Seiichiro Kamisaka

Evaluation of the three-dimensional clinostat as a simulator of weightlessness

Abstract. Concerns regarding the reliability of slow- and fast-rotating uni-axial clinostats in simulating weightlessness have induced the construction of devices considered to simulate weightlessness more adequately. A new three-dimensional (3-D) clinostat equipped with two rotation axes placed at right angles has been constructed. In the clinostat, the rotation achieved with two motors is computer-controlled and monitored with encoders attached to the motors. By rotating plants three-dimensionally at random rates on the clinostat, their dynamic stimulation by gravity in every direction can be eliminated. Some of the vegetative growth phases of plants dependent on the gravity vector, such as morphogenesis, are shown to be influenced by rotation on the 3-D clinostat. The validity of 3-D clinostatting has been evaluated by comparing structural parameters of cress roots and Chara rhizoids obtained under real microgravity with those obtained after 3-D clinostatting. The parameters analyzed up to now (organization of the root cap, integrity and polarity of statocytes, dislocation of statoliths, amount of starch and ER) demonstrate that the 3-D clinostat is a valuable device for simulating weightlessness.

Osmotic Stress Suppresses Cell Wall Stiffening and the Increase in Cell Wall-Bound Ferulic and Diferulic Acids in Wheat Coleoptiles

The relationship between the mechanical properties of cell walls and the levels of wall-bound ferulic (FA) and diferulic (DFA) acids was investigated in wheat (Triticum aestivum L.) coleoptiles grown under osmotic stress (60 mM polyethylene glycol [PEG] 4000) conditions. The cell walls of stressed coleoptiles remained extensible compared with those of the unstressed ones. The contents of wall-bound FA and DFA increased under unstressed conditions, but the increase was substantially reduced by osmotic stress. In response to PEG removal, these contents increased and reached almost the same levels as those of the unstressed coleoptiles. A close correlation was observed between the contents of FA and DFA and the mechanical properties of cell walls. The activities of phenylalanine ammonia-lyase and tyrosine ammonia-lyase increased rapidly under unstressed conditions. Osmotic stress substantially reduced the increases in enzyme activities. When PEG was removed, however, the enzyme activities increased rapidly. There was a close correlation between the FA levels and enzyme activities. These results suggest that in osmotically stressed wheat coleoptiles, reduced rates of increase in phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities suppress phenylpropanoid biosynthesis, resulting in the reduced level of wall-bound FA that, in turn, probably causes the reduced level of DFA and thereby maintains cell wall extensibility.

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.

Growth promotion and an increase in cell wall extensibility by silicon in rice and some other Poaceae seedlings.

Abstract The effect of silicon on organ growth and its mechanisms of action were studied in rice (Oryza sativa L. cv. Koshihikari), oat (Avena sativa L. cv. Victory), and wheat (Triticum aestivum L. cv. Daichino-Minori) seedlings grown in the dark. Applying silicon in the form of silicic acid to these seedlings via culture solution resulted in growth promotion of third (rice) or second (oat and wheat) leaves. The optimal concentration of silicon was 5–10 mM. No growth promotion was observed in early organs, such as coleoptiles or first leaves. In silicon-treated rice third leaves, the epidermal cell length increased, especially in the basal regions, without any effect on the number of cells, showing that silicon promoted cell elongation but not cell division. Silicon also increased the cell wall extensibility significantly in the basal regions of rice third leaves. These results indicate that silicon stimulates growth of rice and some other Poaceae leaves by increasing cell wall extensibility.

Increased molecular mass of hemicellulosic polysaccharides is involved in growth inhibition of maize coleoptiles and mesocotyls under hypergravity conditions.

Zeamays L. cv. Cross Bantam T51) coleoptiles and mesocotyls was suppressed by hypergravity at 30 g and above. Acceleration at 300 g significantly decreased the mechanical extensibility of cell walls of both organs. Hypergravity increased the amounts of hemicellulose and cellulose per unit length in mesocotyl walls, but not in coleoptile walls. The weight-average molecular masses of hemicellulosic polysaccharides were also increased by hypergravity in both organs. On the other hand, the activities of β-glucanases extracted from coleoptile and mesocotyl cell walls were decreased by hypergravity. These results suggest that the decreased activities of β-glucanases by hypergravity cause an increase in the molecular mass of hemicellulosic polysaccharides of both organs. The upshift of molecular mass of hemicellulosic polysaccharides as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of maize coleoptiles and mesocotyls.

Graviperception in growth inhibition of plant shoots under hypergravity conditions produced by centrifugation is independent of that in gravitropism and may involve mechanoreceptors.

Hypergravity caused by centrifugation inhibits elongation growth of shoots by decreasing the cell wall extensibility via suppression of xyloglucan breakdown as well as by the thickening of cell walls. The mechanism of graviperception in hypergravity-induced growth inhibition was investigated in Arabidopsis [A. thaliana (L.) Heynh.] hypocotyls and azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. Hypergravity caused growth suppression in both sgr1-1 and pgm1, which are Arabidopsis mutants deprived of gravitropism, as in wild-type plants, suggesting that the graviperception in hypergravity-induced growth inhibition of shoots is independent of that in gravitropism. Hypergravity had no effects on growth of azuki bean epicotyls or Arabidopsis hypocotyls in the presence of lanthanum or gadolinium, which are blockers of mechanoreceptors. Moreover, lanthanum or gadolinium at the same concentration had no influence on gravitropism of azuki bean epicotyls and Arabidopsis hypocotyls. Hypergravity had no effects on cell wall extensibility and affected neither xyloglucan metabolism nor the thickness of cell walls in the lanthanum- or gadolinium-treated azuki bean epicotyls. Lanthanum or gadolinium inhibited the hypergravity-induced increase in the pH of the apoplastic fluid in the epicotyls, which is involved in the processes of the suppression of xyloglucan breakdown due to hypergravity. These findings suggest that plants perceive the hypergravity stimuli by mechanoreceptors in the plasma membrane, and utilize the perceived signal to regulate the growth rate of their shoots.

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.

Morphogenesis of rice and Arabidopsis seedlings in space.

Oryza sativa L.) and Arabidopsis (A. thaliana L.) were cultivated for 68.5 hr in the RICE experiment on board during Space Shuttle STS-95 mission, and changes in their growth and morphology were analyzed. Microgravity in space stimulated elongation growth of both rice coleoptiles and Arabidopsis hypocotyls by making their cell walls extensible. In space, rice coleoptiles showed an inclination toward the caryopsis in the basal region and also a spontaneous curvature in the same direction in the elongating region. These inclinations and curvatures were more prominent in the Koshihikari cultivar compared to a dwarf cultivar, Tan-ginbozu. Rice roots elongated in various directions including into the air on orbit, but two thirds of the roots formed a constant angle with the axis of the caryopsis. In space, Arabidopsis hypocotyls also elongated in a variety of directions and about 10% of the hypocotyls grew into the agar medium. No clear curvatures were observed in the elongating region of Arabidopsis hypocotyls. Such a morphology of both types of seedlings was fundamentally similar to that observed on a 3-D clinostat. Thus, it was confirmed by the RICE experiment that rice and Arabidopsis seedlings perform an automorphogenesis under not only simulated but also true microgravity conditions.

Mechanoreceptors rather than sedimentable amyloplasts perceive the gravity signal in hypergravity-induced inhibition of root growth in azuki bean.

Elongation of primary roots of azuki bean (Vigna angularis Ohwi et Ohashi) was suppressed under hypergravity conditions produced by centrifugation, such that the growth rate decreased in proportion to the logarithm of the magnitude of the gravity. The removal of the root cap did not influence the hypergravity-induced inhibition of root growth, although it completely inhibited the gravitropic root curvature. Lanthanum and gadolinium, blockers of mechanoreceptors, nullified the growth-inhibitory effect of hypergravity. These results suggest that the gravity signal for the hypergravity-induced inhibition of root growth is perceived independently from that of gravitropism, which involves amyloplasts as statoliths. Horizontal and basipetal hypergravity suppressed root growth as did acropetal hypergravity, all of which were nullified by the presence of lanthanum or gadolinium. These findings suggest that mechanoreceptors on the plasma membrane perceive the gravity signal independently of the direction of the stimuli and roots may utilise it to regulate their growth rate.

Effect of ferulic and p-coumaric acids on Oryza coleoptile growth and the mechanical properties of cell walls

Summary Ferulic or p-coumaric acids applied to Oryza seedlings grown under water decreased the rate of coleoptile elongation and increased the content of these monophenols and lignin in cell walls. Cell wall extensibility represented by an increase in the minimum stress-relaxation time and the relaxation rate decreased in response to the application of these phenolic acids. These facts suggest that the application of ferulic or p-coumaric acids increases the content of cell wall-bound phenolic compounds, which in turn decreases cell wall extensibility, resulting in inhibited cell growth.