Inhibition of reproductive growth of Arabidopsis in airtight vessels

Airtight vessels have various advantages for space experiments. However, Arabidopsis thaliana plants scarcely produced seeds when grown in such vessels. The mechanism by which reproductive growth is inhibited in airtight vessels was studied. The length of the flower stalk was shorter when the plants were grown in airtight vessels. Thus, there was a possibility that the inhibition of reproductive growth was due to the inhibition of vegetative growth. However, even when the plants which has grown under non-airtight conditions and has reached to the flowering stage were transferred to airtight vessels, silique formation was inhibited, suggesting that the airtight environment directly influences reproductive growth. In airtight vessels, anther dehiscence was inhibited, which appears to be the cause of inhibition of silique formation and seed development. Reproductive growth recovered when silica gel was added to the vessels. These results suggest that in airtight vessels, high humidity causes a suppression of anther dehiscence, resulting in the inhibition of reproductive growth. Therefore, the control of humidity by ventilation should be taken into consideration in designing a growth chamber for space experiments.     

Gravitational force regulates elongation growth of Arabidopsis hypocotyls by modifying xyloglucan metabolism.

Growth of dark-grown Arabidopsis hypocotyls was suppressed under hypergravity conditions (300 g), or was stimulated under microgravity conditions in space (Space Shuttle STS-95). The mechanical extensibility of cell walls decreased and increased under hypergravity and microgravity conditions, respectively. The amounts of cell wall polysaccharides (pectin, hemicellulose-I, hemicellulose-II and cellulose) per unit length of hypocotyls increased under hypergravity conditions, and decreased under microgravity conditions. The amount and the molecular mass of xyloglucans also increased under the hypergravity conditions, while those decreased under microgravity conditions. The activity of xyloglucan-degrading enzymes extracted from hypocotyl cell walls decreased and increased under hypergravity and microgravity conditions, respectively. These results indicate that the amount and the molecular mass of xyloglucans are affected by the magnitude of gravity and that such changes are caused by changes in xyloglucan-degrading activity. Modifications of xyloglucan metabolism as well as the thickness of cell walls by gravity stimulus may be the primary event determining the cell wall extensibility, thereby regulating the growth rate of Arabidopsis hypocotyls.     

Effects of gravistimuli on osmoregulation in azuki bean epicotyls

The effects of hypergravity on growth and osmoregulation were examined in dark-grown azuki bean epicotyls. Elongation growth of epicotyls was promptly suppressed by hypergravity at 300g. On the contrary, the increase in fresh weight of epicotyls during incubation was not suppressed by hypergravity at 300g at least up to 6 h. Also, the level of total osmotic solutes increased during epicotyl growth for 6 h, which was not affected by hypergravity. These results suggest that azuki bean epicotyls are capable of maintaining osmoregulation even under 300g conditions for a short period. On the other hand, the increase in fresh weight of epicotyls was suppressed, in addition to suppression of elongation growth, when seedlings were treated with 300g for 24 h. The increase in level of total osmotic solutes was also inhibited by 24 h hypergravity treatment, which was accounted by the reduced levels of organic solutes, such as sugars and amino acids. Furthermore, the dry weight of seeds decreased during incubation for 24 h, but the decrease was inhibited by hypergravity at 300g. Hypergravity treatment at 300g for 24 h also increased the pH value of apoplastic solution in epicotyls. Taken together, these results suggest that the translocation of organic solutes from the seed to epicotyls is inhibited by prolonged hypergravity treatment, which may underlie the suppression of epicotyl growth, and that the breakdown of H⁺ gradient across the plasma membrane in epicotyl cells may be at least partly involved in the reduction of organic solute accumulation under hypergravity conditions.     

Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls.

Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.     

Growth restoration in azuki bean and maize seedlings by removal of hypergravity stimuli.

Hypergravity stimuli, gravitational acceleration of more than 1 x g, decrease the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls by decreasing the cell wall extensibility via an increase in the molecular mass of matrix polysaccharides. An increase in the pH in the apoplastic fluid is hypothesized to be involved in the processes of the increase in the molecular mass of matrix polysaccharides due to hypergravity. However, whether such physiological changes by hypergravity are induced by normal physiological responses or caused by physiological damages have not been elucidated. In the present study, we examined the effects of the removal of hypergravity stimuli on growth and the cell wall properties of azuki bean and maize seedlings to clarify whether the effects of hypergravity stimuli on growth and the cell wall properties are reversible or irreversible. When the seedlings grown under hypergravity conditions at 300 x g for several hours were transferred to 1 x g conditions, the growth rate of azuki bean epicotyls and maize coleoptiles and mesocotyls greatly increased within a few hours. The recovery of growth rate of these organs was accompanied by an immediate increase in the cell wall extensibility, a decrease in the molecular mass of matrix polysaccharides, and an increase in matrix polysaccharide-degrading activities. The apoplastic pH also decreased promptly upon the removal of hypergravity stimuli. These results suggest that plants regulate the growth rate of shoots reversibly in response to hypergravity stimuli by changing the cell wall properties, by which they adapt themselves to different gravity conditions. This study also revealed that changes in growth and the cell wall properties under hypergravity conditions could be recognized as normal physiological responses of plants. In addition, the results suggest that the effects of microgravity on plant growth and cell wall properties should be reversible and could disappear promptly when plants are transferred from microgravity to 1 x g. Therefore, plant materials should be fixed or frozen on orbit for detecting microgravity-induced changes in physiological parameters after recovering the materials to earth in space experiments.     

Improvements in and actual performance of the Plant Experiment Unit onboard Kibo,the Japanese experiment module on the international space station

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.