Chapter 5 Plant Responses to Simulated Microgravity

Chapter 5

PLANT RESPONSES TO SIMULATED MICROGRAVITY

Yoshio Masuda. Seiichiro Kamisaka. Ryoichi Yamamoto. Takayuki Hoson. and Kazuhiko Nish itan i

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 I1. Three-Dimensional Clinostat Experiments . . . . . . . . . . . . . . . . . . . 112 A . TheInstrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 B . Root Growth on the Clinostat . . . . . . . . . . . . . . . . . . . . . . . . 114 C . Shoot Growth on the Clinostat . . . . . . . . . . . . . . . . . . . . . . . 115 D . Mechanical Properties of the Cell Wall . . . . . . . . . . . . . . . . . . . 116 E. Chemical Composition of the Cell Wall . . . . . . . . . . . . . . . . . . 117 In. Buoyancy Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 A . Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 B . Mechanical Properties of the Cell Wall . . . . . . . . . . . . . . . . . . . 119 C . Chemical Composition of the Cell Wall . . . . . . . . . . . . . . . . . . 120 111. Hypergravity Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 n! Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Advances in Space Biology and Medkine V d u w 4. pages 111.1245 Copyright 8 19w by JAI Press Inc AM rights of repdudion in any form reserved ISBN: 1-55938411-5 111

.

.

.

112

MASUDA, KAMISAKA, YAMAMOTO, HOSON, and NISHITANI

I. INTRODUCTION Green plants are essential for the survival of human beings and other living organisms on Earth as well as in space. During the past three decades space has been explored by manned space missions and much information has been obtained on the survival of earthly organismsin space.Much attentionhas been directedto the study of the behavior and survival of living organisms in space, particularly under microgravity conditions. In view of the fact that in hture long-term space missions astronauts will need plants as a source of food, it is of crucial importance: to know how plants grow and complete their life cycle under microgravity conditions. In general, plants are extremely sensitive to environmental factors, such as light or gravity. Plant response to gravity has been an important subject of study in plant physiology for a long time. Like other tropistic responses. gravitropic responses in plants consist of three stages, namely, (1) gravity perception, (2) transformationof the gravitational stimulus, and (3) gravitropic response. In roots, the mechanism of gravipercqtion is basically understood.' However, it is not yet well understood in shoots, stems, and coleoptiles. A major difficulty in the study of gravimpism is the fact that plants are constantly exposed to the 1-Gcondition on Earlh. For over a century attempts have been made to compensate the 1-G vector. The most widely used method is the use of the clinostat.U A standard clinostat consists of a plant container rotating around a horizontal axis at 2-3 rpm.Although the standard one-axis clinostat has bcen useful, we felt that an improved system is needed to provide a better simulation of microgravity conditions on Earth. We have therefore developed a new type of clinostat, which has two axes at right angle to each other, each axisrotating in random fashion! In the following, we describe the advantagesof this so-called threedimensional clinostat for the simulation of microgravity conditions for plant seedlings. Buoyancy provided by water immersion has been used for the study of the effects of the microgravity condition in humans. This technique has not been used in plant biology, since land plants are usually unable to surviveand grow under water. However, therc is an exceptionalland plant, namely rice, which grows better under water than in air.Its growthand metabolism in air and under water have been extensivelycompared.sIn this chapter we describe the results of some experiments with rice seedlings. In addition,we have studied the growth of plant seedlings in hypergravity by the use of a specially designed centrifuge."

I I . THREE-DIMENSIONAL CLINOSTAT EXPERIMENTS A. The Instrument

The threedimensional clinostat with its two right-angle axes is shown in Figure 1. The rotation around the two axes is provided by two geared stepping motors

Plant Responses to Simulated Microgravity

113

figure 1. The three-dimensional clinostat apparatus. L: illumination lamp, M:motor with encoder, SR: slipring, S: sample stage.

(UPD534-MHG-2B, General Motor Co.). A 660 x 340 mm sample stage and an illumination apparatus are attached to opposite sides of an inner frame at distances of 220 mm from the vertical rotation axis. This inner frame is rotated around the vertical axis by the first motor. It is attached to an outer frame, which is rotated around the horizontal axis by the second motor. The rotation of the motors is observed with encodersand an angle sensor attached to them. The electrical connections to motors,lamps, encoders,and a video terminal is provided by 6-way and 12-way sliprings. Onset, rate, and duration of rotation of the motors are controlled and observed by means of a personal computer. When the two motors operate at the same constant rate, plant samples seen from above decline up to 90°, but they are never reversed. The movement of samples observed from the side follows a similar trace. Under this condition, the unilateral influence of gravity is not compensated. When the two motors are rotated at the rate of 1:2,

114

MASUDA, KAMISAKA, YAMAMOTO, HOSON, and NlSHlTANI

the plant samples are reversed and the effect of gravity is compensated. However, the plant samples move only along a certain path. True compensation of the effect of gravity in three-dimensional terms can be achieved when the rate of rotation of the two motors is varied at regular intervals in random fashion according to a table of random numbers. The movement of samples observed from the side follows a similar trace. In the present study, the rotation rate of the two motors is changed at random from 2 to -2 rpm every 30 seconds. Because the average and the farthest distances between plant samples and rotation axis are 140 and 220 mm, respectively, the mean and the maximum centrifugal accelerationsgiven to the materials are 6.3 x l@ and 9.8 x 1@G, respectively. These values are below the graviperception threshold of primary roots of cress.'' The imbalance of the gravity distribution was less than 0.1% after 3 hours of rotation under these conditions.

B. Root Growth on the Clinostat The plants used in the 3-D clinostat experimentswere cress, maize, rice, pea, and azuki. Germination and growth rates of roots and shoots were not significantly influencedby the clinostat conditions,except that the growth of roots and epicotyls of azuki beans was slightly increased. Growth direction of shoots and roots of the different plants varied depending upon the plant species (Fig. 2). Cress seedlings of 16 hours after water imbibition showed a positive gravitmpic response and grew parallel to the gravity vector when placed on the ground. On the 3-D clinostat, on the other hand, germinated radicles curved spontaneously toward the seeds and maintained this arc shape for 2 to 3 hours. The arc was then opened gradually during the succeeding growth period. After about 30 hours following water imbibition,the roots formed a straight line in the direction of the root primordia. This spontaneous curvature of cress roots on the clinostat was observed without exception, regardless of the position and direction in which the seeds had been placed initially. Other plant seedlings behaved differently. In the germinated caryopses of maize and rice or the seeds of pea and azuki bean the roots also grew downward when placed on the ground, but on the 3-D clinostat the roots of maize grew in random directions both in a front view and in a side view of the embryos. In a front view, the angle of the rice roots on the clinostat shows a bimodal distribution with the 0'. The peak at 90' and 270'. In a side view, the rice roots mainly elongated at 9 data indicatethat rice roots rotated on the 3-D clinostatgrew parallel to the direction of the tip of the mot primordia. Roots of pea and azuki bean, placed on the 3-D clinostat. grew parallel to the direction of the tip of the primordia in the early stage of growth; later they grew in random directions both in front view and in side view of the embryos.

Plant Responses to Simulated Microgravity

Cress

Maize

115 Rice

Pea

Azuki

Figure 2. Effect of clinostat-rotationon the growth direction of etiolated seedlings of five different species.

C. Shoot Growth on the Clinostat

Coleoptiles of maize and rice, and epicotyls of pea and azuki bean, grew upward when placed on the ground. However, in a side view of the embryos, coleoptiles of maize and epicotyls of pea and azuki bean show a slight abaxial curvature (away from the caryopsis or seed) under this condition. On the 3-D clinostat, on the other hand, coleoptiles and epicotyls of these plant seedlings grew mostly along the direction of the tip of the primordia in front view of the embryos. In the side view (Fig. 2), maize coleoptiles exhibited an abaxial curvature, but at 72 hours the direction of the maize coleoptiles became dispersed because of an irregular bending of the coleoptile nodes. Rice coleoptiles showed an ad-axial curvature (toward the caryopsis) in a side view. Epicotyls of pea and azuki bean, like maize coleoptiles, exhibited an abaxial curvature. The curvature of coleoptiles and epicotyls increased with further growth. This behavior was observed without exception. regardless of the position and direction in which the seeds had been placed. The four species of plants can thus be divided into two groups in terms of the direction of growth on the 3-D clinostat. In rice, roots elongated in the direction of the tip of the mot primordia and the coleoptiles bent ad-axially. In contrast, roots of maize, pea, and azuki bean elongated in random directions,and their coleoptiles or epicotyls showed an ab-axial curvature. These results indicate that the direction of growth of plant organs changes on the 3-D clinostat. the direction of growth being an autotropism. A similar spontaneous autotropism has been observed on a horizontal clinostat in roots or coleoptiles of different However, autotropism has mostly been found in short-term

116

MASUDA, KAMISAKA, YAMAMOTO, HOSON, and NlSHlTANl

rotation of seedlings that were grown vertically on Earth, and the processes involved in this situation have not been analyzed in detail. Autotropism is often recognized as the reverse curvature which follows after other types of tropism." Nick and SchaferI3 made an additional analysis of the autotropism of maize coleoptiles that were placed on a horizontal clinostat. The curvature consists of an early abaxial phase followed by a long-lasting ad-axial phase. However, on the 3-D clinostat we observed only an ab-axial curvatureof maize coleoptiles from the early growth stage on. The mechanism by which autotropism is induced has not yet been clarified. The spontaneous curvature of the roots on the 3-Dclinostat occurred mainly in the transition zone between roots and shoots, while that of the shoots was observed in the elongation region. It thus appears that the mechanism of autotropism differs for roots and shoots. The trans-organ gradient of the orientation of cortical microtubules is not associated with the spontaneous curvature of maize coleoptiles on a horizontal clino~tat.'~ We have recently found some differences in the mechanical properties of the cell wall between the concave and the convex sides of maize coleoptiles grown on the 3-D clinostat4 D. Mechanical Properties of the Cell Wall

The mechanical properties of coleoptile cell walls were determined with a tensile-tester (RTM-25, Toyo Baldwin Co., Tokyo) connected to a computer (PC9801, NEC, Tokyo). Coleoptiles are fixed in methanol and rehydrated. They are then fastened between the upper and lower movable clamps, the distance between the two clampsbeing 3 mm. They are stretchedby lowering the clamp at 20 mdmin to produce a stress of 10 g. The clamp is stopped after the stress has reached the chosen value of 10 g. The stress is then allowed to decay until it is reduced to a certain value. This stress-relaxation process can be analyzed as a general Maxwell viscoelastic m0de1'~*''according to the equation:

S = R . log (r + Tm)/(r+To) + C where S is stress, t is time in sec., R and Care constants.'8 To, the minimum stress relaxation time, and R, the stress relaxation rate, are thought to represent the capacity of the cell wall to extend, i.e., cell wall loosening.'' Cell wall extensibility ( d g ) is determined%and recorded by the computer.Rice coleoptiles are cut into sections from tip to base, and the cell wall extensibilityof different coleoptile zones is determined. The results are shown in Table 1. The To and R values, representing the capacity of the cell wall to extend, were each the same in the ventral and dorsal flanks of the control maize coleoptiles. On the other hand, in clinostat coleoptiles which show autotropismthe cell wall of the convex, growing flank shows smaller values for To and R than the concave flank.

117

Plant Responses to Simulated Microgravity

Table 1. Effect of Clinostat Rotation Causing Autotropism on the Mechanical Properties and Composition of the Cell Wall Conrrnl Ventral

Dorsal

Clinostat Convex

Concave

16.1 2.9 0.83

16.2 2.9 0.84

14.4 2.7 0.91

17.5 3.2 0.84

13.2 97.2 54.4

11.0 78.7 44.4

8.0 86.1 53.9

9.3 85.3 53.9

10.0 1.3 9.7 5.4

10.0 1.1 7.9 4.4

10.0 0.8 8.6 5.4

10.0 0.9 8.5 5.4

7.8 59.3 32.9

8.2 58.7 33.1

5.4 58.1 36.5

6.3 57.4 36.3

25.3 31.2 4.5 8.5 30.5

25.6 30.8 4.4 8.7 30.5

26.5 32.4 3.8 8.6 28.7

25.5 32.3 3.7 8.2 30.3

To

R Ext

CWPS (pglsegment) Pectin

HC Cellulose

cwps (Fdrn) Length (rn) Pectin

HC CeUulOsc cwps @RD) Peain

HC Cellulose Sugar composition ( w e ) Am XYl

Man Gal

Glc Nou:

-

-

-

Abbreviclrionr: 2 . minimum strcs.~relaxation timc in msec: R stress relaxationrate in %; EXI cell wed exundbility in mmll0g; CWPS a l l wall polysacchmidcs; HC - hemicellulou; Am d i n o r ; X y l Gal @UOSC; Glc glwm. XYIOSC; Mm

-

-

-

-

-

This indicates that the growing side of the cell wall is loosened in the clinostat coleoptiles. This phenomenon may thus be involved in the autotropismmechanism. E. Chemical Composition of the Cell Wall

Changes in the chemical nature of the cell wall may be one of the factors that induce autotropism in a microgravity environment. Cell wall polysaccharides are fractionated in the hemicellulose and cellulose fractions by a modification of the method of Nishitani and Ma~uda.2~ The results are shown in Table 1. The cell wall composition was not significantly different for the two sides, both in the control coleoptiles and in the clinostat coleoptiles. This suggests that the cell wall components determined are not involved in the autotropism mechanism, that the changes in the mechanical property of the cell wall in coleoptiles showing

118

MASUDA, KAMISAKA, YAMAMOTO, HOSON, and NlSHlTANl

autotropism may be due to some unknown metabolic changes in the cell wall components.This needs further investigations. In conclusion, microgravity simulated by means of the 3-D clinostat strongly influenced the direction of growth of plant organs, although it did not affect their gennination rate or growth rate. The patterns of growth direction changes on the clinostat differ for roots and shoots and for different plant species. In order to understand the role of gravity in the regulation of plant growth and development, the mechanism by which such a spontaneous curvature of roots and shoots is induced needs to be clarified. The 3-D clinostat has proved to be a very useful tool for this purpose.

111. BUOYANCY EXPERIMENTS A. Growth Seeds of rice (Oryza sativa L. cv. Sasanishiki) were sterilized in 4% sodium hypochlorite for 1 hour, and soaked for 2 days in water at 30 "Cin darkness. They were then germinated and grown in darkness at 30 "C under three different conditions: 1. under sterilized water of 10 cm depth in a polyvinyl cylinder (water type);

I5 cm high 11.5 cm diameter

2. under sterilized water with constant air bubbling (bubbling-type); and 3. on gauze moistened with sterilized water (air type). From day 2 through day 5 after germination, the coleoptile length was measured every day, and a number of coleoptiles were excised. In some experiments, the coleoptiles were uniformly subdivided from the tip to the base-the first section 7 mm from the tip and then each 10 mm. As shown in Figure 3, the coleoptiles growing under water reached the greatest length with a maximum of 8 1.2 mm on day 5. Growth of the bubbling and air-type coleoptiles was much less: their maximum length was 23.5 mm and 12.4 mm at days 4 and 5 , respectively. Growth of the water and bubbling type coleoptiles was rapid between days 2 and 4, but the growth rate decreased after day 4. The growth rate of the air type coleoptileswas relatively steady and slow. Differences in growth between bubbling and air, approximately 11 mm at days 4 and 5, could be due to the buoyancy condition. Rice coleoptile growth has been known to be regulated by several factors; namely, oxygen pressure and hormones, particularly ethylene which, contrary to ordinary land plants, stimulates rice coleoptile growth?zu

Plant Responses m Simulated Microgravity

1

2

119

3

4

5

Days Figure3. Kinetics of rice coleoptile elongation under water, water with air bubbling, and in air.

6. Mechanical Properties of the Cell Wall

The mechanical properties of the coleoptile cell walls were determined as described in Section KD,except that for water-type coleoptiles a stress of 2 g was used. Rice coleoptiles were cut into sections from the tip to the base, and the cell wall extensibility of different coleoptile zones was determined (Fig. 4). In general, the extensibility of the air and bubbling type coleoptiles was lower than that of the water-type. The slightly higher extensibility of the water-type coleoptiles is probably due to buoyancy. Aging decreased the cell wall extensibility of air and bubbling type coleoptiles. The extensibility decreased towards the base at days 4 and 5 . In water-type coleoptiles,on the other hand, the extensibility was consistently lowest in the middle zones and highest at the tip.

MASUDA, KAMISAKA, YAMAMOTO, H O S O N , and NISHITANI

120

lay 5

lay 4

\

0.3-

.

\

0)

E E

s 0

0.2-

4 - H

P

In

C

0)

3

c

0.1 A

-

03-A

0

~~

Tip

___t

Base

Tip

Base

T@

Base

Figure 4. Changes in the cell wall extensibility in the different regions of rice coleoptiles growing under water, water with air bubbling, and in air.

C. Chemical Composition of the Cell Wall

Cell wall polysaccharides were fractionated in hemicellulose and cellulose fractions by a modification of the method of Nishitani and Masuda.21 The amounts of cellulose (Fig. 5 ) and hemicellulose (Fig. 6) per unit length were much smaller in the water-type coleoptiles than in the air or bubbling-type coleoptiles. Both of these polysaccharides increased towards the base of the coleoptile. Aging increased the contents of the cell wall polysaccharides in all regions of the coleoptile. Hemicellulosic arabinose content per unit length had a similar distribution as that of hemicellulose, indicating that the arabinoxylans increased as the coleoptile aged and that their content in the sugars per unit length was higher at the base than at the tip. The amount of cellulose was similar both in air- and bubbling-type coleoptiles, but it was much less in water-type ones. The amount of hemicellulose and hemicellulosic arabinoseper unit length showed a similar trend in terms of growth condition and age. However, the amount was much less in bubbling-type coleoptilcs than in air-type ones, suggesting that hemicellulosc, rather than cellulose, is more closely related with growth and buoyancy? Phenolic acids were liberated from the cell wall matcrial by treatment with 0.1 M NaOH. After acidification of the solution to about pH 3 with HCl, the phenolic acids were extracted with ethyl acetate. The ethyl acetate extract was air-dried and stored in darkness. Ferulic and diferulic acids were analyzed according to the method of Shib~ya,’~ as previously reported by Kamisaka et a1.= The amount of

figure 5. Changes in the amount of cellulose in the different regions of rice coleop tiles growing under water, water with air-bubbling, and in air.

' E E

a-

6-

cn

0 3

= I

.-8 4 -

$

0 2-

/

/

Figure 6. Changes in the amount of hemicellulose in the different regions of rice coleoptiles growing under water, water with air-bubbling, and in air. 121

MASUDA, KAMISAKA, YAMAMOTO, HOSON, and NlSHlTANl Day 3

Day 4

Tp -Base Figure 7. Changes in the amount of ferulic acid per mole arabinose in the hemicellulose fraction in the different regions of rice coleoptiles growing under water, water with air bubbling, and in air.

phenolic acids was determined with a digital integrator using trans-ferulic and trans,trans-diferulicacids as standards. Trans,trans-diferulicacid was synthesizd by the method of Richtzenhain.26The amount of ferulic and diferulic acids was calculated as the mean of triplicate samples in each experiment. The amounts of ferulic acid per arabinose unit were higher in air- and bubblingtype coleoptiles than in water-type coleoptiles, that of the bubbling-type being slightly less than that of the air-type (Fig. 7). In air-type coleoptiles the ferulic acid content per unit length increased towards the base at days 4 and 5 , but in bubblingtype coleoptiles at day 4 there was no substantial gradient in the contents of ferulic and diferulic acids. In water-type coleoptiles the ferulic acid content was consistently higher in the middle zones than at tip and base. Aging somewhat increased the amounts of these phenolic acids per arabinose unit in coleoptiles grown under any one of the three different conditions. The diferulic acid content generally changed in parallel with the ferulic acid content. The approximate 5: 1 ratio of the ferulic acid to diferulic acid contents per hemicellulose unit was almost constant, regardless of type, age, mne, and growth conditions of the coleoptile?’ Summarizing, the growth of the rice coleoptile appears to be promoted by buoyancy, although oxygen and ethylene are more dominant environmental factors for its growth. This view has been supported by the parameters we have determined: growth, cell wall extensibility, and the amounts of cell wall polysaccharides, particularly hemicellulose and phenolic compounds. The underwater condition has

Table 2. Effect of Hypergravity on the Mechanical Properties and Compositionof the Cell Wall in Cress Hypocotyls Control (24 h)

Initial

tength (mm) To

R

Ext

Uwer

20.5

8.5 108 6.9 0.75

Laver

UPWr

135 X G (24 h)

Lower 11.9

66 6.1 0.97

102 1.3 0.64

78 5.4

on

133 6.7 0.48

1.3 2.8 7.5 9.6

2.0 2.5 6.0 8.8

1.1 3.2 6.3 7.5

1.6 2.3 6.1 10.2

10.3 0.13

10.3 0.19 0.24 0.59 0.86

6.0 0.19

6.0

0.54

0.38

CWPS W x m n t )

Ponin HC-I HC-II Cellulose

1.2 3.0 8.0 10.9

m s 6l€hln) Lcogrh(mm) Pectin HC-I HC-II CclllllOSC

8.5 0.14 0.36 0.94 1.23

0.n 0.73 0.94

1.M 1.26

0.27 1.a2

1.71

CWPS (a)

Pcdin HC-I

HC-II CClllllOSC

5.2 13.2 34.6 47.0

6.1 13.2 35.4 45.3

10.2 13.0 31.2 45.6

6.3 17.8 34.5 41.4

8.0 11.2 30.2 50.6

11.0 46.1 10.2 3.0 27.9 1.8

13.7 49.9 11.3 2.8 21.4 0.9

15.5 25.2 9.3 8.2 37.7 3.8

12.8 41.8 10.1 5.6 26.6 3.1

13.4 24.7 7.2 7.9 43.5 3.3

5.4 2.6 18.3 22.3 4.5 17.9 29.0

4.7 2.2 13.7 23.8 6.4 17.5 31.7

3.8 1.9 5.6 25.8 8.8 18.0 36.1

4.2 1.9 14.2 22.9 5.2 20.5 31.1

4.0 2.0 5.9 26.3 5.8 18.2 37.8

Sugar camposition (wt%)

P& Rha Am

XYl Mall Gal Glc HW+Q Rha FUC

Am XYI MM Gal Glc

123

124

MASUDA, KAMISAKA, YAMAMOTO, HOSON, and NlSHlTANl

proved to be a useful system for the study of simulated microgravity in the rice coleoptile.

I II. H YPERGRAVITY EXPERIMENTS Acommercially availablecentrifuge (Kubota 8700) was equipped with a swinging bucket rotor for cultivating plant seedlingsat hypergravity and with a temperaturecontrolled chamber for 1-G controls. Cress seedlings, germinated and grown in darkness for 20 hours, were transplanted on a 1.2 % agar medium in a tube. After transplantation, the seedlings were incubated for an additional 16 hours under the same conditions. Hypergravity (135 x G) by centrifugal rotation was applied to seedlings placed in the swinging bucket of the centrifuge. Control seedlings were placed in the 1-G chamber of the centrifuge. The hypocotyl length of the seedlings was measured at 12-hour intervals. The growth of cress hypocotyl was suppressed by increased gravity. It should be noticed that the growth was more effectively reduced during the first 12 hour incubation period than during the second 12-hour incubation period.The mechanical properties of the cell wall was measured by the stress-relaxationmethod.28The initial stress value for cress hypocotyls was 10 g. The mechanical properties of the cell wall of the cress hypocotyls was determined by means of the methods described in Section 1I.D. The results are shown in Table 2. The To value was found to be smaller in the upper growth zone than in the lower region. Hypergravity caused a slight but significant increase in the Tovalues for the upper and lower regions. No such clear causal relationship was found in the R value. On the other hand, cell wall extensibility, which represents the achievement of extension growth, was lower in hypergravity.These results suggest that the growth capacity was reduced by the lower cell wall extensibility after cultivation in h ypergravity." The chemical composition of the cress hypocotyl cell wall was determined as described in Section 1I.E. We looked for changes in the contents of cell wall polysaccharides and in their neutral sugar composition. In general, cell wall polysaccharide contents per unit length of the hypocotyl were larger in the hypergravity condition than in the 1-G condition.The enhanced accumulation of cell wall polysaccharides under the hypergravity condition apparently makes the cell wall thick and mechanically rigid. These findings were more or less similar to those for pea seedlings reported by Waldron and Brett.B

IV. CONCLUDING REMARKS In studies of plant response to microgravity,more information is needed on various physiologicalparameters related to growth. It is necessary to know the involvement of phytohomnes in growth regulation in microgravity as well as under 1-G

Plant Responses to Simulated Microgravity

125

conditions. For example, phytohormone content in rice coleoptiles should be studied in clinostat and buoyancy experiments. Wadas reported that the content of indoleacetic acid (IAA) oxidase inhibitor was higher in water-type coleoptiles than in air-type coleoptiles. This suggests that the greater coleoptile elongation under water may be due to an increased level of IAA. However, OhwakiMfound that the IAA levels were about the same in water-type and air-type coleoptiles. Recently, Hoson et aL3' have reportedthat the IAAlevels, as measured by gas chromatographymass spectrometry, showed a significant gradient in air-type coleoptiles. but that there was no such gradient in water-type coleoptiles. In addition, the IAA level per coleoptile increased during growth in air-type coleoptile, but remained constant at a lower level during growth in water-type coleoptiles. It would be valuable to determine the IAAlevels in air-type, bubbling-type and water-type rice coleoptiles to increase our understanding of the role of this phytohormone in the effects of microgravity on the growth of rice coleoptiles. There was no change in germination rate and growth rate by rotation on the 3-D clinostat except for azuki bean epicotyls. This suggests that hormone relations are not modified significantly by the simulated microgravity produced by rotation on the clinostat. However, since the tropistic response is disturbed by these simulated microgravity conditions, it seems possible that the polar transport system of IAA may be modified by this treatment. Determination of the level as well as the transport of this hormone in plant seedlings under 1 G and on the 3-Dclinostat would be desirable.

REFERENCES 1. Sieven. A. Gravity Sensing Mechanisms in Plant Cells. ASGSB Bullerin4:4?-50, 1991. 2. Pfeffcr. W. Pflanzenphysiologie. Ein Handbwh. Zweiter Band. Verlag von Wilhebn EngelmaM. LcipLig. 1881. 3. Sachs. J. brlesungen uberPflanzenphyswlogie. Verlag von Wilhelm Engelmann. Leipig. 1882.

4 . Hoson.T. M a s & , Y., Yamashita. M. Changes in Plant Growth Rocesses under Microgravity ConditionsSimulated by aThree-Dimensional Clinostat. Botanical Magazine Tokyo. 10553-70, 1992. 5. Furuya. M., Masuda. Y.. Yamamoto. R. Effects of E n v i r m n t a l Factors on Mechanical Properties of the Cell Wall in Rice Coleoptiles. Developrnenf.Gmwrh & D@erenriution, 1495105. 1972. 6. Hoson. T.. Wada. S. Role of Hydroxyprolin-Rich Cell Wall Protein in Growth Regulation of Rice ColeoptilesGrown on or Under Water. Plant Cell Physiology, 2151 1-524. 1980. 7. Nagao, M.. Ohwaki. Y.The Effect of Some Inhibitors of Alcoholic Fermentation and R c s ~ r a t h ~ on the Growth of the Coleoptile in Oryza Sativa and Avena Sativa. Science Report of T o h h Universiry. IV. Saies. 20:5&71, 1953. 8. Wada. S. Growth Panems of Rice Coleoptiles Grown on Water and Under Warn. Science Repon of Tohoku University, IV Series, 2 7 199-207.1%1. 9. Zana. I., Masuda. Y.Growth and Cell Wall Changes in Rice Coleoptiles G m h g under Diffaclu Conditions. I. changes in -or Pressure and Cell Wall Polysaccharidcs during Intact Growth. Phnt Cell Physiology, u):1117-1124. 1979.

126

MASUDA, KAMISAKA, YAMAMOTO, HOSON, and NISHITANI

10. Nishitani. K, H w m . T., Kamisaka, S., Masuda. Y.. Yamashita. M. Effect of Gravity on Plant Growth and Cell Wall Properties. Proceedings of 18th Intermtional Symposium on Space TechnologyandScience.kkgoshima. pp. 2109-2114. 1992. 11. Sobick, V.. Sievers. A. Responses of Roots to Simulated Weightlessness on the Fast Rotating Clinostat. Life Sciences and Space Research 1728S290,1979. 12. Brcmckamp. C.E.B. Das Verhahen dcr Grasskeimlingcauf &m Klimtate. Berichre der Deuschen Botanischen GeselLschqft,43:159-165, 1925. 13. Nick. P.. Schafcr, E. Nastic Response of Maize (Zca mays L.) Coleoptilcs during ClinOstat Rotation. P h a . 1W12%131.1989. 14. Firs R.D. Mym. AB. Plant Movemnts Caused by Differential Growth:Unity M Diversity of Mechanism? In: Difemnfid Growth in Planfs (P.W. Barlow. Ed.), pp. 47-55. P e r g m m Prrss, Oxford 1989. IS. Nick, P.. F m y a . M. Schafer, E. Do Mkrotubula Control Growth in Trqisrn? Expcrhents with Maize Cdeoptiks. P h Cell Physiology, 32:999-1006. 1991. 16. Masuda. Y. Auxin-Induced Cell Wall Loosening. Botanical Maguzine Special Issue. 1:103-123,

1978. 17. Masuda.Y. Awn-Induced Cell Elongation and Cell Wall Changes. Botanical Maguzine Tokyo. 103:345-370.1990. 18. Yamamoto, R.. Fujihnra S.. Masuda. Y. Measurement of Stress-Relaxation Properties of Plant Cell Walk. In: Plant Growth Substances. pp. 798-805.Hirokawa Publishing Company. 1974. 19. Yamamoto, R.. Kawamura. H. Masuda. Y. Stress Relalation Properties of the Cell Wall of Growing Intact Plants. Plant Cell Physiology, 151073-1082. 1974. 20. Olson.A.C.. Bonncr. I, Mom, DJ.Face Extension Analysis of Avcna Coleoptik Cell Walls. Plonm. 66:126134,1%5. 21. Nishitani. K., Masuda. Y. Growth and Cell Wall Changes in Azuki Bean Epicotyls. I. Changes in Wall Polyssccharides during Intact Growth. Plant Cell Physiology, W63-74. 1979. 22. M a w N.. Suge. H. Does Ethykne Induce Ebngation of thc Rice Coltoptile thmrgh Auxin? P h t Cell Physiology. u):1147-1 150. 1979. 23. Ku, H.S..Suge. H.. Rappapwt. L. Stimulation of Rice Coleoptile Growth by Ethylene. Planfa, 90:333-339. 1970. 24. Shibuya, N. Phenolic Acids and their Carbohydrate Esters in Ria Colmptde Cell Walls. Phyrochcmbrry. 232233-2237.1984. 25. Kamisak S.. ’IhIBkeda. S.. Tahahashi. K.. Shibata. K. Diferulic and Faulic Acids in thc Cell Wall of Avena Cokoptiles. Their Relatiwhips to Mechanical FYopedes of thc Cell Wall. Physiologia P h a r u m . 78:l-7. 1990. 26. Richtzcnhain. H. Enzymatkche Versuck zur Entstchung dcs Lignim. IV. Dehydrimgen in der Guajacolrcihe. Chemicche Berichre. tX24447453.1949. 27. Tan.K.-S.. H o s a ~ T .M.asuda.Y..K&aka. S.Comlation&twee.nCell WallExtensibility and the Cootent of Ferulic and Diferulic Acids in Cell Walls of Sativa Coleopales Grown under Water and in Air. Physwlogia Plantarum. a397-403, 1991. 28. Yamamoto, R.. Shinozaki. K.. Masuda. Y.S!ms-Relaxah’cm Roperties of Plant Cell Walls with Special Reference to A w n Action. Plant Cell Physiology, 1197-956,1970. 29. Waldron. K.W.. Barctt, C.T. Effects of Extreme A c c e k d o n on thc Gamination, Growth and Cell Wall Composition of Pea Epicotyls. Journal of Experimental Botany. 41:71-77.1990. 30. Ohwaki. Y. Diffusible and Exmtable Auxins in Relation to the Growth Pattern of Rice Coleoptiles. Science Report of Tohoku Universiry,N.Series 3591-99.1970. 31. H o s m T.,Masuda. Y.. Pilet. P.E. Auxin Content in Air and Water Grown Rice Coleoptilcs. Journal of Plant Physiology, 139685-689.1992.