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Ground-based studies of tropisms in hardware developed for the European Modular Cultivation System (EMCS)
Advances in Space Research 36 (2005) 1203–1210 www.elsevier.com/locate/asr
Ground-based studies of tropisms in hardware developed for the European Modular Cultivation System (EMCS) Melanie J. Correll a, Richard E. Edelmann a, Roger P. Hangarter b, Jack L. Mullen b, John Z. Kiss a,* a
Department of Botany, Miami University, Pearson Hall, Oxford, OH 45056, USA b Department of Biology, Indiana University, Bloomington, IN 47405, USA
Received 2 July 2004; received in revised form 7 October 2004; accepted 2 November 2004
Abstract Phototropism and gravitropism play key roles in the oriented growth of roots in flowering plants. In blue or white light, roots exhibit negative phototropism, but red light induces positive phototropism in Arabidopsis roots. The blue-light response is controlled by the phototropins while the red-light response is mediated by the phytochrome family of photoreceptors. In order to better characterize root phototropism, we plan to perform experiments in microgravity so that this tropism can be more effectively studied without the interactions with the gravity response. Our experiments are to be performed on the European Modular Cultivation System (EMCS), which provides an incubator, lighting system, and high resolution video that are on a centrifuge palette. These experiments will be performed at lg, 1g (control) and fractional g-levels. In order to ensure success of this mission on the International Space Station, we have been conducting ground-based studies on growth, phototropism, and gravitropism in experimental unique equipment (EUE) that was designed for our experiments with Arabidopsis seedlings. Currently, the EMCS and our EUE are scheduled for launch on space shuttle mission STS-121. This project should provide insight into how the blue- and red-light signaling systems interact with each other and with the gravisensing system. 2004 Published by Elsevier Ltd on behalf of COSPAR. Keywords: Arabidopsis; Gravitropism; Phototropism; Phytochrome; Spaceflight hardware
1. Introduction Plants have numerous selective and sensitive mechanisms to perceive and respond to various aspects of their environment. Of these environmental signals, gravity and light play key roles in plant development, but other factors such as temperature and moisture are also important. Plants continuously integrate all of the information received by their sensory systems to adjust their growth to their present environmental conditions (Hangarter, 1997; Correll and Kiss, 2002). *
Corresponding author. Tel.: +1 513 529 5428; fax: +1 513 529 4243. E-mail address: [email protected] (J.Z. Kiss). 0273-1177/$30 2004 Published by Elsevier Ltd on behalf of COSPAR. doi:10.1016/j.asr.2004.11.003
On earth, gravity is a ubiquitous and constant signal. In germinating seedlings, gravity is important for orienting the plant so that shoots grow upwards and the roots grow downwards. The primary shoot and root of young seedlings have been the main subject of studies of gravitropism (directed growth in response to gravity). However, in mature plants, lateral organs typically display a characteristic angle of orientation relative to the gravity vector (Digby and Firn, 1995), and this angle can be modified by light conditions (Hangarter, 1997). Gravity and light often interact in influencing the developmental pattern of plants. In particular, several studies have shown that light can modulate gravitropic behavior through the photosensitive pigment phytochrome (reviewed in Hangarter, 1997; Correll and Kiss,
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2002). Light-dependent modulation of gravitropism may affect the orientation of plant organs, and, thus, the final shape of a plant can be determined by interaction of gravitropism and phototropism (directed growth in response to light) in either a synergistic or antagonistic manner (Mullen and Hangarter, 2003). While gravitropism can readily be studied on earth in the absence of light, it is difficult to investigate phototropism in the absence of the effects of gravity. Thus, the microgravity conditions of space provide the needed opportunity to study phototropism without the ‘‘complications’’ of unidirectional gravity. It has been shown that in stem-like organs, interactions between positive phototropism and negative gravitropism determine the direction of growth in young seedlings (Hangarter, 1997). Similarly, in roots of some species (e.g., Arabidopsis), it appears that there is also a comparable interaction between tropisms in orienting root growth (Okada and Shimura, 1992), but, in the case of roots, the reported interaction was between negative phototropism and positive gravitropism. While phototropism in stems and roots is primarily a blue-light induced response (controlled by the phototropins; Briggs and Christie, 2002), we have recently discovered a positive red-light-induced phototropism mediated by phytochromes A and B in roots (Kiss et al., 2003). This red response of roots is the major focus of our upcoming experiments to be performed in microgravity on the International Space Station (ISS). This spaceflight project is in the final stages of development and scheduled to be launched on the space shuttle for the ISS. These experiments will be performed in the European Modular Cultivation System (EMCS), which is a multi-user facility for biological research that was designed by the European Space Agency (ESA). The EMCS has an incubator, atmospheric control, and a high-resolution video camera system that are needed for this project (Brinckmann, 1999; Brinckmann and Schiller, 2002). The entire system is on a variable centrifuge rotor so that a 1g control can be performed and fractional g-levels can be selected as well. We have been working with NASA to design experimental unique hardware (EUE) to study tropisms of Arabidopsis seedlings in EMCS. In this paper, we will give an overview of the EUE designed for our research focusing on plant tropisms and provide some examples of the groundbased testing that is needed to ensure the success of a spaceflight project.
in Kiss et al., 2003) derived from this strain were used in these studies. Prior to placement in 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 contained one-half-strength Murashige–Skoog salts with 1% (w/v) sucrose and 1 mM MES at pH 5.5 (Kiss et al., 2003). For experiments with EMCSÕs experimental containers (ECÕs), the growth medium was used to soak the filter paper described below, and allowed to dry in the laminar flow hood. In addition, for experiments with ECÕs, illumination was provided by LEDÕs, and for the growth phase of the seedlings, a white LED array to produce a fluence rate of approx. 40–50 lmol m 2 s 1 was used. [For some other ground-based experiments, illumination of 30–40 lmol m 2 s 1 was obtained from 34 W ‘‘cool light’’ fluorescent lamps.] The red light to induce seed germination was from a 660 nm LED at a fluence rate of 10 lmol m 2 s 1. The red light for the phototropism experiments was from the same LED at the same fluence rate. For the blue light phototropism experiments, a 450 nm LED was used at 10 lmol m 2 s 1. The LEDÕs are all spaceflight-certified and are manufactured by Purdy Electronics (Sunnyvale, CA, USA). Growth substrates used in tests included Whatman #3 and #17 filter papers and a germination paper (no. BB69; Anchor Paper Co., St. Paul, MN, USA). The black gridded membrane to which seeds are affixed (and is used to enhance contrast for photography) is made of mixed cellulose esters (no. 66585; Pall Corp., Ann Arbor, MI, USA). Additional details of other materials used in the spaceflight hardware and the experimental time line are provided in results and discussion. 2.2. Analysis of growth and curvature Seedlings were photographed with a digital camera equipped with a macro lens, and measurements of curvature and growth were made with the image analysis program Image-Pro Plus (version 4.5; Media Cybernetics, Silver Spring, MD, USA) on a Pentium PC computer. Seedlings were excluded from measurement if their roots contacted neighboring plants. These experiments were repeated at least three times, and values are reported as the mean ±standard error (SE).
3. Results and discussion 2. Materials and methods 2.1. Plant material and culture conditions
3.1. Hardware has been developed for the study of tropisms in the EMCS
Arabidopsis thaliana (Landsberg, Ler, ecotype) WT and several phytochrome-deficient mutants (described
Since the EMCS is a multi-user facility, individual scientists in conjunction with their space agency need
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to develop EUE which will fit into the standard experimental container (EC), which has the dimensions of 160 (length) · 60 (width) · 60 mm (height). Four ECÕs fit on one EMCS rotor, and there are two rotors in the EMCS (Brinckmann and Schiller, 2002). Details about atmospheric control, air exchange rate, ethylene removal, and other capabilities of the EMCS are provided in Brinckmann (1999) and updated on a Web site of ESA (www.spaceflight.esa.int/users/file.cfm?filename=fac-issdest-emcs). The EUE for growing Arabidopsis seedlings is based on our design as modified by NASA engineers, and the hardware (termed TROPI, for the study of tropisms) is illustrated in Fig. 1. The TROPI hardware is designed to fit into the standard EC of the EMCS and includes five growth cassettes per EC (Fig. 1). Each cassette has two sets of LEDÕs: one set of white LEDÕs along the longer side of the cassette and another set consisting of red and blue LEDÕs on one end of the shorter side of the cassette. The white LEDÕs are termed ‘‘growth’’ LEDÕs since they are used in the first part of the experiment to ensure for the ‘‘oriented’’ growth of seedlings, and the blue/red LEDÕs are termed ‘‘stimulation’’ LEDÕs since they are used to study effects of phototropic stimulation. The individual seedling cassettes of the EUE are illustrated in Fig. 2, and the design is based on the concepts successfully used in our previous spaceflight experiments with Arabidopsis seedlings on two Biorack missions (Katembe et al., 1998; Kiss et al., 1998, 1999). Seedlings (typically 14 per cassette) grow on a ‘‘sandwich’’ of a
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black membrane and a sheet of Whatman #17 filter paper that is held together by six retaining clips at the base plate and a layer of 1% (w/v) gum guar (Fig. 2). Dry seeds are placed in containers, and the experiment is initiated when the seeds are hydrated. Prior to attachment to the membrane with gum guar (Katembe et al., 1998), seeds are surface sterilized in ethanol. The transparent plastic cover on the cassette allows for video observations during the flight and has a transparent anti-fogging heater membrane attached so that condensation does not interfere with video observations (Fig. 2). The plastic cover has four slots with a gas permeable membrane covering them to allow for gas exchange with minimal moisture loss. The Whatman #17 filter paper, which is impregnated with Murashige– Skoog media and 1% sucrose, absorbs and holds the water while the black gridded membrane is used to enhance the contrast of the seedlings during the video observations. The cassette base plate has two circular slots – one for a thermistor for temperature sensing and the other serves as a hydration port. These two slots are covered with a 0.22 lm nitrocellulose filter paper to reduce microbial contamination. Dry seeds will be in hardware during launch on the space shuttle, and the experiment will be initiated at a later date on the ISS. 3.2. Biocompatibility tests of EUE components and other tests for spaceflight experiments Since spaceflight opportunities for biological research are infrequent and limited, it is important to
Fig. 1. Experimental Unique Equipment (EUE) for the tropism experiment (TROPI) shown in the experimental container (EC) of the EMCS. Five cassettes (C1–C5) for seedling growth are present in each EC. The white LEDÕs (W LEDÕs) are for growing seedlings, and the blue and red LEDÕs (B/ R LEDs) for phototropic stimulation experiments are at an axis perpendicular to the white LEDs. The direction of the g-vector resulting from the EMCS centrifuge is indicated by the large arrow. BP, base plate of the EC; F, fan for cooling of the EC. Bar = 2 cm.
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Fig. 2. An individual seedling cassette of the TROPI EUE. There are five cassettes per EC (see Fig. 1). Arabidopsis seedlings grow on a ‘‘sandwich’’ of a black membrane and a sheet of Whatman #17 filter paper. Additional details are provided in the text.
perform extensive ground-based testing to help ensure the success of the project during flight. A comprehensive series of biocompatibility and other related tests have been performed since this project was selected for definition and development by NASA. Additional tests were conducted because we designed new EUE and also because this is the first project scheduled on the EMCS. These tests examined numerous factors such as: types of materials used in the EUE, optimal LED illumination (quality and quantity), temperature (and temperature tolerance), humidity in the EC, placement of red illumination period to optimize seed germination, video compression ratios during downlinks, quality of optical surfaces used in imaging, and numerous other parameters. In this section, we give two examples of the biocompatibility tests that we have performed. One of the most important aspects of a successful experiment is to maintain optimal hydration of the seeds and the developing seedlings. The current hardware design allows for one hydration pulse, so we investigated several types of substrates for holding water and allowing for seedling growth. Before settling on the current Whatman #17 filter paper, we tested additional substrates such as a standard seed germination paper and two layers of Whatman #3 filter paper (Fig. 3), which was the design used in our previous Biorack experiments (Katembe et al., 1998; Kiss
et al., 1999). With both types of paper, a nutrient medium consisting of half strength Murashige and Skoog medium, 1 mM MES (pH 5.5), and 1% (w/v) sucrose (Kiss et al., 2003) was embedded in the paper and then air dried. After the seeds were sown, the papers were hydrated with sterile water. While the germination paper retained a greater volume of water compared to the filter paper (750 and 550 ll, respectively), seedling and root growth was greater (with fewer root hairs) on the two layers of filter paper (Fig. 3(a)). This may be due to an inhibitory effect caused by the germination paper or some other factor, but in any case, it was important to establish the more optimal substrate to be used in the spaceflight experiment. Another test (Fig. 3(b)) examined root growth of seedlings on two layers of Whatman #3 paper (hydrated with 550 ll of water) versus a single layer of Whatman #17 paper (with 750 ll of water). Since growth was similar under both conditions, we chose a design of one layer of Whatman #17 since it is easier to assemble the cassettes with a single layer of filter paper. One major advantage of performing spaceflight experiments with dry seeds is that they can retain their viability for long periods. Also, seeds can remain at ambient conditions prior to the launch of the space craft without harm to the experiment. Because of the technical requirements of the launch of our experiment on the
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Fig. 3. Tests of seedling growth with various substrates. Bars = SE. (a) growth was improved when Whatman filter was used relative to germination paper. N = 80–85 and (b) growth characteristics were similar with both a single layer of Whatman #17 filter paper and two layers of Whatman #3. N = 30–43.
shuttle, the EMCS facility and our seeds in EUE may have to be launched in a Multi-Purpose Logistics Module (MPLM), which is used to transport supplies and materials between Earth and the ISS. In contrast to the space shuttle mid-deck, supplies and material on the MPLM, which is integrated into the cargo bay of
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the space shuttle, need to be loaded months before the launch. Thus, we needed to investigate the viability of seeds in the MPLM for relatively long time periods. Seed viability also needed to be studied since the experiment may be in storage on the ISS prior to its activation. For the seed viability studies, we measured seed germination after 110 days under four conditions: surface-sterilized seeds at 22 C, surface-sterilized seeds at 37 C, non-sterilized seeds at 22 C, and non-sterilized seeds at 37 C (Fig. 4). While experiments with seedlings are normally performed at 22 C, we analyzed germination at 37 C since it was possible that temperatures in the MPLM could reach this higher temperature during ‘‘ambient’’ conditions. The time of 110 days was selected since this was the earliest time prior to launch that EUE would need to be stored in the MPLM. Since seeds are surface sterilized prior to placement on the membrane in the cassettes, it was important to assay the viability of sterilized seeds that were stored for 110 days. Under all four conditions tested, seed germination was good to excellent after three days (Fig. 4). The highest germination was with the non-sterilized seeds at either temperature regime, and there was a decrease in germination if sterilized seeds were used in the experiment. This decrease can be attributed to storing seeds with a weakened seed coat (due to the sterilization procedure), but if these seeds are stored at room temperature, germination was approx. 80%, and this is acceptable for a spaceflight experiment in which maximal germination is an important goal. In addition, after five days (data not shown), there was 100% germination in the nonsterilized seeds at either temperature regime, and there was a slight decrease in germination if sterilized seeds were used in the experiment. In our previous spaceflight experiments with sterilized seeds stored for a period of a 2–3 weeks, we had excellent seed gemination, typically greater than 90% (Kiss et al., 1998, 1999, 2000).
Per cent seed germination
100
80
60
40
20
0 Surface Sterilized RoomTemp
Surface Sterilized 37C
Non Sterilized Room Temp
Non Sterilized 37C
Fig. 4. Percent seed germination after storage period of 110 days at the conditions indicated. Germination was measured three days after hydration of seeds, and experiments were replicated three times with 45 seeds per treatment; bars = SE.
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3.3. Seedlings exhibit robust tropistic curvature in the hardware Arabidopsis seedlings grow well in the cassettes designed for the TROPI EUE (Fig. 5), and they exhibit robust tropisms (Figs. 5 and 6) For instance, in terms of blue-light-induced phototropism in seedlings, hypocotyls exhibit a strong positive phototropic response while roots show a distinct negative curvature in response to unilateral blue illumination from the stimulation LEDÕs (Fig. 5). Furthermore, the kinetics, growth, and magnitude of the phototropic response in the spaceflight hardware (Fig. 6) are similar to our ground-based studies on agar in Petri plates (e.g. Correll et al., 2003). In addition, studies of gravitropism of Arabidopsis seedlings (not shown) in the hardware also demonstrate that the kinetics and magnitude of the gravitropic response are similar to the gravitropism exhibited to other ground-based studies that we have published (e.g. Kiss et al., 1997). 3.4. Time line of TROPI experiments to be performed in the EMCS Our spaceflight experiment on tropisms is scheduled to be launched on STS-121 for the ISS. This experiment is scheduled to be launched with the EMCS facility, and
Fig. 6. Kinetics of root phototropism (in unilateral blue illumination) of WT Arabidopsis seedlings in TROPI EUE. The kinetics of negative phototropism in roots in hardware (on a substrate of cellulose esters) is similar to that of experiments performed in standard laboratory conditions (e.g., Petri dishes on agar). N = 44, and bars = SE.
we will be the first group to use this facility. Following its launch on the shuttle, the EMCS will be installed in the Destiny module of the ISS (Brinckmann and Schiller, 2002). Thus, our experiments will be performed on the ISS, and data (videotapes and frozen seedlings; see below) will be returned on a later shuttle flight.
Fig. 5. Images of a phototropism experiment in seedling cassette of the TROPI EUE. At time 0, seedlings were given blue illumination from the left side of the cassettes. Note that by 24 h, the hypocotyls (arrow) grow toward the blue light while the roots (asterisks) grow away from the light. Distance between lines on gridded membrane is 3 mm.
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Fig. 7. Time line of spaceflight experiments. The experiment is activated by hydration of seeds. Following a dark period, seeds will be grown at 1-g in unilateral white light and then phototropism experiments will be performed using red or blue LEDÕs. The photostimulation experiments will be performed in microgravity as illustrated on the time line. In addition to the lg experiment shown, fractional g-levels (ranging from approx. 0.1 to 0.9 g) and a 1g (control) will be performed. Data from these experiments will consist of frozen seedlings and video tapes, which are returned post-flight.
As stated above, the major goal of our project on STS-121 is to determine the influence of gravity on light perception and to better characterize red-light-dependent root phototropism (Kiss et al., 2003). Based on extensive ground-based testing under normal laboratory conditions and in spaceflight hardware, a time line for this experiment has been developed, and it is illustrated in Fig. 7. The sequence shown (Fig. 7) represents a programmed series of steps since the EMCS is automated due to the limited crew time available in the early utilization phase of the ISS (Brinckmann and Schiller, 2002). Dry seeds in hardware will be at ambient temperatures during the launch of the space craft, and the experiment will be activated by hydrating the seeds in ECÕs after they are placed into the EMCS (Fig. 7). [For hydration, we will use 820 ll of water per cassette which provides a slight excess of water.] After imbibition, the seeds in ECÕs will be given a 4 h treatment of red illumination from light emitting diodes (LEDÕs) to stimulate germination. Seedlings then will be grown on the centrifuge at 1g with white LED illumination parallel to the gvector for approx. three days to ensure straight, oriented growth of the plants. Next, the centrifuge will be turned off (seedlings will be in microgravity) and, following a 6 h dark period, seedlings will be provided with unilateral red or blue light treatments. We plan to perform the red and blue illumination portion of the experiments at varying g-levels (lg, fractional-g, and 1g). The data to be obtained consists primarily of video images of seedling growth and tropistic curvature. While there is some limited opportunity for real-time or near real-time downlinks of images, most of the data will be stored on video tapes which will be recovered post-flight. At the end of the tropism experiments, the seedlings will be frozen and stored at 80 C, and these seedlings will be used for gene profiling experiments with microarrays (Moseyko et al., 2002). The overall rationale for these types of experiments is to obtain a better understanding of the relationship between light (phototropism) and gravity (gravitropism) in plant development (Hangarter, 1997; Correll
and Kiss, 2002). In addition, the variable g-accelerations will provide needed information about the g-threshold for gravitropism, which will be important for growing plants during future long-term human space missions. 3.5. Conclusions and outlook Results of our ground-based studies suggest that there are two photosensory systems that elicit phototropic responses in roots: the previously identified bluelight photoreceptor system and a red light phytochrome-based mechanism that we have recently discovered (Kiss et al., 2003). The phototropic response in roots is much weaker compared to the graviresponse, which on earth competes with and often masks the phototropic response. Because of the interfering effect of the strong gravitropic response in roots, microgravity conditions are needed to effectively study this novel red-lightdependent phototropic response. These experiments scheduled for the EMCS will provide a detailed characterization of root phototropism, provide insight into how red light enhances blue-light dependent phototropism in hypocotyls, and provide a better understanding of how plants integrate sensory input from multiple light and gravity perception systems.
Acknowledgements We thank Robert Bowman for providing the image of the EUE used in Fig. 1 and the entire NASA Ames team (M. Eodice, M. Steele, A. Kakavand, E. Houston, and others) for their help with this project. Financial support was provided by NASA Grant NCC2-1200.
References Brinckmann, E. Spaceflight opportunities on the ISS for plant research – the ESA perspective. Adv. Space Res. 24, 779–788, 1999.
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Brinckmann, E., Schiller, P. Experiments with small animals in BIOLAB and EMCS on the International Space Station. Adv. Space Res. 30, 809–814, 2002. Briggs, W.R., Christie, J.M. Phototropins 1 and 2: versatile plant bluelight receptors. Trends Plant Sci. 7, 204–210, 2002. Correll, M.J., Kiss, J.Z. Interactions between gravitropism and phototropism in plants. J. Plant Growth Regul. 21, 89–101, 2002. Correll, M.J., Coveney, K.M., Raines, S.V., et al. Phytochromes play a role in phototropism and gravitropism in Arabidopsis roots. Adv. Space Res. 31, 2203–2210, 2003. Digby, J., Firn, R. The gravitropic set-point angle (GSA): the identification of an important developmentally controlled variable governing plant architecture. Plant Cell Environ. 18, 1434–1440, 1995. Hangarter, R.P. Gravity, light and plant form. Plant Cell Environ. 20, 796–800, 1997. 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., Brinckmann, E., Brillouet, C. Development and growth of several strains of Arabidopsis seedlings in microgravity. Int. J. Plant Sci. 161, 55–62, 2000.
Kiss, J.Z., Edelmann, R.E., Wood, P.C. Gravitropism of hypocotyls of wild-type and starch-deficient Arabidopsis seedlings in spaceflight studies. Planta 209, 96–103, 1999. Kiss, J.Z., Guisinger, M.M., Miller, A.J., et al. Reduced gravitropism in hypocotyls of starch-deficient mutants of Arabidopsis. Plant Cell Physiol. 38, 518–525, 1997. 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 phototropis in roots. Plant Physiol. 131, 1411–1417, 2003. Moseyko, N., Zhu, T., Chang, H.-S., et al. Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays. Plant Physiol. 130, 720–728, 2002. Mullen, J.L., Hangarter, R.P. Genetic analysis of the gravitropic setpoint angle in lateral roots of Arabidopsis. Adv. Space Res. 31, 2229–2236, 2003. Okada, K., Shimura, Y. Mutational analysis of root gravitropism and phototropism of Arabidopsis thaliana seedlings. Aust J. Plant Physiol. 19, 439–448, 1992.
Ground-based studies of tropisms in hardware developed for the European Modular Cultivation System (EMCS) Melanie J. Correll a, Richard E. Edelmann a, Roger P. Hangarter b, Jack L. Mullen b, John Z. Kiss a,* a
Department of Botany, Miami University, Pearson Hall, Oxford, OH 45056, USA b Department of Biology, Indiana University, Bloomington, IN 47405, USA
Received 2 July 2004; received in revised form 7 October 2004; accepted 2 November 2004
Abstract Phototropism and gravitropism play key roles in the oriented growth of roots in flowering plants. In blue or white light, roots exhibit negative phototropism, but red light induces positive phototropism in Arabidopsis roots. The blue-light response is controlled by the phototropins while the red-light response is mediated by the phytochrome family of photoreceptors. In order to better characterize root phototropism, we plan to perform experiments in microgravity so that this tropism can be more effectively studied without the interactions with the gravity response. Our experiments are to be performed on the European Modular Cultivation System (EMCS), which provides an incubator, lighting system, and high resolution video that are on a centrifuge palette. These experiments will be performed at lg, 1g (control) and fractional g-levels. In order to ensure success of this mission on the International Space Station, we have been conducting ground-based studies on growth, phototropism, and gravitropism in experimental unique equipment (EUE) that was designed for our experiments with Arabidopsis seedlings. Currently, the EMCS and our EUE are scheduled for launch on space shuttle mission STS-121. This project should provide insight into how the blue- and red-light signaling systems interact with each other and with the gravisensing system. 2004 Published by Elsevier Ltd on behalf of COSPAR. Keywords: Arabidopsis; Gravitropism; Phototropism; Phytochrome; Spaceflight hardware
1. Introduction Plants have numerous selective and sensitive mechanisms to perceive and respond to various aspects of their environment. Of these environmental signals, gravity and light play key roles in plant development, but other factors such as temperature and moisture are also important. Plants continuously integrate all of the information received by their sensory systems to adjust their growth to their present environmental conditions (Hangarter, 1997; Correll and Kiss, 2002). *
Corresponding author. Tel.: +1 513 529 5428; fax: +1 513 529 4243. E-mail address: [email protected] (J.Z. Kiss). 0273-1177/$30 2004 Published by Elsevier Ltd on behalf of COSPAR. doi:10.1016/j.asr.2004.11.003
On earth, gravity is a ubiquitous and constant signal. In germinating seedlings, gravity is important for orienting the plant so that shoots grow upwards and the roots grow downwards. The primary shoot and root of young seedlings have been the main subject of studies of gravitropism (directed growth in response to gravity). However, in mature plants, lateral organs typically display a characteristic angle of orientation relative to the gravity vector (Digby and Firn, 1995), and this angle can be modified by light conditions (Hangarter, 1997). Gravity and light often interact in influencing the developmental pattern of plants. In particular, several studies have shown that light can modulate gravitropic behavior through the photosensitive pigment phytochrome (reviewed in Hangarter, 1997; Correll and Kiss,
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2002). Light-dependent modulation of gravitropism may affect the orientation of plant organs, and, thus, the final shape of a plant can be determined by interaction of gravitropism and phototropism (directed growth in response to light) in either a synergistic or antagonistic manner (Mullen and Hangarter, 2003). While gravitropism can readily be studied on earth in the absence of light, it is difficult to investigate phototropism in the absence of the effects of gravity. Thus, the microgravity conditions of space provide the needed opportunity to study phototropism without the ‘‘complications’’ of unidirectional gravity. It has been shown that in stem-like organs, interactions between positive phototropism and negative gravitropism determine the direction of growth in young seedlings (Hangarter, 1997). Similarly, in roots of some species (e.g., Arabidopsis), it appears that there is also a comparable interaction between tropisms in orienting root growth (Okada and Shimura, 1992), but, in the case of roots, the reported interaction was between negative phototropism and positive gravitropism. While phototropism in stems and roots is primarily a blue-light induced response (controlled by the phototropins; Briggs and Christie, 2002), we have recently discovered a positive red-light-induced phototropism mediated by phytochromes A and B in roots (Kiss et al., 2003). This red response of roots is the major focus of our upcoming experiments to be performed in microgravity on the International Space Station (ISS). This spaceflight project is in the final stages of development and scheduled to be launched on the space shuttle for the ISS. These experiments will be performed in the European Modular Cultivation System (EMCS), which is a multi-user facility for biological research that was designed by the European Space Agency (ESA). The EMCS has an incubator, atmospheric control, and a high-resolution video camera system that are needed for this project (Brinckmann, 1999; Brinckmann and Schiller, 2002). The entire system is on a variable centrifuge rotor so that a 1g control can be performed and fractional g-levels can be selected as well. We have been working with NASA to design experimental unique hardware (EUE) to study tropisms of Arabidopsis seedlings in EMCS. In this paper, we will give an overview of the EUE designed for our research focusing on plant tropisms and provide some examples of the groundbased testing that is needed to ensure the success of a spaceflight project.
in Kiss et al., 2003) derived from this strain were used in these studies. Prior to placement in 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 contained one-half-strength Murashige–Skoog salts with 1% (w/v) sucrose and 1 mM MES at pH 5.5 (Kiss et al., 2003). For experiments with EMCSÕs experimental containers (ECÕs), the growth medium was used to soak the filter paper described below, and allowed to dry in the laminar flow hood. In addition, for experiments with ECÕs, illumination was provided by LEDÕs, and for the growth phase of the seedlings, a white LED array to produce a fluence rate of approx. 40–50 lmol m 2 s 1 was used. [For some other ground-based experiments, illumination of 30–40 lmol m 2 s 1 was obtained from 34 W ‘‘cool light’’ fluorescent lamps.] The red light to induce seed germination was from a 660 nm LED at a fluence rate of 10 lmol m 2 s 1. The red light for the phototropism experiments was from the same LED at the same fluence rate. For the blue light phototropism experiments, a 450 nm LED was used at 10 lmol m 2 s 1. The LEDÕs are all spaceflight-certified and are manufactured by Purdy Electronics (Sunnyvale, CA, USA). Growth substrates used in tests included Whatman #3 and #17 filter papers and a germination paper (no. BB69; Anchor Paper Co., St. Paul, MN, USA). The black gridded membrane to which seeds are affixed (and is used to enhance contrast for photography) is made of mixed cellulose esters (no. 66585; Pall Corp., Ann Arbor, MI, USA). Additional details of other materials used in the spaceflight hardware and the experimental time line are provided in results and discussion. 2.2. Analysis of growth and curvature Seedlings were photographed with a digital camera equipped with a macro lens, and measurements of curvature and growth were made with the image analysis program Image-Pro Plus (version 4.5; Media Cybernetics, Silver Spring, MD, USA) on a Pentium PC computer. Seedlings were excluded from measurement if their roots contacted neighboring plants. These experiments were repeated at least three times, and values are reported as the mean ±standard error (SE).
3. Results and discussion 2. Materials and methods 2.1. Plant material and culture conditions
3.1. Hardware has been developed for the study of tropisms in the EMCS
Arabidopsis thaliana (Landsberg, Ler, ecotype) WT and several phytochrome-deficient mutants (described
Since the EMCS is a multi-user facility, individual scientists in conjunction with their space agency need
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to develop EUE which will fit into the standard experimental container (EC), which has the dimensions of 160 (length) · 60 (width) · 60 mm (height). Four ECÕs fit on one EMCS rotor, and there are two rotors in the EMCS (Brinckmann and Schiller, 2002). Details about atmospheric control, air exchange rate, ethylene removal, and other capabilities of the EMCS are provided in Brinckmann (1999) and updated on a Web site of ESA (www.spaceflight.esa.int/users/file.cfm?filename=fac-issdest-emcs). The EUE for growing Arabidopsis seedlings is based on our design as modified by NASA engineers, and the hardware (termed TROPI, for the study of tropisms) is illustrated in Fig. 1. The TROPI hardware is designed to fit into the standard EC of the EMCS and includes five growth cassettes per EC (Fig. 1). Each cassette has two sets of LEDÕs: one set of white LEDÕs along the longer side of the cassette and another set consisting of red and blue LEDÕs on one end of the shorter side of the cassette. The white LEDÕs are termed ‘‘growth’’ LEDÕs since they are used in the first part of the experiment to ensure for the ‘‘oriented’’ growth of seedlings, and the blue/red LEDÕs are termed ‘‘stimulation’’ LEDÕs since they are used to study effects of phototropic stimulation. The individual seedling cassettes of the EUE are illustrated in Fig. 2, and the design is based on the concepts successfully used in our previous spaceflight experiments with Arabidopsis seedlings on two Biorack missions (Katembe et al., 1998; Kiss et al., 1998, 1999). Seedlings (typically 14 per cassette) grow on a ‘‘sandwich’’ of a
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black membrane and a sheet of Whatman #17 filter paper that is held together by six retaining clips at the base plate and a layer of 1% (w/v) gum guar (Fig. 2). Dry seeds are placed in containers, and the experiment is initiated when the seeds are hydrated. Prior to attachment to the membrane with gum guar (Katembe et al., 1998), seeds are surface sterilized in ethanol. The transparent plastic cover on the cassette allows for video observations during the flight and has a transparent anti-fogging heater membrane attached so that condensation does not interfere with video observations (Fig. 2). The plastic cover has four slots with a gas permeable membrane covering them to allow for gas exchange with minimal moisture loss. The Whatman #17 filter paper, which is impregnated with Murashige– Skoog media and 1% sucrose, absorbs and holds the water while the black gridded membrane is used to enhance the contrast of the seedlings during the video observations. The cassette base plate has two circular slots – one for a thermistor for temperature sensing and the other serves as a hydration port. These two slots are covered with a 0.22 lm nitrocellulose filter paper to reduce microbial contamination. Dry seeds will be in hardware during launch on the space shuttle, and the experiment will be initiated at a later date on the ISS. 3.2. Biocompatibility tests of EUE components and other tests for spaceflight experiments Since spaceflight opportunities for biological research are infrequent and limited, it is important to
Fig. 1. Experimental Unique Equipment (EUE) for the tropism experiment (TROPI) shown in the experimental container (EC) of the EMCS. Five cassettes (C1–C5) for seedling growth are present in each EC. The white LEDÕs (W LEDÕs) are for growing seedlings, and the blue and red LEDÕs (B/ R LEDs) for phototropic stimulation experiments are at an axis perpendicular to the white LEDs. The direction of the g-vector resulting from the EMCS centrifuge is indicated by the large arrow. BP, base plate of the EC; F, fan for cooling of the EC. Bar = 2 cm.
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Fig. 2. An individual seedling cassette of the TROPI EUE. There are five cassettes per EC (see Fig. 1). Arabidopsis seedlings grow on a ‘‘sandwich’’ of a black membrane and a sheet of Whatman #17 filter paper. Additional details are provided in the text.
perform extensive ground-based testing to help ensure the success of the project during flight. A comprehensive series of biocompatibility and other related tests have been performed since this project was selected for definition and development by NASA. Additional tests were conducted because we designed new EUE and also because this is the first project scheduled on the EMCS. These tests examined numerous factors such as: types of materials used in the EUE, optimal LED illumination (quality and quantity), temperature (and temperature tolerance), humidity in the EC, placement of red illumination period to optimize seed germination, video compression ratios during downlinks, quality of optical surfaces used in imaging, and numerous other parameters. In this section, we give two examples of the biocompatibility tests that we have performed. One of the most important aspects of a successful experiment is to maintain optimal hydration of the seeds and the developing seedlings. The current hardware design allows for one hydration pulse, so we investigated several types of substrates for holding water and allowing for seedling growth. Before settling on the current Whatman #17 filter paper, we tested additional substrates such as a standard seed germination paper and two layers of Whatman #3 filter paper (Fig. 3), which was the design used in our previous Biorack experiments (Katembe et al., 1998; Kiss
et al., 1999). With both types of paper, a nutrient medium consisting of half strength Murashige and Skoog medium, 1 mM MES (pH 5.5), and 1% (w/v) sucrose (Kiss et al., 2003) was embedded in the paper and then air dried. After the seeds were sown, the papers were hydrated with sterile water. While the germination paper retained a greater volume of water compared to the filter paper (750 and 550 ll, respectively), seedling and root growth was greater (with fewer root hairs) on the two layers of filter paper (Fig. 3(a)). This may be due to an inhibitory effect caused by the germination paper or some other factor, but in any case, it was important to establish the more optimal substrate to be used in the spaceflight experiment. Another test (Fig. 3(b)) examined root growth of seedlings on two layers of Whatman #3 paper (hydrated with 550 ll of water) versus a single layer of Whatman #17 paper (with 750 ll of water). Since growth was similar under both conditions, we chose a design of one layer of Whatman #17 since it is easier to assemble the cassettes with a single layer of filter paper. One major advantage of performing spaceflight experiments with dry seeds is that they can retain their viability for long periods. Also, seeds can remain at ambient conditions prior to the launch of the space craft without harm to the experiment. Because of the technical requirements of the launch of our experiment on the
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Fig. 3. Tests of seedling growth with various substrates. Bars = SE. (a) growth was improved when Whatman filter was used relative to germination paper. N = 80–85 and (b) growth characteristics were similar with both a single layer of Whatman #17 filter paper and two layers of Whatman #3. N = 30–43.
shuttle, the EMCS facility and our seeds in EUE may have to be launched in a Multi-Purpose Logistics Module (MPLM), which is used to transport supplies and materials between Earth and the ISS. In contrast to the space shuttle mid-deck, supplies and material on the MPLM, which is integrated into the cargo bay of
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the space shuttle, need to be loaded months before the launch. Thus, we needed to investigate the viability of seeds in the MPLM for relatively long time periods. Seed viability also needed to be studied since the experiment may be in storage on the ISS prior to its activation. For the seed viability studies, we measured seed germination after 110 days under four conditions: surface-sterilized seeds at 22 C, surface-sterilized seeds at 37 C, non-sterilized seeds at 22 C, and non-sterilized seeds at 37 C (Fig. 4). While experiments with seedlings are normally performed at 22 C, we analyzed germination at 37 C since it was possible that temperatures in the MPLM could reach this higher temperature during ‘‘ambient’’ conditions. The time of 110 days was selected since this was the earliest time prior to launch that EUE would need to be stored in the MPLM. Since seeds are surface sterilized prior to placement on the membrane in the cassettes, it was important to assay the viability of sterilized seeds that were stored for 110 days. Under all four conditions tested, seed germination was good to excellent after three days (Fig. 4). The highest germination was with the non-sterilized seeds at either temperature regime, and there was a decrease in germination if sterilized seeds were used in the experiment. This decrease can be attributed to storing seeds with a weakened seed coat (due to the sterilization procedure), but if these seeds are stored at room temperature, germination was approx. 80%, and this is acceptable for a spaceflight experiment in which maximal germination is an important goal. In addition, after five days (data not shown), there was 100% germination in the nonsterilized seeds at either temperature regime, and there was a slight decrease in germination if sterilized seeds were used in the experiment. In our previous spaceflight experiments with sterilized seeds stored for a period of a 2–3 weeks, we had excellent seed gemination, typically greater than 90% (Kiss et al., 1998, 1999, 2000).
Per cent seed germination
100
80
60
40
20
0 Surface Sterilized RoomTemp
Surface Sterilized 37C
Non Sterilized Room Temp
Non Sterilized 37C
Fig. 4. Percent seed germination after storage period of 110 days at the conditions indicated. Germination was measured three days after hydration of seeds, and experiments were replicated three times with 45 seeds per treatment; bars = SE.
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3.3. Seedlings exhibit robust tropistic curvature in the hardware Arabidopsis seedlings grow well in the cassettes designed for the TROPI EUE (Fig. 5), and they exhibit robust tropisms (Figs. 5 and 6) For instance, in terms of blue-light-induced phototropism in seedlings, hypocotyls exhibit a strong positive phototropic response while roots show a distinct negative curvature in response to unilateral blue illumination from the stimulation LEDÕs (Fig. 5). Furthermore, the kinetics, growth, and magnitude of the phototropic response in the spaceflight hardware (Fig. 6) are similar to our ground-based studies on agar in Petri plates (e.g. Correll et al., 2003). In addition, studies of gravitropism of Arabidopsis seedlings (not shown) in the hardware also demonstrate that the kinetics and magnitude of the gravitropic response are similar to the gravitropism exhibited to other ground-based studies that we have published (e.g. Kiss et al., 1997). 3.4. Time line of TROPI experiments to be performed in the EMCS Our spaceflight experiment on tropisms is scheduled to be launched on STS-121 for the ISS. This experiment is scheduled to be launched with the EMCS facility, and
Fig. 6. Kinetics of root phototropism (in unilateral blue illumination) of WT Arabidopsis seedlings in TROPI EUE. The kinetics of negative phototropism in roots in hardware (on a substrate of cellulose esters) is similar to that of experiments performed in standard laboratory conditions (e.g., Petri dishes on agar). N = 44, and bars = SE.
we will be the first group to use this facility. Following its launch on the shuttle, the EMCS will be installed in the Destiny module of the ISS (Brinckmann and Schiller, 2002). Thus, our experiments will be performed on the ISS, and data (videotapes and frozen seedlings; see below) will be returned on a later shuttle flight.
Fig. 5. Images of a phototropism experiment in seedling cassette of the TROPI EUE. At time 0, seedlings were given blue illumination from the left side of the cassettes. Note that by 24 h, the hypocotyls (arrow) grow toward the blue light while the roots (asterisks) grow away from the light. Distance between lines on gridded membrane is 3 mm.
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Fig. 7. Time line of spaceflight experiments. The experiment is activated by hydration of seeds. Following a dark period, seeds will be grown at 1-g in unilateral white light and then phototropism experiments will be performed using red or blue LEDÕs. The photostimulation experiments will be performed in microgravity as illustrated on the time line. In addition to the lg experiment shown, fractional g-levels (ranging from approx. 0.1 to 0.9 g) and a 1g (control) will be performed. Data from these experiments will consist of frozen seedlings and video tapes, which are returned post-flight.
As stated above, the major goal of our project on STS-121 is to determine the influence of gravity on light perception and to better characterize red-light-dependent root phototropism (Kiss et al., 2003). Based on extensive ground-based testing under normal laboratory conditions and in spaceflight hardware, a time line for this experiment has been developed, and it is illustrated in Fig. 7. The sequence shown (Fig. 7) represents a programmed series of steps since the EMCS is automated due to the limited crew time available in the early utilization phase of the ISS (Brinckmann and Schiller, 2002). Dry seeds in hardware will be at ambient temperatures during the launch of the space craft, and the experiment will be activated by hydrating the seeds in ECÕs after they are placed into the EMCS (Fig. 7). [For hydration, we will use 820 ll of water per cassette which provides a slight excess of water.] After imbibition, the seeds in ECÕs will be given a 4 h treatment of red illumination from light emitting diodes (LEDÕs) to stimulate germination. Seedlings then will be grown on the centrifuge at 1g with white LED illumination parallel to the gvector for approx. three days to ensure straight, oriented growth of the plants. Next, the centrifuge will be turned off (seedlings will be in microgravity) and, following a 6 h dark period, seedlings will be provided with unilateral red or blue light treatments. We plan to perform the red and blue illumination portion of the experiments at varying g-levels (lg, fractional-g, and 1g). The data to be obtained consists primarily of video images of seedling growth and tropistic curvature. While there is some limited opportunity for real-time or near real-time downlinks of images, most of the data will be stored on video tapes which will be recovered post-flight. At the end of the tropism experiments, the seedlings will be frozen and stored at 80 C, and these seedlings will be used for gene profiling experiments with microarrays (Moseyko et al., 2002). The overall rationale for these types of experiments is to obtain a better understanding of the relationship between light (phototropism) and gravity (gravitropism) in plant development (Hangarter, 1997; Correll
and Kiss, 2002). In addition, the variable g-accelerations will provide needed information about the g-threshold for gravitropism, which will be important for growing plants during future long-term human space missions. 3.5. Conclusions and outlook Results of our ground-based studies suggest that there are two photosensory systems that elicit phototropic responses in roots: the previously identified bluelight photoreceptor system and a red light phytochrome-based mechanism that we have recently discovered (Kiss et al., 2003). The phototropic response in roots is much weaker compared to the graviresponse, which on earth competes with and often masks the phototropic response. Because of the interfering effect of the strong gravitropic response in roots, microgravity conditions are needed to effectively study this novel red-lightdependent phototropic response. These experiments scheduled for the EMCS will provide a detailed characterization of root phototropism, provide insight into how red light enhances blue-light dependent phototropism in hypocotyls, and provide a better understanding of how plants integrate sensory input from multiple light and gravity perception systems.
Acknowledgements We thank Robert Bowman for providing the image of the EUE used in Fig. 1 and the entire NASA Ames team (M. Eodice, M. Steele, A. Kakavand, E. Houston, and others) for their help with this project. Financial support was provided by NASA Grant NCC2-1200.
References Brinckmann, E. Spaceflight opportunities on the ISS for plant research – the ESA perspective. Adv. Space Res. 24, 779–788, 1999.
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Brinckmann, E., Schiller, P. Experiments with small animals in BIOLAB and EMCS on the International Space Station. Adv. Space Res. 30, 809–814, 2002. Briggs, W.R., Christie, J.M. Phototropins 1 and 2: versatile plant bluelight receptors. Trends Plant Sci. 7, 204–210, 2002. Correll, M.J., Kiss, J.Z. Interactions between gravitropism and phototropism in plants. J. Plant Growth Regul. 21, 89–101, 2002. Correll, M.J., Coveney, K.M., Raines, S.V., et al. Phytochromes play a role in phototropism and gravitropism in Arabidopsis roots. Adv. Space Res. 31, 2203–2210, 2003. Digby, J., Firn, R. The gravitropic set-point angle (GSA): the identification of an important developmentally controlled variable governing plant architecture. Plant Cell Environ. 18, 1434–1440, 1995. Hangarter, R.P. Gravity, light and plant form. Plant Cell Environ. 20, 796–800, 1997. 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., Brinckmann, E., Brillouet, C. Development and growth of several strains of Arabidopsis seedlings in microgravity. Int. J. Plant Sci. 161, 55–62, 2000.
Kiss, J.Z., Edelmann, R.E., Wood, P.C. Gravitropism of hypocotyls of wild-type and starch-deficient Arabidopsis seedlings in spaceflight studies. Planta 209, 96–103, 1999. Kiss, J.Z., Guisinger, M.M., Miller, A.J., et al. Reduced gravitropism in hypocotyls of starch-deficient mutants of Arabidopsis. Plant Cell Physiol. 38, 518–525, 1997. 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 phototropis in roots. Plant Physiol. 131, 1411–1417, 2003. Moseyko, N., Zhu, T., Chang, H.-S., et al. Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays. Plant Physiol. 130, 720–728, 2002. Mullen, J.L., Hangarter, R.P. Genetic analysis of the gravitropic setpoint angle in lateral roots of Arabidopsis. Adv. Space Res. 31, 2229–2236, 2003. Okada, K., Shimura, Y. Mutational analysis of root gravitropism and phototropism of Arabidopsis thaliana seedlings. Aust J. Plant Physiol. 19, 439–448, 1992.