Journal of Experimental Botany, Vol. 47, No. 297, pp. 513-517, April 1996Journal ofExperimentalBotany

Effects of hypergravity on growth and cell wallproperties of cress hypocotyls1

T. Hoson2'6, K. Nishitani3, K. Miyamoto4, J. Ueda4, S. Kamisaka2, R. Yamamoto5 and Y. Masuda5

department of Biology, Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558, JapanzDepartment of Biology, College of Liberal Arts and Sciences, Kagoshima University, Kagoshima 890, Japan

^College of Integrated Arts and Sciences, University of Osaka Prefecture, Sakai 593, Japan5Tezukayama College, Nara 631, Japan

Received 9 May 1995; Accepted 8 December 1995

Abstract

Elongation growth of etiolated hypocotyls of cress(Lepidium sativum L.) was suppressed when they wereexposed to basipetal hypergravity at 35 x g and above.Acceleration at 135xg caused a decrease in themechanical extensibility and an increase in the min-imum stress-relaxation time of the cell wall. Suchchanges in the mechanical properties of the cell wallwere prominent in the lower regions of hypocotyls.The amounts of cell wall polysaccharides per unitlength of hypocotyls increased under the hypergravitycondition and, in particular, the increase in the amountof cellulose in the lower regions was conspicuous.Hypergravity did not influence the neutral sugar com-position of either the pectin or the hemicellulose frac-tion. The amount of lignin was also increased byhypergravity treatment, although the level was low.The data suggest that hypergravity modifies the meta-bolism of cell wall components and thus makes thecell wall thick and rigid, thereby inhibiting elongationgrowth of cress hypocotyls. These changes may con-tribute to the plants' ability to sustain their structuresagainst hypergravity.

Key words: Cell wall extensibility, cellulose, hypergravity,Lepidium sativum L, lignin.

Introduction

The cell wall plays a principal role in sustaining the aerialparts of a plant under I x g o n earth, as does the boneand muscle in an animal body. The chemical and mechan-

ical properties of the cell wall appear to be modifiedunder altered gravity conditions. In fact, the amounts ofcertain components of the cell wall, such as cellulose andlignin, were reduced under a microgravity environmentin space (Cowles et al., 1984; Nedukha, 1996). Thedecrease in levels of cell wall polysaccharides and phen-olics was also observed under microgravity conditionssimulated by water submergence (Masuda et al., 1994a).Such quantitative (and probably also qualitative) changesin cell wall components may cause a modification of themechanical properties of the cell wall, leading to changesin the growth and development of higher plants. However,relatively few studies on cell wall properties have beencarried out under a microgravity environment, because itis difficult to produce real microgravity conditions forenough time on' earth and the opportunity for spaceexperiments is still limited.

Hypergravity conditions can be produced on earth bycentrifugation. Acceleration due to centrifugation hasbeen used to analyse the mechanism of gravity perception(Sievers and Heyder-Caspers, 1983; Wendt and Sievers,1986) and gravicurvature (Lee et al., 1990). Changes inelongation growth (Waldron and Brett, 190; Kasaharaet al., 1995a, b), auxin transport (Ouitrakul and Hertel,1969), circumnutation (Brown and Chapman, 1977), andstarch metabolism (Brown and Piastuch, 1994) have alsobeen studied under hypergravity conditions. As to theproperties of the cell wall, Waldron and Brett (1990) firstanalysed the cell wall composition of epicotyls and rootsof pea (Pisum sativum L.) grown under hypergravityconditions and observed some changes in the amounts ofcell wall polysaccharides and lignin. Recently, Kasahara

1 This paper is dedicated to Professor Andreas Sievers, Universitat Bonn, on his 65th birthday.6 To whom correspondence should be addressed. Fax: +81 6 605 2522. E-mail: h1817@ocugw.cc.osaka-cu.ac.jp

© Oxford University Press 1996

514 Hoson et al.

et al. (1995a) reported an increase in cell wall polysaccha-rides per unit length of hypocotyl of radish (Raphanussativus L.) and cucumber (Cucumis sativus L.). However,changes in the physical properties of the cell wall underhypergravity conditions have not yet been studied. In thepresent study, the effect of hypergravity on growth, themechanical properties of the walls, and the levels of cellwall polysaccharides and lignin in upper and lower halvesof cress hypocotyls were examined. Preliminary results ofthe study have been cited in reviews (Masuda et al.,\994a, b).

Materials and methods

Plant material and culture conditionsSeeds of cress (Lepidium sativum L.) were germinated on filterpaper moistened with water at 25 °C for 20 h in the dark.Germinated seedlings were planted in a small cavity on 1.2%agar in a centrifuge tube and incubated at 24 °C for anadditional 16 h in the dark. The tube was roughly covered withaluminium foil to maintain moisture but to allow free gasexchange. Seedlings were exposed to various degrees ofhypergravity in the basipetal (rooting in) direction with aremodelled centrifuge (model 8700, Kubota, Tokyo, Japan), therotation room of which had been expanded so as to secureshelf space for \xg control seedlings. Seedlings in the tubewere exposed to hypergravity in a swinging bucket rotorattached to the centrifuge at 25 °C in the dark, while controlseedlings were attached vertically to the fixed shelf in the samerotation room. The magnitude of acceleration was regulated bychanging the speed of rotation. Seedlings were collected every12 h after the start of rotation and the length of the hypocotylswas measured. Each growth experiment was repeated at leastthree times.

Mechanical properties of the cell wallCress hypocotyls were separated into upper and lower halvesand then fixed in boiling methanol. After rehydration themechanical properties of the cell wall were measured by thestress-relaxation method with a tensile tester (Tensilon RTM-25, Toyo Baldwin, Tokyo, Japan) connected to a personalcomputer. The segments were fixed between two clamps andstretched at 0.33 mm s"1 until a stress of 10 g was produced inthe samples. The tension as a function of time was recorded atms intervals. The mechanical extensibilty (strain/stress) wascalculated from the total extension of the samples and theextension rate. The minimum stress-relaxation time wascalculated by the method of Yamamoto et al. (1970).

Analyses of cell wall componentsFixed hypocotyl halves were ground in a mortar, and thenwashed twice each with cold water, acetone, a mixture ofmethanol and chloroform ( 1 : 1 . v/v), and ethanol. The cell wallmaterial was treated with 10 units ml"1 a-amylase (EC 3.2.1.1,from porcine pancreas, Sigma, St Louis. MO, USA) for 3 h at37 "C, and thoroughly washed with water. The cell wallpreparation was successively extracted four times (1 h each) at70 °C with 20 mM ammonium oxalate (pH4.0), three times(18 h each) at 25 C with 4% (w/v) KOH and three times (18 heach) at 25 C with 24% (wv) KOH. These extractions yieldedthe pectin, the hemicellulose I and the hemicellulose II fractions.The hemicellulose fractions were neutralized with acetic acid

and dialysed against water. The residues after 24% KOHextraction were washed with 0.1 M acetic acid and water(cellulose fraction). The cell wall fractions were lyophilized. Thecellulose fraction was dissolved by treatment in 72% (v/v)sulphuric acid for 1 h at 25 °C with stirring. Sugar wasdetermined by the phenol-sulphuric acid method (Duboiset al., 1956) and expressed as glucose equivalents.

The lyophilized pectin and hemicellulose fractions werehydrolysed with 2 M trifluoroacetic acid for 1 h at 121 °C. Afterthe trifluoroacetic acid had been removed by evaporation undera stream of air at 50 °C, the sugars were reduced with sodiumborohydride in 1 M ammonia, and acetylated with aceticanhydride at 121 °C for 3 h. The acetylated alditols wereanalysed by a gas chromatograph (GC-14A, Shimadzu, Kyoto,Japan) according to the method of Albersheim et al. (1967).

The cellulose fraction was dissolved in 25% (v/v) acetylbromide in acetic acid and then kept at 70 °C for 30 min in acapped test tube. After cooling, the same volume of 2 M NaOHwas added. The mixture was centrifuged at 3 000 xg for 10 min.7.5 M hydroxylamine was added to the supernatant, and theamount of lignin was determined with an absorbance of thefinal solution at 280 nm (Jaegher et al., 1985).

Results

Growth and the mechanical properties of the cell wall

Elongation growth of cress hypocotyls was suppressed byincreasing gravity. The suppression of elongation for thefirst 12 h was detected at 10 xg and became significant at35 xg (Fig. 1). At 135 xg, elongation rate of hypocotylswas only 15% of the 1 x g control. The effect of hypergrav-ity on root growth was not examined in the present study,because roots did not grow straight along the directionof gravity.

50 100

Gravity (x g)150

Fig. 1. Effect of hypergravity on elongation growth of cress hypocotyls.Germinated seedlings were incubated in normal gravity for 16 h andthen under different hypergravity conditions for 12 h at 25 C in thedark. Initial length, 8.5 mm. Values are means ±SE (n = 20).

The mechanical extensiblity and the minimum stress-relaxation time were employed as the measures of thephysiological extensiblity of the cell wall. Upper growingregions of control hypocotyls showed a higher mechanicalextensibilty and a smaller value of the minimum stress-rel-axation time than lower ones (Fig. 2). Hypergravity at135xg caused a decrease in the mechanical extensibilityand an increase in the minimum stress-relaxation time ofthe cell wall, suggesting that hypergravity makes the cellwall mechanically rigid. Such changes in the mechanicalproperties of the cell wall were prominent in the lowerregions of hypocotyls.

Levels of cell wall polysaccharides and lignin

The cell wall material of cress hypocotyls grown either at1 x g or at 135 x g was divided into four fractions and theamount of polysaccharides in each fraction was deter-mined. When expressed on the basis of hypocotyl region,the amount of the hemicellulose I fraction of upperregions and that of the cellulose fraction of lower regionswere increased by hypergravity treatment, whereas otherfractions did not show clear change (data not shown).Hypergravity also did not influence the ratio of neutralto acidic sugars in the pectin fraction. On the other hand,the amounts of polysaccharides per unit length of hypo-cotyls increased in all fractions under the hypergravity

Initial

1 g Upper

1 g Lower

135 g Upper

135 g Lower I

0

Initial

1 g Upper

1 g Lower

135 g Upper

135 g Lower

200 400 600 800 1000

Mechanical extensibility (dm 10 g"')

1

4-

-

• * i

0 40 80 120 160

Minimum stress-relaxation time (ms)

Fig. 2. Effect of hypergravity on the mechanical properties of the cellwall. Seedlings were grown as indicated in Fig. 1. After preparation,the mechanical extensibility and the minimum stress-relaxation time ofhypocotyls were measured as indicated in Materials and methods.Values are means +SE (n = 20).

Hypergravity and cell walls 515

condition (Fig. 3). The increase in the amount of cellulosein the lower regions was particularly conspicuous. Underthe hypergravity conditions, the proportion of the cellu-lose fraction among cell wall polysaccharides increasedin the lower regions (data not shown).

The pectin fraction of cress hypocotyls mainly consistedof rhamnose, arabinose, and galactose (Fig. 4). In thepectin fraction, the proportion of arabinose was higherwhile that of galactose was lower in the upper regions ofhypocotyls than the lower ones, suggesting that there aresome differences in chemical structure of arabinogalactansbetween the two regions. Hypergravity did not influencethe neutral sugar composition of the pectin fraction(Fig. 4).

The hemicellulose I and II fractions were combinedand the composition in neutral sugars of this total hemi-cellulose was determined (Fig. 5). The hemicellulose frac-tion was mainly composed of arabinose, xylose, galactose,and glucose. The fraction from the upper regions of thehypocotyls contained a higher ratio of arabinose and a

Pectin

HC-I

Initial1 g Upper

— \ S Lower^ > 135 g Upper

135 g Lower

0 1 2 3

Polysaccharides (</g mm"1)

Fig. 3. Effect of hypergravity on the amounts of cell wall polysaccha-rides. The amounts were expressed on the basis of unit length ofhypocotyls. HC-I, the hemicellulose I; HC-II, the hemicellulose IIfractions. Values are means ±SE (n = 3).

Initial1 S UPP"1 g Lower

« p p135 g Lower

20 40

Sugar composition (%)

60

Fig. 4. The neutral sugar composition of pectin fraction from cresshypocotyls grown under different hypergravity conditions. The composi-tion was determined by gas chromatography. Rha, rhamnose; Fuc.fucose: Ara, arabinose: Xyl, xylose; Man. mannose; Gal. galactose;Glc, glucose.

516 Hoson et al.

Initiala i 1 g Upper1^— 1 g Lower" S 135 g Upper

135 g Lower

10 20

Sugar composition i

30 40

Fig. 5. The neutral sugar composition of hemicellulose fractions fromcress hypocotyls grown under different hypergravity conditions. Thehemicellulose I and II fractions were combined. Otherwise as indicatedin Fig. 4.

lower ratio of xylose and glucose, compared with thelower ones. Thus, some differences were detected in thestructure and the level of hemicellulosic polysaccharidesbetween the upper and the lower regions. However,hypergravity did not influence the neutral sugar composi-tion of the hemicellulose fraction (Fig. 5).

The cell wall of cress hypocotyls contained low butsignificant levels of lignin. The amounts of lignin perhypocotyl region and per unit length were higher in theupper regions than the lower ones (Table 1). Hypergravitycaused an increase in the amount of lignin, especiallywhen expressed on the basis of unit length, in both theupper and lower regions.

Discussion

Elongation growth of cress hypocotyls was significantlysuppressed by basipetal hypergravity at 35 x g and above(Fig. 1). The critical value of acceleration for suppressionof cress hypocotyl growth roughly corresponded to thatreported in epicotyls and roots of pea (Waldron andBrett, 1990) and hypocotyls of radish and cucumber(Kashara et al., 1995a). Hypergravity also caused adecrease in the mechanical extensibilty and an increase inthe minimum stress-relaxation time of the cell wall of

Table 1. Effect of hypergravity on the amount of lignin in thecell wall of cress hypocotyls

Initial1 x g upper1 xg lower135 xg upper135 xg lower

Lignin"

(ng region " ' )

165 + 38165 + 35132 + 43253 + 25175+17

(ngmm ')

19 + 416±313 + 443+4*29 + 3'

° Means+ SE of three replicates.b Mean value significantly different (P<0.05) from 1 xg control.

cress hypocotyls (Fig. 2). These results suggest that hyper-gravity suppresses elongation growth of stems by makingthe cell wall mechanically rigid.

Elongation of cress hypocotyls occurred mostly in theupper regions both under 1 xg and hypergravity condi-tions (data not shown). On the contrary, changes in themechanical properties of the cell wall due to hypergravitywere more clearly observed in the lower regions of hypo-cotyls (Fig. 2). These results indicate that the cell wallchanges induced by hypergravity treatment in the lowerregions are not directly associated with cell elongation.However, the wall stiffening in the lower regions maycontribute to sustain the plant structure against hyper-gravity and maintain growth capacity in the upperregions. In fact, elongation of cress hypocotyls for thenext 12 h (between 12 and 24 h after initiation of hyperg-ravity treatment) was not suppressed until 80xg (datanot shown).

Under the present experimental conditions, the basalregions of hypocotyls were exposed to about 10% higheracceleration compared with apical regions. This might beone of the reasons why changes in the mechanical proper-ties of the cell wall due to hypergravity were prominentin the lower regions. In cucumber hypocotyls, growthsuppression and the cell wall thickening were broughtabout not only by basipetal but by acropetal hypergravity(Kasahara et al., 1995a). However, it was difficult toexpose cress seedlings to acropetal hypergravity in thepresent experimental system.

Hypergravity caused an increase in the amounts of cellwall polysaccharides and lignin per unit length of hypo-cotyls (Fig. 3; Table 1). The plant appears to constructthe thick and tough cell wall in response to hypergravityto support its structure. Of the different cell wall compon-ents, the amount of cellulose was most clearly correlatedwith the mechanical properties of the cell wall. Thecontribution of lignin to the mechanical properties of thecell wall may be small, because the level of lignin is lowand because it is higher in the upper, growing regions(Table 1). The increase in the minimum stress-relaxationtime of the cell wall under the hypergravity conditionappears to be caused by an increase in viscosity (Masuda,1990; Sakurai, 1991). In general, viscosity of the cell wallis determined by the nature of various non-cellulosicpolysaccharides (Masuda, 1990; Hoson, 1991; Sakurai,1991). However, hypergravity did not influence the neut-ral sugar composition of either the pectin or the hemicellu-lose fractions of cress hypocotyls (Figs 4, 5). Similarresults were reported in hypocotyls of radish and cucum-ber (Kasahara et al., 1995a) although, in pea epicotyls,hypergravity caused some changes in sugar composition(Waldron and Brett, 1990). Effects of hypergravity onother biochemical and physicochemical properties of non-cellulosic polysaccharides are under investigations.

In general, gravity stimulates the production of ethylene

which induces lateral expansion and the cell wall thicken-ing of stems (Abeles, 1973). Thus, the wall thickeningobserved under hypergravity could be attributed to theethylene produced. However, the cell wall thickening dueto hypergravity mainly occurred in the lower regions(Fig. 3), whereas the lateral expansion due to ethylene isinduced in the upper ones. Furthermore, no significantdifferences in ethylene concentration were detected in theatmosphere of the culture tube between control andhypergravity treatment (data not shown). These resultssuggest that the above possibility can be ignored in thepresent study.

The cell wall plays a principal role in regulation ofgrowth and development of higher plants. Under microg-ravity conditions simulated by water submergence, plantsform thinner cell wall (Masuda et al., 1994a, b). Althoughthe cell wall extensibility of whole organs was not changedmerely by compensating the unilateral influence of gravityby clinostat rotation (Hoson et al., 1992; Masuda et al.,1994a, b), the spontaneous curvature of shoots and rootson a three-dimensional clinostat can be explained by amodification of cell wall properties (Hoson, 1994; Hosonet al., 1995). Nevertheless, little is known about changesin the cell wall under real microgravity in space. Thepresent results show that in cress hypocotyls hypergravitycauses an accumulation of cell wall polysaccharides,especially cellulose, and thus makes the cell wall thickand rigid. From the trends observed in hypergravityexperiments, it might be possible to deduce that theresults will follow the opposite trend under microgravityconditions. By effective use of centrifugation, it may bepossible to predict events that may be expected to occurduring plant growth in a microgravity environment.

Acknowledgements

We thank Mrs T Kotani for technical assistance. The presentstudy was supported in part by a Grant for Basic Research inSpace Station Utilization from the Institute of Space andAstronautical Science, Japan.

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