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Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station
Available online at www.sciencedirect.com
Advances in Space Research xxx (2012) xxx–xxx www.elsevier.com/locate/asr
Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station Sachiko Yano a,⇑, Haruo Kasahara b, Daisuke Masuda b, Fumiaki Tanigaki a Toru Shimazu c, Hiromi Suzuki c, Ichirou Karahara d, Kouichi Soga e, Takayuki Hoson e, Ichiro Tayama f, Yoshikazu Tsuchiya f, Seiichiro Kamisaka d a Japan Aerospace Exploration Agency, 2-1-1 Sengen, Tsukuba 305-8505, Japan Japan Manned Space Systems Corp, 1-1-26 Kawaguchi, Tsuchiura 300-0033, Japan c Japan Space Forum, 3-2-1 Kandasurugadai, Tokyo 101-0062, Japan d Department of Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan e Department of Biology, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Osaka 558-8585, Japan f Chiyoda Advanced Solutions Corporations, 1-1-25 Shin-Urashima-cho, Yokohama 221-0031, Japan b
Received 25 May 2012; received in revised form 26 September 2012; accepted 1 October 2012
Abstract In 2004, Japan Aerospace Exploration Agency developed the engineered model of the Plant Experiment Unit and the Cell Biology Experiment Facility. The Plant Experiment Unit was designed to be installed in the Cell Biology Experiment Facility and to support the seed-to-seed life cycle experiment of Arabidopsis plants in space in the project named Space Seed. Ground-based experiments to test the Plant Experiment Unit showed that the unit needed further improvement of a system to control the water content of a seedbed using an infrared moisture analyzer and that it was difficult to keep the relative humidity inside the Plant Experiment Unit between 70 and 80% because the Cell Biology Experiment Facility had neither a ventilation system nor a dehumidifying system. Therefore, excess moisture inside the Cell Biology Experiment Facility was removed with desiccant bags containing calcium chloride. Eight flight models of the Plant Experiment Unit in which dry Arabidopsis seeds were fixed to the seedbed with gum arabic were launched to the International Space Station in the space shuttle STS-128 (17A) on August 28, 2009. Plant Experiment Unit were installed in the Cell Biology Experiment Facility with desiccant boxes, and then the Space Seed experiment was started in the Japanese Experiment Module, named Kibo, which was part of the International Space Station, on September 10, 2009 by watering the seedbed and terminated 2 months later on November 11, 2009. On April 19, 2010, the Arabidopsis plants harvested in Kibo were retrieved and brought back to Earth by the space shuttle mission STS-131 (19A). The present paper describes the Space Seed experiment with particular reference to the development of the Plant Experiment Unit and its actual performance in Kibo onboard the International Space Station. Downlinked images from Kibo showed that the seeds had started germinating 3 days after the initial watering. The plants continued growing, producing rosette leaves, inflorescence stems, flowers, and fruits in the Plant Experiment Unit. In addition, the senescence of rosette leaves was found to be delayed in microgravity. Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Arabidopsis; Life cycle; Microgravity; Rosette leaf senescence; Seed-to-seed; Temperature and humidity control
1. Introduction ⇑ Corresponding author.
E-mail address: [email protected] (S. Yano).
Over 40 years have passed since biological experiments began to be conducted in satellites and spaceships (Halstead
0273-1177/$36.00 Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asr.2012.10.002
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
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and Dutcher, 1987; Ferl et al., 2002). A variety of plants, both monocotyledons and dicotyledons, have been grown in space to see the effect of microgravity on plant growth and development. Since 1975, the USSR has grown crops such as wheat, onion, oat, pea, cabbage, radish, and lettuce in its spaceships to explore the possibility of agriculture in space (Milov and Rusakova, 1980). Although Arabidopsis is not a crop plant, it has been often used in recent years for plant experiments carried out onboard space shuttles to study the effect of gravity on plants from the perspective of basic sciences. Arabidopsis also has several other advantages: it is the first higher plant whose genome has been deciphered (Cao et al., 2011); it is a compact plant with a short life cycle; and it offers sample variability in the form of mutants and transgenic strains. The seed-to-seed life cycle under microgravity is important for space agriculture. Of the two stages in the life cycle of a typical higher plant, namely the vegetative stage and the reproductive stage, a great deal of information is available on the vegetative stage in space (Hoson et al., 1999; Kiss et al., 2000; Takahashi et al., 1999; Ueda et al., 1999; Wolverton et al., 1999), but very little is known about the reproductive stage (Link et al., 2003; Merkys and Laurinavichys, 1983; Musgrave et al., 1997), and even those results are controversial. On Salyut-7, the reproductive stage in Arabidopsis was delayed and many empty seeds were produced (Merkys and Laurinavichys, 1983), whereas on the International Space Station (ISS) the plant produced fertile seeds (Link et al., 2003). These experimental data obtained in space have been compared with those obtained in 1 G ground-based experiment. In addition to gravity, other environmental conditions in space, such as radiation and the magnetic field, are different from those on the ground. This fact indicates that a facility for plant growth experiments with a centrifuge to produce artificial gravity in the ISS is a prerequisite to analyzing the effect of microgravity on the full life cycle of a plant. The Japan Aerospace Exploration Agency (JAXA) began developing its Cell Biology Experiment Facility (CBEF) with two working areas, namely a microgravity compartment and an artificial gravity compartment on a turntable (centrifuge), after a few conceptual studies in 1993 and 1994. The facility was completed in 2001 as a common resource to support life science experiments with microorganisms, cells, tissues, small animals, and plants in the Japanese Experiment Module (JEM), named Kibo, of the ISS (Ishioka et al., 2004; Yano et al., 2012). The Plant Experiment Unit (PEU) installed inside the CBEF was designed to support the seed-to-seed life cycle of Arabidopsis (Ishioka et al., 2004). In 1997, JAXA initiated the development of a preliminary design of the PEU to support plant growth for an extended period of more than 2 months after analyzing the experimental design of the Space Seed experiment of 1993. The experimental design was specified in 1999, and the design of the PEU to meet the specifications was completed in 2000. A test model of the PEU was fabricated by 2003. The complete specification
of the model is described in an earlier paper (Ishioka et al., 2004). From 2004 to 2008, further ground-based growth experiments with Arabidopsis were carried out using the model, which was part of the CBEF, or in an incubator under a controlled environment to improve the model. The onboard model of the PEU was completed in 2008. In April 2009, 8 units of the flight model, in which dry Arabidopsis seeds had been glued with gum arabic to the rock wool seedbed, were handed over to the National Aeronautics and Space Administration (NASA). On August 28, 2009, the 8 units were transferred to the ISS by STS-128. In Kibo onboard the ISS, the Space Seed experiment began on September 10, 2009 with an initial supply of 20 ml water to the seedbed and was terminated 2 months later on November 11, 2009. In space, Arabidopsis seeds germinated 3 days after the initial watering and the entire life cycle was completed inside the PEU. Arabidopsis plants harvested in Kibo were brought back to Earth by the space shuttle STS-131 (19A) on April 19, 2010. The present report describes the Space Seed experiment with particular reference to improvements in the PEU and in the CBEF, and to the performance of the model and of the modified CBEF in Kibo while onboard the ISS. 2. Improvement in the PEU The specifications for the onboard model of the PEU were nearly identical to those of the test model (Ishioka et al., 2004); the major difference was that the onboard model had a better system for determination of the water content of the seedbed and a plate to cover the seedbed. The outer dimensions of the onboard model were 95 240 170 mm (H W D) (Fig. 1A and B). The unit consisted of a plant growth chamber made of transparent polycarbonate (H W D = 60 60 50 mm, outer size) (Fig. 2) and illuminated with an LED matrix (red and blue radiation in a ratio of 3:1; red LED with luminous spectrum peak at 660 nm [LA-AS-30236, Iwasaki, Japan]; blue LED with a luminous spectrum peak at 470 nm [LAAS-30257, Iwasaki, Japan]). The illumination level on the surface of the seedbed was 110 lmol m 2 s 1. The chamber was fitted with an automated watering system, a humidity control system, and a CCD camera (Fig. 1B). To obtain images of the inside of the plant growth chamber, white LED with spectra peaks at 440 and 550 nm (NSSW100B, Toa, Japan) were used instead of the red and blue LEDs. From 2004 to 2009, we conducted a series of ground-based experiments using the model of the PEU installed at the CBEF at Tsukuba Space Center or in an incubator (Biotron LPH-200-RDDS; Nihon Medical & Chemical Instruments Co. Ltd., Osaka, Japan) at University of Toyama in order to develop a model of the PEU suitable for onboard use. 2.1. Watering system A block of rock wool (NICHIAS Co. Ltd., Tokyo, Japan) placed in the bottom compartment of the growth
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
S. Yano et al. / Advances in Space Research xxx (2012) xxx–xxx
A
3
B
Fig. 1. Onboard model of the Plant Experiment Unit (PEU). A, the external view of the PEU (H W D = 95 240 170 mm) with the cover; B, the inside view of the PEU without the cover. r, LED plate; s, heat sink; t, CCD camera; u, light diffuser; v, plant growth chamber.
analyzer, which measures the IR rays reflected from a target plate placed in contact with one side of the block (Fig. 3). The IR beam hits the black plastic plate 4 mm above the bottom of the plant growth chamber. To facilitate the entry of water and to avoid root invasion into the IR hit-point through the bottom of the target plate, the basal part of the plate was sealed with water-permeable sponge made of polyvinyl alcohol, Berita (number: EB (D); Aion Co. Ltd., Osaka, Japan) (Fig. 3). 2.2. Cover plate on seedbed
Fig. 2. Onboard model of the plant growth chamber. The inner size of the chamber is H W D = 48 56 46 mm. r, stainless steel cover plate with 24 holes; s, IR-target plate; t, polyvinyl alcohol sponge, Berita.
chamber was used as the seedbed (Fig. 3), which was “irrigated” through a tube inserted into the block. In the test model that had no infrared (IR) target plate, water content in the block of rock wool was estimated with an IR moisture analyzer (IR LED, L7850–01; receiver, G8370–01; Hamamatsu Photonics, Japan) by measuring the intensity of IR rays reflected from the side surface of the block of rock wool. However, the uneven fiber density of the rock wool block seriously affected accurate determination of water content, making it difficult to maintain the water content of the seedbed at the desired level. A new technique was developed to overcome this difficulty. The technique involved measuring the amount of excess water with the
In the test model, a transparent and thin plastic plate with 24 small holes was placed on the surface of the seedbed to reduce the loss of water through evaporation (Ishioka et al., 2004). One dry Arabidopsis seed was sown in each hole. Most of the seedlings emerged through the hole but some seedlings failed to do so because of the wider gap between the surface of the rock wool and the plastic plate due to deformation of the thin plastic plate. Therefore, in later models, the plastic plate was replaced with the one made of stainless steel (Figs. 2 and 3). This modification ensured seedling emergence from the holes, probably through enhancing phototropic growth of the hypocotyl. 2.3. Humidity control inside the CBEF The growth experiments were conducted using the PEU placed either in the CBEF or in the Biotron (temperature, 23 °C; relative humidity, 50%). Seeds of Arabidopsis thaliana (L.) Heynh. ecotype Columbia were surface-sterilized with 70% ethanol. After evaporation of the ethanol, dry seeds were glued with gum arabic (1%, w/v) to the seedbed through the holes of the cover plate. The plant growth chamber contained 20 mg HYPONeX powder (N:P:K = 6.5:6.0:19.9; no other minor nutrients) (HYPONeX JAPAN Corp. Ltd., Osaka, Japan). Temperature
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
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Fig. 3. Onboard model of the seedbed of the plant growth chamber. A, the structure of the bottom case of the plant growth chamber; B, the assembly of the seedbed. r, IR-target plate; s, polyvinyl alcohol sponge; t, water inlet; u, stainless steel cover plate; v, block of rock wool (H W D = 10 52 42 mm).
inside the growth chamber was maintained at 23.5 °C and relative humidity at 70–80%. Germination began on the third day after the initial watering and was followed by the development of rosette leaves. Bolting began about 3 weeks from the start of the experiment; the first flower was observed at about 4 weeks; fruit formation began at about 5 weeks; and fruits began turning from green to brown at about 6 weeks. Plant growth in the CBEF differed from that in the Biotron on several counts (Table 1), even though the two
Table 1 Vegetative and reproductive growth of Arabidopsis plants grown in the CBEF without desiccant or in the Biotron.
Number of germinated seeds Number of rosette leaves per plant Length of inflorescences, mm Number of flowers per plantb Number of fruits per plant Number of seeds per plant Number of seeds per fruit Germination rate,%
CBEF without desiccant
Biotron
21.3 ± 1.5 10.9 ± 1.1 99.8.1 ± 15.2 a 7.7 ± 1.2 9.4 ± 1.2 a 2.1 ± 0.4 a 0.2 ± 0.1 a 21.5 ± 10.5
23.3 ± 0.5 9.1 ± 1.5 78.1 ± 4.2 3.7 ± 0.1 5.4 ± 0.2 89.5 ± 6.6 16.3 ± 1.6 89.7 ± 3.0
Levels of significance were calculated by Student’s t test. a P < 0.05 for CBEF vs. Biotron (n = 6). b Number of flowers is the sum of the number of flowers and the number of fruits.
environments led to no significant differences in the number of germinated seeds, the number of rosette leaves on each plant, the length of the inflorescence, and the number of fruits on each plant. The plants grown in the CBEF produced more flowers but markedly fewer seeds, and the average number of seeds in each fruit was much lower in these plants. Lastly, the viability of seeds was also affected; germination percentage in seeds of plants that had been grown in the CBEF was very low. These results indicated that reproductive growth related to seed formation is suppressed in the CBEF. High humidity is reported to suppress reproductive growth in Arabidopsis (Soga et al., 1999; Weigel and Glazebrock, 2002). The plant growth chamber has two filter ports, one for the air inlet and the other for the air outlet. The outlet port is connected to a ventilation pump by a Teflon tube to remove the humid air inside the plant growth chamber. When the ventilation pump is running, air inside the CBEF comes to the plant growth chamber through the air inlet. Humidity inside the plant growth chamber is controlled by the ventilation pump and by the CBEF’s humidity control system (the humidifier). The ventilation pump is switched on when relative humidity inside the growth chamber reaches 80% and switched off when it falls to 70%. Relative humidity inside the Biotron was kept at 50% throughout the experiment by deploying a humidifier and a dehumidifier to keep relative humidity inside the growth chamber between 70 and 80%. However, unlike the
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
S. Yano et al. / Advances in Space Research xxx (2012) xxx–xxx
B
100 95 90
PEU 85 80
CBEF
75
80
Humidity inside the CBEF or PEU (%)
Humidity inside the CBEF or PEU (%)
A
5
70
PEU 60 50 40
CBEF
30 20
70 0
7
14
21
28
35
42
49
56
0
7
14
21
28
35
42
49
56
Incubation time (days)
Incubation time (days)
Fig. 4. Kinetic changes in humidity inside the plant growth chamber of the PEU and the CBEF. A, humidity change without desiccant boxes containing calcium chloride in the CBEF; B, kinetic changes in humidity inside the plant growth chamber and the CBEF. Desiccant boxes were installed in the CBEF 4 days after the start of the growth experiment. Red lines (PEU) in the figures represent relative humidity inside the growth chamber; black lines (CBEF) represent relative humidity inside the CBEF.
Table 2 Vegetative and reproductive growth of Arabidopsis plants grown in the CBEF with desiccant or in the Biotron.
Number of germinated seeds Number of rosette leaves per plant Length of inflorescences, mm Number of flowers per planta Number of fruits per plant Number of seeds per plant Number of seeds per fruit Germination,%
CBEF with desiccant
Biotron
23.3 ± 1.5 8.0 ± 0.7 65.2 ± 1.5 3.2 ± 0.3 3.0 ± 0.2 27.0 ± 2.7 9.4 ± 0.7 89.9 ± 4.4
23.0 ± 0.6 10.0 ± 0.1 57.3 ± 4.4 5.2 ± 0.9 3.1 ± 0.3 17.8 ± 7.3 5.9 ± 2.6 69.2 ± 22.5
No statistically significant differences in the values for CBEF and Biotron (n = 3). a Number of flowers is the sum of the number of flowers and the number of fruits.
Biotron, the CBEF does not have a dehumidifier. As a result, relative humidity inside the CBEF often exceeded 80% (Fig. 4A) because of the exhaust from the PEUs, leading, in turn, to high humidity inside the plant growth chamber, to more than 90% which may cause sterility in Arabidopsis (Weigel and Glazebrock, 2002). As a countermeasure, four aluminum boxes filled with a desiccant were installed in the CBEF. Each box had 20 desiccant bags, each bag containing 6 g of solid calcium chloride (Hakugen, Tokyo, Japan). The bags are made of a special grade of filter paper that is permeable to gas but impermeable to water. The desiccant boxes lowered the humidity inside the CBEF substantially and maintained it lower than 40% throughout the experiment (Fig. 4B). The growth experiment done to see the effect of desiccant on growth and development of Arabidopsis plants indicated that there was no significant difference in growth parameters such as seed production and the ability of produced seeds to germinate between the Biotron and the CBEF with desiccant boxes (Table 2).
3. Experiments onboard Kibo On April 7 and 8, 2009, at the Tsukuba Space Center, dry seeds were glued with gum arabic to the seedbed made of rock wool in the plant growth chamber and the PEUs with Arabidopsis seeds were transported to the Kennedy Space Center. The equipment for the onboard chemical fixation of Arabidopsis, namely Kennedy Space Center Fixation Tubes (KFT; Bionetics Corporation, FL, USA) containing the fixative, were delivered to NASA on August 26, 2009, and the PEUs and KFTs were carried to the ISS by the space shuttle mission STS-128 (17A) on August 28, 2009. The Space Seed experiment onboard Kibo was monitored and controlled from the Tsukuba Space Center. The PEUs were placed in microgravity compartment on an a centrifuge (350 mm in diameter and turned at 87 ± 1 rpm) in the CBEF (Ishioka et al., 2004). The magnitude of gravity was ca. 1.1 g at the surface of seedbed, and ca. 0.7 g at the top of plant growth chamber. The CBEF controls such environmental factors as temperature and humidity as well the power and signal lines including the video image line. The Space Seed experiment began on September 10 with the initial supply of 20 ml water to the block of rock wool (the seedbed) in the plant growth chamber. 3.1. Temperature control The CBEF has 10 connectors for the PEU, 6 for the microgravity compartment and 4 for the centrifuge compartment. For the Space Seed experiment, 8 connectors were used, 4 each for the microgravity compartment and the centrifuge compartment. Ambient temperature in the microgravity compartment is almost the same as that in the centrifuge compartment (Ishioka et al., 2004). The slight difference in temperature caused by differences in the mounting positions of the PEUs was compensated for
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
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target plate, making it impossible to detect the level of water recognition. In response, a fixed volume of water was supplied every day by uplinking the appropriate program file.
Temperature inside the PEU (Degree C)
24.5
24.0
23.5
3.3. Humidity control 007
004
23.0
22.5 0
7
14
21
28
35
42
49
57
63
Incubation time (days) Fig. 5. Changes in average temperature inside the plant growth chamber of 8 PEUs installed in the CBEF. The six accidental stops of power from the CBEF between days 33 and 44 caused a drop in temperature in the plant growth chamber, but in every case, the temperature soon returned to normal. Gravitational condition of each plant growth chamber: 001 to 004, microgravity compartment; 005 to 008, artificial gravity compartment (ca. 1.1 g at the surface of seedbed and ca. 0.7 g at the top of the plant growth chamber).
by a 0.5 W heater in the PEU. Fig. 5 shows that temperature in each plant growth chamber was maintained at 23.5 ± 0.1 °C throughout the growth period (62 days). Although malfunction stopped the operation of the PEU occasionally (Table 3), the drop in temperature was very small, and the temperature soon returned to 23.5 °C. 3.2. Watering system After the initial watering, which took 20 min, an infrared moisture analyzer was operated every 10 min and the water pump delivered 0.17 ml of water in 10 s only when the excess water was below the stipulated level. Watering was carried out, on average, 6–14 times a day until day 32 and 16–17 times a day from day 33 to day 53. The total volume of water supplied to each plant growth chamber varied slightly (data not presented). A downlinked image taken 3 h after the start of the experiment showed a drop of water, about 5 mm in diameter, on the surface of the cover plate in one of the plant growth chambers in the microgravity compartment. However, the drop was not seen in the next image downlinked 6 h later and may have evaporated or was absorbed by the rock wool. During the experiment, in one of plant growth chambers, roots invaded the IR hit-point of the
Relative humidity inside the CBEF increased gradually from about 30% to 60% until day 21 because of the humid exhaust from the plant growth chamber. Introducing the desiccant boxes into the CBEF on day 20 decreased the humidity dramatically (Fig. 6A) but it began to increase gradually until it reached 70%. On day 35, fresh desiccant boxes were introduced, and this led to a rapid drop in relative humidity until it reached about 60%. However, the effect of desiccant on humidity declined with time. Relative humidity inside the growth chambers was maintained between 70 and 80% until day 27, the average level being 75% (Fig. 6B). After that, humidity inside the plant growth chambers fluctuated greatly. The ground-based experiments had indicated that it is difficult to maintain the relative humidity inside the plant growth chamber at 75% if its level inside the CBEF is about 70%. This turned out to be true also onboard Kibo. We attempted to open the door of the CBEF by requesting an astronaut to do so to exchange the humid air inside the CBEF for the dry air in the Kibo cabin, but this maneuver decreased the relative humidity inside the plant growth chamber only by 1%. 3.4. Malfunctions In the later phase of the Space Seed experiment, power supply to the PEUs on a centrifuge was sometimes interrupted (Table 3). This malfunction suspended operations such as LED lighting, heating, and working of the ventilation pump and the water pump. However, the centrifuge continued to rotate regardless of the malfunction. The cumulative downtime of environmental controls in the PEUs on the centrifuge was approximately 28.5 h. 3.5. Plant growth and development onboard Kibo Downlinked images from Kibo showed that cotyledons first appeared 3 days after the initial watering. Average
Table 3 Actions for PEU malfunction during the Space Seed experiment in Kibo onboard the ISS. Experimental time, days
Times/day
Situation
Primary action
33
Once
Power-off in 2 PEUs on a turntable
The function of the PEUs both in the 1g and lg compartment was restored by restarting the PEU-control system.
36
Twice
No downlinking of images from 2 PEUs on a centrifuge
Image acquisition was given up after 3 trials. Images were taken with a digital camera on the last day of the experiment.
37 40 41
Twice Once Twice
The function of PEUs on a turntable was stopped by the reset error (energized state, automatic operation stopped).
Operation of the PEUs was restarted by commands from the ground.
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
S. Yano et al. / Advances in Space Research xxx (2012) xxx–xxx
A Humidity inside the CBEF (%)
Humidity inside the PEU (%)
B
0
7
14
21
28
35
42
49
56
63
7
90 85
007
004
80
002
75 70
008
65 60 0
7
Incubation time (days)
14
21
28
35
42
49
56
63
Incubation time (days)
Fig. 6. Changes in average relative humidity inside the CBEF and PEUs onboard ISS. A, Desiccant boxes were renewed on days 20 and 34; B, Changes in average relative humidity inside each plant growth chamber. Gravitational condition of each plant growth chamber: 001 to 004, microgravity compartment; 005 to 008, artificial gravity compartment (ca. 1.1 g at the surface of seedbed and ca. 0.7 g at the top of the plant growth chamber).
germination percentage by the end of day 10 was 97%. There was no significant difference in the time taken for germination between the experiments on ground and those onboard Kibo. After germination, the plants produced, in succession, rosette leaves, inflorescence, flowers, and fruits
(Fig. 7). Fruit formation was also observed in Kibo irrespective of the magnitude of gravity. The rosette leaves under microgravity remained dark green for longer as compared with plants grown on the centrifuge (Fig. 7), indicating that microgravity had delayed leaf senescence.
4. Discussion
Fig. 7. Bolting of inflorescences in Arabidopsis plants grown for 28 days in Kibo. A and B, plants grown under microgravity; C and D, plants grown under artificial gravity on a centrifuge. The magnitude of artificial gravity was about 1.1 G at the surface of seedbed, and 0.7 G at the top of the plant growth chamber.
The first Japanese announcement of opportunity (AO) for biological experiments aboard space flights was made in 1992. The purpose of this AO was to develop Japanese experimental facilities and equipment. The Space Seed experiment was selected as the model experiment in plant cultivation by the first Japanese AO in 1993, together with four other plant experiments, namely Rice (Hoson et al., 1999), Auxin (Ueda et al., 1999), Root (Wolverton et al., 1999), and PEGT, an abbreviation for peg Takahashi (Takahashi et al., 1999). Because of the delay in assembling the ISS, these four experiments were carried out using the space shuttle STS-95 in 1998 because the experiments could be completed within the flight time of the space shuttle. The Space Seed experiment, which was to last more than 2 months, could not be carried out until 2009 and had to be postponed until the completion of Kibo onboard the ISS. Some experiments on plant cultivation aboard the ISS include those by Berkovich et al. (2004), Iversen et al. (2002), and Link et al. (2003). JAXA executed two projects (Cell Wall and Resist Wall) related to plant sciences by using the European Modular Cultivation System (EMCS) onboard the ISS in 2008 (Hoson et al., 2007; Koizumi et al., 2007), but the watering system of the EMCS did not work well (Kamada et al., 2009). In the present Space Seed experiment, Arabidopsis plants were grown for 62 days in the PEU installed in the CBEF onboard Kibo. As planned, both temperature and humidity inside the plant growth chambers in the PEUs were maintained at the stipulated level for 33 days after the start of the experiment (Figs. 5 and 6B), that is, until the first malfunction (Table 3). Except for the image downlink of PEUs placed
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
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on the centrifuge, most malfunctions were restored by commands from the ground (Table 3). These malfunctions probably influenced subsequent changes in temperature and humidity inside some plant growth chambers on the centrifuge (Figs. 5 and 6B). It is highly probable that all malfunctions in the PEUs onboard Kibo were caused by malfunctions in the CBEF because the same malfunctions occurred simultaneously in two PEUs on the centrifuge. Power to PEUs on the centrifuge is provided from the CBEF through a slip ring, and reduced voltage in the power supply is believed to cause such malfunctions of PEUs on the centrifuge because voltage reduction from +5 V to less than +2.2 V turned out to induce the same malfunction of the PEUs. In July 2011, the centrifuge used in the Space Seed experiment was returned to Earth by the last flight of the space shuttle (STS-135). The continuity test of the centrifuge incorporated into the CBEF at Tsukuba Space Center indicated a flicker in the power supply of +5 V, indicating that the malfunction in the PEUs is caused by irregular power supply from the CBEF to PEUs through the slip ring. We have a plan to replace the centrifuge onboard Kibo by a new centrifuge with improved slip ring. In addition to the malfunction of environmental controls in the PEU, the increase in relative humidity inside the CBEF in the second half of the experiment after the short-term harvest on day 33 (Fig. 6A) may also have resulted in fluctuations in relative humidity inside some plant growth chambers (Fig. 6B). Relative humidity inside the CBEF was maintained below 50% in the ground-based experiment with desiccant boxes (Fig. 4B), whereas relative humidity inside the CBEF onboard Kibo continued to increase despite introducing fresh desiccant boxes two times (Fig. 6A); this was probably because the capacity of the desiccant to absorb moisture had been lowered by some unknown mechanism. As a countermeasure, in next plant cultivation experiment, we should prepare more desiccant boxes to maintain relative humidity inside plant growth chambers between 70 and 80% during an experiment. Downlinked images showed no significant difference in the time taken for germination among the different gravitational conditions, namely microgravity and artificial gravity in the CBEF onboard the ISS and the control on the ground. Hypergravity has been reported to show no substantial influence on germination in pea (Waldron and Brett, 1990), and clinorotation did not affect germination in Arabidopsis (Ishii et al., 1996). These reports indicate that germination is unaffected by gravity. Downlinked images also showed that Arabidopsis plants produce rosette leaves and elongated the inflorescence with flowers and fruits, indicating that we succeeded to complete seed-to-seed plant cultivation under both microgravity and artificial gravity in CBEF onboard Kibo, Japanese experiment module. The EMCS (Brinckmann, 2005) onboard the ISS has been used for analyzing mechanisms of circumnutation
(Johnsson et al., 2009) and phototropism (Millar et al., 2010) . At the stage of inflorescence bolting, Arabidopsis rosette leaves grown in the PEU onboard Kibo were different in color from those grown in the EMCS onboard ISS: Chlorophyll loss took place in rosette leaves in the PEU (Fig. 7), while the leaves in the EMCS showed green in color (see Fig. 1, Johnsson et al. 2009). In the ground-based experiments with the PEU, yellowing of rosette leaves set in about 20 days after sowing, prior to the initiation of bolting, nevertheless Arabidopsis plants could achieve complete their life cycle in the PEU. This was also the case for growth experiments in Kibo. Such early senescence of the rosette leaves may be a kind of stress response caused by dense planting in the PEU, as compared with in the EMCS (Johnsson et al. 2009). Fig. 7 also showed the delayed senescence of rosette leaves under microgravity. Ethylene is known to be the plant hormone that stimulates leaf senescence (Hoffman and Yang, 1982). Physical stress induces the biosynthesis of ethylene in plants, and the weightless condition under microgravity in the present experiment probably lowered the physical stress on rosette leaves, resulted in decreased biosynthesis of ethylene, and, in turn, delayed senescence. In addition, hypergravity was reported to induce the expression of genes related to ethylene response and biosynthesis (Tamaoki et al., 2009). Further results of physiological, morphological, genetic, and physical analyses of Arabidopsis plants brought back by the space shuttle mission STS-131 (19A) will be reported elsewhere. Acknowledgments We wish to acknowledge the Japan Space Forum and the Japan Manned Space Systems Corp. for their help in research coordination, helpful discussions, and technical support in matters such as the CBEF and telemetry operations, image processing, and file transfer. Thanks are also due to the Chiyoda Advanced Solutions Corporation for hardware design of the PEU and technical support in performing the Space Seed experiment. We are also grateful to NASA for international cooperation and assistance. References Berkovich, Y.A., Krivobok, N.M., Sinyak, Y.Y., et al. Developing a vitamin greenhouse for the life support system of the international space station and for future interplanetary missions. Adv. Space Res. 34, 1552–1557, 2004. Brinckmann, E. ESA hardware for plant research on the international space station. Adv. Space Res. 36, 1162–1166, 2005. Cao, J., Schneeberger, K., Ossowski, S., et al. Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nat. Genet. 43, 956– 963, 2011. Ferl, R., Wheeler, R., Levine, H.G., et al. Plants in space. Current Opinion Plant Biol. 5, 258–263, 2002. Halstead, T.W., Dutcher, F.R. Plants in Space. Annu. Rev. Plant Physiol. 38, 317–345, 1987. Hoffman, N.E., Yang, S.F. Enhancement of wound-induced ethylene synthesis by ethylene in preclimacteric cantaloupe. Plant Physiology. 69, 317–322, 1982.
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S. Yano et al. / Advances in Space Research xxx (2012) xxx–xxx Hoson, T., Soga, K., Mori, R., et al. Morphogenesis of rice and Arabidopsis seedlings in space. J. Plant Res. 112, 477–486, 1999. Hoson, T., Matsumoto, S., Soga, K., et al. The outline and significance of the resist wall experiment: role of microtubule-membrane-cell wall continuum in gravity resistance in plants. Biol. Sci. Space 21, 56–61, 2007. Ishii, Y., Hoson, T., Kamisaka, S., et al. Plant growth processes in Arabidopsis under microgravity conditions simulated by a clinostat. Biol. Sci. Space 10, 3–7, 1996. Ishioka, N., Suzuki, H., Asashima, M., et al. Development and verification of hardware for life science experiments in the Japanese experiment module “Kibo” on the international space station. J. Gravit. Physiol. 11, 81–92, 2004. Iversen, T.H., Svare, H., Fossum, K.R., et al. Development of a plant growth support system for experiments on the ISS. J. Gravit. Physiol. 9, 369–370, 2002. Johnsson, A., Solheim, B.G.B., Iversen, T.-H. Gravity amplifies and microgravity decreases circumnutations in Arabidopsis thaliana stems: results from a space experiment. New Phytologist 182, 621–629, 2009. Kamada, M., Omori, K., Yokoyama, R., et al. Preparation and outline of space-based studies on gravity responses and cell wall formation in plants. Biol. Sci. Space 23, 115–120, 2009. 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. Koizumi, K., Yokoyama, R., Kamada, M., et al. Reverse genetic approach to exploring genes responsible for cell-wall dynamics in supporting tissues of Arabidopsis thaliana under microgravity conditions. Biol. Sci. Space 21, 48–55, 2007. Link, B.M., Durst, S.J., Zhou, W., et al. Seed-to-seed growth of Arabidopsis thaliana on the international space station. Adv. Space Res. 31, 2237–2243, 2003. Merkys, A.J., Laurinavichys, R.S. Complete cycle individual development of Arabidopsis thaliana (L.) Heynh. Plants on board the Salyut-7 orbital station. Dokl Akad Nauk SSSR 271, 509–512, 1983.
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Millar, K.D.L., Kumar, P., Correll, M.J., et al. A novel phototropic response to red light is revealed in microgravity. New Phytologist 186, 648–656, 2010. Milov, M., Rusakova, G. Greenhouses in space-higher plants in closed ecological systems. Aviatsia Ei Cosmonautika 3, 36–37, 1980. Musgrave, M.E., Kuang, A., Porterfield, D.M. Plant reproduction in spaceflight environments. Gravit. Space Biol. Bull. 10, 83–90, 1997. Soga, K., Wakabayashi, K., Hoson, T., et al. Inhibition of reproductive growth of Arabidopsis in airtight vessels. Adv. Space Res. 23, 2037– 2040, 1999. Takahashi, H., Mizuno, H., Kamada, M., et al. A spaceflight experiment for the study of gravimorphogenesis and hydrotropism in cucumber seedlings. J. Plant Res. 112, 497–505, 1999. Tamaoki, D., Karahara, I., Nishiuchi, S., et al. Transcriprtome profiling in Arabidopsis inflorescence stems grown under hypergravity in terms of cell walls and plant hormones. Adv. Space Res. 44, 245– 253, 2009. Ueda, J., Miyamoto, K., Yuda, T., et al. Growth and development, and auxin polar transport in higher plants under microgravity conditions in space. BRIC-AUX on STS-95 space experiment. J. Plant Res. 112, 487–492, 1999. Waldron, K.W., Brett, C.T. Effects of extreme acceleration on the germination, growth and cell wall composition of pea epicotyls. J. Exp. Bot. 41, 71–77, 1990. Weigel, D., Glazebrock, J. Arabidopsis: A Laboratory Manual. CSHL Press, 2002. Wolverton, C., Mullen, J.L., Aizawa, S., et al. Inhibition of root elongation in microgravity by an applied electric field. J. Plant Res. 112, 493–496, 1999. Yano, S., Masuda, D., Kasahara, H., et al. Excellent thermal control ability of Cell Biology Experiment Facility (CBEF) for ground-based experiments and experiments onboard the Kibo Japanese experiment module of International Space Station. Biol. Sci. Space 26, 12–20, 2012.
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Advances in Space Research xxx (2012) xxx–xxx www.elsevier.com/locate/asr
Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station Sachiko Yano a,⇑, Haruo Kasahara b, Daisuke Masuda b, Fumiaki Tanigaki a Toru Shimazu c, Hiromi Suzuki c, Ichirou Karahara d, Kouichi Soga e, Takayuki Hoson e, Ichiro Tayama f, Yoshikazu Tsuchiya f, Seiichiro Kamisaka d a Japan Aerospace Exploration Agency, 2-1-1 Sengen, Tsukuba 305-8505, Japan Japan Manned Space Systems Corp, 1-1-26 Kawaguchi, Tsuchiura 300-0033, Japan c Japan Space Forum, 3-2-1 Kandasurugadai, Tokyo 101-0062, Japan d Department of Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555, Japan e Department of Biology, Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Osaka 558-8585, Japan f Chiyoda Advanced Solutions Corporations, 1-1-25 Shin-Urashima-cho, Yokohama 221-0031, Japan b
Received 25 May 2012; received in revised form 26 September 2012; accepted 1 October 2012
Abstract In 2004, Japan Aerospace Exploration Agency developed the engineered model of the Plant Experiment Unit and the Cell Biology Experiment Facility. The Plant Experiment Unit was designed to be installed in the Cell Biology Experiment Facility and to support the seed-to-seed life cycle experiment of Arabidopsis plants in space in the project named Space Seed. Ground-based experiments to test the Plant Experiment Unit showed that the unit needed further improvement of a system to control the water content of a seedbed using an infrared moisture analyzer and that it was difficult to keep the relative humidity inside the Plant Experiment Unit between 70 and 80% because the Cell Biology Experiment Facility had neither a ventilation system nor a dehumidifying system. Therefore, excess moisture inside the Cell Biology Experiment Facility was removed with desiccant bags containing calcium chloride. Eight flight models of the Plant Experiment Unit in which dry Arabidopsis seeds were fixed to the seedbed with gum arabic were launched to the International Space Station in the space shuttle STS-128 (17A) on August 28, 2009. Plant Experiment Unit were installed in the Cell Biology Experiment Facility with desiccant boxes, and then the Space Seed experiment was started in the Japanese Experiment Module, named Kibo, which was part of the International Space Station, on September 10, 2009 by watering the seedbed and terminated 2 months later on November 11, 2009. On April 19, 2010, the Arabidopsis plants harvested in Kibo were retrieved and brought back to Earth by the space shuttle mission STS-131 (19A). The present paper describes the Space Seed experiment with particular reference to the development of the Plant Experiment Unit and its actual performance in Kibo onboard the International Space Station. Downlinked images from Kibo showed that the seeds had started germinating 3 days after the initial watering. The plants continued growing, producing rosette leaves, inflorescence stems, flowers, and fruits in the Plant Experiment Unit. In addition, the senescence of rosette leaves was found to be delayed in microgravity. Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. Keywords: Arabidopsis; Life cycle; Microgravity; Rosette leaf senescence; Seed-to-seed; Temperature and humidity control
1. Introduction ⇑ Corresponding author.
E-mail address: [email protected] (S. Yano).
Over 40 years have passed since biological experiments began to be conducted in satellites and spaceships (Halstead
0273-1177/$36.00 Ó 2012 COSPAR. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asr.2012.10.002
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
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and Dutcher, 1987; Ferl et al., 2002). A variety of plants, both monocotyledons and dicotyledons, have been grown in space to see the effect of microgravity on plant growth and development. Since 1975, the USSR has grown crops such as wheat, onion, oat, pea, cabbage, radish, and lettuce in its spaceships to explore the possibility of agriculture in space (Milov and Rusakova, 1980). Although Arabidopsis is not a crop plant, it has been often used in recent years for plant experiments carried out onboard space shuttles to study the effect of gravity on plants from the perspective of basic sciences. Arabidopsis also has several other advantages: it is the first higher plant whose genome has been deciphered (Cao et al., 2011); it is a compact plant with a short life cycle; and it offers sample variability in the form of mutants and transgenic strains. The seed-to-seed life cycle under microgravity is important for space agriculture. Of the two stages in the life cycle of a typical higher plant, namely the vegetative stage and the reproductive stage, a great deal of information is available on the vegetative stage in space (Hoson et al., 1999; Kiss et al., 2000; Takahashi et al., 1999; Ueda et al., 1999; Wolverton et al., 1999), but very little is known about the reproductive stage (Link et al., 2003; Merkys and Laurinavichys, 1983; Musgrave et al., 1997), and even those results are controversial. On Salyut-7, the reproductive stage in Arabidopsis was delayed and many empty seeds were produced (Merkys and Laurinavichys, 1983), whereas on the International Space Station (ISS) the plant produced fertile seeds (Link et al., 2003). These experimental data obtained in space have been compared with those obtained in 1 G ground-based experiment. In addition to gravity, other environmental conditions in space, such as radiation and the magnetic field, are different from those on the ground. This fact indicates that a facility for plant growth experiments with a centrifuge to produce artificial gravity in the ISS is a prerequisite to analyzing the effect of microgravity on the full life cycle of a plant. The Japan Aerospace Exploration Agency (JAXA) began developing its Cell Biology Experiment Facility (CBEF) with two working areas, namely a microgravity compartment and an artificial gravity compartment on a turntable (centrifuge), after a few conceptual studies in 1993 and 1994. The facility was completed in 2001 as a common resource to support life science experiments with microorganisms, cells, tissues, small animals, and plants in the Japanese Experiment Module (JEM), named Kibo, of the ISS (Ishioka et al., 2004; Yano et al., 2012). The Plant Experiment Unit (PEU) installed inside the CBEF was designed to support the seed-to-seed life cycle of Arabidopsis (Ishioka et al., 2004). In 1997, JAXA initiated the development of a preliminary design of the PEU to support plant growth for an extended period of more than 2 months after analyzing the experimental design of the Space Seed experiment of 1993. The experimental design was specified in 1999, and the design of the PEU to meet the specifications was completed in 2000. A test model of the PEU was fabricated by 2003. The complete specification
of the model is described in an earlier paper (Ishioka et al., 2004). From 2004 to 2008, further ground-based growth experiments with Arabidopsis were carried out using the model, which was part of the CBEF, or in an incubator under a controlled environment to improve the model. The onboard model of the PEU was completed in 2008. In April 2009, 8 units of the flight model, in which dry Arabidopsis seeds had been glued with gum arabic to the rock wool seedbed, were handed over to the National Aeronautics and Space Administration (NASA). On August 28, 2009, the 8 units were transferred to the ISS by STS-128. In Kibo onboard the ISS, the Space Seed experiment began on September 10, 2009 with an initial supply of 20 ml water to the seedbed and was terminated 2 months later on November 11, 2009. In space, Arabidopsis seeds germinated 3 days after the initial watering and the entire life cycle was completed inside the PEU. Arabidopsis plants harvested in Kibo were brought back to Earth by the space shuttle STS-131 (19A) on April 19, 2010. The present report describes the Space Seed experiment with particular reference to improvements in the PEU and in the CBEF, and to the performance of the model and of the modified CBEF in Kibo while onboard the ISS. 2. Improvement in the PEU The specifications for the onboard model of the PEU were nearly identical to those of the test model (Ishioka et al., 2004); the major difference was that the onboard model had a better system for determination of the water content of the seedbed and a plate to cover the seedbed. The outer dimensions of the onboard model were 95 240 170 mm (H W D) (Fig. 1A and B). The unit consisted of a plant growth chamber made of transparent polycarbonate (H W D = 60 60 50 mm, outer size) (Fig. 2) and illuminated with an LED matrix (red and blue radiation in a ratio of 3:1; red LED with luminous spectrum peak at 660 nm [LA-AS-30236, Iwasaki, Japan]; blue LED with a luminous spectrum peak at 470 nm [LAAS-30257, Iwasaki, Japan]). The illumination level on the surface of the seedbed was 110 lmol m 2 s 1. The chamber was fitted with an automated watering system, a humidity control system, and a CCD camera (Fig. 1B). To obtain images of the inside of the plant growth chamber, white LED with spectra peaks at 440 and 550 nm (NSSW100B, Toa, Japan) were used instead of the red and blue LEDs. From 2004 to 2009, we conducted a series of ground-based experiments using the model of the PEU installed at the CBEF at Tsukuba Space Center or in an incubator (Biotron LPH-200-RDDS; Nihon Medical & Chemical Instruments Co. Ltd., Osaka, Japan) at University of Toyama in order to develop a model of the PEU suitable for onboard use. 2.1. Watering system A block of rock wool (NICHIAS Co. Ltd., Tokyo, Japan) placed in the bottom compartment of the growth
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Fig. 1. Onboard model of the Plant Experiment Unit (PEU). A, the external view of the PEU (H W D = 95 240 170 mm) with the cover; B, the inside view of the PEU without the cover. r, LED plate; s, heat sink; t, CCD camera; u, light diffuser; v, plant growth chamber.
analyzer, which measures the IR rays reflected from a target plate placed in contact with one side of the block (Fig. 3). The IR beam hits the black plastic plate 4 mm above the bottom of the plant growth chamber. To facilitate the entry of water and to avoid root invasion into the IR hit-point through the bottom of the target plate, the basal part of the plate was sealed with water-permeable sponge made of polyvinyl alcohol, Berita (number: EB (D); Aion Co. Ltd., Osaka, Japan) (Fig. 3). 2.2. Cover plate on seedbed
Fig. 2. Onboard model of the plant growth chamber. The inner size of the chamber is H W D = 48 56 46 mm. r, stainless steel cover plate with 24 holes; s, IR-target plate; t, polyvinyl alcohol sponge, Berita.
chamber was used as the seedbed (Fig. 3), which was “irrigated” through a tube inserted into the block. In the test model that had no infrared (IR) target plate, water content in the block of rock wool was estimated with an IR moisture analyzer (IR LED, L7850–01; receiver, G8370–01; Hamamatsu Photonics, Japan) by measuring the intensity of IR rays reflected from the side surface of the block of rock wool. However, the uneven fiber density of the rock wool block seriously affected accurate determination of water content, making it difficult to maintain the water content of the seedbed at the desired level. A new technique was developed to overcome this difficulty. The technique involved measuring the amount of excess water with the
In the test model, a transparent and thin plastic plate with 24 small holes was placed on the surface of the seedbed to reduce the loss of water through evaporation (Ishioka et al., 2004). One dry Arabidopsis seed was sown in each hole. Most of the seedlings emerged through the hole but some seedlings failed to do so because of the wider gap between the surface of the rock wool and the plastic plate due to deformation of the thin plastic plate. Therefore, in later models, the plastic plate was replaced with the one made of stainless steel (Figs. 2 and 3). This modification ensured seedling emergence from the holes, probably through enhancing phototropic growth of the hypocotyl. 2.3. Humidity control inside the CBEF The growth experiments were conducted using the PEU placed either in the CBEF or in the Biotron (temperature, 23 °C; relative humidity, 50%). Seeds of Arabidopsis thaliana (L.) Heynh. ecotype Columbia were surface-sterilized with 70% ethanol. After evaporation of the ethanol, dry seeds were glued with gum arabic (1%, w/v) to the seedbed through the holes of the cover plate. The plant growth chamber contained 20 mg HYPONeX powder (N:P:K = 6.5:6.0:19.9; no other minor nutrients) (HYPONeX JAPAN Corp. Ltd., Osaka, Japan). Temperature
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Fig. 3. Onboard model of the seedbed of the plant growth chamber. A, the structure of the bottom case of the plant growth chamber; B, the assembly of the seedbed. r, IR-target plate; s, polyvinyl alcohol sponge; t, water inlet; u, stainless steel cover plate; v, block of rock wool (H W D = 10 52 42 mm).
inside the growth chamber was maintained at 23.5 °C and relative humidity at 70–80%. Germination began on the third day after the initial watering and was followed by the development of rosette leaves. Bolting began about 3 weeks from the start of the experiment; the first flower was observed at about 4 weeks; fruit formation began at about 5 weeks; and fruits began turning from green to brown at about 6 weeks. Plant growth in the CBEF differed from that in the Biotron on several counts (Table 1), even though the two
Table 1 Vegetative and reproductive growth of Arabidopsis plants grown in the CBEF without desiccant or in the Biotron.
Number of germinated seeds Number of rosette leaves per plant Length of inflorescences, mm Number of flowers per plantb Number of fruits per plant Number of seeds per plant Number of seeds per fruit Germination rate,%
CBEF without desiccant
Biotron
21.3 ± 1.5 10.9 ± 1.1 99.8.1 ± 15.2 a 7.7 ± 1.2 9.4 ± 1.2 a 2.1 ± 0.4 a 0.2 ± 0.1 a 21.5 ± 10.5
23.3 ± 0.5 9.1 ± 1.5 78.1 ± 4.2 3.7 ± 0.1 5.4 ± 0.2 89.5 ± 6.6 16.3 ± 1.6 89.7 ± 3.0
Levels of significance were calculated by Student’s t test. a P < 0.05 for CBEF vs. Biotron (n = 6). b Number of flowers is the sum of the number of flowers and the number of fruits.
environments led to no significant differences in the number of germinated seeds, the number of rosette leaves on each plant, the length of the inflorescence, and the number of fruits on each plant. The plants grown in the CBEF produced more flowers but markedly fewer seeds, and the average number of seeds in each fruit was much lower in these plants. Lastly, the viability of seeds was also affected; germination percentage in seeds of plants that had been grown in the CBEF was very low. These results indicated that reproductive growth related to seed formation is suppressed in the CBEF. High humidity is reported to suppress reproductive growth in Arabidopsis (Soga et al., 1999; Weigel and Glazebrock, 2002). The plant growth chamber has two filter ports, one for the air inlet and the other for the air outlet. The outlet port is connected to a ventilation pump by a Teflon tube to remove the humid air inside the plant growth chamber. When the ventilation pump is running, air inside the CBEF comes to the plant growth chamber through the air inlet. Humidity inside the plant growth chamber is controlled by the ventilation pump and by the CBEF’s humidity control system (the humidifier). The ventilation pump is switched on when relative humidity inside the growth chamber reaches 80% and switched off when it falls to 70%. Relative humidity inside the Biotron was kept at 50% throughout the experiment by deploying a humidifier and a dehumidifier to keep relative humidity inside the growth chamber between 70 and 80%. However, unlike the
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
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Fig. 4. Kinetic changes in humidity inside the plant growth chamber of the PEU and the CBEF. A, humidity change without desiccant boxes containing calcium chloride in the CBEF; B, kinetic changes in humidity inside the plant growth chamber and the CBEF. Desiccant boxes were installed in the CBEF 4 days after the start of the growth experiment. Red lines (PEU) in the figures represent relative humidity inside the growth chamber; black lines (CBEF) represent relative humidity inside the CBEF.
Table 2 Vegetative and reproductive growth of Arabidopsis plants grown in the CBEF with desiccant or in the Biotron.
Number of germinated seeds Number of rosette leaves per plant Length of inflorescences, mm Number of flowers per planta Number of fruits per plant Number of seeds per plant Number of seeds per fruit Germination,%
CBEF with desiccant
Biotron
23.3 ± 1.5 8.0 ± 0.7 65.2 ± 1.5 3.2 ± 0.3 3.0 ± 0.2 27.0 ± 2.7 9.4 ± 0.7 89.9 ± 4.4
23.0 ± 0.6 10.0 ± 0.1 57.3 ± 4.4 5.2 ± 0.9 3.1 ± 0.3 17.8 ± 7.3 5.9 ± 2.6 69.2 ± 22.5
No statistically significant differences in the values for CBEF and Biotron (n = 3). a Number of flowers is the sum of the number of flowers and the number of fruits.
Biotron, the CBEF does not have a dehumidifier. As a result, relative humidity inside the CBEF often exceeded 80% (Fig. 4A) because of the exhaust from the PEUs, leading, in turn, to high humidity inside the plant growth chamber, to more than 90% which may cause sterility in Arabidopsis (Weigel and Glazebrock, 2002). As a countermeasure, four aluminum boxes filled with a desiccant were installed in the CBEF. Each box had 20 desiccant bags, each bag containing 6 g of solid calcium chloride (Hakugen, Tokyo, Japan). The bags are made of a special grade of filter paper that is permeable to gas but impermeable to water. The desiccant boxes lowered the humidity inside the CBEF substantially and maintained it lower than 40% throughout the experiment (Fig. 4B). The growth experiment done to see the effect of desiccant on growth and development of Arabidopsis plants indicated that there was no significant difference in growth parameters such as seed production and the ability of produced seeds to germinate between the Biotron and the CBEF with desiccant boxes (Table 2).
3. Experiments onboard Kibo On April 7 and 8, 2009, at the Tsukuba Space Center, dry seeds were glued with gum arabic to the seedbed made of rock wool in the plant growth chamber and the PEUs with Arabidopsis seeds were transported to the Kennedy Space Center. The equipment for the onboard chemical fixation of Arabidopsis, namely Kennedy Space Center Fixation Tubes (KFT; Bionetics Corporation, FL, USA) containing the fixative, were delivered to NASA on August 26, 2009, and the PEUs and KFTs were carried to the ISS by the space shuttle mission STS-128 (17A) on August 28, 2009. The Space Seed experiment onboard Kibo was monitored and controlled from the Tsukuba Space Center. The PEUs were placed in microgravity compartment on an a centrifuge (350 mm in diameter and turned at 87 ± 1 rpm) in the CBEF (Ishioka et al., 2004). The magnitude of gravity was ca. 1.1 g at the surface of seedbed, and ca. 0.7 g at the top of plant growth chamber. The CBEF controls such environmental factors as temperature and humidity as well the power and signal lines including the video image line. The Space Seed experiment began on September 10 with the initial supply of 20 ml water to the block of rock wool (the seedbed) in the plant growth chamber. 3.1. Temperature control The CBEF has 10 connectors for the PEU, 6 for the microgravity compartment and 4 for the centrifuge compartment. For the Space Seed experiment, 8 connectors were used, 4 each for the microgravity compartment and the centrifuge compartment. Ambient temperature in the microgravity compartment is almost the same as that in the centrifuge compartment (Ishioka et al., 2004). The slight difference in temperature caused by differences in the mounting positions of the PEUs was compensated for
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target plate, making it impossible to detect the level of water recognition. In response, a fixed volume of water was supplied every day by uplinking the appropriate program file.
Temperature inside the PEU (Degree C)
24.5
24.0
23.5
3.3. Humidity control 007
004
23.0
22.5 0
7
14
21
28
35
42
49
57
63
Incubation time (days) Fig. 5. Changes in average temperature inside the plant growth chamber of 8 PEUs installed in the CBEF. The six accidental stops of power from the CBEF between days 33 and 44 caused a drop in temperature in the plant growth chamber, but in every case, the temperature soon returned to normal. Gravitational condition of each plant growth chamber: 001 to 004, microgravity compartment; 005 to 008, artificial gravity compartment (ca. 1.1 g at the surface of seedbed and ca. 0.7 g at the top of the plant growth chamber).
by a 0.5 W heater in the PEU. Fig. 5 shows that temperature in each plant growth chamber was maintained at 23.5 ± 0.1 °C throughout the growth period (62 days). Although malfunction stopped the operation of the PEU occasionally (Table 3), the drop in temperature was very small, and the temperature soon returned to 23.5 °C. 3.2. Watering system After the initial watering, which took 20 min, an infrared moisture analyzer was operated every 10 min and the water pump delivered 0.17 ml of water in 10 s only when the excess water was below the stipulated level. Watering was carried out, on average, 6–14 times a day until day 32 and 16–17 times a day from day 33 to day 53. The total volume of water supplied to each plant growth chamber varied slightly (data not presented). A downlinked image taken 3 h after the start of the experiment showed a drop of water, about 5 mm in diameter, on the surface of the cover plate in one of the plant growth chambers in the microgravity compartment. However, the drop was not seen in the next image downlinked 6 h later and may have evaporated or was absorbed by the rock wool. During the experiment, in one of plant growth chambers, roots invaded the IR hit-point of the
Relative humidity inside the CBEF increased gradually from about 30% to 60% until day 21 because of the humid exhaust from the plant growth chamber. Introducing the desiccant boxes into the CBEF on day 20 decreased the humidity dramatically (Fig. 6A) but it began to increase gradually until it reached 70%. On day 35, fresh desiccant boxes were introduced, and this led to a rapid drop in relative humidity until it reached about 60%. However, the effect of desiccant on humidity declined with time. Relative humidity inside the growth chambers was maintained between 70 and 80% until day 27, the average level being 75% (Fig. 6B). After that, humidity inside the plant growth chambers fluctuated greatly. The ground-based experiments had indicated that it is difficult to maintain the relative humidity inside the plant growth chamber at 75% if its level inside the CBEF is about 70%. This turned out to be true also onboard Kibo. We attempted to open the door of the CBEF by requesting an astronaut to do so to exchange the humid air inside the CBEF for the dry air in the Kibo cabin, but this maneuver decreased the relative humidity inside the plant growth chamber only by 1%. 3.4. Malfunctions In the later phase of the Space Seed experiment, power supply to the PEUs on a centrifuge was sometimes interrupted (Table 3). This malfunction suspended operations such as LED lighting, heating, and working of the ventilation pump and the water pump. However, the centrifuge continued to rotate regardless of the malfunction. The cumulative downtime of environmental controls in the PEUs on the centrifuge was approximately 28.5 h. 3.5. Plant growth and development onboard Kibo Downlinked images from Kibo showed that cotyledons first appeared 3 days after the initial watering. Average
Table 3 Actions for PEU malfunction during the Space Seed experiment in Kibo onboard the ISS. Experimental time, days
Times/day
Situation
Primary action
33
Once
Power-off in 2 PEUs on a turntable
The function of the PEUs both in the 1g and lg compartment was restored by restarting the PEU-control system.
36
Twice
No downlinking of images from 2 PEUs on a centrifuge
Image acquisition was given up after 3 trials. Images were taken with a digital camera on the last day of the experiment.
37 40 41
Twice Once Twice
The function of PEUs on a turntable was stopped by the reset error (energized state, automatic operation stopped).
Operation of the PEUs was restarted by commands from the ground.
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
S. Yano et al. / Advances in Space Research xxx (2012) xxx–xxx
A Humidity inside the CBEF (%)
Humidity inside the PEU (%)
B
0
7
14
21
28
35
42
49
56
63
7
90 85
007
004
80
002
75 70
008
65 60 0
7
Incubation time (days)
14
21
28
35
42
49
56
63
Incubation time (days)
Fig. 6. Changes in average relative humidity inside the CBEF and PEUs onboard ISS. A, Desiccant boxes were renewed on days 20 and 34; B, Changes in average relative humidity inside each plant growth chamber. Gravitational condition of each plant growth chamber: 001 to 004, microgravity compartment; 005 to 008, artificial gravity compartment (ca. 1.1 g at the surface of seedbed and ca. 0.7 g at the top of the plant growth chamber).
germination percentage by the end of day 10 was 97%. There was no significant difference in the time taken for germination between the experiments on ground and those onboard Kibo. After germination, the plants produced, in succession, rosette leaves, inflorescence, flowers, and fruits
(Fig. 7). Fruit formation was also observed in Kibo irrespective of the magnitude of gravity. The rosette leaves under microgravity remained dark green for longer as compared with plants grown on the centrifuge (Fig. 7), indicating that microgravity had delayed leaf senescence.
4. Discussion
Fig. 7. Bolting of inflorescences in Arabidopsis plants grown for 28 days in Kibo. A and B, plants grown under microgravity; C and D, plants grown under artificial gravity on a centrifuge. The magnitude of artificial gravity was about 1.1 G at the surface of seedbed, and 0.7 G at the top of the plant growth chamber.
The first Japanese announcement of opportunity (AO) for biological experiments aboard space flights was made in 1992. The purpose of this AO was to develop Japanese experimental facilities and equipment. The Space Seed experiment was selected as the model experiment in plant cultivation by the first Japanese AO in 1993, together with four other plant experiments, namely Rice (Hoson et al., 1999), Auxin (Ueda et al., 1999), Root (Wolverton et al., 1999), and PEGT, an abbreviation for peg Takahashi (Takahashi et al., 1999). Because of the delay in assembling the ISS, these four experiments were carried out using the space shuttle STS-95 in 1998 because the experiments could be completed within the flight time of the space shuttle. The Space Seed experiment, which was to last more than 2 months, could not be carried out until 2009 and had to be postponed until the completion of Kibo onboard the ISS. Some experiments on plant cultivation aboard the ISS include those by Berkovich et al. (2004), Iversen et al. (2002), and Link et al. (2003). JAXA executed two projects (Cell Wall and Resist Wall) related to plant sciences by using the European Modular Cultivation System (EMCS) onboard the ISS in 2008 (Hoson et al., 2007; Koizumi et al., 2007), but the watering system of the EMCS did not work well (Kamada et al., 2009). In the present Space Seed experiment, Arabidopsis plants were grown for 62 days in the PEU installed in the CBEF onboard Kibo. As planned, both temperature and humidity inside the plant growth chambers in the PEUs were maintained at the stipulated level for 33 days after the start of the experiment (Figs. 5 and 6B), that is, until the first malfunction (Table 3). Except for the image downlink of PEUs placed
Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002
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on the centrifuge, most malfunctions were restored by commands from the ground (Table 3). These malfunctions probably influenced subsequent changes in temperature and humidity inside some plant growth chambers on the centrifuge (Figs. 5 and 6B). It is highly probable that all malfunctions in the PEUs onboard Kibo were caused by malfunctions in the CBEF because the same malfunctions occurred simultaneously in two PEUs on the centrifuge. Power to PEUs on the centrifuge is provided from the CBEF through a slip ring, and reduced voltage in the power supply is believed to cause such malfunctions of PEUs on the centrifuge because voltage reduction from +5 V to less than +2.2 V turned out to induce the same malfunction of the PEUs. In July 2011, the centrifuge used in the Space Seed experiment was returned to Earth by the last flight of the space shuttle (STS-135). The continuity test of the centrifuge incorporated into the CBEF at Tsukuba Space Center indicated a flicker in the power supply of +5 V, indicating that the malfunction in the PEUs is caused by irregular power supply from the CBEF to PEUs through the slip ring. We have a plan to replace the centrifuge onboard Kibo by a new centrifuge with improved slip ring. In addition to the malfunction of environmental controls in the PEU, the increase in relative humidity inside the CBEF in the second half of the experiment after the short-term harvest on day 33 (Fig. 6A) may also have resulted in fluctuations in relative humidity inside some plant growth chambers (Fig. 6B). Relative humidity inside the CBEF was maintained below 50% in the ground-based experiment with desiccant boxes (Fig. 4B), whereas relative humidity inside the CBEF onboard Kibo continued to increase despite introducing fresh desiccant boxes two times (Fig. 6A); this was probably because the capacity of the desiccant to absorb moisture had been lowered by some unknown mechanism. As a countermeasure, in next plant cultivation experiment, we should prepare more desiccant boxes to maintain relative humidity inside plant growth chambers between 70 and 80% during an experiment. Downlinked images showed no significant difference in the time taken for germination among the different gravitational conditions, namely microgravity and artificial gravity in the CBEF onboard the ISS and the control on the ground. Hypergravity has been reported to show no substantial influence on germination in pea (Waldron and Brett, 1990), and clinorotation did not affect germination in Arabidopsis (Ishii et al., 1996). These reports indicate that germination is unaffected by gravity. Downlinked images also showed that Arabidopsis plants produce rosette leaves and elongated the inflorescence with flowers and fruits, indicating that we succeeded to complete seed-to-seed plant cultivation under both microgravity and artificial gravity in CBEF onboard Kibo, Japanese experiment module. The EMCS (Brinckmann, 2005) onboard the ISS has been used for analyzing mechanisms of circumnutation
(Johnsson et al., 2009) and phototropism (Millar et al., 2010) . At the stage of inflorescence bolting, Arabidopsis rosette leaves grown in the PEU onboard Kibo were different in color from those grown in the EMCS onboard ISS: Chlorophyll loss took place in rosette leaves in the PEU (Fig. 7), while the leaves in the EMCS showed green in color (see Fig. 1, Johnsson et al. 2009). In the ground-based experiments with the PEU, yellowing of rosette leaves set in about 20 days after sowing, prior to the initiation of bolting, nevertheless Arabidopsis plants could achieve complete their life cycle in the PEU. This was also the case for growth experiments in Kibo. Such early senescence of the rosette leaves may be a kind of stress response caused by dense planting in the PEU, as compared with in the EMCS (Johnsson et al. 2009). Fig. 7 also showed the delayed senescence of rosette leaves under microgravity. Ethylene is known to be the plant hormone that stimulates leaf senescence (Hoffman and Yang, 1982). Physical stress induces the biosynthesis of ethylene in plants, and the weightless condition under microgravity in the present experiment probably lowered the physical stress on rosette leaves, resulted in decreased biosynthesis of ethylene, and, in turn, delayed senescence. In addition, hypergravity was reported to induce the expression of genes related to ethylene response and biosynthesis (Tamaoki et al., 2009). Further results of physiological, morphological, genetic, and physical analyses of Arabidopsis plants brought back by the space shuttle mission STS-131 (19A) will be reported elsewhere. Acknowledgments We wish to acknowledge the Japan Space Forum and the Japan Manned Space Systems Corp. for their help in research coordination, helpful discussions, and technical support in matters such as the CBEF and telemetry operations, image processing, and file transfer. Thanks are also due to the Chiyoda Advanced Solutions Corporation for hardware design of the PEU and technical support in performing the Space Seed experiment. We are also grateful to NASA for international cooperation and assistance. References Berkovich, Y.A., Krivobok, N.M., Sinyak, Y.Y., et al. Developing a vitamin greenhouse for the life support system of the international space station and for future interplanetary missions. Adv. Space Res. 34, 1552–1557, 2004. Brinckmann, E. ESA hardware for plant research on the international space station. Adv. Space Res. 36, 1162–1166, 2005. Cao, J., Schneeberger, K., Ossowski, S., et al. Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nat. Genet. 43, 956– 963, 2011. Ferl, R., Wheeler, R., Levine, H.G., et al. Plants in space. Current Opinion Plant Biol. 5, 258–263, 2002. Halstead, T.W., Dutcher, F.R. Plants in Space. Annu. Rev. Plant Physiol. 38, 317–345, 1987. Hoffman, N.E., Yang, S.F. Enhancement of wound-induced ethylene synthesis by ethylene in preclimacteric cantaloupe. Plant Physiology. 69, 317–322, 1982.
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Please cite this article in press as: Yano, S., et al. Improvements in and actual performance of the Plant Experiment Unit onboard Kibo, the Japanese experiment module on the international space station. J. Adv. Space Res. (2012), http://dx.doi.org/10.1016/j.asr.2012.10.002