarter, 1997). Similarly, in roots of some species (e.g., A. thaliana), it appears that there is also a comparable interaction between tropisms in orienting root growth (Vitha et al., 2000), but, in the case of roots, the reported interaction was between negative phototropism and positive gravitropism. However, we have recently discovered a positive red-light-induced phototropism in roots that is mediated by photoreceptors in the phytochrome family (Kiss et al., 2003). In most plants, the phytochrome gene family has five members (PHYA–PHYE; Quail, 2002); so in our space experiments, we will investigate phototropism in both the normal WT Arabidopsis seedlings as well as in a series of phytochrome mutants (phyA, phyB, and phyAB). While gravitropism can readily be studied on earth in the absence of light, it is difficult to investigate phototropism without the effects of gravity (Correll and Kiss, 2002). Thus, the microgravity conditions of space can be used to study phototropism without the ‘‘complications’’ of unidirectional gravity. Our spaceflight experiment, termed TROPI for tropisms, will use microgravity as a unique research tool to study an important issue in plant biology. In addition to microgravity treatments, TROPI will also have a 1-g in-flight control from an onboard centrifuge as well as fractional gravity treatments ranging from 0.1g to 0.9g (Brinckmann, 1999). These latter experiments are relevant to the current exploration agenda at NASA since it is necessary to understand how living systems respond to the fractional gravities found on the Moon and Mars. The TROPI experiments will be performed in the European Modular Cultivation System (EMCS), which is a biological research facility that was designed by the European Space Agency (ESA). The EMCS has an incubator, atmospheric control, two centrifuge rotors, and a high-resolution video camera (Brinckmann, 1999; Brinckmann and Schiller, 2002). This system is fairly sophisticated and automated (requiring little crew time). Working with NASA and ESA, we have developed EUE (experimental unique equipment), which was described in our previous publication (Correll et al., 2005), for TROPI. Briefly, the EUE consists of five seedling cassettes with LED lighting and a water delivery system in an Experimental Container (EC). In this paper, we describe several additional biocompatibility experiments which led us to modify the TROPI EUE to include activated carbon filters to remove harmful gases, and this resulted in seedlings with vigorous growth and robust tropistic responses. 2. Materials and methods 2.1. Spaceflight hardware The TROPI experiment will be performed in the EMCS, and this spaceflight research facility is described in papers by Brinckmann (1999) and Brinckmann and Schiller
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(2002). The TROPI hardware consists of five cassettes to support growth of Arabidopsis seedlings and associated components (Fig. 1). Our recent publication provides information on the TROPI system (Correll et al., 2005), but some additional details and modifications to the system are provided in the next paragraph and in Section 3. The TROPI hardware includes several printed circuit boards related to the power supplies of the LEDs and other components. These circuit boards received a conformal coating (part no. 1-2577; Dow Corning Corp., Midland, MI, USA) with a thickness of 0.004–0.005 in. (=100– 130 lm). This procedure was done to comply with NASA safety requirements and to protect the electrical components from moisture, corrosion, abrasion, and other environmental stresses. As discussed below, filters made of activated carbon cloth (part no. FM5K/250; Calgon Carbon Corp., Pittsburgh, PA, USA) were placed on the seedling cassette (over a gas-permeable membrane), at the base of the EC, or in both locations in order to absorb materials that may have been produced as a result of offgassing. One carbon filter was placed at the cassette level, and four filters were placed at the EC base. 2.2. Plant material Seedlings of A. thaliana of the Landsberg (Ler) WT ecotype were used in these studies along with several phytochrome mutants. These mutant strains were phyA-201, phyB-1, and phyA201ÆphyB-1 (double mutant) and were described by Hennig et al. (2002). 2.3. Culture conditions Two general types of culture conditions were used in our experiments: seedlings were grown on nutrient agar plates or were cultured in spaceflight hardware. In the case of the agar plates, seeds were surface sterilized in 70% (v/v) ethanol containing 0.002% (v/v) Triton X-100 for 5 min and two times in 95% (v/v) ethanol for 1 min each (Correll et al., 2005). After several rinses in sterile distilled water, seeds were sown on to a pre-sterilized cellulose film that was placed on top of 1.2% (w/v) agar with one-half-strength Murashige and Skoog salts with 1% (w/v) sucrose and 1 mM MES (pH 5.5) in square (100 · 15 mm) Petri dishes. The dishes were sealed with Parafilm and placed on edge so that the surface of the agar was vertical. Seedlings were grown for 4 days in white light (70 lmol m 2 s 1) obtained from 34 W cool-white fluorescent lamps. In terms of the spaceflight hardware, seeds were surface sterilized in 70% (v/v) ethanol for 5 min followed by several rinses in 95% (v/v) ethanol, and then allowed to air dry in a sterile laminar flow hood. The growth medium described above was used to soak Whatman # 17 filter paper and allowed to dry in a laminar flow hood. Seeds were affixed with 1% (w/v) gum guar (Katembe et al., 1998; Kiss et al., 1998) to a black gridded membrane which is made of mixed cellulose esters (no. 66585; Pall Corp., Ann
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Fig. 1. TROPI hardware (also termed EUE for experimental unique equipment) showing the individual seedling cassette (a) and the experimental container (EC) for the EMCS. (a) Arabidopsis seedlings are shown growing in the cassette after a blue stimulation has been provided from the left of the figure. Distance between lines on gridded membrane is 3 mm. (b) Five cassettes (*) for seedling growth are present in each EC. The arrow indicates the bank of red and blue LEDs that are used for the phototropic stimulation phase of the experiment, and there are white LEDs (perpendicular to the stimulation LEDs) for oriented growth of seedlings. Dimensions of the EC are 186 mm (length) · 100 mm (width) · 90 mm (height).
Arbor, MI, USA). At this point, the black membrane with the seeds was affixed to the top of one layer of filter paper using 1% (w/v) gum guar and placed into the TROPI cassette. The assembled cassettes were placed into ECs, and the automated 6-day time line sequence was run in the EMCS Experimental Reference Model (ERM), which also has been termed the Science Reference Model (Brinckmann, 1999). This 6-day sequence consists of hydration of seeds, growth of seedlings, and phototropism experiments, and the sequence replicates the proposed spaceflight experiments. The time line as well as additional details about the TROPI hardware and materials used is outlined in our previous paper (Correll et al., 2005). 3. Results and discussion 3.1. The TROPI experimental unique equipment (EUE) The EMCS is an ideal facility for our experiments to study the interactions of gravity and phototropism in plant
seedlings, and it was designed specifically for A. thaliana to be used as one of the model organisms (Brinckmann and Schiller, 2002). The operating ‘‘philosophy’’ of EMCS is similar to that of the successful Biorack module, which was also designed by ESA (Katembe et al., 1998). Specifically, the EMCS provides for standard Experimental Containers (ECs), and the individual experiments will have specific hardware, termed experimental unique equipment (EUE), designed for the specific needs of the experiment (Fig. 1). Thus, we have worked closely with NASA and ESA to design the optimal EUE for TROPI. The TROPI EUE consists of five cassettes to support growth of Arabidopsis seedlings (Fig. 1a), and these five cassettes are housed in one EMCS EC (Fig. 1b). We have published a paper on the details of the seedling cassette and the additional components of the hardware (Correll et al., 2005). Briefly, each cassette has two sets of LEDs: one set of white LEDs along the longer side of the cassette and a set of red and blue LEDs on one end of the shorter side of the cassette. The white LEDs provide illumination
J.Z. Kiss et al. / Advances in Space Research 39 (2007) 1154–1160
for oriented growth of seedlings, and the blue/red LEDs are used to study the effects of phototropic stimulation. An important feature of the transparent plastic cover on the cassette is an anti-fogging heater membrane that is used to make sure condensation does not interfere with video observations. Seedlings in the cassette exhibit healthy growth (Fig. 1a) and the hypocotyls exhibit a vigorous positive phototropism in response to unilateral blue illumination provided by the LEDs at the end of the cassette. The final configuration of the TROPI hardware has five seedling cassettes per EC (Fig. 1b). Other major parts of the EUE in the EC include a water delivery system, circulation fans, control circuitry for the anti-fog heater, circuit boards for the LEDs, and other components. 3.2. Conformal coating of circuit boards had a negative impact on seed germination and seedling development Once the final design of the TROPI EUE was tested and approved, the spaceflight-approved hardware circuit boards had to be conformal coated to meet NASA safety requirements. At this point, we performed long-term storage and biocompatibility tests to determine the viability of seeds and growth of seedlings after periods of seed storage of up to 6 months in the final configuration of the TROPI hardware. The 6-month period was used as a stan-
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dard since seeds in TROPI EUE may have to be stored up to this period before the experiment is initiated on the ISS increment. Seeds that were placed in the hardware (and immediately hydrated) germinated and had vigorous growth with a robust tropistic curvature response (Fig. 2a). However, we found poor seed germination and stunted growth of the few seedlings that did germinate after a 6-month period of storage in the TROPI EUE (Fig. 2b). It should be noted that the cover of the EC was sealed so that there was little or no gas exchange with the environment outside of the EC during the storage experiment. A decrease in seed germination and seedling growth after 2 months of storage in the EUE was also observed in a separate test (data not shown). Additional studies were performed to quantify the extent of the effects of storage in TROPI hardware on seed germination on all four seed strains that we plan to use in our experiments on the ISS (Fig. 3). These studies considered three treatments: no storage of seeds with a germination test on agar plates, no storage of seeds with a germination test in the TROPI EUE, and 6-month storage of seeds with a germination test in the TROPI EUE. First, there was no significant difference between the agar plates and EUE in the two treatments with no storage of seeds (Fig. 3). This result indicates that there was no detrimental effect on seed germination when the seeds were placed in
Fig. 2. Seedlings (5 days old) after immediate hydration of seeds (a) and after seeds were stored for 6 months in TROPI EUE (without carbon filters; b). (a) Seeds that were not stored in hardware resulted in seedlings with robust growth and phototropic curvature. Blue light stimulation was provided from the left of the figure. (b) Seeds that were stored for 6 months in hardware (and then hydrated) resulted in poor seed germination and stunted growth of seedlings. Distance between lines on gridded membrane is 3 mm.
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Percent germination
100
WT phyA phyB phyAB
80 60 40 20 0
No Storage-Agar
No Storage-EUE
6 Month StorageEUE
Fig. 3. The effects of different storage systems on seed germination. All four strains of Arabidopsis studied showed close to 100% germination when cultured in Petri dishes with nutrient agar, and the per cent germination also was high for all four strains when seeds were cultured in the TROPI EUE without a storage period. However, there was a large drop in germination when seeds were stored in hardware for 6 months. Sample size was from 168 to 400 seeds.
the hardware with a germination test that was performed immediately. However, there was a major deleterious effect on seed germination when the seeds were stored in the EUE hardware for 6 months prior to hydration for the test. Seed germination decreased from essentially 100% in the control to 8%, 44%, 31%, and 17% after 6 months of storage in the EUE for the Ler WT, phyA, phyB, and phyAB, respectively. Unexpectedly, while the extended storage had negative consequences for seed germination in all strains, the Ler WT had the greatest reduction in germination compared to the other strains. 3.3. Addition of carbon filters improved biocompatibility of the TROPI EUE In order to mitigate the negative effects of seed storage for several months in the TROPI EUE, we placed carbon filter membranes in two places in the EUE (Fig. 4). Carbon filters were added to either the seedling cassette (Fig. 4a) or at the base of the entire EC (Fig. 4b). In some cases, carbon filters were placed both at the cassette level and at the base of the EC.
Fig. 4. Modifications to the TROPI EUE with the addition of carbon filters to seedling cassettes (a) and the EC base (b). Carbon filters (CF) were placed in either the seedling cassette (Cass) and/or in two locations at the base of the entire EC. A nylon mesh (NM) was used to hold the carbon filters (CF) in place. I/O represents the data and electrical input/output interface for the EC with the EMCS unit, and RM is the retaining mask at the seedling cassette level. Dimensions of the EC base (b) are 186 mm · 100 mm.
J.Z. Kiss et al. / Advances in Space Research 39 (2007) 1154–1160
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Fig. 5. The effects of seed storage on growth of seedlings (after 5 days) in TROPI EUE for 2 months with and without added carbon filters. (a) In the EUE with no filters, seed germination was reduced, and the growth of seedlings was reduced. (b) Germination and growth improved when carbon filters were placed at the base of the EC. (c) The best results for seed germination and seedling growth were obtained when carbon filters were placed in both the EC base and at the cassette level. Distance between lines on gridded membrane is 3 mm.
Seed germination and seedling growth were assayed after a 2-month period in which seeds were stored in TROPI EUE (Fig. 5). Unfortunately, in these studies, we were limited to this 2-month period (rather than a more extended 6-month period) due to time constraints for final hardware assembly for spaceflight. Four experimental treatments were considered: a control with no carbon filter, carbon filters at cassette only, carbon filters at the base of the EC only, and carbon filters both at the EC base and at the cassette level. In the control (no filters), seedlings grown in hardware after 2 months of seed storage exhibited reduced germination and growth (Fig. 5a). Addition of carbon filters to the base of the EC improved seed germination and seedling growth considerably (Fig. 5b). The best results in terms of both germination and growth occurred when
carbon filters were added to both the EC base and to the seedling cassette (Fig. 5c). The above qualitative results were corroborated in quantitative studies of seed germination (Fig. 6). These experiments confirmed that while adding carbon filters to either the cassette or the EC base alone improved seed germination, the best results were obtained when filters were added to both the cassette and the EC base. Thus, in the final design of the TROPI EUE for spaceflight, we have incorporated addition of carbon filters to both the seedling cassettes and at the base of the entire EC. The conformal coating of circuit boards is only one potential source of chemical off-gassing that leads to poor seed germination and seedling growth. Other sources of off-gassing in the TROPI EUE include the plastics from
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ware biocompatibility including types of materials used in the EUE, optimal LED illumination, temperature, humidity, and other parameters. Since opportunities for spaceflight experiments are infrequent, it is important that every possible factor that can be tested is carefully examined prior to flight (Correll and Kiss, 2007).
Per cent germination
80
60
40
References 20
0
No filter
Filter-base
Filtercassette
Filter-base + cassette
Fig. 6. The effects of 2-month storage in TROPI EUE (with or without carbon filters) on seedling germination. The lowest per cent germination of seeds was obtained in the control with no filters. Seed germination improved when carbon filters were placed at either the cassette or the EC base, but the highest seed germination was obtained when carbon filters were placed in both the EC base and at the cassette level. Sample size was from 68 to 133 seeds.
fans and cassette covers or the elastomers and adhesives used to secure hardware components. Regardless of the source of off-gassing, the addition of carbon filters allows for absorption of potentially harmful gases, and this modification to our hardware resulted in improved seed germination and growth of seedlings. Also, storing newly constructed hardware in ventilated areas can also improve off-gassing of harmful components of the EUE. Unfortunately, due to time constraints related to the preparation of the hardware for spaceflight, we were unable to determine the relative contribution of storing the new hardware in ventilated locations and the addition of carbon filters to the EUE toward reducing the presence of harmful gases. Nevertheless, in this study and others (Correll et al., 2005), we have considered many aspects of TROPI hard-
Brinckmann, E. Spaceflight opportunities on the ISS for plant research – the ESA perspective. Adv. Space Res. 24, 779–788, 1999. Brinckmann, E., Schiller, P. Experiments with small animals in BIOLAB and EMCS on the International Space Station. Adv. Space Res. 30, 809–814, 2002. Correll, M.J., Edelmann, R.E., Hangarter, R.P., et al. Ground-based studies of tropisms in hardware developed for the European Modular Cultivation System (EMCS). Adv. Space Res. 36, 1203–1210, 2005. Correll, M.J., Kiss, J.Z. Interactions between gravitropism and phototropism in plants. J. Plant Growth Regul. 21, 89–101, 2002. Correll, M.J., Kiss, J.Z. Space-based research on plant tropisms, in: Gilroy, S., Masson, P.H. (Eds.), Plant Tropisms. Blackwell Publishing, Oxford, in press. Hangarter, R.P. Gravity, light and plant form. Plant Cell Environ. 20, 796–800, 1997. Hennig, L., Stoddart, W.M., Dieterle, M., et al. Phytochrome E controls light-induced germination of Arabidopsis. Plant Physiol. 128, 194–200, 2002. Katembe, W.J., Edelmann, R.E., Brinckmann, E., et al. The development of spaceflight experiments with Arabidopsis as a model system in gravitropism studies. J. Plant Res. 111, 463–470, 1998. Kiss, J.Z., Katembe, W.J., Edelmann, R.E. Gravitropism and development of wild-type and starch-deficient mutants of Arabidopsis during spaceflight. Physiol. Plant 102, 493–502, 1998. Kiss, J.Z., Mullen, J.L., Correll, M.J., et al. Phytochromes A and B mediate red-light-induced positive phototropism in roots. Plant Physiol. 131, 1411–1417, 2003. Quail, P.H. Phytochrome photosensory signalling networks. Nat. Rev. Mol. Cell Biol. 3, 85–93, 2002. Sakai, T., Wada, T., Ishiguro, S., et al. RPT2: A signal transducer of the phototropic response in Arabidopsis. Plant Cell 12, 225–236, 2000. Vitha, S., Zhao, L., Sack, F.D. Interaction of root gravitropism and phototropism in Arabidopsis wild-type and starchless mutants. Plant Physiol. 122, 453–462, 2000. Whippo, C.W., Hangarter, R.P. Phototropism: bending towards enlightenment. Plant Cell 18, 1110–1119, 2006.
Biocompatibility studies in preparation for a spaceflight experiment on plant tropisms (TROPI) John Z. Kiss a,*, Prem Kumar a, Robert N. Bowman b, Marianne K. Steele b, Michael T. Eodice b, Melanie J. Correll c, Richard E. Edelmann a a
c
Department of Botany, Miami University, Oxford, OH 45056, USA b NASA Ames Research Center, Moffett Field, CA 94035, USA Department of Agricultural and Biological Engineering, University of Florida, Gainesville, FL 32611, USA Received 31 August 2006; received in revised form 8 December 2006; accepted 12 December 2006
Abstract The interaction among tropisms is important in determining the growth form of a plant. Thus, we have developed a project to study the interaction between two key tropistic responses (i.e., gravitropism and phototropism) to be performed in microgravity on the International Space Station (ISS). Specifically, we are interested in the role of red-light-absorbing phytochrome pigments in modulating tropisms in seedlings of Arabidopsis thaliana. This project, termed TROPI for tropisms, is to be performed on the European Modular Cultivation System (EMCS), which provides an incubator with atmospheric control, lighting, and high-resolution video. The EMCS has two rotating centrifuge platforms so that our experiments can be performed at microgravity, 1g (control), and fractional g-levels. In order to optimize these spaceflight experiments, we have continued ground-based technical tests as well as basic science experiments. Since the seeds will have to be stored for several months in hardware prior to use on the ISS, we tested the effects of long-term storage of seeds in the TROPI EUE (experimental unique equipment) on germination rates and plant growth. The EUE consists of five seedling cassettes with LED lighting and a water delivery system in an Experimental Container (EC). Preliminary studies showed that there were reduced seed germination and plant growth after several months of storage in the EUE. We determined that the likely source of this biocompatibility problem was the conformal coating of electrical components of the EUE, which was required by NASA for safety reasons. In order to alleviate this problem, carbon filters were added to both the seedling cassettes and to the base of the EC. We expect that these improvements to the hardware will result in healthy plants capable of robust tropistic responses in our spaceflight experiments. 2006 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: EMCS; Gravitropism; ISS; Plant space biology; Phototropism; Spaceflight hardware
1. Introduction On Earth it is very difficult to study the interacting effects of gravity and other stimuli such as light on plant growth. However, a project was launched on the space shuttle (missions STS-121 in July 2006 and STS-115 in September 2006) to examine these interactions in seedlings of Arabidopsis thaliana on the International Space Station (ISS). A major goal of these experiments to be performed *
Corresponding author. Tel.: +1 513 529 5428; fax: +1 513 529 4243. E-mail address: [email protected] (J.Z. Kiss).
on the ISS is to gain insights into the cellular and molecular mechanisms of phototropism, the directed growth of plants in response to light. Phototropism in stems has been more extensively studied compared to root phototropism, which when observed in most species is typically away from the light stimulus (i.e., negative phototropism; Whippo and Hangarter, 2006). However, since the phototropic response in roots is weak relative to gravitropism, it remained largely unstudied for decades (Sakai et al., 2000). Several reports have demonstrated that, in stems, interactions between positive phototropism and negative
0273-1177/$30 2006 COSPAR. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.asr.2006.12.017
J.Z. Kiss et al. / Advances in Space Research 39 (2007) 1154–1160
gravitropism determine the direction of growth in young seedlings (e.g., Hangarter, 1997). Similarly, in roots of some species (e.g., A. thaliana), it appears that there is also a comparable interaction between tropisms in orienting root growth (Vitha et al., 2000), but, in the case of roots, the reported interaction was between negative phototropism and positive gravitropism. However, we have recently discovered a positive red-light-induced phototropism in roots that is mediated by photoreceptors in the phytochrome family (Kiss et al., 2003). In most plants, the phytochrome gene family has five members (PHYA–PHYE; Quail, 2002); so in our space experiments, we will investigate phototropism in both the normal WT Arabidopsis seedlings as well as in a series of phytochrome mutants (phyA, phyB, and phyAB). While gravitropism can readily be studied on earth in the absence of light, it is difficult to investigate phototropism without the effects of gravity (Correll and Kiss, 2002). Thus, the microgravity conditions of space can be used to study phototropism without the ‘‘complications’’ of unidirectional gravity. Our spaceflight experiment, termed TROPI for tropisms, will use microgravity as a unique research tool to study an important issue in plant biology. In addition to microgravity treatments, TROPI will also have a 1-g in-flight control from an onboard centrifuge as well as fractional gravity treatments ranging from 0.1g to 0.9g (Brinckmann, 1999). These latter experiments are relevant to the current exploration agenda at NASA since it is necessary to understand how living systems respond to the fractional gravities found on the Moon and Mars. The TROPI experiments will be performed in the European Modular Cultivation System (EMCS), which is a biological research facility that was designed by the European Space Agency (ESA). The EMCS has an incubator, atmospheric control, two centrifuge rotors, and a high-resolution video camera (Brinckmann, 1999; Brinckmann and Schiller, 2002). This system is fairly sophisticated and automated (requiring little crew time). Working with NASA and ESA, we have developed EUE (experimental unique equipment), which was described in our previous publication (Correll et al., 2005), for TROPI. Briefly, the EUE consists of five seedling cassettes with LED lighting and a water delivery system in an Experimental Container (EC). In this paper, we describe several additional biocompatibility experiments which led us to modify the TROPI EUE to include activated carbon filters to remove harmful gases, and this resulted in seedlings with vigorous growth and robust tropistic responses. 2. Materials and methods 2.1. Spaceflight hardware The TROPI experiment will be performed in the EMCS, and this spaceflight research facility is described in papers by Brinckmann (1999) and Brinckmann and Schiller
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(2002). The TROPI hardware consists of five cassettes to support growth of Arabidopsis seedlings and associated components (Fig. 1). Our recent publication provides information on the TROPI system (Correll et al., 2005), but some additional details and modifications to the system are provided in the next paragraph and in Section 3. The TROPI hardware includes several printed circuit boards related to the power supplies of the LEDs and other components. These circuit boards received a conformal coating (part no. 1-2577; Dow Corning Corp., Midland, MI, USA) with a thickness of 0.004–0.005 in. (=100– 130 lm). This procedure was done to comply with NASA safety requirements and to protect the electrical components from moisture, corrosion, abrasion, and other environmental stresses. As discussed below, filters made of activated carbon cloth (part no. FM5K/250; Calgon Carbon Corp., Pittsburgh, PA, USA) were placed on the seedling cassette (over a gas-permeable membrane), at the base of the EC, or in both locations in order to absorb materials that may have been produced as a result of offgassing. One carbon filter was placed at the cassette level, and four filters were placed at the EC base. 2.2. Plant material Seedlings of A. thaliana of the Landsberg (Ler) WT ecotype were used in these studies along with several phytochrome mutants. These mutant strains were phyA-201, phyB-1, and phyA201ÆphyB-1 (double mutant) and were described by Hennig et al. (2002). 2.3. Culture conditions Two general types of culture conditions were used in our experiments: seedlings were grown on nutrient agar plates or were cultured in spaceflight hardware. In the case of the agar plates, seeds were surface sterilized in 70% (v/v) ethanol containing 0.002% (v/v) Triton X-100 for 5 min and two times in 95% (v/v) ethanol for 1 min each (Correll et al., 2005). After several rinses in sterile distilled water, seeds were sown on to a pre-sterilized cellulose film that was placed on top of 1.2% (w/v) agar with one-half-strength Murashige and Skoog salts with 1% (w/v) sucrose and 1 mM MES (pH 5.5) in square (100 · 15 mm) Petri dishes. The dishes were sealed with Parafilm and placed on edge so that the surface of the agar was vertical. Seedlings were grown for 4 days in white light (70 lmol m 2 s 1) obtained from 34 W cool-white fluorescent lamps. In terms of the spaceflight hardware, seeds were surface sterilized in 70% (v/v) ethanol for 5 min followed by several rinses in 95% (v/v) ethanol, and then allowed to air dry in a sterile laminar flow hood. The growth medium described above was used to soak Whatman # 17 filter paper and allowed to dry in a laminar flow hood. Seeds were affixed with 1% (w/v) gum guar (Katembe et al., 1998; Kiss et al., 1998) to a black gridded membrane which is made of mixed cellulose esters (no. 66585; Pall Corp., Ann
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Fig. 1. TROPI hardware (also termed EUE for experimental unique equipment) showing the individual seedling cassette (a) and the experimental container (EC) for the EMCS. (a) Arabidopsis seedlings are shown growing in the cassette after a blue stimulation has been provided from the left of the figure. Distance between lines on gridded membrane is 3 mm. (b) Five cassettes (*) for seedling growth are present in each EC. The arrow indicates the bank of red and blue LEDs that are used for the phototropic stimulation phase of the experiment, and there are white LEDs (perpendicular to the stimulation LEDs) for oriented growth of seedlings. Dimensions of the EC are 186 mm (length) · 100 mm (width) · 90 mm (height).
Arbor, MI, USA). At this point, the black membrane with the seeds was affixed to the top of one layer of filter paper using 1% (w/v) gum guar and placed into the TROPI cassette. The assembled cassettes were placed into ECs, and the automated 6-day time line sequence was run in the EMCS Experimental Reference Model (ERM), which also has been termed the Science Reference Model (Brinckmann, 1999). This 6-day sequence consists of hydration of seeds, growth of seedlings, and phototropism experiments, and the sequence replicates the proposed spaceflight experiments. The time line as well as additional details about the TROPI hardware and materials used is outlined in our previous paper (Correll et al., 2005). 3. Results and discussion 3.1. The TROPI experimental unique equipment (EUE) The EMCS is an ideal facility for our experiments to study the interactions of gravity and phototropism in plant
seedlings, and it was designed specifically for A. thaliana to be used as one of the model organisms (Brinckmann and Schiller, 2002). The operating ‘‘philosophy’’ of EMCS is similar to that of the successful Biorack module, which was also designed by ESA (Katembe et al., 1998). Specifically, the EMCS provides for standard Experimental Containers (ECs), and the individual experiments will have specific hardware, termed experimental unique equipment (EUE), designed for the specific needs of the experiment (Fig. 1). Thus, we have worked closely with NASA and ESA to design the optimal EUE for TROPI. The TROPI EUE consists of five cassettes to support growth of Arabidopsis seedlings (Fig. 1a), and these five cassettes are housed in one EMCS EC (Fig. 1b). We have published a paper on the details of the seedling cassette and the additional components of the hardware (Correll et al., 2005). Briefly, each cassette has two sets of LEDs: one set of white LEDs along the longer side of the cassette and a set of red and blue LEDs on one end of the shorter side of the cassette. The white LEDs provide illumination
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for oriented growth of seedlings, and the blue/red LEDs are used to study the effects of phototropic stimulation. An important feature of the transparent plastic cover on the cassette is an anti-fogging heater membrane that is used to make sure condensation does not interfere with video observations. Seedlings in the cassette exhibit healthy growth (Fig. 1a) and the hypocotyls exhibit a vigorous positive phototropism in response to unilateral blue illumination provided by the LEDs at the end of the cassette. The final configuration of the TROPI hardware has five seedling cassettes per EC (Fig. 1b). Other major parts of the EUE in the EC include a water delivery system, circulation fans, control circuitry for the anti-fog heater, circuit boards for the LEDs, and other components. 3.2. Conformal coating of circuit boards had a negative impact on seed germination and seedling development Once the final design of the TROPI EUE was tested and approved, the spaceflight-approved hardware circuit boards had to be conformal coated to meet NASA safety requirements. At this point, we performed long-term storage and biocompatibility tests to determine the viability of seeds and growth of seedlings after periods of seed storage of up to 6 months in the final configuration of the TROPI hardware. The 6-month period was used as a stan-
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dard since seeds in TROPI EUE may have to be stored up to this period before the experiment is initiated on the ISS increment. Seeds that were placed in the hardware (and immediately hydrated) germinated and had vigorous growth with a robust tropistic curvature response (Fig. 2a). However, we found poor seed germination and stunted growth of the few seedlings that did germinate after a 6-month period of storage in the TROPI EUE (Fig. 2b). It should be noted that the cover of the EC was sealed so that there was little or no gas exchange with the environment outside of the EC during the storage experiment. A decrease in seed germination and seedling growth after 2 months of storage in the EUE was also observed in a separate test (data not shown). Additional studies were performed to quantify the extent of the effects of storage in TROPI hardware on seed germination on all four seed strains that we plan to use in our experiments on the ISS (Fig. 3). These studies considered three treatments: no storage of seeds with a germination test on agar plates, no storage of seeds with a germination test in the TROPI EUE, and 6-month storage of seeds with a germination test in the TROPI EUE. First, there was no significant difference between the agar plates and EUE in the two treatments with no storage of seeds (Fig. 3). This result indicates that there was no detrimental effect on seed germination when the seeds were placed in
Fig. 2. Seedlings (5 days old) after immediate hydration of seeds (a) and after seeds were stored for 6 months in TROPI EUE (without carbon filters; b). (a) Seeds that were not stored in hardware resulted in seedlings with robust growth and phototropic curvature. Blue light stimulation was provided from the left of the figure. (b) Seeds that were stored for 6 months in hardware (and then hydrated) resulted in poor seed germination and stunted growth of seedlings. Distance between lines on gridded membrane is 3 mm.
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Fig. 3. The effects of different storage systems on seed germination. All four strains of Arabidopsis studied showed close to 100% germination when cultured in Petri dishes with nutrient agar, and the per cent germination also was high for all four strains when seeds were cultured in the TROPI EUE without a storage period. However, there was a large drop in germination when seeds were stored in hardware for 6 months. Sample size was from 168 to 400 seeds.
the hardware with a germination test that was performed immediately. However, there was a major deleterious effect on seed germination when the seeds were stored in the EUE hardware for 6 months prior to hydration for the test. Seed germination decreased from essentially 100% in the control to 8%, 44%, 31%, and 17% after 6 months of storage in the EUE for the Ler WT, phyA, phyB, and phyAB, respectively. Unexpectedly, while the extended storage had negative consequences for seed germination in all strains, the Ler WT had the greatest reduction in germination compared to the other strains. 3.3. Addition of carbon filters improved biocompatibility of the TROPI EUE In order to mitigate the negative effects of seed storage for several months in the TROPI EUE, we placed carbon filter membranes in two places in the EUE (Fig. 4). Carbon filters were added to either the seedling cassette (Fig. 4a) or at the base of the entire EC (Fig. 4b). In some cases, carbon filters were placed both at the cassette level and at the base of the EC.
Fig. 4. Modifications to the TROPI EUE with the addition of carbon filters to seedling cassettes (a) and the EC base (b). Carbon filters (CF) were placed in either the seedling cassette (Cass) and/or in two locations at the base of the entire EC. A nylon mesh (NM) was used to hold the carbon filters (CF) in place. I/O represents the data and electrical input/output interface for the EC with the EMCS unit, and RM is the retaining mask at the seedling cassette level. Dimensions of the EC base (b) are 186 mm · 100 mm.
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Fig. 5. The effects of seed storage on growth of seedlings (after 5 days) in TROPI EUE for 2 months with and without added carbon filters. (a) In the EUE with no filters, seed germination was reduced, and the growth of seedlings was reduced. (b) Germination and growth improved when carbon filters were placed at the base of the EC. (c) The best results for seed germination and seedling growth were obtained when carbon filters were placed in both the EC base and at the cassette level. Distance between lines on gridded membrane is 3 mm.
Seed germination and seedling growth were assayed after a 2-month period in which seeds were stored in TROPI EUE (Fig. 5). Unfortunately, in these studies, we were limited to this 2-month period (rather than a more extended 6-month period) due to time constraints for final hardware assembly for spaceflight. Four experimental treatments were considered: a control with no carbon filter, carbon filters at cassette only, carbon filters at the base of the EC only, and carbon filters both at the EC base and at the cassette level. In the control (no filters), seedlings grown in hardware after 2 months of seed storage exhibited reduced germination and growth (Fig. 5a). Addition of carbon filters to the base of the EC improved seed germination and seedling growth considerably (Fig. 5b). The best results in terms of both germination and growth occurred when
carbon filters were added to both the EC base and to the seedling cassette (Fig. 5c). The above qualitative results were corroborated in quantitative studies of seed germination (Fig. 6). These experiments confirmed that while adding carbon filters to either the cassette or the EC base alone improved seed germination, the best results were obtained when filters were added to both the cassette and the EC base. Thus, in the final design of the TROPI EUE for spaceflight, we have incorporated addition of carbon filters to both the seedling cassettes and at the base of the entire EC. The conformal coating of circuit boards is only one potential source of chemical off-gassing that leads to poor seed germination and seedling growth. Other sources of off-gassing in the TROPI EUE include the plastics from
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ware biocompatibility including types of materials used in the EUE, optimal LED illumination, temperature, humidity, and other parameters. Since opportunities for spaceflight experiments are infrequent, it is important that every possible factor that can be tested is carefully examined prior to flight (Correll and Kiss, 2007).
Per cent germination
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Fig. 6. The effects of 2-month storage in TROPI EUE (with or without carbon filters) on seedling germination. The lowest per cent germination of seeds was obtained in the control with no filters. Seed germination improved when carbon filters were placed at either the cassette or the EC base, but the highest seed germination was obtained when carbon filters were placed in both the EC base and at the cassette level. Sample size was from 68 to 133 seeds.
fans and cassette covers or the elastomers and adhesives used to secure hardware components. Regardless of the source of off-gassing, the addition of carbon filters allows for absorption of potentially harmful gases, and this modification to our hardware resulted in improved seed germination and growth of seedlings. Also, storing newly constructed hardware in ventilated areas can also improve off-gassing of harmful components of the EUE. Unfortunately, due to time constraints related to the preparation of the hardware for spaceflight, we were unable to determine the relative contribution of storing the new hardware in ventilated locations and the addition of carbon filters to the EUE toward reducing the presence of harmful gases. Nevertheless, in this study and others (Correll et al., 2005), we have considered many aspects of TROPI hard-
Brinckmann, E. Spaceflight opportunities on the ISS for plant research – the ESA perspective. Adv. Space Res. 24, 779–788, 1999. Brinckmann, E., Schiller, P. Experiments with small animals in BIOLAB and EMCS on the International Space Station. Adv. Space Res. 30, 809–814, 2002. Correll, M.J., Edelmann, R.E., Hangarter, R.P., et al. Ground-based studies of tropisms in hardware developed for the European Modular Cultivation System (EMCS). Adv. Space Res. 36, 1203–1210, 2005. Correll, M.J., Kiss, J.Z. Interactions between gravitropism and phototropism in plants. J. Plant Growth Regul. 21, 89–101, 2002. Correll, M.J., Kiss, J.Z. Space-based research on plant tropisms, in: Gilroy, S., Masson, P.H. (Eds.), Plant Tropisms. Blackwell Publishing, Oxford, in press. Hangarter, R.P. Gravity, light and plant form. Plant Cell Environ. 20, 796–800, 1997. Hennig, L., Stoddart, W.M., Dieterle, M., et al. Phytochrome E controls light-induced germination of Arabidopsis. Plant Physiol. 128, 194–200, 2002. Katembe, W.J., Edelmann, R.E., Brinckmann, E., et al. The development of spaceflight experiments with Arabidopsis as a model system in gravitropism studies. J. Plant Res. 111, 463–470, 1998. Kiss, J.Z., Katembe, W.J., Edelmann, R.E. Gravitropism and development of wild-type and starch-deficient mutants of Arabidopsis during spaceflight. Physiol. Plant 102, 493–502, 1998. Kiss, J.Z., Mullen, J.L., Correll, M.J., et al. Phytochromes A and B mediate red-light-induced positive phototropism in roots. Plant Physiol. 131, 1411–1417, 2003. Quail, P.H. Phytochrome photosensory signalling networks. Nat. Rev. Mol. Cell Biol. 3, 85–93, 2002. Sakai, T., Wada, T., Ishiguro, S., et al. RPT2: A signal transducer of the phototropic response in Arabidopsis. Plant Cell 12, 225–236, 2000. Vitha, S., Zhao, L., Sack, F.D. Interaction of root gravitropism and phototropism in Arabidopsis wild-type and starchless mutants. Plant Physiol. 122, 453–462, 2000. Whippo, C.W., Hangarter, R.P. Phototropism: bending towards enlightenment. Plant Cell 18, 1110–1119, 2006.