Blockage of Brassinosteroid Biosynthesis and Sensitivity ... · Brassinosteroid Mutants of Garden...

Plant Physiol. (1 997) 1 1 3: 31-37

Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea'

Takahito Nomura, Masayoshi Nakayama, James B. Reid, Yasutomo Takeuchi, and Takao Yokota*

Department of Bioproductive Science (T.N.), and Weed Science Center (Y.T.), Utsunomiya University, Utsunomiya 321, Japan; Department of Plant Science, University of Tasmania, C.P.O. Box 252C, Hobart,

Tasmania 7001, Australia (J.B.R.); and Department of Biosciences, Teikyo University, Utsunomiya 320, Japan (M.N., T.Y.)

Endogenous brassinosteroids (BRs) in the dwarf mutants Ika and Ikb of garden pea (Pisum sativum 1.) and comparable wild-type plants were quantified by gas chromatography-selected ion monitoring using deuterated interna1 standards. In young shoots of the Ikb mutant, the levels of brassinolide, castasterone, and 6-deoxocastasterone were 23-, 22-, and 9-fold lower, respectively, than those of wild-type plants. Applications of brassinolide, castasterone, typhasterol, 3-dehydroteasterone, and teasterone nor- malized internode growth of Ikb seedlings. These findings indicate that the Ikb plants are BR-deficient mutants, probably as a consequence of a block in the BR biosynthetic pathway prior to the production of teasterone. Young shoots of Ika plants contained only 50% less brassinolide and 5 times more castasterone than the equivalent wild-type tissues. The Ika seedlings were approximately 100 times less responsive to brassinolide than the Ikb mutant, and application of castasterone had only a marginal effect on Ika internode growth, suggesting that the Ika lesion results in impaired sensitivity to BR.

Since brassinolide was first isolated as a growth- promoting steroid from rape pollen (Grove et al., 1979) a variety of related steroids, termed BRs, have been isolated from higher and lower plants (see Sakurai and Fujioka, 1993; Takatsuto, 1994). Because BRs occur ubiquitously in plants and have strong and unique biological activities, they have been classified as a new group of plant hormones (Yokota and Takahashi, 1986) that affect stem elongation; pollen tube growth; leaf bending, unrolling, and epinasty; proton-pump activation (see Mandava, 1988); and xylem differentiation (Iwasaki and Shibaoka, 1991).

Recent studies on BR biosynthesis pathways with nopaline- and octopine-type crown gall cells of Catharan- thus roseus, as well as in the nontransformed cells, have demonstrated that brassinolide is produced from teasterone via typhasterol and castasterone (Suzuki et al., 1994a) and that 3-dehydroteasterone is an intermediate in the

'This work was supported by a grant-in-aid for cooperative research from the Ministry of Education, Science, and Culture of Japan (grant no. 06305010) and a grant-in-aid (Bio Media Program) from the Ministry of Agriculture, Forestry, and Fisheries of Japan (grant no. BMP-96-V-1).

* Corresponding author; e-mail yokota8nasu.bio.teikyo-u.ac.jp; fax 81-28-627-7187.

31

conversion of teasterone to typhasterol (Suzuki et al., 1994b). Furthermore, pathways from campesterol to teasterone have been investigated in nopaline-type crown gall cells of C. roseus (Suzuki et al., 1995b), and it has been shown that 6-deoxocastasterone is converted to castasterone and brassinolide (Choi et al., 1996). Metabolism of teasterone to castasterone and / or brassinolide has also been shown to occur in seedlings of C. roseus, Nicotiana tabacum, and Oryza sativa (Suzuki et al., 1995a). These findings are summarized in the BR biosynthesis pathway illus- trated in Figure 1.

To elucidate further the role of BRs in plant growth and development, we attempted to find BR-deficient mutants of the garden pea (Pisum sativum L.). It was assumed that BR deficiency would be associated with a dwarf phenotype, since a previous study showed that peas treated with the triazole growth retardant uniconazole (Sumitomo Chemi- cal Co., Osaka, Japan) exhibited dwarfism that was associated with reduced endogenous levels of not only GAs, but also of castasterone, a brassinolide precursor (Yokota et al., 1991). A number of dwarf pea mutants are known to have a block in their GA biosynthesis pathway (Reid, 1993). However, many short-stature mutants are not deficient in endogenous GAs and the cause of their dwarfism is not fully understood (Lawrence et al., 1992). This group in- cludes the Ika and Ikb mutants of P. sativum, both of which have normal GA biosynthesis (Lawrence et al., 1992) and respond only marginally to exogenous GA (Reid and Ross, 1989). Both mutants are characterized by reduced stem elongation, peduncle length, and basal branching, as well as a ridged or corrugated surface, a condition referred to as "banding" (Ross and Reid, 1986; Reid and Ross, 1989), and the overall phenotypes are different from those of GA- deficient plants (Reid and Ross, 1989). Although both the lka and lkb mutants have a lower endogenous IAA content compared with wild-type plants, their phenotypes cannot be explained solely on the basis of IAA deficiency (Reid and Davies 1992; McKay et al., 1994). It was against this background that the lka and lkb P. sativum mutants were selected for study as potential BR biosynthesis mutants. In the present investigation endogenous levels of brassinolide, teasterone, typhasterol, castasterone, and 6-

Abbreviations: BR(s), brassinosteroid(s); SIM, selected ion monitoring.

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3 2 Nomura et al. Plant Physiol. Vol. 11 3 , 1997

brassinolide was confirmed i n studies wi th seed-

Figure 1. Biosynthetic pathways of brassinolide elucidated using normal and crown gall cells of C. roseus. The pathway from teasterone to

HO * HOh & ..........................................

HOV (Unknown pathway)

6-Deoxocastasterone lings of some higher plants. Campesterol

i t t

Teasterone 3-Dehydroteasierone Typhasterol Castastero ne Brassinolide

deoxocastasterone in lka, lkb, and wild-type shoots were determined and the responsiveness of the mutants to exogenous BRs was also investigated.

MATERIALS A N D METHODS

Plant Materials for Analysis of Endogenous BRs

The pure lines of garden pea (Pisum safivum L.) used in this study were cv Torsdag (wild type, tall) and the two single-gene mutant lines derived from this cultivar, NGB5865 (lka) and NGB5862 (Ikb). Thirty-six-day-old shoots (Table I) were excised at the soil leve1 from plants seeded in pots on December 20, 1993, and grown in a greenhouse without heating before harvesting on January 25, 1994. The top four internodes were excised from 6-month-old plants that had been seeded on October 27, 1992, and grown under natural conditions before harvesting on April 12, 1993, when the heights of lka, Zkb, and wild-type plants were about 0.5, 0.5, and 1 m, respectively. The fresh weights of the 20 shoots of wild-type, lka, and lkb plants were 560, 437, and 437 g, respectively.

Extraction and Purification

Plant materials were extracted with methanol, and 2H,- labeled interna1 standards were added to the extract prior to reduction to an aqueous residue. With the 36-d-old seedlings, 0.25 pg of [2H,]brassinolide and 0.5 pg each of [2H,]castasterone, [2H,]typhasterol, [2H,]teasterone, and [2H,]6-deoxocastasterone were added to the extracts. In the case of the 6-month-old plants, 0.2 pg of [2H,]brassinolide and 1 pg each of [2H,]castasterone, [2H,]typhasterol, and [2H,]teasterone were used. The aqueous residue was partitioned three times against chloroform. The chloroform phases were combined and evaporated to dryness and then

partitioned between hexane and 80% methanol. The 80% methanol phase was evaporated to dryness and the residual solid was purified on a column of silica gel (4 g) eluted with chloroform, and with chloroform containing 1,3,5,7,10,20, and 50% methanol. To monitor the biological activity of BRs, sample aliquots were assayed using the rice lamina inclination test (Arima et al., 1984). Eluates obtained with 3 to 7% methanol in chloroform were combined, dissolved in 60% aqueous methanol, and loaded onto a column of charcoal (chromatography grade, 4 g; Wako Pure Chemicals, Osaka, Japan) that was eluted with methano1:water (6:4 and 8:2, v / v), methanol, and methano1:chloroform (9:1, 7:3, 5:5, and 3:7, v/v). The last three fractions were combined and chro- matographed on a column of Sephadex LH-20 (bed volume, 500 mL) using methanokchloroform (4:1, v/v) as a mobile phase. Ten-milliliter fractions were collected. Fractions 33 to 37 were combined and subjected to reverse-phase HPLC on a Senshu Pak ODS 3251-D column (8 x 250 mm; Senshu Scientific Co., Ltd., Tokyo, Japan) eluted with an acetonitrile to water gradient. The mobile phase was programmed as follows: 0 to 20 min, 45% acetonitrile; 20 to 40 min, 45 to 100% acetonitrile; 40 to 45 min, 100% acetonitrile. The flow rate was 2.5 mL min-' and fractions were collected every minute. The column oven temperature was maintained at 40°C. The following fractions, corresponding to BR retention times, were collected for analysis by GC-SIM: fractions 14 and 15 (brassinolide), 21 and 22 (castasterone), 30 to 32 (teasterone), 35 to 37 (3-dehydroteasterone and typhasterol), and 38 to 40 (6-deoxocastasterone).

CC-SIM

Samples were converted to methaneboronates or methaneboronate-trimethylsilyl ethers and then subjected to GC-SIM using a JEOL JMS AX 505 instrument equipped

Table 1. Data on 36-d-old excised P. sativum shoots analyzed for the endogenous BRs

Data are means 2 SE of 25 plants randomly selected.

Cenotype No. of Plants Shoot Height No. of Expanded Leaves

per Shoot Total Weight Averaged Weight per Segment

cm g g

W i l d type 92 30.2 i 0.7 8.06 ? 0.12 129 1.40 Ika 72 17.3 2 0.4 9.34 2 0.15 97 1.34 Ikb 87 16.2 t 0.4 8.68 t 0.16 118 1.36



Brassinosteroid Mutants of Garden Pea 3 3

Table 11. Endogenous levels of BRs in shoots of the P. sativum mutants Ika and Ikb, and wild-type plants Data are expressed as ng kg-' fresh weight (data expressed as nanograms per shoot are shown in parentheses).

36-d-01d Plants (shoots) 6-Month-Old Plants (top four internodes) BR

Wild tvoe Ika Ikb Wild tvDe Ika Ikb

Brassinolide 164 (0.23) 96 (0.1 3) 7 (0.01) 840 (24) 245 (5.5) 350 (9.0) Castasterone 355 (0.50) 1935 (2.6) 16 (0.02) 2360 (66) 31 70 (69) 60 (1.5) Typhasterol - - - 995 (28) 285 (6.3) 11 O (2.9) Teasterone n.d.b n.d. n.d. n.d. n.d. n.d. 6-Deoxocastasterone 31 33 (4.4) 71 07 (9.5) 355 (0.48) n.a.' n.a. n.a.

a -, No reliable data were obtained. n.d., Not detectable. n.a., Not analyzed.

with a DB-5 column (J&W Scientific, Folsom, CA; 0.25 mm X 15 m; 0.25-mm film thickness) under the conditions described by Yokota et al. (1994). The 'H, and 'H, ions monitored to quantify individual BRs were m / z 155 and 161 (fragment ions of methaneboronate of brassinolide), m / z 512 and 518 (M+ ions of methaneboronate of castasterone), m / z 498 and 504 (M' ions of methaneboronate of 6-deoxocastasterone), and m/z 544 and 550 (Mt ions of methaneboronate-trimethylsilyl ether of typhasterol and teasterone).

Deuterated Standards

'H,-labeled brassinolide, castasterone, typhasterol, and teasterone, synthesized according to Takatsuto and Ikekawa (1986), were supplied by Dr. S. Takatsuto of Joetsu University of Education (Joetsu, Japan). ['H6]-6- Deoxocastasterone (melting point, 238°C) was synthesized from [2H6]castasterone using the method of Mori et al. (1984).

Application of BRs

Pea seeds were sown in a tray filled with moist vermic- ulite and placed in growth cabinets (Nihon Ikakikai, Osaka, Japan) fitted with fluorescent lights (40-W daylight-white tube, Toshiba, Tokyo, Japan; approximately 240 Fmol m-' s-' at top of plant). The temperature was maintained at 25°C for the first 3 d, then lowered to 20°C for further growth. Brassinosteroids were applied to the surface of the expanding third leaf in 10 pL of ethanol containing 0.15% Tween 20 about 8 d after planting. Control seedlings were treated with the same solvent containing no BR. As seen in Tables IV and V, significant growth differences were observed regardless of approximately identical light conditions; however, these differences had little effect on the results.

Measurement of Cell length and Width

Three days after BR treatment, epidermal replicas were obtained from the 4th internode of five seedlings using a transparent manicure diluted 3-fold with ethyl acetate. Five imprints of cells per plant were randomly selected and the lengths and widths were measured under a micro- scope. The number of cells per internode was calculated by dividing the internode length by the cell length.

RESULTS AND DISCUSSION

The endogenous brassinolide castasterone and 6-deoxocastasterone levels in 36-d-old shoots of lkb seedlings were approximately 23-, 22-, and 9-fold lower, respectively, than those found in equivalent wild-type plants (Table 11). In the top four internodes of 6-month-old lkb plants, the levels of brassinolide, castasterone, and typhasterol were 2.4-, 39-, and 9-fold lower, respectively, than those obtained for wild-type plants (Table 11). These findings indicate that the Ikb mutant is BR-deficient. However, the leve1 of brassinolide in the top four internodes of the 6-month-old plants was not reduced as much. This may be ascribed to the environmental conditions used for cultivating plant materials, because the two plant materials analyzed were culti- vated under quite different conditions, as described in "Materials and Methods." It is also remarkable that the levels of brassinolide and castasterone in the top four internodes of the 6-month-old lkb plants were much higher than those in the whole aerial parts of the 36-d-old shoots (Table 11). This finding may imply that younger tissues contain higher levels of BRs.

Dose-response data for brassinolide (Fig. 2) indicated

15 - E - E - 5 - g 10-

4 - E :

- - - M

- - O

W 3 5 - + 1 ng brassinolide -+- 10 ng brassinolide --t 100 ng brassinolide

0 O 1 2 3

Days after treatment

Figure 2. Time course of brassinolide effects on the internode length of Ikb plants of P. sativum. The nearly expanded third leaves of Ikb plants were treated with brassinolide and the lengths of the immediately upper (4th) internodes were recorded. The internode lengths of wild-type plants are shown for comparison. Bars indicate SE values (n = 10). WT, Wild-type plants.



34 Nomura et al. Plant Physiol. Vol. 113, 1997

Figure 3. Effects of brassinolide on the growthof Ika and Ikb plants of P. sativum. Plants weregrown for 3 d after the application of 100 ng ofbrassinolide to the third leaves, indicated byarrows. A, Ika plant (control); B, brassinolide-treated Ika plant; C, Ikb plant (control); D,brassinolide-treated Ikb plant; and E, wild-typeplant.

B

that application of as little as 1 ng to the third leaves cancause elongation of the 4th internode of 1kb plants to thelength of nontreated wild-type plants. Brassinolide alsorestored the growth of 1kb petioles as shown in Figure 3.When the response of wild-type and Ikb plants to brassino-lide was compared, it was found that application of 10 to100 ng of brassinolide masked the differences in internodelength of the wild-type and mutant plants (Fig. 4). Thebrassinolide-induced elongation of the Ikb internode iscaused by an increase in cell extension rather than by anincrease in cell number (Table III). Internodes of Ikb plantsare normally thicker than those of the wild type. The 4thinternode of Ikb plants remained thick after brassinolidetreatment (Fig. 3). This may be due to ethylene evolutiontriggered by treatment with exogenous brassinolide (Ar-teca and Bachman, 1987). Epinasty was observed when 100ng or more of brassinolide was applied, and this too maybe a consequence of the induction of ethylene productionby brassinolide. A dosage of 1 /u,g of brassinolide per plantcaused no further elongation and resulted in malforma-tions due to stem twisting. The effect of brassinolide onbanding was not clear because no banding was observed inthe young Ikb plants that we used as controls.

25:

20:

15:

10:

0 1 10 100 1000Dosage (ng brassinolide/plant)

Figure 4. Effects of brassinolide on the internode length of wild-typeand Ikb plants of P. sativum. The expanded third leaves of wild-typeand Ikb plants were treated with brassinolide and after 3 d the lengthof the 4th internode immediately above the third leaf was recorded.Bars indicate SE values (n = 10). WT, Wild-type plant.

The lower endogenous BR levels and the ability ofbrassinolide to enhance internode elongation implies thatthe dwarf phenotype of the Ikb mutant is likely to be aconsequence of BR deficiency. This view is supported bythe finding that all of the brassinolide biosynthesis pre-cursors tested except 6-deoxocastasterone exhibited sig-nificant biological activity when applied to Ikb seedlings(Table IV). Brassinolide and castasterone were extremelybiologically active, causing marked elongation of the 4thand even the 5th internodes. The order of activity wasbrassinolide —> castasterone —> typhasterol approximately3-dehydroteasterone —» teasterone. This is the reverse ofthe expected BR biosynthesis pathway (see Fig. 1). Be-cause brassinolide is the most potent BR (Yokota andMori, 1992), the activities of the precursors may be deter-mined by the efficiency of their metabolism to brassinol-ide, as well as the efficiency of their uptake by the plants.The data obtained on biological activity and endogenousBR levels indicate that the dwarfism of the Ikb mutant isdue to a reduced brassinolide content that is a conse-quence of a lesion in the BR biosynthesis pathway that islikely to reside prior to the production of teasterone.Although metabolic studies of both wild-type and mutantseedlings are required, the weak biological activity of6-deoxocastasterone (Table IV) indicates that it is lesslikely to be converted to brassinolide by Ikb plants. Since6-deoxocastasterone also shows a very low activity in therice lamina inclination bioassay (Arima et al., 1984), awidely used BR assay system (Yokota and Mori, 1992), wehave suggested that this compound is an inactivationproduct of BR metabolism rather than a precursor ofbrassinolide (Yokota et al., 1987). However, a recent study(Choi et al., 1996) showed that 6-deoxocastasterone was aprecursor of castasterone and brassinolide in crown gallcells of C. roseus. Thus, it would be necessary to furtherinvestigate metabolic fates of 6-deoxocastasterone as wellas of 6-dihydrocastasterone, a putative intermediate be-tween 6-deoxocastasterone and castasterone.

Although the data presented above indicate thatbrassinolide deficiency has a central role in the reducedgrowth of Ikb plants, it should be noted that the Ikb pheno-type is at least partially attributable to a reduction in freeIAA levels (McKay et al., 1994; Yang et al., 1996). Promo- www.plantphysiol.orgon June 2, 2020 - Published by Downloaded from

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Brassinosteroid Mutants of Garden Pea 35

Table I I I . Effects of brassinolide on the epidermal cells of the 4th internode of Ikb plants of P. sativum In each instance five epidermal cells were measured from each of five plants and data are expressed

as means t SE (n = 5). Epidermal Cells

Cenotype lnternode Length Lenath Width No. in internode

mm Pm Pm Wild type 14.8 ? 0.8 195 ? 6.0 28.5 ? 0.6 76.7 t 0.7 Ikb control 8.8 i 0.6 105 t 2.9 30.2 2 0.5 82.7 t- 6.4 lkb treated with 100 ng 25.0 t 2.1 351 t 1 1 32.9 L 0.6 74.7 ? 8.8

of brassinolide

tion of the growth of pea plants by exogenous IAA has been demonstrated (Yang et al., 1993), and there is a syn- ergistic interaction between IAA and BRs in the promotion of cucumber hypocotyl sections (Katsumi, 1985). Further- more, since application of brassinolide increases the endogenous IAA content of squash seedlings (Eun et al., 1989), it is evident that further studies are necessary at the biochemical leve1 to determine the IAA-BR relationship in the lkb mutant and other plant species.

The brassinolide content of 36-d-old shoots and of the top four internodes of 6-month-old lka plants was 1.7- and 3.4-fold lower, respectively, than that of the wild-type plants (Table 1s). This deficiency, then, is less marked than that in lkb plants. In contrast, there was a distinct 5.5-fold increase in the castasterone content of the 36-d-old lka shoots compared with the wild type, although the effect was less evident in the top four internodes of the lka plants. The levels of 6-deoxocastasterone in the 36-d-old shoots and typhasterol in the 6-month-old tissue were 2.3-fold higher and 3.4-fold lower, respectively, than those found in the wild type. Application of 100 ng of brassinolide to lka plants caused the 4th internode to elongate to the length of the nontreated wild-type plants (Fig. 3; Table V), although no significant effect was observed with the 5th internode. Therefore, the lka plants are approximately 100 times less sensitive to brassinolide than the Ikb mutant. Application of 1 pg of brassinolide caused no further elongation. The biological activity of castasterone was very weak, much lower than that of brassinolide (Table V). In view of its poor response to exogenous brassinolide, the most likely explanation for the dwarf phenotype of the lka seedling is

that it is a BR response mutant. Accumulation of higher levels of castasterone in the lka seedlings may be explain- able by hypothesizing that conversion of castasterone or some dead-end products might be regulated by the BR response pathway.

In conclusion, our results suggest that a deficiency of brassinolide causes dwarfism in the lkb mutant of P. sativum, whereas a reduced response to brassinolide lies behind the dwarf phenotype of lka seedlings. These findings provide strong support for the view that BRs are important endogenous plant growth regulators and that the control of brassinolide levels is an essential requirement for normal plant growth. It has recently been proposed that the dwarf Arabidopsis mutants with a constitutive de-etiolation phenotype, det2 (Li et al., 1996) and cpd (Szekeres et al., 1996), have lesions in their BR biosynthesis pathways. In darkness these mutants exhibit a number of features, such as a short- ened hypocotyl and opening of the apical hook, which are usually associated with de-etiolation processes induced by red light. Both of these aspects of the mutant phenotype were reversed by brassinolide application, raising the pos- sibility of the existence of regulatory interactions between light and BR signaling. However, Reid and Davies (1992) observed that the response of lka and lkb mutants to light was similar to that of wild type, revealing that, in the dark, the Ika and lkb peas do not show the de-etiolation character- istics that occur in the Arabidopsis mutants. In the present study (data not shown), we also examined phenotypes of both the lka and lkb mutants grown in the dark. Although they were shorter in the dark than wild-type plants, no marked de-etiolation was observed, at least from the super-

Table IV. Effects of brassinolide and its biosynthetic intermediates on the internode length of Ikb plants of P. sativum

BRs. Data are means t SE (n = 5). The lengths of the 4th and 5th internodes were measured 3 d after the third leaves were treated with

Genotype Compound Tested Dosage 4th lnternode Length 5th lnternode Length

Pdplant mm

Wild type N o n e Ikb None

Brassinolide 0.1 Castasterone 0.1 Typhasterol 5 3-De h ydroteasterone 5 Teasterone 5 6-Deoxocastasterone 5

17.7 ? 0.6 8.8 ? 0.6

22.3 t- 1.2 23.9 ? 1.1 17.5 t- 3.0 20.2 t- 0.9 13.9 ? 0.8 9.7 t 0.4

11.9 t 1.1 7.7 ? 0.4

18.6 t 2.1 12.4 ? 1.9 6.3 t 0.4 7.2 t 0.6 7.2 t 0.6 7.5 t- 0.3



36 Nomura et al. Plant Physiol. Vol. 11 3, 1997

Table V. Effects of brassinolide and castasterone on the internode length of lka plants of P. sativum

BRs. Data are means 2 SE fn = 5). The lengths of the 4th and 5th internodes were measured 3 d after the third leaves were treated with

Genotype Compound Tested Dosaae 4th Internode Lenath 5th Internode Leneth

ng/p/ant

Wild type None lka None

Brassinol ide 10 1 O0

1 O00 Castasterone 10

1 O0 1 O00

13.2 2 0.4 7.3 2 0.3 8.2 2 0.3

12.4 2 0.8 9.8 t 0.5 7.9 i 0.3 9.3 2 0.5 8.7 t 0.3

mm

8.4 2 0.4 6.6 2 0.5 6.9 2 0.6 7.2 t 0.7 7.1 2 0.4 6.8 2 0.4 6.8 i 0.6 6.6 2 0.5

ficial appearance (e.g. major leaf expansion). Thus, further anatomical and biochemical investigation will be necessary to clarify whether BRs are involved i n photomorphogenesis.

ACKNOWLEDGMENTS

We thank Ryuji Okuda for preparation of deuterated 6- deoxocastasterone, Eiichi Suzuki, Akira Osawa, Junpei Takahashi, and Akihiko Saito for technical assistance, Dr. M. Aburatani (Fuji Chemical Industries, Takaoka, Japan) for the gifts of teasterone and typhasterol, and Dr. S. Takatsuto (Joetsu University of Edu- cation) for supplying deuterated BRs. We also thank Dr. Alan Crozier (Glasgow University, UK) for his critica1 reading of the manuscript.

Received July 17, 1996; accepted August 29, 1996. Copyright Clearance Center: 0032-0889/97/113/0031/07.

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